CA1270467A - System for metering nutrient media to cell culture containers and method - Google Patents
System for metering nutrient media to cell culture containers and methodInfo
- Publication number
- CA1270467A CA1270467A CA000536719A CA536719A CA1270467A CA 1270467 A CA1270467 A CA 1270467A CA 000536719 A CA000536719 A CA 000536719A CA 536719 A CA536719 A CA 536719A CA 1270467 A CA1270467 A CA 1270467A
- Authority
- CA
- Canada
- Prior art keywords
- media
- bag
- flow conduit
- conduit
- flexible
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/14—Bags
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/58—Reaction vessels connected in series or in parallel
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
- C12M33/07—Dosage or metering devices therefore
Abstract
SYSTEM FOR METERING NUTRIENT MEDIA
TO CELL CULTURE CONTAINERS AND METHOD
ABSTRACT OF THE DISCLOSURE
A system for metering nutrient media to cell culture containers includes a media flow conduit which defines at one end means for substantially aseptic communication with the source of the media. In a preferred embodiment, sterile connectors may be used from essentially sterile conditions. A branch conduit extends from the flow conduit at a location spaced from the ends. The branch conduit communicates with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end. The flow conduit also defines a portion downstream from the branch conduit, which communicates with at least one cell culture container, each container being a flexible bag. At least part of the flexible bag is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized, scaled bag having polyethylene walls 0.013 inch thick.
TO CELL CULTURE CONTAINERS AND METHOD
ABSTRACT OF THE DISCLOSURE
A system for metering nutrient media to cell culture containers includes a media flow conduit which defines at one end means for substantially aseptic communication with the source of the media. In a preferred embodiment, sterile connectors may be used from essentially sterile conditions. A branch conduit extends from the flow conduit at a location spaced from the ends. The branch conduit communicates with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end. The flow conduit also defines a portion downstream from the branch conduit, which communicates with at least one cell culture container, each container being a flexible bag. At least part of the flexible bag is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized, scaled bag having polyethylene walls 0.013 inch thick.
Description
r~CllNICAL FIELD
In the culturing of cells, for example hybridoma cells ior manufacturing monoclonal antibodies or the like, the typical cell container is a roller bottle which is placed in a machine to gently agitate the contents during cell culturing operation.
The systems of the prior art are fairly labor intensive and wasteful of space. Also, prior art systems may be "open" systems, in that their interiors must be opened at some time during use, creating the possibility of bacterial contamination. Thus, a laminar flow hood may be needed to use the systems.
Spinner flasks with paddles, and stationary flasks in incubators, are also used to culture cells.
ilowever, such systems are also open systems.
Likewise, all of the above systems are quite labor intensive in their use, thus being expensive, inconvenient, and unreliable for maintaining aseptic conditions.
In many current culturing techniques for gas exchan~e in a conventional incubator, the cap of the flaslc must be left partly open, with a resulting risk of contamination by unwanted microorganisms.
It has been suggested in the patent literature and elsewhere ~o use bags made of gas permeable plastic to culture tissue and the like (see for example U.S. Patent iios. 3,941,662, 3,102,082, and 4,142,940). Advantages that flexible, oxygen permeable containers may have are that they can provide a closed system, while the gas permeability of the container walls permits the use of conventional cell culture incubators without requiring the opening ~' ` ' ~, , ;, , :
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of the cell cul~ure containers. Also, agitators and gas sparging may be eliminated hy the use of such bags. The bags of course take vp less incubator space; will not break if dropped; and are disposable/ eliminating thP
need to clean and resterilize glassware. Also, bays are generally cheap and disposable, which avoids the need to resterilize and depyrogenate reusable containers, as in the prior art.
U.S. patent no. 4,140,162 discloses special gas-permeable bags, these bags being in commercial use for the storage of platelets.
In accordance with this invention, various systems are pr~vided to permit aseptic and even sterile filling of gas permeable bags with cell growth media, to facilitate cell culturing processes making use of such bags. There always is the serious need to preserve substantially aseptic conditions during ~ell culturing.
With this invention, the number of cell cultures which are lost due to a break in aseptic conditions may be reduced, while significant labor savings are provided as well.
SUMMARY OF THE DnnENTION
Various aspects of the invention are as follows:
A method for metering nutrient media into at least one cell culture container in substantially a~eptic manner, which method comprises passing liquid nutrient media through a media flow conduit which is in substantially aseptic connection at one end with a source o~ said media; passing said media from the flow conduit through a branch conduit extending from said flow conduit at a location spaced from its ends, into means for receiving a predetermined-volume aliquot of media; passing said predetermined-volume aliquot of nutrient media again through the branch conduit into and ~27~
- 2a -through a downstream por~ion of said media flow conduit, into at least one attached, flexible, flat-collapsible bag, at least part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at leas~ 2 times ~he carbon dioxide diffusion between the bag interior and ex~erior, compared with an identically-sized, sealed bag having polyethylene walls 0.013 inch thick.
A method for metering nutrient media into at least one cell culture container in substantially aseptic manner, which method comprises passing liquid r.utrient media through a media flow conduit which is in substantially aseptic connection at one end with a source of said media, and metering a~ least ~ne aliquot of said media through a downstream portion of said media flow conduit into at least one attached, flexible bag, at least part of which is made of a material capable of providing at least 2 times the oxygen diffucion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with identically-sized sealed bag having polyethylene wall 0.013 inch thick, and culturing human white blood cells in the nutrient media within said flexible bag.
A system for metering nutrient media to at least one cell culture container in substantially aseptic manner, which comprises:
a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media: a branch conduit extending from said flow conduit at a location spaced from its ends, said branch conduit communicating with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end; said flow conduit defining a portion downstream from said branch conduit which communicates with said cell culture container, each container being a flexible bag, at least ~7~3~
- ~b -part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide di~fusion betwPen the bag interior and exterior, compared with an idsntically -sized, sealed bag having polyethylene walls 0.013 inch thick, said bag containing a flexible, open cell structure to increase the sur~ace area within each bay for cell adhesion, said flexible bag containing an access port, said accPss port carrying tubular coupler means defining a pointed inner end and carryiny needle pierceable, resealable injection site means on its ouker end, to permit inoculation by a needle and syringe of culture media in said bag with desired cells in aseptic manner.
A system for metering nutrient media to a plurality of cell culture containers in substantially aseptic manner, which comprises:
a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media; a branch conduit extending from said flow conduit at a location spaced from its ends;
said branch conduit communicating with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end, said flow conduit defining a branched portion downstream from said branch conduit which communicates with said cell culture containers, ea~h container being a flexible bag, at least part of which i5 made of a material capable of providing at least ~ times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized, sealed bag having polyethylene walls 0.013 inch thick, each bag containing a flexible, open cell structure to increase the surface area within each bag for cell adhesion.
~27~.~gk~7 - 2c -A system for metering nutrient media to at least one cell culture contain0r in sub~tantially aseptic manner, which comprises:
a container of said nutrient media; a media flow conduit communicating at one end with the interior of said container in substantially aseptic mann2r; means for passing aliquots o~ said nutrient media of predetermined volume through said media flow conduit, said flow conduit defining a portion adjacent its end opposed to said one end which communicates with at least one cell culture container, said cell culture container being a flexible bag, at least part of which is made of a material providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized sealed bag having polyethylene walls 0.013 inch thick, said bag containing a flexible, open cell sitructure to increase the surface area within each bag for cell adhesion.
D~SCRIPTIO~ OF THE INVENTION
In this invention, a system is provided for metering nutrient media to at least one cell culture container in substantially aseptic manner. The system i`~ !
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comprises a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media. Typically, a branch conduit extends from the flow conduit at a location spaced from its ends. The branch conduit communicates with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end. ~lternatively, other flow metering means may be provided.
The flow conduit defines a portion downstream from the branch conduit which is adapted to communicate with the cell culture container or containers. ~ach container is a flexible bag, at least part of which is made of a material providing at least 2 times the oxygen diffusion between the bag interior and exterior, when compared with an idell~ically-sized, sealed bag having polyethylene walls 0.013 inch thick.
Thus, a large container of nutrient media mny be connected to the system, and one or more aliquots of predetermined volumes of media may flow through the media flow conduit, with the aliquots being formed by an appropriate means. Thereafter, the individual aliquots may be sent to individual, flexible bags so that desired volumes of media may be inserted into any number of said flexible ba~s under aseptic conditions. Because the bags have adequate oxygen and carbon dioxide diffusion through their walls, they may be used for cell cul~uring while providing the advantages described above.
The large, nutrient media container may have gas and water-impermeable walls for stable storage of the media, contrary to the flexible bags used herein.
In one instance, the means for receiving the , ~ . "
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predetermined-volume aliquots may be a container or chamber such as a burette, or even a flexible, collapsible bag connectcd to the branch conduit, often with a 3-way valve being positioned at the junction of the media flow conduit and branch conduit to control flow into and out of the branch conduit. In another example, the branch conduit may connect with a s~ringe, the plunger of which may be used to draw the predetermined-volume aliquots, one after another, into the syringe. The flow conduit in this circumstance ` may define one-way valve means which are positioned upstream and downstream from the branch conduit, to permit only downstream flow as controlled by the syringe. Thus, one may draw a predetermined amount of media into the syringe by pulling the plunger out, and then expelling it back into the flow conduit. ~ecause of the one-way valve system, that volume of fluid is forced downstream to provide a connected, flexible bag with a predetermined-volume aliquot of media.
