US20070042488A1

US20070042488A1 - Cell spraying device, method and sprayed cell suspension

Info

Publication number: US20070042488A1
Application number: US11/518,012
Authority: US
Inventors: Reinhard Bornemann
Original assignee: Individual
Current assignee: Renovacare Sciences Corp
Priority date: 2001-02-07
Filing date: 2002-02-07
Publication date: 2007-02-22
Also published as: US9029140B2; ES2747300T3; US20190307550A1; US20180098840A1; US9078741B2; US20150182739A1; US10631974B2; US20210169636A1; EP2957288A1; ES2534353T3; US20020106353A1; US10729536B2; AUPR298901A0; US20110311497A1; US20100196334A1; US9867692B2; US20110150848A1; EP2957288B1; EP3632452A1; PT2343079E

Abstract

The invention provides a device and methods suitable for producing a cellular spray of cells. The sprayed cells are of interest for covering and growing on a surface, including a skin wound. In applying the method and/or using the device, cells for grafting onto a patient are dispersed in a solution and sprayed with the device for distribution over the recipient's graft site.

Description

FIELD OF THE INVENTION

This invention relates to a simple, rapid, and cost effective technique for the grafting of cells, in particular to a device for spraying a cell suspension from a tissue sample obtained from a donor site and distributing that cell suspension to a recipient site.

BACKGROUND

Spraying of cells may be of interest for the distribution of cell suspensions onto a tissue wound. This can be applied, e.g., in general surgery to help regenerate tissue trauma. There are many methods for treating skin wounds known to those skilled in the art. For example, skin grafting techniques exist, which aim to reconstruct skin areas of the body that have suffered either damage or defects to the skin. In general, these types of grafts are classified according to their host-donor relationship and by their thickness. The most clinically applied graft is the autologous graft, whereby tissue is taken from one area of the body and applied to another area. The grafted tissue then develops a new blood supply and attaches to the underlying tissues. There are several types of skin grafts presently used, including split-thickness, full-thickness grafts, and micro-grafting. Each of these graft types must be prepared using certain techniques, and each one has its inherent advantages and disadvantages. Split-thickness grafts often require considerable skill, time and expensive equipment. Further, donor sites are painful, result in scarring and limit the coverable area. Although split-thickness grafts may be more successful than full-thickness grafts, they are usually cosmetically less attractive. Full-thickness grafts require less skill or expensive equipment, and their cosmetic appearance is better than that of split-thickness grafts. However, full-thickness grafts do not “take” as well as split-thickness grafts. Micro-grafts are more easily accomplished and require no special instruments. However, their cosmetic appearance is not as good as other techniques, as the resulting scarring is unacceptable.
A variation to the above grafting techniques is the mesh graft, which is a type of split-thickness or full-thickness skin graft in which parallel rows of slits are cut into the treated tissue. Some of the advantages of mesh grafts include: greater coverage of the effected area, drainage of blood or serum from beneath the graft, and increased conformity of the graft to uneven recipient areas. This technique has been very successful, with 90 to 100 percent “take” after the grafts have been applied on healthy granulation beds.
An alternative to split-skin grafting is to form a blister under suction at a donor site, then remove the skin above the blister and transplant it onto the recipient site. The production of blisters to treat wounds has been used since the 1960s. The blisters are produced by a suction device, such as Dermavac™, at a suction pressure of approximately 250-300 mmHg for 1-2 hours. The blisters are then cut off and placed on the wound. The healing time is around 10-14 days. There are several disadvantages to this method such as the amount of time required to prepare the graft is too long and the graft may not result in re-pigmentation of the area; or uneven pigmentation is common around the edges of the area of treatment.
Micro-grafting has become a more common approach for large area cover and involves the “snipping off” of a number of very small sections of tissue from a donor site and applying them to a dressing that is which is in turn applied to the wound area.
Another technology for the generation of tissue in vitro is to culture epidermis. Cultured epithelial autografts (CEA), provided in confluent grown cell sheets, are an important adjunct in the coverage of burns and other situations in which large areas of the body's surface experience skin loss. There are many centres throughout the world with tissue culture facilities whose aim is to produce autologous epithelial grafts for use in a wide variety of applications. The usefulness and application of CEA is related to its ability to achieve confluent cells sheets suitable for grafting. This technique overcomes many of the disadvantages of the previous treatments described above. For example, cultured epithelial autografts reduce the demand for donor sites. However, these autografts are slow growing and require time to cultur, which often exceeds the preparation time of the recipient's sites. Moreover, blister formation by wound secretion below the sheet grafts hinder grafting.
Navarro et al. (2000) and Wood et al. (2003) describe the use of single cells suspended in Hartmans's solution and distributed over the wound, thus avoiding the sheets. The cell suspension may be delivered via a pipette, common “eye-droppers,” syringe and needle, and/or other similar devices to place small quantities of cellular suspension on a graft site. As method of choice a mechanical hand driven spray technique is described (see references).
The spray technique adresses some afore mentioned problems in the field. A hand driven spray method and subsequently the distribution of the cells, however, is not performed in a controlled manner and results in uneven cell distribution.
The present invention provides a method, a device, and a cell suspension generated by using the method, each of which seeks to ameliorate some of the disadvantages associated with prior art CEA grafting technology.

