Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/02—Preservation of living parts
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/02—Preservation of living parts
  • A01N1/0236—Mechanical aspects
  • A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/02—Preservation of living parts
  • A01N1/0236—Mechanical aspects
  • A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
  • A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F25—REFRIGERATION OR COOLING; COMBINED HEATING AND REFRIGERATION SYSTEMS; HEAT PUMP SYSTEMS; MANUFACTURE OR STORAGE OF ICE; LIQUEFACTION SOLIDIFICATION OF GASES
  • F25D—REFRIGERATORS; COLD ROOMS; ICE-BOXES; COOLING OR FREEZING APPARATUS NOT OTHERWISE PROVIDED FOR
  • F25D25/00—Charging, supporting, and discharging the articles to be cooled

Definitions

• This invention relates to apparatus for and methods of cryopreservation.
• Cryopreservation is the process whereby material, particularly biological material, suspended in an appropriate medium (typically, buffered saline) containing a cryoprotectant, is cooled to a low temperature so as to preserve the sample.
• an appropriate medium typically, buffered saline
• cryoprotectant typically, a cryoprotectant
• the cooling rate the biological material is subjected to is critical to successful cryopreservation, and that reproducibility of the desired optimal cooling rates has a significant bearing on the overall success of the cryopreservation process.
• a linear cooling rate i.e. a substantially constant fall in temperature per unit time
• a certain degree of departure from such a linear rate inevitably occurs during freezing of the sample, due to the transition of the sample from an essentially liquid phase to the solid phase.
• sperm or egg samples are typically packaged in synthetic plastics straws (0.25 - 5ml in volume) and cooled using varying methods. Following cooling to the desired sub-zero temperature, the straws are removed and plunged into liquid nitrogen (“LN”) and then transferred to canisters for storage in an LN Dewar vessel until required. The plunging of cooled samples into LN is a process called "purging".
• CRCs controllable rate coolers
• Passive coolers The optimal linear cooling rates can only be achieved by the use of CRCs.
• CRCs are expensive and comparatively complex pieces of apparatus, in which the rate of cooling is determined by expensive electronic equipment which controls the rate at which LN coolant is pumped from an LN reservoir into a cooling chamber containing the sample to be frozen.
• Passive coolers typically comprise a Dewar vessel containing a static reservoir of LN.
• the LN vessel has an opening through which samples are introduced, the opening defining the upper end of a neck region.
• the lower end of the neck region is defined by a more or less pronounced shoulder.
• Use of such coolers generally involves the suspension of samples at a pre -determined height above the LN reservoir, the cooling being achieved by vapour from the LN.
• This arrangement has the advantage of simplicity of construction and comparative cheapness.
• the simplicity of passive coolers means that they can be readily portable, and may thus be used under field conditions (for several days if need be). However, in such coolers the cooling rates are dependent on the difference between ambient (LN vapour) and sample temperatures.
• results obtained with conventional passive coolers are not reproducible. Cooling rates may vary both between individual samples cooled in a single batch and between different batches. This lack of reproducibilty is a considerable problem, as it prevents cryopreservation of different samples with a predictable degree of viability, which in turn leads to uncertainty when samples are retrieved from storage. As a result, frequently more stored material will be used than may be strictly necessary, in order to ensure that at least some of the material used is viable. This, in turn, means that more material must be subjected to cryopreservation to begin with.
• cryopreservation has also been attempted, especially under field conditions away from a laboratory.
• dry ice blocks have been used but these have a very short life span (1-2 days) and this can limit the range of field cryopreservation.
• the cooling rates during the cryopreservation are not linear, and there is a complete lack of reproduciblilty in the results obtained under such conditions.
• the invention provides a cooling assembly for use in a cryopreservation cooler, the assembly comprising: a plurality of individual sample holders, each sample holder being capable of accepting a sample receptacle containing a sample to be preserved; and sample holder retaining means for retaining the sample holders in a spaced apart relationship, such that a coolant fluid may pass between the sample holders.
• the cooling assembly is used with a conventional cryopreservation cooler, preferably a passive cooler.
• the coolant fluid will be liquid nitrogen (LN) vapour from a static reservoir of LN provided in the cooler vessel.
• the cooling assembly is held, in use, in the neck region of the cooler.
• the cooling assembly is provided with an assembly support means, such as arms or a flange, to suspend the cooling assembly from the opening of the LN cooler vessel in the neck region thereof.
• the cooling assembly support means may make releasable engagement with a collar (preferably adjustable) provided on die outside of the cooler.
