JPH09110601A - Cryopreservation of cultured epithelial cell sheet - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable utilization as skin wound bandage having survivability and colony-forming ability of a cell by treating a culturing sheet of epithelial cell cooled to a specific temperature or below and impregnating a cryoprotecting agent therein. SOLUTION: A culturing sheet of epithelial cell in which a cryoprotecting agent is impregnated at about at about <=-180 deg.C carries out two stage treatment raising the temperature in a range of -120 to -80 deg.C (at a changing rate of 20-100 deg.C per min) and raising the temperature to 20-37 deg.C. A method for raising the sheet temperature to about -80 deg.C by exposing the sheet with a gas in rise of temperature and heating the sheet to 20-37 deg.C in a solution is preferably used as the method for raising the sheet temperature. Further, as addition step, it is desired to remove a cryoprotecting agent immersed in the sheet by incubating the sheet in a balanced salt solution (especially suitably lactic acid Ringer's solution) over 4hr.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to cryopreservation and long-term preservation of a cultured epithelial tissue sheet which has cell viability and colony forming ability and is useful as a dressing for skin wounds.

[0002]

2. Description of the Related Art The development of skin wound dressings that promote the growth of new cells while preventing the leakage of serum and infection of wounds on the skin due to burns, ulcers, surgical excision, etc. is of great importance to the medical community. Has been done. Conventional dressings cannot adequately protect widespread wounds, and several alternatives have been developed. These replacement methods include a method of transplanting cut or completely thick cadaver skin or pig skin, human allograft, and human autograft.
Unless immunosuppressive therapy was employed, rejection, except autologous transplantation, was in most cases unsuccessful. Furthermore, conventional autograft techniques have not been feasible for severe burns that cover a large area.

Green et al. Have developed a method for culturing epithelial cell sheets with several cell thicknesses for treating burns, ulcers, and other skin wounds. U.S. Patent 4,
No. 016,036 discloses a method of continuously culturing keratinocytes to produce a layered sheet of epithelium. US Pat. No. 4,304,866 discloses a method of culturing keratinocytes and producing an implantable cell sheet by peeling the cell sheet from the adherent support using an enzyme such as despase. US Pat. No. 4,456,687 discloses agents useful in promoting epithelial cell growth. The disclosures of these patents are incorporated herein by reference. In the culture system developed by Green et al., Epithelial cells rapidly divide on the surface of a tissue culture dish or flask, eventually forming a confluent, moderately layered, tightly bonded sheet of cells. . These confluent cultures can be published as a bound cell sheet, for example, by treatment with the enzyme despase (see US Pat. No. 4,304,866). The culture sheet is attached to petrolatum-immersed gauze or other non-adhesive support, transferred to the operating room in the culture solution, and used for treating the patient.

A wide range of burns can be covered with autograft materials prepared by these methods.
However, autologous transplantation requires time for culture. While culturing cells for autologous transplantation, the wound can be protected by an allogeneic transplant, which is effective as a temporary wound dressing. The allograft material promotes the healing of chronic skin ulcers and scars on the exfoliated skin graft supplier.
Cultured autologous material prepared by the method of Green et al. Is available from BioSurface Technology, Inc., Cambridge, Mass. Allograft material is available for experimental and clinical trial use.

A significant practical problem in the use of cultured epithelial cell transplants is their inherently short shelf life. The viability and colony forming ability of the sheet are rapidly lost when leaving the medium in which the sheet is growing. This limits the time and distance that the sheet travels from the production facility to the operating room. Cell sheets are exceptionally brittle. This cell sheet can maintain the function of growth and colony forming ability when treated on a wound surface at most within 6 to 8 hours after exfoliation with dispase. Time constraints hinder securing large cell sheets. The development of cryopreservation techniques to extend the storage time of culture sheets will enable the maintenance of large numbers of cell sheets that can be transported worldwide.

The prior art is replete with descriptions of various tissue preservation methods including cryopreservation, the use of specific cell culture media, reliable packaging techniques, and the like. Cryopreservation allows a substance to be frozen and stored for long periods of time in the presence of a cryoprotectant. The drug eliminates aqueous material inside and outside the cells and prevents the formation of ice crystals. There are a number of disclosed protocols regarding the nature and amount of cryoprotectants and / or the time, course, temperature, etc. of the freezing step in an attempt to maintain cell viability after a freeze-thaw cycle. See, for example, U.S. Pat. Nos. 4,559,298 and 4,688,387.

