CA2012757A1 - Method of revitalizing cells prior to cryopreservation - Google Patents
Method of revitalizing cells prior to cryopreservationInfo
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- CA2012757A1 CA2012757A1 CA002012757A CA2012757A CA2012757A1 CA 2012757 A1 CA2012757 A1 CA 2012757A1 CA 002012757 A CA002012757 A CA 002012757A CA 2012757 A CA2012757 A CA 2012757A CA 2012757 A1 CA2012757 A1 CA 2012757A1
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- Prior art keywords
- tissue
- cryopreservation
- cells
- incubated
- hours
- Prior art date
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Classifications
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- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Abstract
ABSTRACT
A method of revitalizing cells or tissues that are to be cryopreserved for storage at ultracold temperatures, e.g. -196°C is disclosed which comprises preincubation of the cells or tissue from about 5 minutes to about 24 hours. The pre-incubation may be conducted at a temperature ranging from about 27°C to about 42°C, after which the tissue or cells are cryopreserved.
A method of revitalizing cells or tissues that are to be cryopreserved for storage at ultracold temperatures, e.g. -196°C is disclosed which comprises preincubation of the cells or tissue from about 5 minutes to about 24 hours. The pre-incubation may be conducted at a temperature ranging from about 27°C to about 42°C, after which the tissue or cells are cryopreserved.
Description
2 ~ r~
METHOD OF REVITALIZING CEL~S PRIOR TO CRYOPRESERVATION
TECHNICAL FIELD
The present invention relates to a method for revitalizing cells or tissues that are to be cryopreserved for storage at ultracold temperature.
Hence, the metabolic energy status and the tissue or cellular function are preserved, and maximized upon subsequent thawing and transplantation.
More particularly, the process described herein results in increased cell viability and functional capacity upon thawing. Tissue cryopreserved after this revitalization process is of much higher quality for transplantation than tissue cryopreserved without use of the revitalization process described herein.
BACKGROUND OF THE INVENTION
Current medical technology allows the use of several different types of tissue for transplantation to correct congenital, diseased-induced or degenerative failure of a recipient's tissue. Some examples include allograft human heart valves, veins, corneas, bone marrow, etc. Investigators have generally agreed that fresh tissue gives improved performance over old or dead tissue.
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Human tissue remains viable :in vitro for short periods of time, e.g., usually less than t~o to three days. Storage periods of this limited duration are usually inadequate for most tissue types due to the complexities in assuring the best match of donor to recipient (e.g. such factors as relative size of a graft, human leukocyte antigen and ABO blood group), as well as the time needed to test the tissue for pathogens. Consequently, much of the available donor tissue is unused due to the severe loss of cell viability over time. These shortcomings may be circumvented by the viable cryopreservation and ultracold storage of the tissue.
Ultracold storage of cells and tissues became possible after the discovery in 1949, by Polge, Smith and Par~s, that glycerol could be used to protect cells from injury due to freezing. With the advent of 1QW
temperature biology, workers in medical and biological fields have been seeking better ways to maintain the viability of frozen donor cells or tissues.
Several methods for freezing cells and cell aggregates have been repor~ed. For example, U.S. Patent No. 3,303,662 discloses a process for cell preservation that utilizes a cryoprotectant in the freezing process.
The performance of a cryopreserved, transplantable tissue correlates directly with the viability of that tissue upon thawing. One parameter that provides an assessment of the cellular viability of tissue is the general metabolic energy status of the cells. In order for transplanted cells to perform their critical roles in the recipient, these cells must have sufficient metabolic capacity to carry out key energy-dependent processes. For example, one such process that is dependent on cellular metabolic energy is the biosynthesis of proteins. Furthermore, essentially any cellular, tissue or organ function is ultimately dependent on energy derived from cellular metabolism.
~ 3 ~ ~ v ~ r 7 Cells that are metabolically and functionally suppressed after thawing may not recover sufficiently to endure the shock of transplantation into a donor, and thus may not survive.