SUMMARY OF THE INVENTION

The present invention provides a method and/or device suitable for producing a transplantable cellular spray of living cells suitable for grafting to a patient. In applying the method and/or in using the device, cells suitable for grafting to a patient are dispersed in a solution and sprayed with the device for distribution over the recipient graft site.
According to the invention a method is provided for spraying a cell suspension through a controlled spray head suitable for application to a patient utilizing a spray device, which method comprises the steps of: (a) subjecting a tissue sample including cells suitable for grafting to a patient, to at least a physical and/or chemical dissociating means capable of dissociating cells in the tissue sample; (b) taking the cells suitable for grafting on to a patient into a saline solution, (c) filtering the cellular suspension produced to remove large cellular conglomerates; and spraying the cell suspension through a spray head.
According to the invention an electronically controlled apparatus is provided as a medical device for distribution of tissue regenerating cells in a sterile suspension over a tissue surface via electronic controlled compressed gas and/or pump driven spraying through a sterilizeable spray head, providing continuous force application in a single shot and generating suspension drops containing cells.
According to the invention there is provided a cell suspension produced according to the above-described method. Preferably the cells in the suspension are autologous cells (i.e. they are isolated from the patient requiring an autograft), or stem cells.
According to another aspect of the invention a method is provided to treat a patient in need of graft surgery.
Other aspects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing description, which proceeds with reference to the following descriptions and drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 compares two application modes of skin cells to a patient. Application of the method and/ or device described in this text at hand, spraying skin cells onto a skin wound surface, is illustrated on the left side. This can be compared to the state of the art medical treatment with skin cell application using confluently grown keratinocyte sheets (right). Using sprayed cells result in the need of fewer cells while in a larger treatment surface can be enabled for therapy. Blister formation is avoided by the use of single cells without forming a closed sheet. Reducing the cell number speeds up application time by avoiding an in vitro cell expansion. This reduces in vitro differentiation and therefore better preserves basal keratinoyte progenitor cells in the cell suspension.