Preserving tissues by cryopreservation is
It is a complex and expensive process, which makes it possible to produce highly variable results. However, no other method has extended the storage viability of animal tissues for a very short time, for example, more than about 8 hours. For example, Pittelkow
Other examples (J. Invest. Dermatol .4 : 410-17,413-14, 1986)
Please refer to. For a long time, skin banks have used frozen human skin for burns as a temporary allograft coating. However, this freeze is not very practical.

Preserved skin is metabolically active, but it is not certain that epithelial cells can self-renew (Hemi
bach, D., et al., Artificial Dermis for Major Burn
s: "aMulti-Ceter Randomized Clinical Trial", Ann, S
urg. 313-320, September 1988).

As described by Green et al.
Large-scale production of cultured epithelial autologous transplants facilitates permanent widespread coverage of the patient's own skin. Although large quantities of cultured epithelium are produced for patients, the epithelial life span is limited and presents a major problem. As a result of this, Canceleda and Delkka are 10%
We have developed a method to freeze confluent sheets of cultured keratinocytes in culture medium containing glycerol (EP
0 296 475). However, experience with this method has confirmed that the rate of recovery of the cells fluctuates and is generally very low. Furthermore, the range of incubation times was very narrow and proved impractical for large-scale production. Moreover, transplants frozen by some protocol and stored and thawed in liquid nitrogen are often cracked. Wound covering may be possible, but at the cellular level, problems remain regarding the active state of the confluent sheet.

Early studies using high molecular weight cryoprotectants have confirmed that only polyvinylpyrrolidone (PVP) and dextran (molecular weight 30-100Kd) prevent red blood cell destruction during cryopreservation. (Pegg, DE, "Banking of
Cells, Tissues and Organsat Low Tempratures ", Cur
rent Trends in Cryobiology, edited by A. Smith, 1970). The combination of glycerol with dextran and hydroxylethyl starch partially maintains the automatic force of freeze-thawing of sheep sperm (Schmehl et al., "The Effects of Nonpenetr.
ating Cryoprotectants Added to Test-Yolk-Glycerol
Extender on the Post-Thaw Motility of Ram Spermato
zoa "23 Cryobiol. 6: 512-17, 1986).

It has been confirmed that, when trypan blue dye is excluded as an index of viability, only hydroxylethyl starch (HES) promotes the viability of cryopreserved cells which are hematopoietic sources. However, it is only effective if a cryoprotectant is added and then slowly removed in a very time-consuming manner (Conscience, Fischer, "An.
Improved Preservation Technique for Cells of Hemap
oetic Origin "22 Cryobiol. 5: 495-98, 1985). This viability parameter only provides information on short term membrane stability, not on tissue growth potential or long term viability. Not the authors of this document, HE
S is said to be ineffective for cryopreservation of epithelial tissue-derived cells. In another study, HES was effective at cryopreserving cells that are hematopoietic sources, as determined by cell proliferation analytes, a parameter that better reflects long-term viability of cells. (Wang, et al., Cryobiol, 2
4: 229-237, 1987).

Studies using high molecular weight non-permeable (glass moldable) agents as cryoprotectants tend to concentrate on floating cells such as the above-mentioned red blood cells and lymphocytes. However, the need to maintain the integrity of the cohesive sheet of cells during cryopreservation severely limits the recovery of viable cells (ie cells with the ability to regenerate tissue).

[0013]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a cryopreservation methodology capable of preserving a living culture sheet of epithelial cells. In this methodology, the method preserves the structural integrity of the sheet to enable the formation of epithelial tissue useful for treating wounds, and also maintains the mitotic capacity of the cells of the sheet. It is. A novel method of cryopreserving a confluent sheet of live cultured epithelial cells has been discovered so that it remains useful as a wound dressing for the skin. According to this method, the colony forming ability of the cells in the sheet can be maintained, that is, a significant number of the cells in the epithelial cell sheet can be mitotically separated so that healthy epithelium can be regenerated. Live cells can be stored. The method further allows the cultured epithelial sheet to be harvested when the sheet is matured and stored in a cryopreserved state for future use.

[0014]

The method comprises the following steps. 1) Immerse the sheet in a cryopreservation solution containing at least a non-cell penetrating glass forming agent and preferably a cell penetrating glass forming agent. 2) Cool the sheet to at least -65 ° C, more preferably -120 ° C (glass transition temperature of water) or less, and particularly preferably -180 ° C or less, which is a long-term storage temperature, and freeze the sheet. As a preferable cooling method, it is desirable to cool in the range of about -180 to -196 ° C under the vaporized atmosphere of liquid nitrogen. 3) As the next step, store the sheet in the range of at least -65 ° C to about -180 ° C. 4) At least 35% colony forming ability, often
Thaw to prepare a complete sheet of cells that are 40% and often more than 50%.