• the cooling assembly support means comprises two support arms, one either side of the cooling assembly which extend above the assembly substantially parallel to the long axis thereof.
• the arms preferably comprise a portion which projects radially outwards, which projecting portion releasably engages an accommodating slot provided in the collar.
• adjustment of the collar in one manner allows for vertical movement of the cooling assembly relative to the cooler, and adjustment of the collar in another manner varies the diameter thereof, such that the collar may be fitted to coolers of different sizes.
• the sample holders are arranged substantially vertically within the cooling assembly.
• the sample holders are substantially tubular, each sample holder being capable of accepting a substantially tubular sample receptacle, such as a straw, of the type commonly used in cryopreservation.
• the sample holders are made of material with a high thermal conductivity (such as copper, aluminium or brass).
• the cooling assembly will comprise between 10 and 80 sample holders, particularly between 12 and 70 sample holders.
• the arrangement will be such that the straws or other receptacles containing the samples are thermally insulated from the sample holders to a small degree (compared to the intimate thermal contact provided between the coolant fluid and the external surface of the sample holders).
• the small degree of thermal insulation is afforded by a thin air-jacket around each sample receptacle.
• the purpose of providing a small degree of thermal insulation between the sample receptacle and die sample holder is to prevent me establishment of a temperature gradient along the sample receptacle, and so to provide for more uniform cooling.
• samples are placed in receptacles for cryopreservation, each receptacle being supported by sample receptacle support means which prevents the receptacle from making substantial contact with the wall of the sample holder, tfiereby forming a thin air-jacket around each sample receptacle.
• the receptacle is a straw (e.g. reference numeral 13, Figure 4) of the conventional type, the sample receptacle support means comprising a projection located at the bottom of a tubular support holder, me projection being of appropriate dimensions as to become inserted a short distance into the straw, thereby preventing the straw from touching the bottom or sides of the sample holder.
• the sample receptacle support means comprises a material of comparatively poor thermal conductivity, such as PVC, which helps to maintain a small degree of thermal insulation between die sample receptacle and die sample holder.
• the sample holder retaining means comprises a base plate, with a plurality of holes drilled dierethrough, each hole serving to receive and retain a respective sample holder.
• the spacing between the holes in the base plate thus determines me separation between sample holders.
• the sample holder retaining means will further comprise a top plate, of similar character to the base plate.
• the top plate will typically comprise a plurality of holes, corresponding to me holes driled through the base plate.
• the sample receptacle when positioned in a respective sample holder, will desirably project through the holes in the top plate (to facilitate manipulation of the sample receptacles).
• the top plate will thus, in preferred embodiments, serve to support the upper end of the sample receptacles and help to prevent them from touching the sides of me sample holders.
• the cooling assembly support arms will be rigidly connected (e.g. welded) to die base plate, and preferably also to d e top plate, thereby providing considerable strength to the assembly.
• me receptacle support means may also serve to hold die cooling assembly together and dius provide additional strength.
• the receptacle support means may be positioned so as to pass dirough the hole in the base plate, from the underside, and _ ⁇ en engage the sample holder in a screw ireaded engagement so as to assist in holding d e cooling assembly together and retaining the sample holder in position.
• the separation between the base plate and die top plate is preferably such as to allow for removal of the sample holders from the assembly, if desired, by removal of me sample receptacle support means.
• top plates may be used in conjunction with the cooling assembly.
• the top plate welded to d e cooling assembly support arms is provided with lugs or holes which may be engaged by corresponding projections provided on one or more different "adaptor" top plates.
• the adaptor plates are substantially similar, but the holes drilled dierethrough will be of a different diameter in different adaptor plates, such that different adaptor plates will be intended for use widi sample receptacles of different sizes.
• Advantageously die cooling assembly will be formed with a channel therethrough or form such as a channel in use, the channel being of sufficient dimensions to permit the passage of a sample receptacle.
• the cooling assembly will be provided wi h such a channel, but as an alternative, the channel may be formed, in use, between the cooling assembly and the inner wall of the cooler vessel.
• me top and base plates of the cooling assembly are both equipped with a central aperture, which apertures are aligned, a channel being defined between the apertures by the surrounding sample holders.
• This arrangement allows the samples to be purged without requiring a second LN vessel in which to plunge the samples. Moreover, the cooled samples are in contact with the LN vapour at nearly all times, so that the samples cannot start to warm up in the period between cooling and purging. This is particularly important when attempting to cryopreserve material under field conditions in hot (e.g. tropical) climates.