DESCRIPTION OF THE INVENTION

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variation and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification. Functionally equivalent products, compositions and where appropriate methods are clearly within the scope of the invention as described herein.
Throughout this specification and the claims that follow, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Having regard to the above, this invention provides a unique method and/or device suitable for producing a transplantable cellular suspension of living tissue suitable for grafting to a patient. In applying the method and/or in using the device cell preparations of different origin may be used. This includes stem cell preparations and patient autologous cells, whereas donor tissue is harvested and subjected to a tissue dissociating means. Cells suitable for grafting to a patient, or back to a patient, are dispersed in a solution that is suitable for immediate dispersion over the recipient graft site.
The subject invention has many advantages over the prior art some of which are described in the following paragraphs.
1. It provides a time efficient method for supplying a cellular cover to a tissue in a clinical setting. That is, cells are finely and evenly distributed over a wound, avoiding the use of cell sheets. This is achievable because there is a controlled procuring of the cell suspension with a method provided by an apparatus, thus allowing cell spraying to be performed more evenly than the mechanical hand operated methods of the prior state of the art.
2. It provides a method and an apparatus, which avoids the blister formation associated with the use of conventional CEA's.
3. It aids in the achievement of rapid cell coverage in areas of tissue wounds, tissue trauma/injury and donor sites. It provides a means for reducing the size of skin cell donor sites—the biopsy donor site is markedly smaller than a split skin graft donor site and reduces or eliminates the use of split skin graft donor sites; improves the expansion rate of cell coverage; improves the rate of healing of small burns; is useful for small areas of skin reconstructions, such as scars; and improves scar quality.
4. It provides a means for the treatment of various skin disorders or diseases. For example, it may be used for the following: epidermal resurfacing, replacement after skin loss, site match-up during re-pigmentation of an area of skin, treatment of burn wounds, leukoderma, vitiligo, piebaldism, in the treatment of scars—for example, caused through incorrect wound healing, improper scar direction or scar distortion from wound contraction, acne scars; resurfacing cosmetic dermabrasion, resurfacing after laser treatment and in association with dermal reconstruction. Additionally the method may be used for cell replacement therapy, including, for example, nerve cell replacement treatment, epithelial cell (such as urothelial cell, buccal mucosal cell and respiratory epithelial cell) replacement treatment, endothelial cell replacement treatment and osteogenic precursor cell replacement treatment. The method may also be used to stimulate tissue regeneration in surgically induced wounds.
5. It provides a means to produce a suspension of cells in a ratio to each other comparable with those seen in situ. That is, due to the manner of preparation of the cellular suspension, cells such as keratinocyte basal cells, Langerhans cells, fibroblasts and melanocytes typically have enhanced survival rates in comparison to standard tissue culture techniques, whereby selective cell culture can result in the loss of certain cell types. This has the advantage of allowing for the correct re-pigmentation of skin after a skin graft.
7. It allows faster surgery and healing—thereby reducing trauma for patients during the phase of their medical care.
The invention relates to at least two distinct cell sources, all suitable for use in resurfacing and regeneration of damaged tissue: (i) non-autologous cells, including stem cells, and (ii) autologous cells, including the patient's own progenitor cells.
The invention provides a method for preparing an autologous cell suspension. According to this method, tissue is harvested from a patient by means known in the art of tissue grafting. Preferably this is achieved by taking a tissue biopsy. With the harvesting of the biopsy consideration must be given to the depth of the biopsy and size of the surface area. The depth and size of the biopsy influence the ease at which the procedure can be undertaken and the speed with which a patient recovers from the procedure. In a highly preferred form of the invention the chosen donor site should appropriately match the recipient site, for example post-auricular for head and neck, thigh for lower limbs, inner-upper-arm for upper limbs, or palm for sole or vice-versa.
Once a biopsy has been harvested from a patient the tissue sample is subjected to physical and/or chemical dissociating means capable of dissociating cellular stratum in the tissue sample. Methods for dissociating cellular layers within the tissues are well known in the field. For example, the dissociating means may be either a physical or a chemical disruption. Physical dissociation means might include, for example, scraping the tissue sample with a scalpel, mincing the tissue, physically cutting the layers apart, or perfusing the tissue. Chemical dissociation means might include, for example, digestion with enzymes such as trypsin, dispase, collagenase, trypsin-edta, thermolysin, pronase, hyaluronidase, elastase, papain and pancreatin. Non-enzymatic solutions for the dissociation of tissue can also be used. Preferably, dissociation of the tissue sample is achieved by placing the sample in a pre-warmed enzyme solution containing an amount of enzyme sufficient to dissociate cellular stratum in the tissue sample.
After the tissue sample has been immersed in the enzyme solution for an appropriate amount of time, the sample is removed and washed with nutrient solution.
The saline/nutrient solution used in the method should be capable of significantly reducing and more preferably removing the effect of the enzyme either by dilution or neutralization. The nutrient solution used in the method will also preferably have the characteristics of being (i) free of at least xenogenic serum, (ii) capable of maintaining the viability of the cells until applied to a patient, and (iii) suitable for direct application to a region on a patient undergoing tissue grafting. After application of a suitable saline/nutrition solution to the tissue sample, the cellular stratum of the sample is separated permitting the cells capable of reproduction to be removed from the cellular material and suspended in the nutrient solution. Where the tissue sample is skin, the dermis and epidermis are preferably separated to allow access to the dermal-epithelial junction of both surfaces.