There are several steps in the handling of human tissue for cryopreservation that can decrease the metabolic energy status and depress the energy-dependent functions of the cells. The time between death and the harvest of the tissue (warm ischemia) and the time from harvest until cryopreservation (cold ischemia) are most influential. Prolonged warm and/or cold ischemia results in cells that are severely metabolically and functionally depressed.
Cryopreservation itself appears to reduce cellular energy and metabolic capacity, and to reduce energy-dependent functions at least minimally. Hence, there is a long-standing need for a method of maintaining tissue viability post-implant and for revitalizing cells in the tissue post-harvest, such that the cells essentially completely recover from the transient metabolic lesions and loss of function induced by warm and cold ischemia.
The invention therefore fulfills this long-term need for greatly improved viability, and maximizes the functional capacity of cryopreserved cells upon thawing and transplantation. In addition, revitalized cells are better able to withstand the rigors of cryopreservation.
Tissues are currently placed into solutions such as tissue culture media, Lactated Ringers, saline or Collins solution on wet ice for shipping. The concentration of compounds contained in these solutions, the time period during which the tissues are retained therein, and the temperature at which the tissues are shipped can vary widely. Due to the combined effects of these variables, and due to variations in the times of warm and cold ischemia, it is difficult to predict the degree of metabolic and functional depression for any given tissue. One important feature of the present 7 ~ ~
invention is that the method improves the metabolic status, and hence the capacity to function of tissues upon transplant, even with widely varying degrees of metabolic and functional suppression.
Accordingly, it is one object of the present invention to provide a method for revitalizing cells or tissues prior to cryopreservation.
It is another object of the present invention to provide a method of enhancing transplant cell viability and functional capacity upon thawing.
It is yet another object of the present invention to provide a method that improves the ability of a cryopreserved tissue or cell to survive and function upon thawing and transplantation.
It is yet another object of the present invention to provide a method for cell revitalization that can be used concomitantly with other procedures, such as antibiotic sterilization, which may be necessary in the preparation of transplantable tissue for cryopreservation.
These and other objects, features, a~d advantages of the present invention will become apparent after review of the following detailed description of the disclosed embodiments and the appended claims.
SUMMARY OF THE INVENTION
The present invention encompasses a method for optimizing cell revitalization comprising placing cells into a nutrient medium and incubating said cells prior to cryopreservation, at a temperature and for a period of time effective for optimizing cell revitalization.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 discloses the cellular energy and functional status of cells following preincubation, cryopreservation, thawing and post-thaw incubation. The Figure compares cellular energy and functional status of tissue that is held at 4C prior to cryopreservation to that of tissue that is revitalized (preincubated at 37C) in accordance with the preferred embodiment of the present invention. Time o for preincubation represents the hypothetical decrease of cellular metabolic and functional status to 50% of that seen for tissue in situ. This decrease is attributed to the combined effects of warm and cold ischemia, prior to preincubation.
Figure 2 shows continuous records of heat dissipation by heart valve leaflets. Revitalized leaflet halves ( ) were incubated at 37 C for six hours and then cryopreserved and thawed as described in Example 2. The control half leaflets were (+) held at 4 C for six hours and then cryopreserved and thawed as described in Example 2. Revitalized leaflets produce heat at a rate more than 2-times that of control, nonrevitalized leaflets.
DETAILED DESCRIPTION OF T~E PREFERRED EMBODIMENT
The present method for revitalizing tissue or cells assures increased cellular viability and functional capacity upon thawing. The tissues, which are typically metabolically and functionally depressed by exposure to various durations of warm and cold ischemia, are treated in a manner that fosters recovery from these transient, ischemia-induced lesions. Since it is usually not practical to remove tissue immediately after donor death, nor is it usually possible to cryopreserve the tissue immediately after procurement, periods of warm and/or cold ischemia are unavoidable in the processing of human tissue for viable cryopreservation. By treating the tissue prior to cryopreservation, the method of the present invention assures that the metabolic energy status and functional capacity of the tissue are restored.