• sample receptacle delivery means which serves to guide die cooled sample receptacles into the storage canister.
• the sample receptacle delivery means may comprise an elongate member, beatable in the channel formed in the cooling assembly (or in a channel formed between me cooling assembly and die cooler vessel), and preferably extending into the storage canister positioned beneath, thereby guiding sample receptacles, placed in the channel, into the storage canister.
• a storage canister can be manoeuvered from the side of die LN vessel to a position beneadi die cooling assembly to receive the sample receptacles.
• the sample receptacle delivery means is located in die channel in the assembly so as to extend in to me storage canister.
• the sample receptacle delivery means is removed and the canister may be re-positioned at the side of die vessel.
• anodier storage canister can be positioned beneadi die cooling assembly to accept these when cooled to die correct temperature, and the sample recepucle delivery means replaced.
• an LN vessel can perform the functions of both cooling and storing samples simultaneously.
• Storage canisters are typically provided widi a long thin handle, extending through the neck region and out of die opening of the LN vessel in which they are positioned, which handles facilitate manipulation of me canisters.
• the collar provided on die outside of die cooler will desirably comprise a plurality of slots, vertically disposed around its circumference, to accommodate me storage canister handles.
• die invention provides a cryopreservation cooler, comprising the cooling assembly defined above.
• die cooler is a passive cooler comprising a static reservoir of LN and is capable of achieving a substantially linear cooling rate.
• the cooler can perform the functions of both cooling and storing samples simultaneously. Further, the cooler is able to give highly reproducible results.
• the invention provides a mediod of subjecting material to a substantially linear cooling rate in a passive cryopreservation cooler, die method comprising use of die cooling assembly defined above.
• Figure la shows a side elevation of an embodiment of die cooling assembly of the invention
• Figure lb shows die same embodiment in plan view
• Figure lc shows, in longitudinal section, to a different scale, a passive cryopreservation cooler comprising the cooling assembly of the invention
• Figure 2 shows an exploded perspective view of die cooling assembly shown in Figure lc and to die same scale thereof;
• Figure 3 shows, to a different scale, an exploded perspective view of a similar embodiment of the cooling assembly of die invention
• Figure 4 shows an enlarged longitudinal sectional view of a sample receptacle in a sample holder, together widi sample receptacle support means, and part of me sample holder retaining means;
• Figure 5 shows a collar for use in suspending d e cooling assembly of the invention in an LN vessel
• Figures 6a - 6c show, for information, graphs of cooling rate (temperature against time) obtained for samples cooled in a controlled rate cooler (a), a passive cooler comprising a cooling assembly in accordance widi die intention (b) and a passive cooler used without the cooling assembly of the invention (c); and
• Figure 7 shows graphs of cooling rates obtained in four different runs using a passive cryopreservation cooler comprising a cooling assembly in accordance widi die present invention.
• die cooling assembly 10 comprises eighteen sample holders 12.
• the cooling assembly 10 is designed for use widi a ten litre LN Dewar vessel.
• Each sample holder 12 consists of a copper mbe 114mm long, having an internal diameter of 3.8mm, sufficient to accommodate a conventional-size sample receptacle (a straw), which straw is 134mm long and has a volume of about 0.5ml (reference numeral 13, Figure 4).
• the eighteen sample holders 12 are arranged vertically in two concentric circles, die inner circle comprising six sample holders 12, the outer circle comprising twelve sample holders 12.
• sample holders 12 are retained by sample holder retaining means, which comprises a disk-like base plate 14 and a disk-like top plate 16, both 38mm in diameter.
• sample holder retaining means which comprises a disk-like base plate 14 and a disk-like top plate 16, both 38mm in diameter.
• the 1C assembly 10 is also provided widi a pair of support arms 18 which extend above the top plate 16 and d en project outwards at right angles.
• the suppo ⁇ arms 18 are 163mm long and the right-angled projection is 57mm long.
• the support arms 18 are welded at opposed positions on the circumference of me base plate 14 and the top plate 16.
• the base plate 14 and the top plate 16 are both provided widi an array of eighteen circular holes 20.
• Each hole 20 in the base plate 14 is associated wid a respective sample holder and a respective sample receptacle support means 22.
• the receptacle support means 22 is made of PVC and comprises a projection 25 having at one end a tapered po ⁇ ion 24 and, at die other end, a flanged po ⁇ ion 26.
• the projection 25 is 8mm long.