The cryoprotective solution contains the following components in weight percent. Non-cell penetrating glass forming agent about 5-20
%, Preferably 15%, and about a cell-penetrating glass former.
Ten%. Glycerol, propylene glycol, ethylene glycol, dimethyl sulfoxide (DMSO) or a mixture or derivative thereof is used as a cell-permeable glass forming agent. Glycerol is particularly preferred as the cell permeable glass former. Dextran, polyvinylpyrrolidone, hydroxylethyl starch, chondroitin sulfate, polyethylene glycol, or a mixture or derivative thereof is used as the non-cell penetrating glass forming agent. Dextran and hydroxyl starch are particularly preferred.

In a preferred embodiment, the freezing step requires gentle cooling of the sheet to at least about -4 ° C to about -80 ° C. Then, it is cooled to a temperature in the range of about -180 ° C to about -196 ° C, which is the vapor temperature of liquid nitrogen. Slow cooling is performed at a cooling rate of about 1 ° C./min. Even more preferably, the melting step requires warming the sheet from low storage temperature to -120 ° C to approximately -80 ° C for 1 to 5 minutes using air or other gas. That is, the heating rate is about 20 ° C / min to about 100 ° C / min. In addition, the sheets can be incubated in a water bath or elevated to temperatures of about 20 ° C to about 37 ° C.

Prior to use as a skin wound dressing, the thawed sheet allows cells to be temporarily stored prior to use.
Rinse without lyoprotectant at physiological pH, eg isotonic buffer, preferably Ringer's lactate. The epithelial cells of the sheet used in this method are preferably cultured keratinocytes. Confluent sheets are several layers of cells thick and consist of different layers.

The cultured human epithelial cell sheet can serve as a permanent autograft material for the treatment of skin wounds. As a temporary allograft material, this sheet can be used as a treatment for chronic skin ulcers and resected graft donors, and also as a highly effective burn dressing it can. Sheets can be prepared with a culture system developed by Reinwald and Green. In this system, continuously cultured epithelial cells rapidly divide on the surface of a tissue culture dish or flask, eventually forming a loose, layered, polarized sheet of cells that are tightly bound to one another. A micrograph of a cross section of the culture sheet is shown in FIG. The stratified epithelial cell culture is treated with an enzyme such as despase, peeled off as a binding cell sheet, fixed on a non-adhesive material such as vaseline or vaseline soaked gauze, and fixed in the operating room. Carry in the culture solution,
Treat the patient.

A significant limitation in the use of cultured epithelial cell transplants is their extreme brittleness and short shelf life. When the explants were separated from their culture supports for more than 8 hours, the cell viability of the explants was determined by measuring the ability of detached cells to recover growth and colony under optimal cell conditions. The experimental result that it is decreasing is obtained. For this reason, sales of cultured epithelial cell sheets have been limited locally. That is, the hospital is close enough to the production center to prepare, transport and administer the graft within at least 8 hours after the first addition of despase to the culture to start the isolation in the culture facility. There was a need. The actual time of transfer was only 2.3 hours due to the time required to prepare the graft. Operating room schedules and graft arrival times had to be carefully coordinated. Preservation of the sheets was not possible at any time, which made it impossible to keep inventory of timed products.

It is clear from the observation that the reason why the viability of the separated epithelial cell sheet is so short is explained. That is, epithelial cells are
It was found that single cells separated from the culture by TA treatment lose their potential for division and undergo final differentiation when temporarily held under conditions that prevent them from recombining with the surface. .

Experiments measuring viability for more than 8 hours revealed the following. That is, the temperature at which the despase-treated graft is retained is a temperature that is extremely critical to cell viability as measured by the colony-forming ability (CFE) and the total number of colony-forming cells that have recovered (CFC or recovered total cells x CFE). It was When the cultured epithelial sheet is stored at a temperature that is equal to or slightly lower than the physiological temperature condition, the CFE of fresh tissue is
Can't hold. Similarly, carefully control the medium conditions, pH, carbon dioxide / oxygen balance, etc.
Preservation at did not have a clearly confident effect. If the culture sheet is at the temperature limit of 10 ℃ to 25
CF when held at ℃, preferably 13 ℃ to 23 ℃
E can hold for approximately 20-30 hours or longer. However, at present, there is no method for storing the culture sheet for more than one day while maintaining its usefulness as a wound dressing. A freshly prepared product for wound healing applications cannot be preserved as a preservative.