As used herein, the term "revitalization" means h ~' t i~ .J
restoration or optimization of cellular or tissue metabolic processes and energy-dependent functions after cryopreservation, accomplished by treating the cells or tissue prior to cryopreservation. Tissue and cellular function are ultimately dependent upon cellular metabolism. Hence, cell/tissue viability and functional capacity are optimized upon transplant to maximize the transplant success rate. Revitalization measurement as used herein is not limited to specified metabolic effects or pathways, and any measure of metabolic activity or tissue or cellular function can be used.
For example, protein synthesis, vascular constriction, nerve conductivity, muscle contractility, radioactive precursor uptake, radioactive or fluorescent metabolite production as well as other measures of metabolic activity or processes dependent on metabolic energy can be evaluated.
The methods described herein apply to cells and tissue as well as organs which are to be transplanted, and are not limited to specific forms of transplantable tissue. Examples include bone marrow cells, tendon, ligament, islet cells, fibroblasts, cornea, blood vessel, heart, liver, embryo, etc. The terms "cells,"
"tissue" and "organ" when referring to the transplantable material described herein are therefore used in the most general sense, and are used interchangeably to refer to cells, tissues, organs, etc.
Upon receipt in the laboratory, the tissue of choice is dissected away from any unwanted tissue and placed into a suitable tissue culture medium, in a container that allows for sufficient oxygenation of the medium. The tissue and medium are placed into an incubator, or a shaking water bath for an effective length of time and at an effective temperature to optimize or maximize cell or tissue viability after the sample has undergone cryopreservation and has thawed and ?., ~ J ~ ~J~l been prepared for transplant into a patient in need for such treatment.
A number of tissue and/or cell culture media can be used successfully in practicing the present invention.
Media, such as balanced tissue culture media or simple phosphate buffered saline (supplemented with a nutrient such as glucose), can be used for most tissue types. In addition, a protein suspension, such as blood serum or artificial serum may he present in the media.
Revitalization is conducted for an effective time period and at a temperature which is effective for revitalizing the tissue, there~y maximizing transplant effectiveness.
The treatment time required for revitalization may range from about 5 minutes to about 24 hours, with the preferred time being about 30 minutes to 9 hours. The most preferred time for revitalization is about 6 hours.
The temperature at which the tissue is treated ranges from about 27C to about 42C, with about 35C to about 40C being preferred. The optimal temperature for revitalization is 37C.
Following the revitalization procedure, the tissue is cryopreserved following the sLandard methods, in the chosen solutions, and optionally in the presence of the cryoprotectant(s) that have been shown to be optimal for each given tissue.
The frozen tissue is stored at ultracold temperatures (e.g. -196C in liquid nitrogen). The thawing and dilution steps typically are those which have heen shown to be optimal for the given tissue. The metabolic and functional advantage (relative to non-revitalized tissue) gained by revitalizing the tissue prior to cryopreservation will be maintained after the thawing and dilution steps.
One of thP advantages of the present invention is that regardless of the cryopreservation procedure followed, treating the tissue prior to freezing results i e ' i in improved tissue quality (relative to non-revitalized tissue) upon thawing.
The following specific examples will illustrate the invention as it applies to the revitalization of human heart valve tissue prior to cryopreservation, and the maintenance of this metabolic and functional advantage after thawing and dilution. However, as described above, it will be appreciated that these teachings apply to all transplantable tissues; various alternatives will be apparent to those of ordinary skill in the art from the teachings herein, and the invention is not limited to the specific illustrative examples.
Hearts were procured in toto and shipped to the laboratory. In preparation for transport, each heart was placed into a sterile intestinal bag with about 350 ml of Lactated Ringers, saline or Collins solution. The bag was secured with a plastic band or umbilical tape and was placed into a second intestinal bag, which was likewise secured. The heart, which is thus double bagged, was placed in a plastic container and the lid secured. The container was then put into a third sterile intestinal bag and put into a styrofoam shipping container with wet ice. Upon receipt, the aortic and/or pulmonary valves were dissected and placed in the original shipping solution.