• the tapered po ⁇ ion 24 is 2mm long and tapers from 4mm wide at its base to just 1.5mm wide at its apex.
• the flanged po ⁇ ion 26 is provided wid an indent suitable for receiving the blade of a screwdriver.
• the diameter of the flanged po ⁇ ion 26 is greater ti an the diameter of the hole 20 in the base plate 14, such that the receptacle suppo ⁇ means 22 can be secured by a screw diread engagement with the sample holder 12.
• the hole 20 in the top plate 16 has a wide diameter at its lower surface, of appropriate dimension to accommodate die upper end of die sample holder 12.
• the hole 20 in the top plate 16 has a shoulder, at which point the diameter of the hole is reduced to 3.5mm, preventing passage of the sample holder 12 through the hole 20 in the top plate 16.
• he reduced diameter is still sufficiently large to allow passage therethrough of a typical 0.5ml straw.
• the top plate 16 and die base plate 14 both possess a circular central aperture 28. between which apertures 28 a channel is defined by the walls of the inner circle of sample holders 12.
• the channel dius formed through the cooling assembly 10 is wide enough to permit the passage therethrough of a sample receptacle.
• FIG. lc illustrates die cooling assembly in use in conjunction with a passive cryopreservation cooler.
• the cooler comprises a Dewar vessel 30 containing a reservoir of LN (not shown).
• the Dewar vessel 30 has a neck region 31, the upper end of which is defined by the opening of the vessel 30, die lower end being defined by a pronounced shoulder 33.
• the cooling assembly 10 is suspended in die neck region 31 of die vessel 30 by means of the suppo ⁇ arms 18, which releasably engage an adjustable collar 32 placed around die outside of die opening of the vessel 30. Adjustment of die collar 32 can be performed to raise or lower the cooling assembly relative to the vessel 30. A different adjustment of the collar 32 varies the diameter thereof, such that the collar 32 can be fitted to vessels 30 widi different size openings.
• LN vapour from the LN reservoir passes through the neck region of the vessel 30, tiuis acting to cool the cooling assembly 10 and e samples placed dierein.
• the LN vapour is free to pass between die gaps between the sample holders 12, tiius making immediate and intimate contact with every sample holder 12. This arrangement minimises variation between individual samples and allows for a substantially linear cooling rate.
• a storage canister 34 ( Figure lc) is rotated dirough 180°, thus being moved from the side of die vessel 30 to a position beneadi me central aperture 28 in the cooling assembly 10. Straws may then be removed from the sample holders 12 and deposited through the channel defined in the assembly 10 and into the storage canister 34. When finished, me storage canister 34 is replaced at ae side of die vessel 30. If desired, further straws can be placed in the cooling assembly 10 for cooling. When cooled, tiiese further straws may be placed in the same or a different storage canister 34 (several such canisters 34 being provided witiiin the vessel 30).
• Figure 3 shows an exploded view of a slightiy different embodiment of die cooling assembly 10.
• the assembly 10 comprises a disk-like sample holder alignment plate 35, which is positioned substantially mid-way along the sample holders 12 and serves to align die sample holders 12 between the corresponding holes 20 provided in die top plate 16 and the base plate 14.
• the alignment plate 35 is provided with holes 20 and a central aperture 28, corresponding to those in the top plate 16 and the base plate 14.
• a small hole 36 On each side of die central apermre 28 in the top plate 16 diere is provided a small hole 36, which receives a corresponding pin provided on the underside of a disk-like adaptor plate 38.
• the adaptor plate 38 is essentially similar to the top plate 16, with an array of eighteen holes 20 and a central aperture 28 corresponding to those in the top plate 16.
• the holes 20 provided in the adaptor plate 38 are of smaller diameter than those in the top plate 16, and are intended to suppo ⁇ die upper ends of 0.25ml volume straws, such that by adding or removing the adaptor plate 38 as appropriate, die cooling assembly 10 can be used for eitiier 0.25 or 0.5ml volume straws as desired.
• d e sample holders 12 are inse ⁇ ed at a slight angle into the holes 20 in die top plate 16, which allows sufficient clearance for the sample holders 12 to dien be positioned ve ⁇ ically between die holes 20 in me top plate 16 and die corresponding holes 20 in d e base plate 14.
• Each sample holder 12 is secured in position by inse ⁇ ion of a respective sample receptacle suppo ⁇ means 22, which enters into a screw threaded engagement with a screw threaded po ⁇ ion provided at die lower end of die sample holder 12.