Looking at the history of cryopreservation methods, the optimization of the protocol for cryopreserving specific cells can be done with other types of cells, with cells of the same type from different species, or with other types of tissue. It can be seen that the results are not always good when using cells. The method of freezing keratinocyte suspensions has yielded unsatisfactory results when applied to complete sheets. Freezing the complete tissue for actual use in transplantation has not been successful, to our knowledge, of any tissue type (see EP 0296475 by Cancerda et al.).
The method disclosed herein for cryopreserving a confluent sheet of living cultured epithelial cells after separation from the culture substrate can be used to maintain mitotic or cell-forming ability at acceptable levels or to maintain sheet integrity. Hold. In summary, the method consists of 4 steps. First, the cell sheet is allowed to equilibrate in the cryoprotective solution for a time sufficient to thoroughly mix or replace the intracellular and intercellular water with the cryoprotectant. Second, the sheet cools the cryoprotected cells at a slow enough rate of temperature drop to avoid ice crystal formation and subsequent damage, preferably from about -180 ° C to -196 ° C.
Frozen sheets can be stored at about -180 ° C for long periods and at higher temperatures for short periods, for example at -65 ° C. Prior to use, the sheet is warmed with air or other gas within 1-3 minutes at room temperature and then quickly warmed, for example in a water bath, to completely melt it. Fourth, the cryoprotectant is removed by rinsing the cultured epithelial cell sheet with an isotonic buffer such as lactated Ringer's solution.

[0023]

DETAILED DESCRIPTION OF THE INVENTION The detailed steps are disclosed below. Preparation of Cultured Epithelial Cell Sheet Epithelial cells (keratinocytes) are seeded in a T150 culture flask (Costar) at a specific gravity that reaches confluence in 10-12 days. Cultures of frozen cell suspensions prepared from several epithelial cell lines can be used for allogeneic transplantation. Cells obtained by biopsy from burn patients can be used for autologous transplantation. Culture is performed at 37 ° C using a gas-tight flask.
FAD medium (Hams F12 medium with adenine 1 part, Dulbecco's modified Eagle medium (DME) 3 parts, 10% fetal bovine serum (FBS)
, 0.4 μg / ml hydrocortisone, 1 × 10 ⁻¹⁰ M cholera toxin, 2 × 10 ⁻⁹ M triiodothyronine). It is further grown in the presence of deadly emitted 3T3 fibroblasts. See U.S. Pat. No. 4,016,036. 10 ng / ml epidermal growth factor was included in the first inoculation.

When the cell culture reaches confluence,
The cell culture is used to prepare explants. The supernatant medium is aspirated, 40 ml of Desase II (Boehringer Mannheim) are added to the flask to a final concentration of 2.5 mg / ml (about 1.2 U / ml) and incubated at 37 ° C. When the edges of the sheet are peeled off (45 minutes), bake the top of the flask with a soldering iron to remove.

The enzyme solution is replaced with 20 ml of DME medium. The epithelial cell sheet is then rinsed once more with 20 ml DME medium.
Leave 3-4 ml of the second rinse, aspirate the whole solution, and apply 5 × 10 cm gauze (cheese blowpons) impregnated with Vaseline (registered trademark) to the surface cells facing the gauze bandage. Place it on the peeled cell sheet so that it overlaps with the cell sheet. The binding cell sheet is then bandaged with 12-15 staples (Rigaclip Ethicon / J & J).

Sheets are cut wide, fixed, and the grafts are transferred epithelial cell side up to 100 mm dishes. Use a rubber policeman to hold the end of the graft against the dish to prevent it from floating. Add 12 ml of DME gently and then transfer the dish to a storage container. (Fig. 1 shows a cross-sectional view of a typical culture sheet obtained by this method.) Cell recovery (R) and colony forming ability analysis (CFE) show the total number of mitotic cells (CFC). Do to confirm.
In this procedure, the optimal conditions that protect viability during the cryopreservation step are determined. The conditions are the composition of the cryoprotective medium, the equilibration time in the cryoprotective medium prior to freezing, the freezing rate, the storage temperature, the melting step, the rinsing step, the subsequent storage and the like.

The colony forming ability (CFE) analysis was carried out by
After storage for 3 days, the implants prepared as in the example below are used. The unfrozen graft is analyzed as a control. The fixation clip is removed using forceps, and the prepared cell sheet is trypsin (0.05%) and EDTA (0.01%).
Is separated into a single cell suspension of isotonic buffer containing The enzymatic reaction is stopped by the addition of bovine serum, and then 0.5 ml of cell suspension is added to 4.5 ml of FAD to make a 10-fold dilution, which is repeated twice. An aliquot of the first cell suspension is counted using a hemocytometer. Final cell density 1000-200
Cell suspension was prepared to 0 cells / ml, and 1 ml of cell suspension was added to 12 ml of FAD containing 3T3 cells killed by irradiation.
Inoculate the 100 mm dish containing the medium.