The downstream portion of the flow conduit may define a plurality of branching conduit end portions, each end portion being connected in substantially aseptic manner to one of said flexible bags. The branching conduit end portions and bags may cach define sterilizable connection means/ permitting the establisllment of sterile connection between said bags and branching conduit end portions without subjecting the whole system to sterilizing conditions.
Such sterilizable connection means have been approved and used in the manufacture of medical sets by Travenol Laboratories, Inc. of Deerfield, Illinois, and other ~nown sterilizable systems exist as well.
See in particular U.S. Patent No. Rc. 32,056, Lilcewise, similar sterilizable connection :, ' means for forming a sterilized connection with the media source may be used to obtain aseptic connection at the upstream end of the system~ without subjecting the whole system to sterilizing conditions.
Additionally, the flow conduit may carry an access site between the one end at its upstre~m end, and the branch conduit. Ior example, this access site may be used to add serum, when that is a desired component, of each aliquot of media liquid by first passing some oi the aliquot of media liquid through the branch conduit; then adding some serum through the access site; and finally adding the final amount of media liquid to f~ll the predetermined aliquot, which is then passed once again through the branch conduit downstream to a Elexible bag.
Additionally, the flexible bag of this invention may define an outer enclosing wall, and an inner, open cellular structure which is compatible with growing cells and serves as a site for cellular adherence. Preferably, the inner, open cellular structure is rather flexible, so that the bag may be provided in initial condition where it is wrapped in shrink wrap means under compressive pressure, causing the ce]lular structure within the bag to be in a relative]y collapsed configuration, to cause the bag to be thinner than in its configuration of use.
Accordingly, when the shrink wrap is removed, the bag and cellular structure can expand to assume their normal condition of use.
~s a result of this, the flexible bags of this invention may have very high interior æurface area, serving as adherence sites for cells that are cultured while adhering to a surface. The nature Or the material Or the cellular structure may be of any .
~7~ 7 appropriate and compatible material for culturing cells. Preferably, the cellular structure may be of the honeycomb type with hexagonal channels passing through it. The hexagonal (or other shaped) honeycomb cell passage ways preferably are aligned to terminate adjacent the access port means in the bag. Lilcewise plastic open cell foams, fibrous materials including hollow fibers, or any other open-cell type materials may be used as the cellular structure to provide adherence areas to cells that adhere to a surface during culturing.
In addition to the open cellular structure, other means may be provided inside the flexible bag to increase tlle area of cell adherence. For example, a microcarrier such as small glass spheres or sodium alginate may be employed to increase surface area for cell adherence.
Alternatively, bags with open, empty interiors may be used for culturing cells which do not adhere to surfaces, for example, suspension cells such as mast hybridoma cells and human monocytes, in a bag made of a nonadherent material.
The other parts of the system of this invention may be made of conventional plastic materials for such products as is currently available.
The tubular conduit mernbers may be made of a conventional polyvinyl chloride formulation, but preferably, the flexible bag may be made of an oxygen and carbon dioxide permeable material of a formula~ion as described in U.S. Patent No. 4,1~,162~
~dditionally, thin-walled polyethy]ene, or polytetrafluoroethylene, may be used for such bags, as described in the prior art. Other formulations as well may be used for gas-permeal)le bag walls, for exarnp]e .
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poly(ethylene-Yinyl acetate) 7 silicones, or a hydrocarbon resin sold under the trademark TPX.
DESCRIPTION OF TIIE D~AI~INGS
In the drawings, Fig~ 1 is an elevational view of a system for metering nutrient media to a cell culture container in substantially aseptic Manner.
Fig. lA is a detailed perspective Yiew of the connectors used for connecting the media flow conduit with a flexible bag in accordance with this invention.
Fig. 2 is an elevational view of an alternative embodiment of media flow conduit.
Fig. 3 is an elevational view of an alternative embodimellt oi media flow conduit which may be used in conjunction with the media storage bag and the cell culture container of Fig. 1, to provide liquid media to said cell culture continer.
Fig. 4 is an elevational view of another alternatiYe embodiment oi media flow conduit which may be used in conjunction with a media storage bottle and the cell culture container of Fig. 1, to provide liquid media ~o said cell culture container.
Fig. 5 is an exploded view of another system in accordance with this invention for transferring in sterile manner media from a supply container to a plurality of flexible cell culture containers, including ancillary equipment that may be used in conjullction therewitll.
Fig. 6 is an elevational view of another embo(liment o~ cell culture container in accordance i.
~ . .
with this invention.
Fig. 7 is an elevational view of the bag of Fig. 6, rotated 90 degrces about its longitudinal axis, witll portions brolcen away, and showing how it may be packaged in a shrink wrap package.
Fig. 8 is a fragmentary, longitudinal sectional view taken along line 8-g of lig. 7.
Fig, 9 is an elevational view, with portions brokcn away, of the bag of lig. 6 after the shrinlc wrap has been removed.
Fig. 10 is a sectional view taken along line lO-l0 of Fig. 9~ showing the expanded, honeycomb nature of the cellular structure within the bag.
DESCRIPTION OF SPECIFIC EMBODI~IENTS
Referring to Figs. 1 and la, a first cmbodiment of this invention is disclosed. A source of media lO is disc]osed as a flexible bag of nutrient media for the growth of a certain cell. The cells may be hybridomas from ~hich monoclonal antibodies may be obtained by culturing.
I~lternatively, the cells may be white blood cells such as lymphocytes from a cancer patient. In this circumstance, as is taught in the literature, culturing of the lymphocyte with a lymphokine such as interleukin-2 can provide an activated lymphocyte which is more active in the process of identifying and killing tumor cells. These activated lymphocytes may then be returned to the patient for treatment of the cancer.
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In accordance with this invention, individual bags 12 for the culturing of cells are ~illed in a substantially asep~ic manner with at 1 ast one predetermined-volume aliquot of media through a media flow conduit 14.
Thereafter, the cells for culturing are placed into bag 12. The specific media used will be any appropriate type of media desired, depending on the particular cells to be cultured~ Man~ different varieties of media are taught in the prior art. It ~hould also be known that the verb "culture" may refer to the maintenance of cells and their multiplication by growth, but alternatively, it can apply to situations where the cells do not multiply but simply are treated (for example, with a lymphokine) to change their characteristics. One cell medium tha~ may be used for lymphocytes is a medium consisting of RPMI 1640 (low endotoxin; M.A. Bioproducts of Walkersville, Md.~ also including 10 units per ml of penicillin, 10 micrograms/ml. o~ streptomycin sulfate, 2 ml. o~
glutamine, 5 micrograms per ml. of gentamicin, and 2 percent by weight of heat-inactivated human AB serum.
This formulation is taught in Rosenberg U.S. Patent 4,690,915. Appropriate media for hybridoma cells are widely available in the literature.
Media flow conduit 14 dafines at one end 16 a conventional spiXe for penetration of media bag 10. If a media bottle is used with a conventional plug in its mouth of a design used with parenteral solution bottles, spike 16 may be a conventional vented spike of known design.
Media flow conduit 14 carries a conventional injection site 18 into which supplemental materials ~ ~7~ -r~à, - 1 o -may be inserted by a needle syringe in a gPnerally aseptic manner, for e~amplc, serum such as fetal bovine serum, amino acids, vitamins, or antibiotics, which typically are not stored with the artificial nutrient media within bag ]0.
i~ledia flow conduit 14 comprises flexible tubing typically of polyvinylchloride or any other conventional tubing of the type used in parenteral solution administration and the like. I~oller clamp 20 may be provided to control flow through the tubing, and may be of conventional design.
Media flow conduit 14 may have a dividable connection comprising a pair of luer lock connectors 22,24 which are connected together with an aseptic seal, being initially provided in sterile condition as long as protective caps 26, 28 have not been previously removed. Designs of Luer connectors are l;nown to the art, some of them being disclosed in Dennehey et al., U.S. Patent No. 4,346,703 and Ruschke et al. U.S. patent No. 4,452,473.
Liquid nutrient media may flow from bag 10, through the connection of members 22, 24 into 3-way valve 30, which is also of conventional design9 being commercially sold on various parenteral solution sets and the like by various companies. The rotatable handle 32 of valvc 30 may be in a position so that the flow travels upwardly through branch conduit 34, which may connect through a burette 36, carrying a vent 38 and an injection site 40. Such burettes are also in commercial use in the parenteral solution field, One such burette is described in U.S, patent No.
4,322,247. This particular burette may be connccted to branch conduit 34 by the action of connectors 42, 44 of design similar to conncctors 22, 24.
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Accordingly, a predetermined-volume aliquot of nutrient media from bag lU may flow through media flow conduit 14 into burette 36, which has volume measuring indiccs, until the predetermined volume of the aliquot has been reached. lhen, the user can shift 3-way valve 32 to cause the flow to return from burette 36, through branch conduit 34, into the portion 14a of conduit 14 which is downstream from branch conduit 34, Clamp 46, of conventional design, may be loosened to permit such flow. Downstream portion 14a may be connected to 3-way valve 30 by more connectors 4~, 50 of similar design to connectors 22, 24.
In those circumstances where the predetermined-volume aliquot is of a constant volume every time, burette 36 may be replaced with a simple, collapsible bag having a filled volume which corresponds to the desired volume of the aliquot. The burette is useful when variable volume aliquots are intended to be drawn from cell culture container 10.
A pressure head may be provided to the system by raising media bag 10 above branch conduit 34, the volume metering means 36 such as the burette being raised over bag 12 as well.