• a screw driver is preferably employed to tighten die engagement.
• the resulting cooling assembly 10 is substantially cylindrical.
• Samples for cryopreservation are diluted widi an appropriate cryodiluent (examples of which are well-known to those skilled in me art) and drawn into 0.25 or 0.5ml synthetic plastics straws.
• the straws are then sealed widi colour-coded powder or beads and initially chilled: hydrated polyacrylamide gel pellets are frozen by immersion in LN and mixed widi unfrozen gel pellets to form a gel/ice mixture.
• the gel/ice mixture is used for initial chilling of die straws. Referring to Figure 4, die straws 13 are then wiped dry and one straw placed in each sample holder 12.
• the tapered region 24 of the receptacle suppo ⁇ means 22 becomes inse ⁇ ed a shon distance into the lower end of die straw 13 and serves to prevent the straw from making substantial contact with the sample holder 12.
• a thin air-jacket 15 is formed around each straw 13 within the sample holder 12, which air-jacket 15 inhibits the establishment of a temperature gradient ve ⁇ ically along the length of the straw 13 when the cooling process commences.
• the straw 13 is suppo ⁇ ed at its upper end by passage through the hole 20 in die top plate 16.
• the sample holder 12 is of such a length as to ensure that a po ⁇ ion of the straw projects above die top plate 16, which enables the straw to be grasped and facilitates retrieval of the straw from the cooling assembly 10.
• FIG 5 shows an enlarged perspective view of a collar 32 for use in conjunction with the cooling assembly of the invention.
• the collar 32 is a substantially annular metallic ring, defining a central apermre of sufficient diameter to allow location of the collar around die outside of the opening of a Dewar vessel (such as 30 in Figure lc).
• the lower surface of die collar 32 is provided with six identical slots 40 positioned at regular intervals around die circumference thereof. Each slot 40 accommodates d e handle of a respective storage canister (34 in Figure lc).
• the upper surface of the collar 32 is provided wid tiiree pairs of slots 42 positioned at regular intervals around the circumference thereof, each member of any pair of slots 42 being capable of accommodating the radially projecting po ⁇ ion of a respective cooling assembly suppo ⁇ arm 18.
• Each pair of slots 42 is cut to a different depth, such tiiat, by altering the slots 42 used to suspend die cooling assembly 10, the position of the cooling assembly 10 can be varied relative to the Dewar vessel 32.
• Figure 6 shows tiiree sets of graphs showing cooling rates.
• Figure 6a shows the graphs of temperature (20°C to -80°C) against time (minutes) obtained when cooling samples in a controllable rate cooler ("CRC").
• CRC controllable rate cooler
• the cooling rate is substantially linear between a temperature of 20 °C and -50°C, widi a minor departure from the straight line during freezing of d e sample (at about - 10°C).
• Figure 6c shows the graphs obtained using a conventional passive cooler. As can be readily seen, tiiere is considerable variation between different samples, and the pre-freezing and post-freezing cooling rates are very different.
• Figure 6b shows the cooling rates obtained when using me cooling assembly of the invention in conjunction with a passive cooler. It can be observed that the cooling rate is substantially linear from 20 °C to -50 °C (as with die CRC) but declines slightly at lower temperatures. The only departure from the straight line in the temperature range 20 to -50°C is during freezing of die samples. The samples in question were samples of milt from the Tilapia Oreochronus niloticus.
• Figure 7 shows similar cooling rates obtained widi die same apparatus under field conditions in a hot country (Mexico).
• the mean pre-freezing cooling rate (of four runs) was 5.3°C/minute (SeM 0.4), and die mean post-freezing rate was 4.3°C/minute (SeM 0.25), giving an overall mean cooling rate of 4.8°C/minute (SeM 0.5).
• SeM 0.4 die mean post-freezing rate
• SeM 0.25 die mean post-freezing rate
• SeM 0.5 overall mean cooling rate of 4.8°C/minute
• cooling assembly is designed for use with a Dewar vessel of greater than ten litre capacity, the cooling assembly comprising seventy sample holders.

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Cited By (9)

Citations (9)

• 1994
  • 1994-07-16 GB GB9414428A patent/GB9414428D0/en active Pending
• 1995
  • 1995-07-17 WO PCT/GB1995/001677 patent/WO1996002801A1/en active Application Filing
  • 1995-07-17 AU AU29335/95A patent/AU2933595A/en not_active Abandoned

Patent Citations (9)

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