After 10-14 days, the cultured cells were fixed with 10% formalin in phosphate buffered saline and 1% rhodamine.
Stain with a mixed solution of% Nile Blue A. Colonies are counted using a dissecting microscope and scored as growing or stunted. CFE is calculated as follows. CFE = total colonies / plated cells

The percent recovery and percent CFE are calculated as follows: (R _FZ / R _FS ) × 100 = Recovery% (FE _FZ / CFS _FS ) × 100 = CFE% Here, FZ means frozen and FS means fresh.

Total recovery of colony forming cells (CFC) is calculated as follows. CFC = ratio of total recovered cells × recovered colony forming ability (CFE)
Ratio CFC = (CFE _FZ × R _FZ / CFS _FS × R _FS ) × 100

CF when using the protocol of the present invention
E% is usually above 35%, generally above 40%, and often reaches 50% or more.

Cryopreservation Protocol The development of cryopreservation methods involves stacking independent studies, connecting optimal elements, and optimizing each element of the process to identify the final step. is necessary. Optimal freezing, compatible with maintaining maximum viability,
Each of the steps of storage, melting and rinsing needs to be specified. These elements are identified by colony forming ability analysis as described above.

The standard cryopreservation medium consists of physiological balanced salt solution (eg cell culture medium) supplemented with bovine serum, glycerol and a cell-penetrating glass forming agent. Cryopreservation of cells in suspension is successful, but the ability to preserve the viability of cells removed from the cell sheet is not well understood. FIG. 2 shows the beneficial effects of supplementing a standard cryopreservation medium with additional components, a non-cell permeable glass former (such as dextran). It should be noted that the use of non-cell penetrating agents with cell penetrating agents significantly improves cell recovery and thus viability (CFC) compared to using cell penetrating agents alone. I have to.

FIG. 3 shows that the non-cell permeable glass former was used to improve viability. These drugs have a complex carbohydrate polymer structure. The non-cell-penetrating glass forming agent preferably comprises about 50-500 kilodalton (kd) polymeric dextran, preferably 50-70 kd chondroitin sulphate, polovinylpyrrolidone, polyethylene glycol, mutated starch such as hydroxyethyl starch. I can give you. Glycerol is preferred as the cell-penetrating glass forming agent, but propylene glycol, ethylene glycol, dimethyl sulfoxide, and other penetrating glass forming agents known from the prior art can also be used.

At the beginning of the cryopreservation step, the cell sheet for freezing in the cryoprotective medium must be soaked for a sufficient time to allow the cells to equilibrate with the cryoprotectant. FIG. 4 shows the effect on viability when a cultured epithelial sheet was equilibrated for a long time with a cryoprotectant before freezing. The data show that the sheets were cryopreserved prior to freezing without affecting the viability of cells in the cryopreserved sheets.
It shows that it can be equilibrated to time.
Equilibration is typically done for approximately 30-60 minutes, 17
C. to 25.degree. C., usually at room temperature, in cryoprotective solution in thin storage dishes.

After equilibration, the sheet and the dish containing the cryoprotective solution are sealed so that gas and water are impermeable. Sheets in sealed containers should be at least-
Cool to 65 ° C (using dry ice), preferably -120 ° C, and approximately -180 ° C to -196 ° C for long-term storage. The cooling rate is slow from 0 ° C to -80 ° C (1 ° C / min or less). This helps to suppress the formation of ice crystals. Preferably, cooling is from -40 ° C to -100 ° C
The temperature is preferably -80 ° C to -85 ° C by a cooling ratio adjusting cooling device such as a commercially available program cell freezer (No. 1010/2700, manufactured by Climond). It is then transferred to a liquid nitrogen storage container and maintained in liquid nitrogen vapor to further reduce its temperature.

In the preferred freezing protocol, the sheet is cooled in a sealed container until the tissue is approximately 4 ° C. The sheet is then cooled at 1 ° C. per minute. After reducing the sheet temperature to at least -65 ° C, preferably -85 ° C, transfer the container to a liquid nitrogen freezer and store at about -180 ° C (nitrogen vapor) or -196 ° C (liquid nitrogen). .