Connection to bag 12 by conduit 14a may be obtained by the use of similar connectors 52, 54 for placing the predetermined-volume aliquot of nutrient media into bag 12. 3ag 12 per se is known to the art, being described in U.S. Patent Nos. Re. 31,135 and 30 4,140,162. The particu]ar material of bag 12 has a high capability for oxygen and carbon dioxide diffusion, very substantially over that of po]yvinylchloride and other materials in prior art bags. Also, fewer lcachable materials are present in :
,.
~7~Æ~7 the bag materia].s of the eited patent no. 4,148,162.
'I`ypically, bag 12 is filled with nutrient media and cells to no more than about one quarter to one half of its LuJ1 capacity, to provide a relatively high ratio of internal surface area to vo].ume of the media and cells, so that abundant oY.ygen ean diffuse into the bag and carbon dioxide can diffuse out of the bag~, to faciliLate cell metabolism and xrowth.
Thus, by the system of this invention, one or more cell culture l-ags 12 ean be prepared for receiving their cells for eulturing by reeeiving one or more ali~uots of nutrient media of predetermined volume, under substantially aseptie eonditions to minimize tlle eurrent problem eommonly eneountered in eell culturing today of eontamination of many of the ce]~ culture samples.
The cells themselves may be inserted in~o bags 12 by removal of conventiona].ly designed plastie port proteetors 56 whieh elose access port 58. The ce].1s may be added througll tubular sampling si.te eoupler 60, whicll penetrates an internal diaphragm in port 68 to gain aecess to the eontainer. Coupler 60 may have a conventional injection site 61 on its ouLer end. If desired, coupler 60 may remain within port 58 to elose it during ~:he culturinæ operation.
Alternative].y, an added closure mAy be provided for port 58. If desired, a needle syringe may be used to pierce injecti.on site 61 to inject the eells into bag 12. ()therwise, bag ]2 may carry its own injection site.
To separate bag 12 from its media fl.ow conduit set ]4, 14a, one may p]aee a heat seal or other seal aeross a portion of the tubing 14a, whieh may be made of polyvinylcllloride, or another -thermoplastic material. Alternatively, short length of tubing 62 may be transverse]y sealed. One then cuts through the seal while maintaining its integrity on preferably both sides so that bag 12, with its aliquot of nutrient media, can be separated and taken or use in a cell cultureO
Turning to Fig. 2, a simplified media flow conduit 64 is provided, being the length of ~lexible ~ubing 6~ whicll is shown to serve as roller pump t:ubing in a conventional ro]ler pllmp G8, which is only partially and schematically shown, and connector 67 o conduit 64 may be a connector of conventional design, similar for examp~e, to connector 22. End 68a provides a connector which may be similar to connector 24.
Conduit 64 ~serves as a substitute for branch conduit 34, 3-way valve 30, an~ burette 36. It may be connected by connectors 67, 68a to media flow conduit portions 14, 14a to provide an assembly that is otherwise suhstantially equivalent to that shown in Fig. ], where the individua] flliquots of nutrient media f]ow in their predeternlined volumes in a manner as governed by rol]er pump 68.
Turning to ~ig. 3, another embodiment is djsclose~, serving as a suhstitute for branch conduit 34, 3-way valve 30, and burette 36. ~ differing brallcll con(luit 63 is provi(led in a set portion 65 which carries connectors 67a, 69 on their respective en(ls for connecting with otiler portions 14, 14a of the me(lia flow conduit. Clalnp 70 nlay also bc provided as shohn.
A~ljacent to branch conduit housing 72 lie a pa;r of one way valves 74, 76 of conventional design Lo assure that flow througll conduit 65 goes only in one direc~ion from connector 67a to connector 69.
Connector 67a may connect w].th media flow conduit ~ortion 14, while connec-or ~3~ connects with portion 14a. A syringe 78, o conventional design, may be connecte~ by a simple luer connection to branch conduit 63 (removing respective end caps 8Q, 82).
8yringe 7~ carries vo]ume indicating indicia.
I~ccordingly, when the entire set is set up, with the structure of i:i.g. 3 replacing one-way valve 3(), branch contluit 34, and burette 36, one can simply use syringe 78 as a pump to draw from container the predetern)ined-volllme al.iquot, and then to force it downstream to conduit portion ]4a and ba~ or bags 12, due to the action of one way valves 74, 76. ~lany designs of appropriate one way valve are known such as duckbill valves, flapper va]ves antl the like. One particular one way valve that may be used for valves 74, 76 is a valve sold by Travenol Laboratories, Inc.
as part of a parenteral solution set.
Referring to Fig. 4, anothcr system in accordance with the system is provided for transferring liquid nutrient media from a rigid media bottle lOa to a culture container 12a, which may be a Ele~:ible bag made in accordance witll conventional Lechnology.
Stopper 84 fits in the neclc of bottle ].Oa, and derincs n port 8~3 for spike penetration. Threclded cap ~8 is fi~te(l onto the neck of bottle lOa to retain stopper 84 i.n position.
Integral airway spike 90, of a commerciAl.
design for penetration of bottles of paren~eral.
so].uti.on is provided, being conrlected to a first length ')~ of a media f]ow condui.t. Conduit 92 ~er~linates in molrled asselllbly 94, which includes brancll contlui.t (~fi an(l a pai.r of onc-way va]ves 98, 100 .. ..
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respectively positioned upstream and downstream from branch condlIit (~6. A more compact design of valves for tlle same purpose combined into a single structure is disc]osed in U.S. paLent No. 4,084,606.
Syringe ]~2, of conventional design, may connect to branch conduit 96 so that as the syringe is pumped, media solution flows downstream in pre(IeterminecI-volume aliquots as controlled by each stroke of the syringe handle 104.
~ssembly 94 also carries male luer connector 106, which is adapted to connect with female luer 108, w}lich, in turn, is carried by media flow conduit section 110. Conduit section 110, in turn, is controlled hy a roller clamp 112, and communicates througi- Y-connector 114 and conduit section 116 to cu]ture container l~a. Container 12a may be a collapsible bag made in accordance with U.S.Pat. No.
4,14~,162 or any other flexible sheeting materia]
having the required high oxygen and carhon dioxide dirfusioIl, when compared with a bag having walls of polyethylenc.
The remaining branch tube 118 is controlled by another roller clamp 120, and may terminate in a male needle adapter 122 or another conventional colIllecLer.
In use, sampling site coup]er 123 may be a holloh~ tube with a pointed end plus a needle-piercab]e situs 124, and may be used to penetrate access port l~G of container 12a t:o insert the cells for cu]turing, typically arter filling of container 12a with the predetermined-vo]unIe aliquot Or media solution via syringe 1()2, After such filling, in a manner previolIsly describe(I, rol]er c]nmp 1]2 may be closed an(I connectors ]06, 108 separated to remove the ~ : ,"
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container to its cuituring environlnent, or tube 110 may be heat sealed shut. Added nutrients or other agents mfly be adde(l Lhrollgh access port 128 as desired. Situs ]24 may be repeatedly pierced as de~sired f()r addin~ or removi 11~ media or other agents.
After the culturing phase, further processing steps may be perfornled. In the case of suspended cells where protein production is an end goa], the product-ricll supernatent may be sep~rated from the cells and harvesLed, such as with a n centrifugaLion or membrane system. Alternatively, the contents of the culture container may be transferred to other containers for further processing through tubings 116, 118 and adal)ter 122.
~ig. 5 is an explode(i view of anotiler system in accordance with this inventioll in which sterility of Lhe materials processed may be assured with a higher (legree oL relial)ility. Nutrient media container l~b, of a design similar to either previous embodiment, may be connected to a media flow conduit 130 by means of a pair of identical sterile connector devices 132, a specific embodiment of which has been demonstr.l~e(l as a product by Travenol Laboratories, Inc. and which operate in accordance with the principles described in U.S. I'at. No. ~e. 32,~56.
l`hesr-~ connectors are ]ocked together, and then irratllated wiLh intense light or infrared radiation, resulting in a sterile, fluid pathway being formed between the two connectors 132.
As in an earlier embodiment, three-way valve 134 is provi(le(l, carrying branch conduit ]36 whicll conllects with a burette ]3~. Alternative]y, burette ~3~ may be replaced with n sma]], connected bag if desired for cost savings with the maxirnllnl fluid volume ~' .
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of the bag being identicai to the desired, predetermined-volume aliquot.
I)ownstream portion 14() of the flow conduit is branclled a~s shown to provi(le a series of branches 5 ]42, each of which is terminated with a sterile connecter 132a, corre~poll(ling in desip,n to connectors 132, }.ach connecter ]32fl may be connected in sterile manner with a sterile connector 132h of one of the serie~ of ~lexible bags 134a, In this eireumstance, 10 hy appropriate clamping of lines 142 (which may be equippecl witll slide clamps or the like), the desired predetern\ined-vo]ulne aliguots of media solution may be metered from container l()b, through braneh eonduit 136 alld burette 13~, sequentially into each of ba~s 134 15 after the sterile connection bet-~leen connectors 132a and 132b has heen made.
If desired, added connection sites may be provide(l by Y-connector 136 which includes a Y-shaped arrangement of conduits, each terminated with a 20 sterile connector 132c, similar in design to sterile eonnectors ]32a. Ey the use of members ~36, the nllmber of availal)]e sites for connection with bags 134a may be inereased by any desirecl number.
Added aneillary equipment 139 eonstitutes a 25 y-eonnector having a filter. Sterile eonneetors 132d may be eonnectecl into line llith eonneetors 132a or 132c, Lor example, so that the media material may be filtered. Alternntively, arter eulturing of the eel~s hns taken plaee in bag 134a, the eontents of the ba~
30 r,~ay he eonneeted in sterile manner to eonneetor 132cd through its seeond eonneetor 132e, and the eontents of thc~ bag fi]terecl. I`he filtrate then ean pass throu~h anotller eonllector ]32f to another contniner sucll as riltrate eontainc r 133 with connectors 132j, for .....
