US5145770A
Expired / Lapsed
US
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Cryopreservation of cultured epithelial sheets
Assignee: Genzyme Corp
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Filing: 1990-06-04
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Granted: 1992-09-08
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Expires: 2010-06-04
Abstract
Abstract Disclosed is a method of cryopreserving a sheet of living, cultured epithelial cells for use as a skin wound dressing. The method ensures that the integrity of the sheet will be maintained, and it preserves a significant number of cells in a mitotically competent, physiologically healthy form. It involves use of a non cell penetrating cryoprotectant and certain freezing and thawing conditions.
Inventors
- 👤 Ross A. Tubo
- 👤 Susan F. Schaeffer
- 👤 Alexander Schermer
- 👤 Richard Odessey
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 14:56
CPC Classifications
A; A01; A01N; A01N1/00; A01N1/10; A01N1/12; A01N1/122; A01N1/125
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
129 words
An improved method for maintaining a cohesive sheet of living epithelial cells in a cryopreserved state, comprising the steps of: A. providing a cultured, stratified sheet of epithelial cells separated from its substratum; B. immersing said cultured sheet for greater than 15 minutes in a cryoprotective solution comprising a non-cell-penetrating, glass-forming agent which is removable by dilution and rinsing upon thawing, in combination with a cell-penetrating, glass-forming agent; C. cooling said sheet in the cryoprotective solution to a temperature of or below approximately -65° C.; D. storing the cooled sheet at a temperature at or below -65° C.; and E. thawing the cryopreserved sheet to produce an intact, mitotically competent sheet of viable cells capable of stratified differentiation, wherein said intact sheet is useful as a skin wound dressing.
Claim 2
Dependent Claim
129 words
An improved method for maintaining a cohesive sheet of living epithelial cells in a cryopreserved state, comprising the steps of: A. providing a cultured, stratified sheet of epithelial cells separated from its substratum; B. immersing said cultured sheet for greater than 15 minutes in a cryoprotective solution comprising a non-cell-penetrating, glass-forming agent which is removable by dilution and rinsing upon thawing, in combination with a cell-penetrating, glass-forming agent; C. cooling said sheet in the cryoprotective solution to a temperature of or below approximately -65° C.; D. storing the cooled sheet at a temperature at or below -65° C.; and E. thawing the cryopreserved sheet to produce an intact, mitotically competent sheet of viable cells capable of stratified differentiation, wherein said intact sheet is useful as a skin wound dressing.
Claim 3
Dependent Claim
23 words
The method of claim 1, wherein the cryoprotective solution comprises from about 5% to about 20% by weight of said non-cell-penetrating, glass-forming agent.
Claim 4
Dependent Claim
32 words
The method of claim 1, wherein the non-cell-penetrating, glass-forming agent is selected from the group consisting of dextran larger than 70 kilodaltons, polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, chondroitin sulfate and mixtures thereof.
Claim 5
Dependent Claim
19 words
The method of claim 1, wherein the non-cell-penetrating, glass-forming agent is dextran larger than 70 kilodaltons or hydroxyethyl starch.
Claim 6
Dependent Claim
22 words
The method of claim 4, wherein the agent is dextran having a molecular weight from approximately 70 kilodaltons to approximately 500 kilodaltons.
Claim 7
Dependent Claim
15 words
The method of claim 5, wherein said dextran is combined with the cell-penetrating agent, glycerol.
Claim 8
Dependent Claim
30 words
The method of claim 1, wherein the cryoprotective solution comprises about 10% by weight of a cell-penetrating, glass-forming agent selected from the group consisting of glycerol, dimethylsulfoxide and mixtures thereof.
Claim 9
Dependent Claim
12 words
The method of claim 6, wherein the cell-penetrating, glass-forming agent is glycerol.
Claim 10
Dependent Claim
23 words
The method of claim 1, wherein the immersion step is performed for a time sufficient to equilibrate the sheet with the cryoprotective solution.
Claim 11
Dependent Claim
22 words
The method of claim 9, wherein the immersion step is conducted for a time period of between 15 minutes and 180 minutes.