The valves were stored at 4~C for 4 to 72 hours.
Following this storage period, the valve leaflets were dissected out, and each leaflet was cut into two equal parts. The valve leaflet pieces were placed into Dulbecco's Modified Eagle's Medium ("DMEM") (low glucose, with 10% fetal calf serum) in sterile tissue culture tubes and stored on ice. One half of each leaflet was left in this solution on ice. The other half of the leaflet was transferred into a sterile tissue culture tube, which contained the same solution, P1 3 r,~
but which had been warmed to 37C. The sterile tubes containing these half leaflets were placed into a 37~C
incubator. After six hours all leaflet halves were assayed for cell viability and functionality.
The assay measured the incorporation of 3H 2-deoxyglucose into 2-deoxyglucose 6-phosphate by leaflet cells. This assay determines the integrity of the cell membrane, the functional capacity of the transmembrane glucose transport proteins, the integrity of the hexokinase enzymes and the general energy status of the cellO The last parameter is important because ATP is needed for the 2-deoxyglucose to be phosphorylated.
The half leaflets were placed into approximately 2 ml of sterile Hanks solution at room temperature for 3-5 minutes. They were then transferred into 0.5 ml of Hanks (at 37C in a heating block) containing 10 uCi/ml H 2-deoxyglucose. After a 30 minute incubation, the half leaflets were immediately transferred to approximately 10 mls of ice cold Hanks solution. The solution was aspirated off with a pippette and another 10 mls of Hanks added. This washing procedure, which removes any extracellular 2-deoxyglucose and washes out any intracellular 2-deoxyglucose that is not phosphorylated, was repeated 4 more times. The half leaflets were then placed in 0.5 ml of 1 M NaOH and incubated at 60~C for 30 minutes. The tissue was then homogenized by sonication, and the resulting homogenate was centrifuged for 10 minutes in a table top Eppendorf centrifuge. The disintegrations per minute ("3H DPM") in the resulting supernatant were determined by liquid scintillation counting. All values for 3H DPM were normalized for the amount of protein present in the supernatant. The results are given in Table A.
TABLE A
Comparison of 2-deoxyglucose incorporation (DPM / mg protein) by half leaflets given a six hour incubation at f?, ~JJ ~ 2 ~ 7 37C to the incorporation by half leaflets held at 4C
for six hours.
DPM Values for Tissue Preincubated at: % 37C vs. 4C
4C 37C (37C/4C x 100) 138,431 318,742 230 132,568 364,123 274 261,695 302,192 115 258,983 430,819 166 197,876 451,193 228 202,518 318,179 157 116,452 144,760 124 109,274 307,184 281 131,514 367,644 280 x + SE = 206 + 21 %
The data demonstrates that there is an approximately 2-fold improvement in cell viability and functional capacity when the tissue is revitalized by incubation at 37C relative to tissue incubated at 4C.
This difference is presented diagrammatically at point A. Such results show that post-ischemic revitalization at 37C leads to recovery of the cells from transient, ischemia-induced metabolic lesions and from the concomitant depression of cellular function. Thus, the data indicate that revitalization at 37C markedly improves the metabolic energy status and function of the cells, and hence the overall quality of the fresh human heart valve leaflets.
Heart valve half leaflets were prepared and incubated at 4C or 37C as described in Example 1.
These half leaflets were then cryopreserved, essentially via the method described in U.S. application serial no.
000,095. The frozen leaflets were stored at -196'C for at least 16 hours. The leaflets were thawed, and the cryoprotectant was diluted as described in the aforementioned patent application. Immediately after thawing and dilution, the half leaflets were assayed for 2-deoxyglucose incorporation as described in Example 1.
The results are shown in Table B.
TABLE B
Comparison of 2-deoxyglucose incorporation (DPM / mg protein) by half leaflets given a six hour incubation at 37C vs. half leaflets given a six hour incubation at 4C. After the incubations all leaflets were cryopreserved, thawed, diluted and immediately assayed.