, . . . . .
7~ 7 storage or f~lrther processing. By this manner, ~i]Lrate whic}l mny carry a biochemical product manufactured by tlle cel~s, may be separated from the cells in reliably steri.le manner, once all the components have been initially steri].izecl.
Kecep~acle container 141 may also be used in any desired manner witll its connector 132g, for extlmple to connect iLs connector 132g with with the remaining outlet connector 132i of fi].ter member 139.
~s may be desired serum may be added to the media by means of serum container 142a, carrying its sterile connector 132h. It may be used to make connection with the second connector 132 of media bag l~b so that the serum and media may be mixed prior to the metering of the various a].iquots i.nto the respective bags 134a. Alternat:ively, connector 132h may be connected through the use of ancillary device 13'.~.
~ther par~s o tlle set suc]l as injection site 144 and clamps l4(), 1.4~ may be l)rovided as deemed appropriate.
Turning now to Figs. 6-10, a specific type of flexible bag 15() is disclosed for use in this invelltion, being analagous in function and outer structure to bags l~, ].2a, and 134a. Bag 15~ may be made of f]exih]e, plastic sheeting having the desired niill oXygCIl and carbon di.oxide diffusion chartlcteristics as discussed above. Ilowever, bag 150 is providetl with a aci.1ity or culturing cclls that need to adhere to a surface. ~y thi.s invention, a bag havillg an ultrahi~ll surface aren in its interior is provided so that a large number of such cells may be cul.LIlre(l in the bag under conditi.oll.s thclt are optimal for their growtll and deve~opnnent.
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~ s shown, flexible bag ]50 mny be made of a film wall materia.l haYing the desired, adequate oxygen and carbon dioxi.de diffu.sioll capability to permit the culturing of ce]ls tllereill under sealed, substantia].ly asel)tic cond]t.i.ons. ~ag 150 defines an outer enclosing ~all 152 which may, for example, be the remainder Or n blo-- "~01 ded parison in accordance with l)lo~ mo~dinc, technology, or may be made of a pair of peri[)hera].ly seale(l sheets in accordnnce with hcat or 10 1'.1~. sealing technologies. Within bag 150 is an e inner, open cellular struc~ure whicll is eapable of supporting growing cells, and serves as a site for eell adherence. ~ee particlllar]y Figs. 9 and 10 for broken away and sectionnl views of the cel.]ular 15 s~ructure 154, An enlarged view of the hexa~onnl cells 156 is shown in ~ig. 10. Each cell 156 is of hexagoncll cross seetion, and is typieally open at its respective ends, rnther in the form of a honeycomh shape, with the individual ee].]s 154 being aligne(l 20 ~ith one end of eaeh honeyeomb eell passageway 158 termi~ ting adjaeent the access ports 16~ of said bag.
The open eellular strllcture may be made of ally desired mrlteria]. suital)le for the adllesioll and llealtlly maintenance of the ce].ls, particularly 25 relative].y hi.gh surface enerl;y po]ymers, preferably ha~ing n surfclce energy of at least ahout that of eellulose for example. ~xnml)les of such materials ine~ude cellulose, polyestèr materials, high surfaee energy n~lon (such as thnt so]d by the llexcel Co. of 30 Ca].ifornia), corona discllarge treated polyolefin, or the like.
I'he open cellular material may, in the alternative, coml)rise ho].lo~ fibers, particular].y tho.se extending i.n a manner analagous to the direction - : .
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ol ex~ension of open ce]ls 15~. 'I'hc rnaterial may be an open-celled foam, or a fibrous mass, either woven or nonwove~ he tcrm cel]ular is intended to imply open porosity, not necessarily indicating that rliscree~ cells are present as in an operl-cel]ed foain7 althougil such is inten(led to be included as one possiblc type of opcn- celle(l nnaterial.
l`he flexible bag 150 may be compressed in packaging by wrapping in a known hent-shrink w~ap.
The advantage of packaging bag 150 by a shrink wrap technique as shorn in Figs. 7 and ~ is primarily to save space until bag ]50 is ready for use.
Sterile filter vent 164 may be provided for communication between the interior and exterior of the bag to facilitate this process. Specifically, the vent may contain a filter of hydrophobic material in accordance with known technology which exhibits strong bacteria bloclcing characteristics.
As shown in lig. 8, cellular structure 156 may be compressed by the compressive action of the heat shrink wrap 162, for efficient shipping arl(l storage.
When it is desired to make use of bag 150, shrink wrap 162 may be torn a~ray, permitting bag 150 to expand ~y natural expansive action of open cellular material 154, t:o the configuration shown in Figs. 9 and ]0. Alternatively, this expansion may take place upon softening of cellular material 154 as it is wel:ted rith liquid nutrient media.
l~uring tlle culturing of cells in a bag simi]ar to bag 150, :it, may be (Iesire(l to provide a mecllanica] means to gent],y knead the bclg to assure circulation of nutrient media throug}l the open cel]s o[ material ]54, so that oxygen and carbon dioxide ma~
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~;Z7Q ~6 d diffuse into and out of bag wa]l 152, and no stagnant l)ockets of nutrient media will form, Wi)iCII may interfere with cel] viabi]ityO
'l'he abovc has been offered for illustrative pur~oses only, and is not intcnded to limit thc scope of the invention of this application, whicll is a~s dcf:incd in the claim.s bclow.
, ,
In the culturing of cells, for example hybridoma cells ior manufacturing monoclonal antibodies or the like, the typical cell container is a roller bottle which is placed in a machine to gently agitate the contents during cell culturing operation.
The systems of the prior art are fairly labor intensive and wasteful of space. Also, prior art systems may be "open" systems, in that their interiors must be opened at some time during use, creating the possibility of bacterial contamination. Thus, a laminar flow hood may be needed to use the systems.
Spinner flasks with paddles, and stationary flasks in incubators, are also used to culture cells.
ilowever, such systems are also open systems.
Likewise, all of the above systems are quite labor intensive in their use, thus being expensive, inconvenient, and unreliable for maintaining aseptic conditions.
In many current culturing techniques for gas exchan~e in a conventional incubator, the cap of the flaslc must be left partly open, with a resulting risk of contamination by unwanted microorganisms.
It has been suggested in the patent literature and elsewhere ~o use bags made of gas permeable plastic to culture tissue and the like (see for example U.S. Patent iios. 3,941,662, 3,102,082, and 4,142,940). Advantages that flexible, oxygen permeable containers may have are that they can provide a closed system, while the gas permeability of the container walls permits the use of conventional cell culture incubators without requiring the opening ~' ` ' ~, , ;, , :
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of the cell cul~ure containers. Also, agitators and gas sparging may be eliminated hy the use of such bags. The bags of course take vp less incubator space; will not break if dropped; and are disposable/ eliminating thP
need to clean and resterilize glassware. Also, bays are generally cheap and disposable, which avoids the need to resterilize and depyrogenate reusable containers, as in the prior art.
U.S. patent no. 4,140,162 discloses special gas-permeable bags, these bags being in commercial use for the storage of platelets.
In accordance with this invention, various systems are pr~vided to permit aseptic and even sterile filling of gas permeable bags with cell growth media, to facilitate cell culturing processes making use of such bags. There always is the serious need to preserve substantially aseptic conditions during ~ell culturing.
With this invention, the number of cell cultures which are lost due to a break in aseptic conditions may be reduced, while significant labor savings are provided as well.
SUMMARY OF THE DnnENTION
Various aspects of the invention are as follows:
A method for metering nutrient media into at least one cell culture container in substantially a~eptic manner, which method comprises passing liquid nutrient media through a media flow conduit which is in substantially aseptic connection at one end with a source o~ said media; passing said media from the flow conduit through a branch conduit extending from said flow conduit at a location spaced from its ends, into means for receiving a predetermined-volume aliquot of media; passing said predetermined-volume aliquot of nutrient media again through the branch conduit into and ~27~
- 2a -through a downstream por~ion of said media flow conduit, into at least one attached, flexible, flat-collapsible bag, at least part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at leas~ 2 times ~he carbon dioxide diffusion between the bag interior and ex~erior, compared with an identically-sized, sealed bag having polyethylene walls 0.013 inch thick.
A method for metering nutrient media into at least one cell culture container in substantially aseptic manner, which method comprises passing liquid r.utrient media through a media flow conduit which is in substantially aseptic connection at one end with a source of said media, and metering a~ least ~ne aliquot of said media through a downstream portion of said media flow conduit into at least one attached, flexible bag, at least part of which is made of a material capable of providing at least 2 times the oxygen diffucion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with identically-sized sealed bag having polyethylene wall 0.013 inch thick, and culturing human white blood cells in the nutrient media within said flexible bag.
A system for metering nutrient media to at least one cell culture container in substantially aseptic manner, which comprises:
a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media: a branch conduit extending from said flow conduit at a location spaced from its ends, said branch conduit communicating with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end; said flow conduit defining a portion downstream from said branch conduit which communicates with said cell culture container, each container being a flexible bag, at least ~7~3~
- ~b -part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide di~fusion betwPen the bag interior and exterior, compared with an idsntically -sized, sealed bag having polyethylene walls 0.013 inch thick, said bag containing a flexible, open cell structure to increase the sur~ace area within each bay for cell adhesion, said flexible bag containing an access port, said accPss port carrying tubular coupler means defining a pointed inner end and carryiny needle pierceable, resealable injection site means on its ouker end, to permit inoculation by a needle and syringe of culture media in said bag with desired cells in aseptic manner.