Cells capable of reproduction are then removed from the separated stratum by any means known in the art. Preferably, the reproductive cells are scraped off the surface of the stratum using an instrument such as a scalpel. Cells capable of reproduction within the dermal-epithelial junction include but are not limited to keratinocyte basal cells, Langerhans cells, fibroblasts and melanocytes. Following release of the cells from the tissue sample they are suspended in the saline/nutrient solution.
The invention provides simultaneously a method for using a non-autologous cell suspension. To procure cells of any source, the cells are suspended in an aquaeus saline/nutrition solution. The solution may be anything physiological from a basic salt solution to a more complex nutrient solution. Preferably, the nutrient solution is free of all serum but contains various salts that resemble the substances found in body fluids; this type of solution is often called physiological saline. Phosphate or other non-toxic substances may also buffer the solution in order to maintain the pH at approximate physiological levels. Suitable nutrient solutions that are preferred base on Ringer-lactate solutions, including Hartmann's solution, dialysis solutions, and on peripheral intravenous nurtition solutions.
Preferably only a small volume of solution is applied to the tissue sample after the harvesting steps, or by suspending non-autologous cells, otherwise the suspension may become too fluid therein providing difficulties in applying the suspension to the graft.
The cell suspension is then applied by using the spray device, described in the claims. To avoid excessively large cellular congregates in the cellular suspension the suspension is preferably filtered, either prior to using the suspension with the device, or by a specific feature of the device.
Prior to application with the device or immediately after filtering, the cellular suspension may be diluted to produce an appropriate cell density suitable for the purpose with which the suspension is to be used.
According to the invention there is provided a sprayed aqueous cell suspension, highly suitable for tissue regeneration and grafting techniques, produced by the method described. An important advantage of the invention is an even cell distribution.
An important aspect of utilizing such a suspension in grafting technology is that it can be used to greatly expand the area or volume of a wound that can be treated quickly by in situ multiplication of a limited number of cells. Cellular multiplication is encouraged on the patient rather than in an in vitro system, as provided by the state of the art CEA method.
The number and concentration of cells seeded onto graft site may be varied by modifying the concentration of cells in suspension, or by modifying the quantity of suspension that is distributed onto a given area or volume of the graft site.
Another unique feature of the cell suspension produced according to the method of the invention is that the composition of cells in the cellular preparation is comparable to that seen in situ compared to prior art CEA cellular preparation. Importantly, it contains the basal keratinocytes and skin progenitor cells for skin regeneration, which are typically lost in the CEA method. In this prior art, culture of the cellular preparation utilizes selective culture for keratinocytes, therefore the loss of cellular constituents such as skin progenitor cells, fibroblasts and melanocytes occurs, whereas the cellular suspension produced by the method of the invention has a cell composition comparable to the in situ cell population.
According to a further aspect of the invention there is provided a method of treatment of the patient requiring a tissue graft. By this method the cellular suspension produced according to the invention is applied to a graft site.
According to the invention there is provided an apparatus containing a spray head to distribute the cells. The suspension may be sprayed through any type of nozzle that transforms liquid into small airborne droplets.
According to the invention there is provided an electronically controlled apparatus as a medical device to operate the spraying through a sterilizeable spray head. Preferrably the apparatus enables a distribution of cells using a 0.5-60±20 ml sterile cell suspension through a spray head. Preferrably, the apparatus transfers the cell suspension from a medical grade disposable sterilizeable syringe, including 0.5-60 ml sterile Luer-lock syringes.
The apparatus can be operated preferrably basing on producing compressed gas, e.g. air, for the spray head, or forcing the cell suspension pump driven through the nozzle, e.g. by motor operated pushing of a sterile Luer-lock syringe containing the cell suspension, without mixing with gas. The apparatus preferrably provides continuous force application over a range of 0.5-10±1.0 minutes for in a single shot, or several shots, and generates suspension drops containing cells in the range of 30-500+200 millimeter.
The apparatus may provide means to measure and control parameters such as flow, pressure, and/or temperature.
The apparatus preferrably also transfers the cell suspension from a medical grade sterilizeable container to the sterilizeable spray head via a disposable filter capable of separating large cellular congregates from a cellular suspension. Any filter capable of separating excessively large cellular congregates from the suspension may be used. In a highly preferred form of the invention the filter exibits a cut off of approximately 5-100 cells, preferably 20-60 cells and most preferred 40 cells.
The apparatus may comprise a first and second member wherein: (i) the first member includes power supply, gas/air supply and electronic controls, and (ii) the second member includes a sterilizeable spray head and the container with the cell suspension. In that case both members are connected through a cable/wire/tube sensor/effector connector which may be sterilizeable or can be covered with a sterile operation foil hose and has suitable connectors to the memebr (i) and (ii).
The apparatus may comprise a first and second member wherein both members are wirelessly connected for data exchange, including blue tooth technology, to connect sensor/effector controls in the first and second member.
The apparatus may also feature battery operation, facilitating an easy use in operation theaters. In that preferred case, the apparatus comprises an all-in one device for hand-held operation.
After the cell suspension has been applied to the recipient graft site, the wound may be covered with a wound dressing. Preferably, the healing of the wound is followed up by standard protocols for graft treatment known to those skilled in the art.