Preservation of tissues at -180 ° C or lower can maintain the colony forming ability of cells rather than storing sheets at higher temperatures, as shown in FIG. Storage at -180 ° C with liquid nitrogen vapor is superior to storage at -80 ° C using a mechanical freezer and storage at -77 ° C with dry ice. The data make it clear. Viability is dramatically reduced during the first 2 or 3 days of storage at -80 ° C or -77 ° C. In contrast, viability at -180 ° C is longer-term stable. The implants are preferably shipped at -180 ° C.

To melt the sheet, remove the sealed container from the liquid nitrogen freezer and hold in air at room temperature and for 1 minute to approximately 3-5 minutes. This produces a heating rate of between 20 ° C / min and 100 ° C / min. then,
The graft is warmed to room temperature regardless of the heating rate. Preferably, in the final step, the sealed container is submerged in the water bath until the graft is thawed. Thereby, the frozen sheet does not crack. Melting can be performed by immersing in a water bath at 37 ° C. for 1.25 minutes.
If the temperature of the water bath is 25 ° C, the melting will be approximately
It takes 1.5 minutes. The water bath can be omitted and the sheet can be melted at room temperature instead. However, this requires 27 minutes and often reduces cell viability.

The thawed sheet is removed from the cryoprotectant within 1 hour, preferably as soon as possible. Once the sheet has thawed, open the container and bring the cryopreservation solution to a physiological pH (approximately 6.8-7.4) to dilute the cryoprotectant.
Replace with isotonic buffer solution, preferably FAD medium or Ringer's lactate solution. Table 1A shows that not all isotonic buffers at physiological pH help to dilute the cryoprotectant. Phosphate buffered saline and standard saline, when judged by CFE,
Clearly reduces viability. The thawed sheet is preferably equilibrated with buffer for 15 minutes. Also up to 4 hours CFE
Can be gently lowered and left to stand. (Table 5)

[0041]

[Table 1] Removal of cryoprotective medium from cryopreserved culture epithelial transplants after thawing Use isotonic buffer at physiological pH as rinsing buffer Buffer colony forming cells (%) rCFE FAD 9.0 35.0 PBS 3.4 13.1 Normal physiology Salt 3.0 11.5

The explants were removed from the cryoprotective medium, placed in the rinse solution and left at room temperature for 15 minutes. Colony forming ability analysis was performed and CFE recovery (rCFE) was calculated as% of unfrozen, non-preserved control implants. FAD; F12 / DME keratinocyte growth medium, PBS; phosphate buffered saline, normal saline; 0.9% saline

[0043]

TABLE 2 Melting graft rinsing duration colony forming cells number of times increase (%) rCFE 1 hour 6.4 71.1 2 hours 5.2 57.8 4 hours 4.5 50.0

The explants were removed from the cryoprotective medium and the FA
D. Place in rinse solution and let stand at room temperature for the period indicated above. Colony forming ability analysis was performed and CFE recovery (rCFE) was calculated as% of unfrozen, non-preserved control implants.

Those that limit the final step of cryopreservation were tested and are shown in FIG. For the production of intact and restored normal metabolic function cell sheets, cultured epithelial sheets were equilibrated in cryoprotectant for up to 1 hour before freezing, and frozen, thawed and left in cryoprotectant for 1 hour. The data show that over time, and finally in the lactated Ringer's solution, rinsing occurs for up to 2 hours.

Sheets prepared as disclosed above surgically cover clean skin wounds, such as burns and ulcers, with a developing layer in contact with the surface of the wound and using a gauze lining as a bandage. be able to. The allograft sheet actually falls off, but helps safe and effective wound protection before rejection. Autograft sheets are generally permanent and differentiate until they form completely layered skin.
The high CFC and CFE values obtained by this cryopreservation method means that the majority of the individual cells in the sheet are mitotic and numerous epithelial cell colonies are formed in the wound area.

The invention is further described by the following non-limiting examples.

[0048]

Example 1 Collected culture explants were treated with 15% dextran (7
(0,000 mw) and 10% glycerol in a culture medium containing a cryoprotectant. The container should be gas and water impermeable and well filled with solution to leave little space and allow the graft to equilibrate at room temperature for 30 minutes. This sheet has about 4 tissue pieces
Cool in sealed container to ℃. The sheet is then cooled to approximately -85 ° C, 1 ° C per minute. When the temperature of the sheet reaches -85 ° C, the container is transferred to a liquid nitrogen freezer and stored in liquid nitrogen vapor (about -180 ° C).

To melt the sheet, the sealed container is removed from the liquid nitrogen freezer and left at room temperature in air for approximately 1 minute. It is then placed in a 37 ° C. water bath for about 75 seconds to thaw. After thawing, open the container and place the sheet in Ringer's lactate solution at physiological pH for 5 minutes. The buffer is then changed and the sheet is allowed to equilibrate in the medium for at least 10 minutes.