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DPM Values for T.issue Preincubated at- % 37 C vs. 4C
4~C 37C (37 C/4 C x 100) 27,281 70,372 258 34,219 118,682 347 33,944 86,439 255 132,447 185,887 140 82,604 320,953 389 162,623 201,013 124 3949 17,215 436 3990 14,366 360 13,975 28,888 207 14,960 38,016 254 16,052 17,130 107 5193 18,951 365 17,320 29,482 170 2489 20,533 825 x + SE = 304 + 44 %
The data demonstrates that the revitalization of tissue by incubation at 37C for six hours, prior to cryopreservation, results in approximately a 3-fold greater cellular viability and functional capacity than that noted when tissue is given a pre-cryopreservation incubation at 4C. This difference is presented diagrammatically at point B. The revitalization at 37C
r, leads to recovery of the cells from transient, ischemia-induced metaboli~ lesions and functional depression, and the improved metabolic state and functional capacity are maintained after cryopreservation and thawing. Thus, revitalization at 37 C markedly improves the metabolic energy status and function, and hence the overall quality of the cryopreserved and thawed tissue.
Human heart valve leaflets were treated as described in Example 2, except that after thawing and dilution, all of the leaflet halves were placed in DMEM
(low glucose with 10% fetal calf serum) and given a 6 hour post-thaw incubation at 37C. After this incubation period, the half leaflets were assayed for 2-deoxyglucose incorporation as described in Example 1.
The results are shown in Table C.
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~14-TABLE C
Comparison of 2-deoxyglucose incorporation (DPM /
mg protein) by half leaflets given a six hour incubation at 37C vs. half leaflets given a six hour incubation at 4OC. After the incubations, all leaflets were cryopreserved, thawed, diluted and then given a 6 hour post-thaw incubation at 37C. After this incubation, the half leaflets were assayed for 2-deoxyglucose incorporation.
DPM Values for Tissue Preincubated at: % 37C vs. 4C
43C37C (37C/4C x 100) 109,009 171,963 158 79,676 103,058 129 148,937 176,034 118 127,787 459,873 360 150,302 239,906 160 119,150 224,751 189 x + SE = 186 ~ 33 %
The data shows that the revitalization of tissue by incubation at 37C prior to cryopreservation, results in approximately a two fold greater cellular viability than that noted when tissue is given a pre-cryopreservation incubation at 4~C, even after the tissue has heen given a six hour post-thaw incubation at 37C. This J~
difference is presented diagrammatically at point C.
Such results show that post-ischemic revitalization at 37C leads to recovery of the cells from transient, ischemia-induced metabolic lesions and functional depression, and that the improved metabolic state and functional capacity are maintained after cryopreservation, thawing, and incubation. Thus, the data indicates that revitalization at 37C markedly improves the metabolic energy status and functional capacity, and hence the overall quality of the cryopreserved and thawed tissue. Furthermore, these results show that revitalized tissue would have a marked advantage over non-revitalized tissue during the first six hours after transplantation.
Human heart valve leaflets were treated as described in Example 2, except that after thawing and dilution, all of the leaflet halves were placed in DMEM
(low glucose with 10% fetal calf serum) and given a 16 hour post-thaw incubation at 37C. After this incubation period, the half leaflets were assayed for 2-deoxyglucose incorporation as described in Example 1.
The results are shown below in Table D.
TABLE D
Comparison of 2-deoxyglucose incorporation (DPM / mg protein) by half leaflets given a six hour incubation at 37C vs~ half leaflets given a six hour incubation at 4C. After the incubations all leaflets were cryopreserved, thawed, diluted and then given a 16 hour post-thaw incubation at 37C. After this incubation, the half leaflets were assayed for 2-deoxyglucose incorporation.
DPM Values for Tissue Preincubated at: % 37C vs. 4C
4OC 37C (37C/4C x 100) 35,988 112,025 311 72,313 88,043 122 295,436 479,791 162 x + SE = 198 + 47 %
These results indicate that revitalized heart valve leaflets maintain their functional advantage over nontreated tissue even after 16 hours, post-thaw, at 37C. This difference is presented diagrammatically at point D.