EXAMPLE

If not otherwise indicated, all materials were purchased from Biochrom AG, Berlin, Germany. Media were supplemented with antibiotics (Penicillin/Streptomycin, 120 μ/g/ml) and antimycotics (Amphotericin B, 2.5 μg/ml). A 1 cm²skin biopsy was obtained after obtaining informed consent of the donor and cut into 2 mm²pieces. The method is described in more detail in Johnen C, et al., Burns. 2006; 32(2). Prior to separation of epidermis and dermis the pieces were exposed to 0.4% collagenase (Serva Electrophoresis GmbH, Heidelberg, Germany) in DMEM at 37° C. Separated epidermis was incubated with 0.05% trypsin/0.02% EDTA-solution for 15 minutes. The single cell suspension was cultivated in a standard culture flask with serum free culture medium (EpiLife, TEBU, Offenbach, Germany). Cells were incubated at a cell density of 10⁴per cm², using a CO₂-incubator (Heraeus BB 6060, Kendro, Langenselbold, Germany) at 37° C. in a humidified atmosphere with 5% CO₂. Medium was changed every two days. As 80% confluence was reached, cells were detached by trypsinization and used with the above described compressor operated spray device prototype. Operation partameter were set to an air flow of 3.7 l/min and a fluid flow of 4.2 cc/min. This adjustment resulted in a spray pressure of 8.2 mmHg. The cells were sprayed into a non medium filled standard cell culture dish at a density of 10⁴cells per cm². As control cells from the same suspension were cultivated, under the above described culture conditions after pipetting into a medium filled culture flask with the same density. Cell morphology was monitored by light microscopy (Zeiss, Axiovert 25). Sprayed and non-sprayed cells showed similar morphologic appearance in light- and phase-contrast microscopy, they also showed comparable follow up culture behavior.
Further examples will be given in a manuscript, to be submitted after filing of the application at hand to Elsevier, N.Y., USA for consideration in the book “Principles of Regenerative Medicine” edited by Antony Atala et al., to be published in 2007.
Modifications and variations of the described methods and device of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant field in which this invention resides are intended to be within the scope of the described claims.