A system for metering nutrient media to a plurality of cell culture containers in substantially aseptic manner, which comprises:
a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media; a branch conduit extending from said flow conduit at a location spaced from its ends;
said branch conduit communicating with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end, said flow conduit defining a branched portion downstream from said branch conduit which communicates with said cell culture containers, ea~h container being a flexible bag, at least part of which i5 made of a material capable of providing at least ~ times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized, sealed bag having polyethylene walls 0.013 inch thick, each bag containing a flexible, open cell structure to increase the surface area within each bag for cell adhesion.
~27~.~gk~7 - 2c -A system for metering nutrient media to at least one cell culture contain0r in sub~tantially aseptic manner, which comprises:
a container of said nutrient media; a media flow conduit communicating at one end with the interior of said container in substantially aseptic mann2r; means for passing aliquots o~ said nutrient media of predetermined volume through said media flow conduit, said flow conduit defining a portion adjacent its end opposed to said one end which communicates with at least one cell culture container, said cell culture container being a flexible bag, at least part of which is made of a material providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized sealed bag having polyethylene walls 0.013 inch thick, said bag containing a flexible, open cell sitructure to increase the surface area within each bag for cell adhesion.
D~SCRIPTIO~ OF THE INVENTION
In this invention, a system is provided for metering nutrient media to at least one cell culture container in substantially aseptic manner. The system i`~ !
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comprises a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media. Typically, a branch conduit extends from the flow conduit at a location spaced from its ends. The branch conduit communicates with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end. ~lternatively, other flow metering means may be provided.
The flow conduit defines a portion downstream from the branch conduit which is adapted to communicate with the cell culture container or containers. ~ach container is a flexible bag, at least part of which is made of a material providing at least 2 times the oxygen diffusion between the bag interior and exterior, when compared with an idell~ically-sized, sealed bag having polyethylene walls 0.013 inch thick.
Thus, a large container of nutrient media mny be connected to the system, and one or more aliquots of predetermined volumes of media may flow through the media flow conduit, with the aliquots being formed by an appropriate means. Thereafter, the individual aliquots may be sent to individual, flexible bags so that desired volumes of media may be inserted into any number of said flexible ba~s under aseptic conditions. Because the bags have adequate oxygen and carbon dioxide diffusion through their walls, they may be used for cell cul~uring while providing the advantages described above.
The large, nutrient media container may have gas and water-impermeable walls for stable storage of the media, contrary to the flexible bags used herein.
In one instance, the means for receiving the , ~ . "
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predetermined-volume aliquots may be a container or chamber such as a burette, or even a flexible, collapsible bag connectcd to the branch conduit, often with a 3-way valve being positioned at the junction of the media flow conduit and branch conduit to control flow into and out of the branch conduit. In another example, the branch conduit may connect with a s~ringe, the plunger of which may be used to draw the predetermined-volume aliquots, one after another, into the syringe. The flow conduit in this circumstance ` may define one-way valve means which are positioned upstream and downstream from the branch conduit, to permit only downstream flow as controlled by the syringe. Thus, one may draw a predetermined amount of media into the syringe by pulling the plunger out, and then expelling it back into the flow conduit. ~ecause of the one-way valve system, that volume of fluid is forced downstream to provide a connected, flexible bag with a predetermined-volume aliquot of media.
The downstream portion of the flow conduit may define a plurality of branching conduit end portions, each end portion being connected in substantially aseptic manner to one of said flexible bags. The branching conduit end portions and bags may cach define sterilizable connection means/ permitting the establisllment of sterile connection between said bags and branching conduit end portions without subjecting the whole system to sterilizing conditions.
Such sterilizable connection means have been approved and used in the manufacture of medical sets by Travenol Laboratories, Inc. of Deerfield, Illinois, and other ~nown sterilizable systems exist as well.
See in particular U.S. Patent No. Rc. 32,056, Lilcewise, similar sterilizable connection :, ' means for forming a sterilized connection with the media source may be used to obtain aseptic connection at the upstream end of the system~ without subjecting the whole system to sterilizing conditions.
Additionally, the flow conduit may carry an access site between the one end at its upstre~m end, and the branch conduit. Ior example, this access site may be used to add serum, when that is a desired component, of each aliquot of media liquid by first passing some oi the aliquot of media liquid through the branch conduit; then adding some serum through the access site; and finally adding the final amount of media liquid to f~ll the predetermined aliquot, which is then passed once again through the branch conduit downstream to a Elexible bag.
Additionally, the flexible bag of this invention may define an outer enclosing wall, and an inner, open cellular structure which is compatible with growing cells and serves as a site for cellular adherence. Preferably, the inner, open cellular structure is rather flexible, so that the bag may be provided in initial condition where it is wrapped in shrink wrap means under compressive pressure, causing the ce]lular structure within the bag to be in a relative]y collapsed configuration, to cause the bag to be thinner than in its configuration of use.
Accordingly, when the shrink wrap is removed, the bag and cellular structure can expand to assume their normal condition of use.
~s a result of this, the flexible bags of this invention may have very high interior æurface area, serving as adherence sites for cells that are cultured while adhering to a surface. The nature Or the material Or the cellular structure may be of any .
~7~ 7 appropriate and compatible material for culturing cells. Preferably, the cellular structure may be of the honeycomb type with hexagonal channels passing through it. The hexagonal (or other shaped) honeycomb cell passage ways preferably are aligned to terminate adjacent the access port means in the bag. Lilcewise plastic open cell foams, fibrous materials including hollow fibers, or any other open-cell type materials may be used as the cellular structure to provide adherence areas to cells that adhere to a surface during culturing.
In addition to the open cellular structure, other means may be provided inside the flexible bag to increase tlle area of cell adherence. For example, a microcarrier such as small glass spheres or sodium alginate may be employed to increase surface area for cell adherence.
Alternatively, bags with open, empty interiors may be used for culturing cells which do not adhere to surfaces, for example, suspension cells such as mast hybridoma cells and human monocytes, in a bag made of a nonadherent material.
The other parts of the system of this invention may be made of conventional plastic materials for such products as is currently available.
The tubular conduit mernbers may be made of a conventional polyvinyl chloride formulation, but preferably, the flexible bag may be made of an oxygen and carbon dioxide permeable material of a formula~ion as described in U.S. Patent No. 4,1~,162~
~dditionally, thin-walled polyethy]ene, or polytetrafluoroethylene, may be used for such bags, as described in the prior art. Other formulations as well may be used for gas-permeal)le bag walls, for exarnp]e .
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poly(ethylene-Yinyl acetate) 7 silicones, or a hydrocarbon resin sold under the trademark TPX.
DESCRIPTION OF TIIE D~AI~INGS
In the drawings, Fig~ 1 is an elevational view of a system for metering nutrient media to a cell culture container in substantially aseptic Manner.
Fig. lA is a detailed perspective Yiew of the connectors used for connecting the media flow conduit with a flexible bag in accordance with this invention.
Fig. 2 is an elevational view of an alternative embodiment of media flow conduit.
Fig. 3 is an elevational view of an alternative embodimellt oi media flow conduit which may be used in conjunction with the media storage bag and the cell culture container of Fig. 1, to provide liquid media to said cell culture continer.
Fig. 4 is an elevational view of another alternatiYe embodiment oi media flow conduit which may be used in conjunction with a media storage bottle and the cell culture container of Fig. 1, to provide liquid media ~o said cell culture container.
Fig. 5 is an exploded view of another system in accordance with this invention for transferring in sterile manner media from a supply container to a plurality of flexible cell culture containers, including ancillary equipment that may be used in conjullction therewitll.
Fig. 6 is an elevational view of another embo(liment o~ cell culture container in accordance i.
~ . .
with this invention.
Fig. 7 is an elevational view of the bag of Fig. 6, rotated 90 degrces about its longitudinal axis, witll portions brolcen away, and showing how it may be packaged in a shrink wrap package.
Fig. 8 is a fragmentary, longitudinal sectional view taken along line 8-g of lig. 7.
Fig, 9 is an elevational view, with portions brokcn away, of the bag of lig. 6 after the shrinlc wrap has been removed.
Fig. 10 is a sectional view taken along line lO-l0 of Fig. 9~ showing the expanded, honeycomb nature of the cellular structure within the bag.
DESCRIPTION OF SPECIFIC EMBODI~IENTS
Referring to Figs. 1 and la, a first cmbodiment of this invention is disclosed. A source of media lO is disc]osed as a flexible bag of nutrient media for the growth of a certain cell. The cells may be hybridomas from ~hich monoclonal antibodies may be obtained by culturing.
I~lternatively, the cells may be white blood cells such as lymphocytes from a cancer patient. In this circumstance, as is taught in the literature, culturing of the lymphocyte with a lymphokine such as interleukin-2 can provide an activated lymphocyte which is more active in the process of identifying and killing tumor cells. These activated lymphocytes may then be returned to the patient for treatment of the cancer.
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In accordance with this invention, individual bags 12 for the culturing of cells are ~illed in a substantially asep~ic manner with at 1 ast one predetermined-volume aliquot of media through a media flow conduit 14.
Thereafter, the cells for culturing are placed into bag 12. The specific media used will be any appropriate type of media desired, depending on the particular cells to be cultured~ Man~ different varieties of media are taught in the prior art. It ~hould also be known that the verb "culture" may refer to the maintenance of cells and their multiplication by growth, but alternatively, it can apply to situations where the cells do not multiply but simply are treated (for example, with a lymphokine) to change their characteristics. One cell medium tha~ may be used for lymphocytes is a medium consisting of RPMI 1640 (low endotoxin; M.A. Bioproducts of Walkersville, Md.~ also including 10 units per ml of penicillin, 10 micrograms/ml. o~ streptomycin sulfate, 2 ml. o~
glutamine, 5 micrograms per ml. of gentamicin, and 2 percent by weight of heat-inactivated human AB serum.