Claim 12
Dependent Claim
29 words
The method of claim 9, wherein the sheet is immersed for as long as 120 minutes in said cryoprotective solution at 17°-30° C. and then cooled to 4° C.
Claim 13
Dependent Claim
33 words
The method of claim 1, wherein the cooling step is conducted at a rate of about 1° C. per minute to cool the sheet from about 4° C. to below about -80° C.
Claim 14
Dependent Claim
26 words
The method of claim 1, wherein the sheet is cooled slowly to approximately -80° C. and then cooled to a temperature at or below -180° C.
Claim 15
Dependent Claim
21 words
The method of claim 1, wherein the cooling and storing steps are done to a temperature at or below -120° C.
Claim 16
Dependent Claim
21 words
The method of claim 1, wherein the cooling and storing steps are conducted in a rigid, transparent, sterile, gas impervious container.
Claim 17
Dependent Claim
27 words
The method of claim 1, wherein the storing step is performed at a temperature at or below approximately -180° C., whereby the storage interval may be increased.
Claim 18
Dependent Claim
58 words
The method of claim 1, wherein the sheet is cooled to a temperature below about -180° C. and the thawing of the sheet is accomplished by first incubating the sheet in air until the sheet reaches a temperature of between about -120° C. and -80° C. and then immersing the sheet in liquid until the sheet is thawed.
Claim 19
Dependent Claim
40 words
The method of claim 1, wherein the thawing step comprises allowing the sheet to warm up to about -80° C. over a period of time between about 1 minute and 5 minutes and then warming the sheet to room temperature.
Claim 20
Dependent Claim
47 words
The method of claim 1 comprising the additional step of: F. removing the cryoprotective from the thawed sheet by transferring said sheet to a rinsing solution selected from the group consisting of FAD medium and lactated Ringer's solution for a period of up to approximately four hours.
Claim 21
Dependent Claim
18 words
The method of claim 1, wherein step C is conducted by exposing the sheet to liquid nitrogen vapors.
Claim 22
Dependent Claim
11 words
The method of claim 1, wherein the epithelial cells are keratinocytes.
Claim 23
Dependent Claim
23 words
The method of claim 1, wherein the intact sheet of viable cells is characterized by a colony forming efficiency of at least 35%.
Claim 24
Dependent Claim
22 words
The method of claim 1, wherein prior to step B, the cultured sheet of epithelial cells is mounted on a supportive backing.
Claim 25
Independent Claim
77 words
A method of warming a cryoprotectant impregnated, cultured sheet of epithelial cells from a temperature below about -180° C. to prepare it for application to a patient as a skin wound dressing, the method comprising of step of: raising the temperature of the sheet to between about -120° C. and -80° C. at a rate of about 20° C. to about 100° C. per minute and thereafter increasing the temperature to between 20° C. and 37° C.
Claim 26
Independent Claim
77 words
A method of warming a cryoprotectant impregnated, cultured sheet of epithelial cells from a temperature below about -180° C. to prepare it for application to a patient as a skin wound dressing, the method comprising of step of: raising the temperature of the sheet to between about -120° C. and -80° C. at a rate of about 20° C. to about 100° C. per minute and thereafter increasing the temperature to between 20° C. and 37° C.
Claim 27
Dependent Claim
36 words
The method of claim 24, wherein the sheet is heated to the temperature of about -80° C. by exposure to a gas and then heated to between about 20° C. and 37° C. in a liquid.
Claim 28
Dependent Claim
36 words
The method of claim 24, comprising the additional step of removing the cryoprotectant impregnating said sheet by incubating said sheet in a rinsing solution selected from the group consisting of FAD medium and lactated Ringer's solution.
Claim 29
Dependent Claim
21 words
The method of claim 1, wherein the temperature for immersing said cultured sheet in step B is below about 30° C.
Claim 30
Dependent Claim
16 words
The method of claim 27, wherein said temperature is between about 17° C. and 30° C.
References Cited (Backward Citations)
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