Claims

1. A device for an electronically flow controlled distribution of tissue regenerating cells, including skin cells, in an approximately 0.5-80 ml, preferably 5-40 ml and most preferably 5-20 ml sterile suspension across an area for further growth, via controlled compressed gas and/or pump driven spraying through an electronically pressure and/or flow controlled spray head, providing continuous application over a range of approximately 0.5-10, preferably 1-5 and most preferably 1-2 minutes in a single shot, or several shots, preferably up to 3 shots, while generating suspension drops containing cells ranging between 30-500±200 mm in size, wherin the spray head may be sterilizeable or can be covered with a sterile surgery foil hose, and wherein the following procedures are enabled: (a) subjecting a tissue sample including cells for grafting onto a patient, to at least a physical and/or chemical dissociating means capable of dissociating cells in the tissue sample; (b) taking the cells, which may also have been expanded after the aforementioned step, for spraying into a saline solution, wherein the solution is (i) free of xenogenic serum, (ii) capable of maintaining the the cells alive until applied, (iii) contains electrolytes in a physiologic composition, and (iv) allows direct application to a biomaterial surface or region on a patient undergoing tissue grafting; (c) filtering the cellular suspension produced according to steps (a) and (b) to remove cellular conglomerates of more than 10-500 cells, preferably 50-300 cells and most preferably 50-100 cells; and (d) distributing the cell suspension through the flow controlled spray head onto the receipient surface.

2. A device according to claim 1 that is driven via a gas compressor.

3. A device according to claim 1 that is driven via a motor.

4. A device according to claims 1 to 3, which contains sensors to measure flow and/or pressure, and/or temperature.

5. A device according to claims 1 to 4, which contains sensors to measure and feedback controls to control flow and/or pressure, and/or temperature.

6. A device according to claims 1 to 5, which transfers the cell suspension from medical-grade disposable sterilizeable syringes, including 0.5-60 ml sterile Luer-lock syringes, preferably 1-50 ml and most preferred 5-20 ml syringes.

7. A device according to claims 1 to 6, which transfers the cell suspension from a medical-grade sterilizeable container, including luer-lock syringes, to the sterilizeable spray head via a disposable filter capable of separating large cellular congregates with a cut off of approximately 5-100 cells, preferably 20-60 cells and most preferred 40 cells from a cellular suspension.

8. A device according to claims 1 to 7, which is battery operated.

9. A device according to claims 1 to 8 for distributing a cell suspension, containing a first and second component wherein: (i) the first component includes the power supply, gas/air supply and electronic controls, and (ii) the second component includes a sterilizeable spray head and the container with the cell suspension; and wherein both components are connected through a cable/wire/tube sensor/effector connector, which may be sterilizeable or can be covered with a sterile operation foil hose and has suitable connectors to the components (i) and (ii).

10. A device according to claim 1 to 9 for distributing a cell suspension, containing a first and second component wherein both components are wirelessly connected for data exchange, including blue tooth technology to connect sensor/effector controls in the first and second component.

11. A device according to claims 1 to 10 which utilizes a solution as an aquaeous solution containing electrolytes in a physiologic composition, including Ringer-Lactate like electrolyte solutions, including Hartman's solution.

12. A cell suspension produced according to the devices and methods of claims 1 to 11.

13. A cell suspension produced according to at least one of the aforementioned claims prepared from autologous cells, including in vitro expanded autologous cells.

14. A cell suspension produced according to at least one of the aforementioned claims prepared from stem cells.

15. A method of utilizing the device according to at least one of the aforementioned claims and producing a cell suspension for treating a patient in need of graft surgery, which is is comprised of the steps: (a) preparing a cell suspension according to the method of claims 1 to 14; and (b) administering the suspension directly to a region on the patient that requires a cell graft in a manner that facilitates spraying of the cell suspension in an even distribution over the graft area.

16. A method of utilizing the device according to at least one of the aforementioned claims and producing a cell suspension for treating a patient in need of graft surgery with a cell suspension according to one of the aforementioned claims, said devices according to claims 1 to 11 and administering the suspension to a region on the patient that requires a cell graft in a manner that facilitates spraying of the cell suspension in an even distribution over the graft area.

17. A method of coating an artificial surface or a biomaterial surface for commercial use with the device according to at least one aforementioned claims, said method is comprised of the steps: (a) preparing a cell suspension according to the method of claims 1 to 16; and (b) administering the suspension directly onto an artificial or a biomaterial that requires a cell coated surface in a manner that facilitates spraying of the cell suspension in an even distribution.