This formulation is taught in Rosenberg U.S. Patent 4,690,915. Appropriate media for hybridoma cells are widely available in the literature.
Media flow conduit 14 dafines at one end 16 a conventional spiXe for penetration of media bag 10. If a media bottle is used with a conventional plug in its mouth of a design used with parenteral solution bottles, spike 16 may be a conventional vented spike of known design.
Media flow conduit 14 carries a conventional injection site 18 into which supplemental materials ~ ~7~ -r~à, - 1 o -may be inserted by a needle syringe in a gPnerally aseptic manner, for e~amplc, serum such as fetal bovine serum, amino acids, vitamins, or antibiotics, which typically are not stored with the artificial nutrient media within bag ]0.
i~ledia flow conduit 14 comprises flexible tubing typically of polyvinylchloride or any other conventional tubing of the type used in parenteral solution administration and the like. I~oller clamp 20 may be provided to control flow through the tubing, and may be of conventional design.
Media flow conduit 14 may have a dividable connection comprising a pair of luer lock connectors 22,24 which are connected together with an aseptic seal, being initially provided in sterile condition as long as protective caps 26, 28 have not been previously removed. Designs of Luer connectors are l;nown to the art, some of them being disclosed in Dennehey et al., U.S. Patent No. 4,346,703 and Ruschke et al. U.S. patent No. 4,452,473.
Liquid nutrient media may flow from bag 10, through the connection of members 22, 24 into 3-way valve 30, which is also of conventional design9 being commercially sold on various parenteral solution sets and the like by various companies. The rotatable handle 32 of valvc 30 may be in a position so that the flow travels upwardly through branch conduit 34, which may connect through a burette 36, carrying a vent 38 and an injection site 40. Such burettes are also in commercial use in the parenteral solution field, One such burette is described in U.S, patent No.
4,322,247. This particular burette may be connccted to branch conduit 34 by the action of connectors 42, 44 of design similar to conncctors 22, 24.
. ~
Accordingly, a predetermined-volume aliquot of nutrient media from bag lU may flow through media flow conduit 14 into burette 36, which has volume measuring indiccs, until the predetermined volume of the aliquot has been reached. lhen, the user can shift 3-way valve 32 to cause the flow to return from burette 36, through branch conduit 34, into the portion 14a of conduit 14 which is downstream from branch conduit 34, Clamp 46, of conventional design, may be loosened to permit such flow. Downstream portion 14a may be connected to 3-way valve 30 by more connectors 4~, 50 of similar design to connectors 22, 24.
In those circumstances where the predetermined-volume aliquot is of a constant volume every time, burette 36 may be replaced with a simple, collapsible bag having a filled volume which corresponds to the desired volume of the aliquot. The burette is useful when variable volume aliquots are intended to be drawn from cell culture container 10.
A pressure head may be provided to the system by raising media bag 10 above branch conduit 34, the volume metering means 36 such as the burette being raised over bag 12 as well.
Connection to bag 12 by conduit 14a may be obtained by the use of similar connectors 52, 54 for placing the predetermined-volume aliquot of nutrient media into bag 12. 3ag 12 per se is known to the art, being described in U.S. Patent Nos. Re. 31,135 and 30 4,140,162. The particu]ar material of bag 12 has a high capability for oxygen and carbon dioxide diffusion, very substantially over that of po]yvinylchloride and other materials in prior art bags. Also, fewer lcachable materials are present in :
,.
~7~Æ~7 the bag materia].s of the eited patent no. 4,148,162.
'I`ypically, bag 12 is filled with nutrient media and cells to no more than about one quarter to one half of its LuJ1 capacity, to provide a relatively high ratio of internal surface area to vo].ume of the media and cells, so that abundant oY.ygen ean diffuse into the bag and carbon dioxide can diffuse out of the bag~, to faciliLate cell metabolism and xrowth.
Thus, by the system of this invention, one or more cell culture l-ags 12 ean be prepared for receiving their cells for eulturing by reeeiving one or more ali~uots of nutrient media of predetermined volume, under substantially aseptie eonditions to minimize tlle eurrent problem eommonly eneountered in eell culturing today of eontamination of many of the ce]~ culture samples.
The cells themselves may be inserted in~o bags 12 by removal of conventiona].ly designed plastie port proteetors 56 whieh elose access port 58. The ce].1s may be added througll tubular sampling si.te eoupler 60, whicll penetrates an internal diaphragm in port 68 to gain aecess to the eontainer. Coupler 60 may have a conventional injection site 61 on its ouLer end. If desired, coupler 60 may remain within port 58 to elose it during ~:he culturinæ operation.
Alternative].y, an added closure mAy be provided for port 58. If desired, a needle syringe may be used to pierce injecti.on site 61 to inject the eells into bag 12. ()therwise, bag ]2 may carry its own injection site.
To separate bag 12 from its media fl.ow conduit set ]4, 14a, one may p]aee a heat seal or other seal aeross a portion of the tubing 14a, whieh may be made of polyvinylcllloride, or another -thermoplastic material. Alternatively, short length of tubing 62 may be transverse]y sealed. One then cuts through the seal while maintaining its integrity on preferably both sides so that bag 12, with its aliquot of nutrient media, can be separated and taken or use in a cell cultureO
Turning to Fig. 2, a simplified media flow conduit 64 is provided, being the length of ~lexible ~ubing 6~ whicll is shown to serve as roller pump t:ubing in a conventional ro]ler pllmp G8, which is only partially and schematically shown, and connector 67 o conduit 64 may be a connector of conventional design, similar for examp~e, to connector 22. End 68a provides a connector which may be similar to connector 24.
Conduit 64 ~serves as a substitute for branch conduit 34, 3-way valve 30, an~ burette 36. It may be connected by connectors 67, 68a to media flow conduit portions 14, 14a to provide an assembly that is otherwise suhstantially equivalent to that shown in Fig. ], where the individua] flliquots of nutrient media f]ow in their predeternlined volumes in a manner as governed by rol]er pump 68.
Turning to ~ig. 3, another embodiment is djsclose~, serving as a suhstitute for branch conduit 34, 3-way valve 30, and burette 36. ~ differing brallcll con(luit 63 is provi(led in a set portion 65 which carries connectors 67a, 69 on their respective en(ls for connecting with otiler portions 14, 14a of the me(lia flow conduit. Clalnp 70 nlay also bc provided as shohn.
A~ljacent to branch conduit housing 72 lie a pa;r of one way valves 74, 76 of conventional design Lo assure that flow througll conduit 65 goes only in one direc~ion from connector 67a to connector 69.
Connector 67a may connect w].th media flow conduit ~ortion 14, while connec-or ~3~ connects with portion 14a. A syringe 78, o conventional design, may be connecte~ by a simple luer connection to branch conduit 63 (removing respective end caps 8Q, 82).
8yringe 7~ carries vo]ume indicating indicia.
I~ccordingly, when the entire set is set up, with the structure of i:i.g. 3 replacing one-way valve 3(), branch contluit 34, and burette 36, one can simply use syringe 78 as a pump to draw from container the predetern)ined-volllme al.iquot, and then to force it downstream to conduit portion ]4a and ba~ or bags 12, due to the action of one way valves 74, 76. ~lany designs of appropriate one way valve are known such as duckbill valves, flapper va]ves antl the like. One particular one way valve that may be used for valves 74, 76 is a valve sold by Travenol Laboratories, Inc.
as part of a parenteral solution set.
Referring to Fig. 4, anothcr system in accordance with the system is provided for transferring liquid nutrient media from a rigid media bottle lOa to a culture container 12a, which may be a Ele~:ible bag made in accordance witll conventional Lechnology.
Stopper 84 fits in the neclc of bottle ].Oa, and derincs n port 8~3 for spike penetration. Threclded cap ~8 is fi~te(l onto the neck of bottle lOa to retain stopper 84 i.n position.
Integral airway spike 90, of a commerciAl.
design for penetration of bottles of paren~eral.
so].uti.on is provided, being conrlected to a first length ')~ of a media f]ow condui.t. Conduit 92 ~er~linates in molrled asselllbly 94, which includes brancll contlui.t (~fi an(l a pai.r of onc-way va]ves 98, 100 .. ..
. ,:
, :~ ' . "' ~ '"':
respectively positioned upstream and downstream from branch condlIit (~6. A more compact design of valves for tlle same purpose combined into a single structure is disc]osed in U.S. paLent No. 4,084,606.
Syringe ]~2, of conventional design, may connect to branch conduit 96 so that as the syringe is pumped, media solution flows downstream in pre(IeterminecI-volume aliquots as controlled by each stroke of the syringe handle 104.
~ssembly 94 also carries male luer connector 106, which is adapted to connect with female luer 108, w}lich, in turn, is carried by media flow conduit section 110. Conduit section 110, in turn, is controlled hy a roller clamp 112, and communicates througi- Y-connector 114 and conduit section 116 to cu]ture container l~a. Container 12a may be a collapsible bag made in accordance with U.S.Pat. No.
4,14~,162 or any other flexible sheeting materia]
having the required high oxygen and carhon dioxide dirfusioIl, when compared with a bag having walls of polyethylenc.
The remaining branch tube 118 is controlled by another roller clamp 120, and may terminate in a male needle adapter 122 or another conventional colIllecLer.