[0050]

Example 2 Grafts were prepared from 15% hydroxyethyl starch (Hes
equilibrate for 15 minutes at room temperature in a shallow storage dish in a cryoprotective solution consisting of pan®) and 10% glycerol. After equilibration, the container is sealed impermeable to gas and water. The sheet in the sealed container is cooled until the tissue is approximately 4 ° C. Following this step, the sheet is cooled to approximately -85 ° C, 1 ° C per minute. When the sheet reaches -85 ° C, transfer it to a liquid nitrogen freezer and store it in liquid nitrogen (approximately -196 ° C).

To melt the sheet, the sealed container is removed from the liquid nitrogen freezer and left at room temperature in air for approximately 1 minute. Then place in a water bath and allow to thaw. After thawing, open the container,
The sheet is soaked in Ringer's lactate solution at physiological pH or serum-free DME / F12 keratinocyte growth medium for 5 minutes. 5
After minutes, the buffer is changed and the sheets are allowed to equilibrate in medium for 4 hours. The present invention can be embodied in other specific forms. Other implementations are within the following claims.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a layered cultured keratinocyte sheet released from a culture flask by despase. 1 is a representative micrograph of a layered cultured keratinocyte sheet using the method of the present invention.

FIG. 2 is a bar graph showing the effect of using a non-cell penetrating agent for cryoprotection. 10% for cultured keratinocyte sheet
6 minutes (gly) in cryopreservation culture (CPM) containing glycerol
c-6), then equilibrate for 30 minutes (gly c-30) or 15%
Equilibrate for 30 minutes in cryopreservation culture (CPM) containing 10% glycerol with dextran (70 kd), then -180 ° C
It was cooled to room temperature and stored at -180 ° C for 2-3 days. After thawing, the viability was measured by evaluating whole cell recovery (R), colony forming ability (CFE), viability of colony forming cells (CFC) after cell sheet degradation, and the like. CFC = recovered cells x recovered cell CFE (non-frozen, non-preserved control sheet expressed as a percentage)

FIG. 3 is a bar graph showing the effect of dextran and hespan (hydroxyl ethyl starch) on the viability of cryopreserved cultured epithelial grafts. Graft is 15% in 10% glycerol
Equilibrate in CPM containing either Hespan, 15% dextran (70 kd) or 15% dextran (500 kd),
It was cooled to -180 ° C and stored for 2 days. Viability after thawing is R
, CFE and CFC were evaluated and measured. Expressed as a percentage of non-frozen, non-preserved control implants.

FIG. 4 is a bar graph showing the effect of cryoprotective medium prior to freezing on the viability of cryopreserved culture epithelial grafts. The implant is
Equilibration was performed on cryopreservation medium containing 15% dextran (70 kd) in 10% glycerol at room temperature for 30, 60 and 120 minutes. After thawing, the graft cells were assayed for R 2, CFE, and CFC. Results are expressed as percent of control implants.

FIG. 5 shows a bar graph of the effect of storage temperature on the viability of cryopreserved cultured epithelial grafts. Explants were cryopreserved medium containing 15% dextran in 10% glycerol at room temperature,
Equilibrate for 30 minutes, freeze at -85 ° C, and -180 in liquid nitrogen vapor.
Store at ℃ or mechanical freezer at -80 ℃, solid carbon dioxide (Dry ice, sublimation point -77 ℃)
Was stored at -65 ° C. R of graft after thawing,
CFE and CFC were measured. Results were expressed as a percentage of non-frozen, non-conserved transplants as control transplants.

FIG. 6 is a bar graph showing the effect of cryopreserved cultured epithelial transplants after thawing. The grafts were equilibrated for 60 minutes in cryopreservation medium before freezing. After freezing at -180 ℃ for 1-3 days,
Implants were thawed immediately. 0, 30, 60 minutes after thawing CP
Remove from M and rinse with lactated Ringer's solution for 1-2 hours. Measure the cell recovery, CFE and CFC of the graft after treatment,
Expressed as a percentage of unfrozen, non-preserved implants as control implants.