In use, sampling site coup]er 123 may be a holloh~ tube with a pointed end plus a needle-piercab]e situs 124, and may be used to penetrate access port l~G of container 12a t:o insert the cells for cu]turing, typically arter filling of container 12a with the predetermined-vo]unIe aliquot Or media solution via syringe 1()2, After such filling, in a manner previolIsly describe(I, rol]er c]nmp 1]2 may be closed an(I connectors ]06, 108 separated to remove the ~ : ,"
,:
:
. , , .. .. .
~2~
container to its cuituring environlnent, or tube 110 may be heat sealed shut. Added nutrients or other agents mfly be adde(l Lhrollgh access port 128 as desired. Situs ]24 may be repeatedly pierced as de~sired f()r addin~ or removi 11~ media or other agents.
After the culturing phase, further processing steps may be perfornled. In the case of suspended cells where protein production is an end goa], the product-ricll supernatent may be sep~rated from the cells and harvesLed, such as with a n centrifugaLion or membrane system. Alternatively, the contents of the culture container may be transferred to other containers for further processing through tubings 116, 118 and adal)ter 122.
~ig. 5 is an explode(i view of anotiler system in accordance with this inventioll in which sterility of Lhe materials processed may be assured with a higher (legree oL relial)ility. Nutrient media container l~b, of a design similar to either previous embodiment, may be connected to a media flow conduit 130 by means of a pair of identical sterile connector devices 132, a specific embodiment of which has been demonstr.l~e(l as a product by Travenol Laboratories, Inc. and which operate in accordance with the principles described in U.S. I'at. No. ~e. 32,~56.
l`hesr-~ connectors are ]ocked together, and then irratllated wiLh intense light or infrared radiation, resulting in a sterile, fluid pathway being formed between the two connectors 132.
As in an earlier embodiment, three-way valve 134 is provi(le(l, carrying branch conduit ]36 whicll conllects with a burette ]3~. Alternative]y, burette ~3~ may be replaced with n sma]], connected bag if desired for cost savings with the maxirnllnl fluid volume ~' .
'' ., .~ .
~7~
of the bag being identicai to the desired, predetermined-volume aliquot.
I)ownstream portion 14() of the flow conduit is branclled a~s shown to provi(le a series of branches 5 ]42, each of which is terminated with a sterile connecter 132a, corre~poll(ling in desip,n to connectors 132, }.ach connecter ]32fl may be connected in sterile manner with a sterile connector 132h of one of the serie~ of ~lexible bags 134a, In this eireumstance, 10 hy appropriate clamping of lines 142 (which may be equippecl witll slide clamps or the like), the desired predetern\ined-vo]ulne aliguots of media solution may be metered from container l()b, through braneh eonduit 136 alld burette 13~, sequentially into each of ba~s 134 15 after the sterile connection bet-~leen connectors 132a and 132b has heen made.
If desired, added connection sites may be provide(l by Y-connector 136 which includes a Y-shaped arrangement of conduits, each terminated with a 20 sterile connector 132c, similar in design to sterile eonnectors ]32a. Ey the use of members ~36, the nllmber of availal)]e sites for connection with bags 134a may be inereased by any desirecl number.
Added aneillary equipment 139 eonstitutes a 25 y-eonnector having a filter. Sterile eonneetors 132d may be eonnectecl into line llith eonneetors 132a or 132c, Lor example, so that the media material may be filtered. Alternntively, arter eulturing of the eel~s hns taken plaee in bag 134a, the eontents of the ba~
30 r,~ay he eonneeted in sterile manner to eonneetor 132cd through its seeond eonneetor 132e, and the eontents of thc~ bag fi]terecl. I`he filtrate then ean pass throu~h anotller eonllector ]32f to another contniner sucll as riltrate eontainc r 133 with connectors 132j, for .....
, . . . . .
7~ 7 storage or f~lrther processing. By this manner, ~i]Lrate whic}l mny carry a biochemical product manufactured by tlle cel~s, may be separated from the cells in reliably steri.le manner, once all the components have been initially steri].izecl.
Kecep~acle container 141 may also be used in any desired manner witll its connector 132g, for extlmple to connect iLs connector 132g with with the remaining outlet connector 132i of fi].ter member 139.
~s may be desired serum may be added to the media by means of serum container 142a, carrying its sterile connector 132h. It may be used to make connection with the second connector 132 of media bag l~b so that the serum and media may be mixed prior to the metering of the various a].iquots i.nto the respective bags 134a. Alternat:ively, connector 132h may be connected through the use of ancillary device 13'.~.
~ther par~s o tlle set suc]l as injection site 144 and clamps l4(), 1.4~ may be l)rovided as deemed appropriate.
Turning now to Figs. 6-10, a specific type of flexible bag 15() is disclosed for use in this invelltion, being analagous in function and outer structure to bags l~, ].2a, and 134a. Bag 15~ may be made of f]exih]e, plastic sheeting having the desired niill oXygCIl and carbon di.oxide diffusion chartlcteristics as discussed above. Ilowever, bag 150 is providetl with a aci.1ity or culturing cclls that need to adhere to a surface. ~y thi.s invention, a bag havillg an ultrahi~ll surface aren in its interior is provided so that a large number of such cells may be cul.LIlre(l in the bag under conditi.oll.s thclt are optimal for their growtll and deve~opnnent.
':'`
. . .
~27el~ ~
~ s shown, flexible bag ]50 mny be made of a film wall materia.l haYing the desired, adequate oxygen and carbon dioxi.de diffu.sioll capability to permit the culturing of ce]ls tllereill under sealed, substantia].ly asel)tic cond]t.i.ons. ~ag 150 defines an outer enclosing ~all 152 which may, for example, be the remainder Or n blo-- "~01 ded parison in accordance with l)lo~ mo~dinc, technology, or may be made of a pair of peri[)hera].ly seale(l sheets in accordnnce with hcat or 10 1'.1~. sealing technologies. Within bag 150 is an e inner, open cellular struc~ure whicll is eapable of supporting growing cells, and serves as a site for eell adherence. ~ee particlllar]y Figs. 9 and 10 for broken away and sectionnl views of the cel.]ular 15 s~ructure 154, An enlarged view of the hexa~onnl cells 156 is shown in ~ig. 10. Each cell 156 is of hexagoncll cross seetion, and is typieally open at its respective ends, rnther in the form of a honeycomh shape, with the individual ee].]s 154 being aligne(l 20 ~ith one end of eaeh honeyeomb eell passageway 158 termi~ ting adjaeent the access ports 16~ of said bag.
The open eellular strllcture may be made of ally desired mrlteria]. suital)le for the adllesioll and llealtlly maintenance of the ce].ls, particularly 25 relative].y hi.gh surface enerl;y po]ymers, preferably ha~ing n surfclce energy of at least ahout that of eellulose for example. ~xnml)les of such materials ine~ude cellulose, polyestèr materials, high surfaee energy n~lon (such as thnt so]d by the llexcel Co. of 30 Ca].ifornia), corona discllarge treated polyolefin, or the like.
I'he open cellular material may, in the alternative, coml)rise ho].lo~ fibers, particular].y tho.se extending i.n a manner analagous to the direction - : .
. ~ . . . . . ..
. ' ' . , . . .
. ''' , ~ ~ ''.
~7~
ol ex~ension of open ce]ls 15~. 'I'hc rnaterial may be an open-celled foam, or a fibrous mass, either woven or nonwove~ he tcrm cel]ular is intended to imply open porosity, not necessarily indicating that rliscree~ cells are present as in an operl-cel]ed foain7 althougil such is inten(led to be included as one possiblc type of opcn- celle(l nnaterial.
l`he flexible bag 150 may be compressed in packaging by wrapping in a known hent-shrink w~ap.
The advantage of packaging bag 150 by a shrink wrap technique as shorn in Figs. 7 and ~ is primarily to save space until bag ]50 is ready for use.
Sterile filter vent 164 may be provided for communication between the interior and exterior of the bag to facilitate this process. Specifically, the vent may contain a filter of hydrophobic material in accordance with known technology which exhibits strong bacteria bloclcing characteristics.
As shown in lig. 8, cellular structure 156 may be compressed by the compressive action of the heat shrink wrap 162, for efficient shipping arl(l storage.
When it is desired to make use of bag 150, shrink wrap 162 may be torn a~ray, permitting bag 150 to expand ~y natural expansive action of open cellular material 154, t:o the configuration shown in Figs. 9 and ]0. Alternatively, this expansion may take place upon softening of cellular material 154 as it is wel:ted rith liquid nutrient media.
l~uring tlle culturing of cells in a bag simi]ar to bag 150, :it, may be (Iesire(l to provide a mecllanica] means to gent],y knead the bclg to assure circulation of nutrient media throug}l the open cel]s o[ material ]54, so that oxygen and carbon dioxide ma~
, ~ .
~;Z7Q ~6 d diffuse into and out of bag wa]l 152, and no stagnant l)ockets of nutrient media will form, Wi)iCII may interfere with cel] viabi]ityO
'l'he abovc has been offered for illustrative pur~oses only, and is not intcnded to limit thc scope of the invention of this application, whicll is a~s dcf:incd in the claim.s bclow.
, ,
Claims (25)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for metering nutrient media into at least one cell culture container in substantially aseptic manner, which method comprises passing liquid nutrient media through a media flow conduit which is in substantially aseptic connection at one end with a source of said media; passing said media from the flow conduit through a branch conduit extending from said flow conduit at a location spaced from its ends, into means for receiving a predetermined-volume aliquot of media; passing said predetermined-volume aliquot of nutrient media again through the branch conduit into and through a downstream portion of said media flow conduit, into at least one attached, flexible, flat-collapsible bag, at least part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized, sealed bag having polyethylene walls 0.013 inch thick.
2. The method of Claim 1 including the step of sequentially and repeatedly passing liquid nutrient media through said branch conduit into the means for receiving a predetermined-volume aliquot of media, and passing each aliquot of media through a downstream, branched portion of said flow conduit sequentially into each of a plurality of said flexible, flat collapsible bags, whereby a plurality of said flat-collapsible bags may be filled with said predetermined-volume aliquot of media in substantially aseptic manner.
3. The method of Claim 1 in which one passes a portion of said predetermined-volume aliquot of media through the media flow conduit, then adding to the media flow conduit a predetermined amount of serum, followed by adding more liquid media through said media flow conduit, to fill said receiving means with a predetermined-volume aliquot comprising a mixture of liquid media and serum, said aliquot being then transferred to said flexible, flat-collapsible bag.
4. The method of Claim 1 including the step of forming a sterile connection between the media flow conduit and a source of nutrient media without subjecting the whole system to sterilizing conditions.
5. The method of Claim 1 including the step of connecting said branching conduit ends with said separate, flexible bags under conditions to form substantially sterile connections without subjecting the whole system to sterilizing conditions.
6. A method for metering nutrient media into at least one cell culture container in substantially aseptic manner, which method comprises passing liquid nutrient media through a media flow conduit which is in substantially aseptic connection at one end with a source of said media, and metering at least one aliquot of said media through a downstream portion of said media flow conduit into at least one attached, flexible bag, at least part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with identically-sized sealed bag having polyethylene wall 0.013 inch thick, and culturing human white blood cells in the nutrient media within said flexible bag.
7. The method of Claim 6 including the step of sequentially and repeatedly passing aliquots of predetermined volume through a downstream, branched portion of said flow conduit sequentially into each of a plurality of said flexible bags, whereby said plurality of bags may be filled with said predetermined-volume aliquots of media in substantially aseptic manner.
8. The method of Claim 6 in which one passes a portion of said aliquot of media through the media flow conduit, then adding to the media flow conduit a predetermined amount of serum followed by adding more liquid media through said media flow conduit and to said flexible bag, whereby said predetermined-volume aliquot of media, containing serum, is provided to said bag.
9. The method of Claim 6 including the step of forming a sterile connection between the media flow conduit and a source of nutrient media without subjecting the whole system to sterilizing conditions.
10. The method of Claim 9 including the step of connecting the conduit end opposed to said one end with at least one of said flexible bags under conditions to form substantially sterile connections without subjecting the whole system to sterilizing conditions.
11. A system for metering nutrient media to at least one cell culture container in substantially aseptic manner, which comprises:
a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media; a branch conduit extending from said flow conduit at a location spaced from its ends, said branch conduit communicating with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end; said flow conduit defining a portion downstream from said branch conduit which communicates with said cell culture container, each container being a flexible bay, at least part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized, sealed bag having polyethylene walls 0.013 inch thick, said bag containing a flexible, open cell structure to increase the surface area within each bag for cell adhesion, said flexible bag containing an access port, said access port carrying tubular coupler means defining a pointed inner end and carrying needle pierceable, resealable injection site means on its outer end, to permit inoculation by a needle and syringe of culture media in said bag with desired cells in aseptic manner.
a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media; a branch conduit extending from said flow conduit at a location spaced from its ends, said branch conduit communicating with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end; said flow conduit defining a portion downstream from said branch conduit which communicates with said cell culture container, each container being a flexible bay, at least part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized, sealed bag having polyethylene walls 0.013 inch thick, said bag containing a flexible, open cell structure to increase the surface area within each bag for cell adhesion, said flexible bag containing an access port, said access port carrying tubular coupler means defining a pointed inner end and carrying needle pierceable, resealable injection site means on its outer end, to permit inoculation by a needle and syringe of culture media in said bag with desired cells in aseptic manner.
12. The metering system of Claim 11 in which a three way valve is positioned at the junction of said media flow conduit and branch conduit to control flow into and out of said branch conduit.
13. The metering system of Claim 11 in which said branch conduit connects with a syringe, said flow conduit defining one way valve means positioned upstream and downstream of said branch conduit to permit only downstream flow as controlled by said syringe.
14. The metering system of Claim 11 in which a plurality of said flexible bag cell culture containers are present, and said downstream portion of the flow conduit defines a plurality of branching conduit end portions, each end portion being connected in substantially aseptic manner to one of said flexible bags.
15. The metering system of Claim 14 in which said branching conduit end portions and bags each define sterilizable connection means permitting the establishment of sterile connection between said bags and branching conduit end portions without subjecting the whole system to sterilizing conditions.
16. The metering system of Claim 11 in which said flow conduit carries an access site between the one end and the branch conduit.
17. The metering system of Claim 11 in which said media flow conduit defines, at said one end, sterilizable connection means for forming a sterile connection with said media source without subjecting the whole system to sterilizing conditions.
18. The metering system of Claim 11 in which said media flow conduit is integrally attached to a container which is said source of media.
19. The system of Claim 11 in which said material is poly(ethylene-vinyl acetate).
20. A system for metering nutrient media to a plurality of cell culture containers in substantially aseptic manner, which comprises:
a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media; a branch conduit extending from said flow conduit at a location spaced from its ends;
said branch conduit communicating with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end, said flow conduit defining a branched portion downstream from said branch conduit which communicates with said cell culture containers, each container being a flexible bag, at least part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized, sealed bag having polyethylene walls 0.013 inch thick, each bag containing a flexible, open cell structure to increase the surface area within each bag for cell adhesion.
a media flow conduit which defines at one end means for substantially aseptic communication with a source of said media; a branch conduit extending from said flow conduit at a location spaced from its ends;
said branch conduit communicating with means for receiving a predetermined-volume aliquot of media through the media flow conduit from said one end, said flow conduit defining a branched portion downstream from said branch conduit which communicates with said cell culture containers, each container being a flexible bag, at least part of which is made of a material capable of providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized, sealed bag having polyethylene walls 0.013 inch thick, each bag containing a flexible, open cell structure to increase the surface area within each bag for cell adhesion.
21. The system of Claim 20 in which said open cell material is in the form of a honeycomb shape, with the cells of said honeycomb material being aligned with one end of each honeycomb cell passageway terminating adjacent said access port means in said bag.
22. A system for metering nutrient media to at least one cell culture container in substantially aseptic manner, which comprises:
a container of said nutrient media; a media flow conduit communicating at one end with the interior of said container in substantially aseptic manner, means for passing aliquots of said nutrient media of predetermined volume through said media flow conduit, said flow conduit defining a portion adjacent its end opposed to said one end which communicates with at least one cell culture container, said cell culture container being a flexible bag, at least part of which is made of a material providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized sealed bag having polyethylene walls 0.013 inch thick, said bag containing a flexible, open cell structure to increase the surface area within each bag for cell adhesion.
a container of said nutrient media; a media flow conduit communicating at one end with the interior of said container in substantially aseptic manner, means for passing aliquots of said nutrient media of predetermined volume through said media flow conduit, said flow conduit defining a portion adjacent its end opposed to said one end which communicates with at least one cell culture container, said cell culture container being a flexible bag, at least part of which is made of a material providing at least 2 times the oxygen diffusion and at least 2 times the carbon dioxide diffusion between the bag interior and exterior, compared with an identically-sized sealed bag having polyethylene walls 0.013 inch thick, said bag containing a flexible, open cell structure to increase the surface area within each bag for cell adhesion.
23. The metering system of Claim 22 in which a plurality of said flexible bags are present, and the portion of said media flow conduit which is adjacent the end opposed to said one end defines a plurality of branching conduit end portions, each end portion being connected in substantially aseptic manner to one of said flexible bags.
24. The system of Claim 23 in which said media flow conduit defines a section of tubing adapted for installation in roller pump means for metering of said nutrient media therethrough.
25. The system of Claim 22 in which said flexible bag carries a tubular access port, said tubular access port carrying therein a tubular coupler defining a pointed inner end and an outer end which carries a needle pierceable, resealable injection site, whereby nutrient media in said bag may be inoculated with cells by means of a needle and syringe which penetrates said injection site.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US862,344 | 1986-05-12 | ||
| US06/862,344 US4829002A (en) | 1986-05-12 | 1986-05-12 | System for metering nutrient media to cell culture containers and method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1270467A true CA1270467A (en) | 1990-06-19 |
Family
ID=25338272
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000536719A Expired - Lifetime CA1270467A (en) | 1986-05-12 | 1987-05-08 | System for metering nutrient media to cell culture containers and method |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4829002A (en) |
| EP (1) | EP0267266A4 (en) |
| JP (1) | JPS63503201A (en) |
| AU (1) | AU607915B2 (en) |
| CA (1) | CA1270467A (en) |
| WO (1) | WO1987006952A1 (en) |
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- 1987-05-07 AU AU74304/87A patent/AU607915B2/en not_active Ceased
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| US6146890A (en) * | 1994-07-03 | 2000-11-14 | Danon; David | Method and system for cultivating macrophages |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0267266A1 (en) | 1988-05-18 |
| AU607915B2 (en) | 1991-03-21 |
| WO1987006952A1 (en) | 1987-11-19 |
| JPS63503201A (en) | 1988-11-24 |
| AU7430487A (en) | 1987-12-01 |
| EP0267266A4 (en) | 1990-01-23 |
| US4829002A (en) | 1989-05-09 |
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