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[Procedure amendment]

[Submission date] October 7, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Continued Front Page (72) Inventor Susan F. Schaeffer United States 01778 Massachusetts, Wayland, Happy Hello Road 5 (72) Inventor Alexander Shermer United States 02178 Massachusetts, Belmont, Frederick Street 15 (72) Inventor Richard Odyssey United States 02162 Masston, Newton Lower Fall C's, Clehua Drive 15

Claims (28)

[Claims] 1. An improved method for maintaining a cohesive sheet of epithelial cells that survives cryopreservation, comprising the steps of: A. Incubate and supply a sheet of epithelial cells separated from its gently stratified culture substrate. B. Immerse the culture sheet in a cryoprotective solution containing a non-cell permeable glass forming agent in combination with a cell permeable glass forming agent. C. Cool the sheet in the cryoprotective solution to a temperature of about -65 ° C or below. D. Store the sheet from step C at a temperature of about -65 ° C or lower. E. The step of thawing a cryopreserved sheet to prepare a complete mitotic sheet of viable cells that is stratifying and capable of differentiation, which is useful as a skin wound dressing. is there. 2. The method of claim 1, wherein the cryoprotective solution comprises about 5% to about 20% of said non-cell permeable glass forming agent. 3. The non-cell penetrating glass forming agent according to claim 1, which is selected from the group consisting of dextran, polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, chondroitin sulfate, and mixtures and derivatives thereof. Method. 4. The method according to claim 1, wherein the non-cell permeable glass forming agent is dextran or hydroxyethyl starch. 5. The method of claim 4, wherein the glass forming agent is dextran having a molecular weight of approximately 70 kilodaltons to 500 kilodaltons. 6. The cryoprotective solution is one which comprises about 10% by weight of a cell-penetrating glass forming agent selected from the group consisting of glycerol, dimethylsulfoxide, and mixtures and derivatives thereof. The method described in 1. 7. The method of claim 6, wherein the cell-permeable glass forming agent is glycerol. 8. The method of claim 1, wherein the soaking step is performed for a time sufficient to equilibrate the sheet to the cryopreservation solution. 9. The method of claim 8 wherein the soaking step is performed for 15 minutes to 180 minutes. 10. The above cryoprotective solution at 17 ° -30 ° C.
10. The method of claim 9 wherein the sheet is soaked for 20 minutes and then cooled to 4 ° C within 60 minutes. 11. A cooling step from about 4 ° C. to about -80 ° C.
The method of claim 1 performed at a rate of about 1 ° C. per minute to cool the sheet to below. 12. The method of claim 1 wherein the sheet is slowly cooled to about -80 ° C and then the temperature range is -180 ° C to below. 13. The method of claim 1 wherein the cooling and storage steps are from -120 ° C to below. 14. The steps of cooling and storing are rigid, transparent,
The method of claim 1 performed in a sterile, gas impermeable container. 15. The method of claim 1, wherein the cooling and storage steps are performed at a temperature of about −180 ° C. or lower, which allows for an extended storage period. 16. Cooling the sheet at a temperature of about −180 ° C. or lower and melting the sheet in the temperature range of −120 ° C. to −80 ° C. by incubating the sheet in a gas, which is followed by The method of claim 1, wherein the thawing is by immersion in a solution. 17. The method of claim 1, wherein the melting step comprises heating the sheet once to −80 ° C. for about 1 to 5 minutes, then raising to room temperature. 18. The method according to claim 1, wherein the following steps are added.
The described method. E. Remove cryoprotectant from thawed sheet to maintain viability of the sheet by transferring to balanced salt solution for up to about 4 hours. 19. The method according to claim 18, wherein the balanced salt solution is a culture medium such as DME / F12 or a lactated Ringer's solution. 20. The method of claim 1, wherein step B is performed by exposing the sheet to liquid nitrogen vapor. 21. The method according to claim 1, wherein the epithelial cells are keratinocytes. 22. The method of claim 1, wherein the complete sheet of viable cells is characterized by a colony forming ability of at least 35%. 23. The method of claim 1, wherein the complete sheet of viable cells is characterized by at least 40% cell recovery. 24. The method of claim 1 including the additional step of placing the sheet on the support prior to step B. 25. A method for treating a culture sheet of epithelial cells impregnated with a cryoprotectant at a temperature of about -180 ° C. or less in preparation for application as a skin wound dressing to a patient. , The method is that the temperature of the sheet is 20 ℃ per minute.
To a temperature of between 120 ° C and -80 ° C at a rate of between 100 ° C and 1 minute, and then a temperature of between 20 ° C and 37 ° C. 26. Approximately -80 by exposing the sheet to gas
26. The method according to claim 25, wherein the temperature is raised to a temperature of 0C and then warmed in the solution to a temperature between 20C and 37C. 27. The method of claim 25, comprising the additional step of removing the cryoprotectant soaking the sheet by incubating the sheet in a balanced salt solution for up to about 4 hours. 28. The method of claim 5, wherein the dextran is in combination with the cell penetrating agent glycerol.