Heart valve leaflets were processed as described in Example 2, except that pre-cryopreservation incubation of three hours duration at 37C was compared to a three hour incubation at 4*C. The results are shown in Table E.
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TABLE E
Comparison of 2-deoxyglucose incorporation (DPM /
mg protein) by half leaflets given a three hour incubation at 37C vs. half leaflets given a three hour incubation at 4C. After the incubations all leaflets were cryopreserved, thawed, diluted and immediately assayed.
DPM Values for Tissue Preincubated at: % 37C vs. 4C
4C 37C (37C/4C x 100) 10,488 26,062 248 19,839 34,172 172 18,873 33,169 176 44,872 54,130 121 58,496 56,509 97 57,270 61,499 107 x + SE = 154 + 21 %
The data show that the revitalization of tissue by incubation at 37C for three hours, prior to cryopreservation, results in approximately 1.5-fold greater cellular viability than that noted when tissue is given a pre-cryopreservation incubation at 4C. Such results show that post-ischemic revitalization at 37C, which can be accomplished in as little as three hours, leads to recovery of the cells from transient ischemia-induced metabolic lesions and functional depression, and that the improved metabolic state and functional capacity are maintained after cryopreservation and thawing. Thus, revitalization at 37C for three hours markedly improves the metabolic energy status and functional capacity, and hence the overall quality of the cryopreserved and thawed tissue.
Human heart valve leaflets were treated as described in Example 4, except that after thawing and dilution, all of the leaflet halves were placed in DMEM (low glucose with 10%
fetal calf serum) and given a 6 hour post-thaw incubation at 37C. After this incubation period, the half leaflets were assayed for 2-deoxyglucose incorporation as described in Example 1. The results are shown in Table F.
TABLE F
Comparison of 2-deoxyglucose incorporation (DPM / mg protein) by half leaflets given a three hour incubation at 37c vs. half leaflets given a three hour incubation at 4C. After the incubations all leaflets were cryopreserved, thawed, diluted and then given a 6 hour post-thaw incubation at 37C.
After this incubation, the half leaflets were assayed for 2-deoxyglucose incorporation.
2~ 757 DPM Values for Tissue Preincubated at: % 37C vs. 4_ 4C 37C (37C/4C x 100) 13,196 20,852 158 11,364 17,760 156 7320 25,237 345 201,917 297,080 147 186,455 280,580 150 185,035 226,723 123 x + SE = 180 + 31 %
The data shows that the revitalization of tissue by incubation at 37 C for three hours prior to cryopreservation results in approximately 2-fold greater cellular viability than that noted when tissue is given a pre-cryopreservation incubation at 4 C, even a~ter the tissue has been given a six hour post-thaw incubation at 37 C. Such results show ~hat post-ischemic revitalization at 37~ C leads to recovery of the cells from transient, ischemia-induced metabolic lesions and functional depression, and that the improved metabolic state, and functional capacity are maintained after cryopreservation thawing, and incubation at physiological temperature. Thus, revitalization at 37 C markedly improves the metabolic energy status and functional capacity and hence, the overall quality of the cryopreserved and thawed human heart valve leaflets.
Furthermore, these results show that revitalized tissue would have a marked advantage over non-revitalized tissue within the t~J
first six hours after transplantation.
~ eart valve leaflets are treated as described in Example 2, except that after thawing and dilution, two revitalized leaflet halves (preincubated at 37 C for six hours prior to cryopreservation), from the same valve are rinsed briefly with Hanks and placed into an open-flow microcalorimeter and perfused with Hanks at a flow rate of 15 ml/minute. The tissue chamber is maintained at 37 C, and heat dissipation by the tissue is recorded continuously for a 2 hour period. The two control leaflet halves (preincubated at 4 C
prior to cryopreservation) are thawed, diluted and treated in an identical manner.
The rate of heat dissipation by the revitalized leaflet halves upon thawing is more than ~-times the rate seen with the control, nonrevitalized leaflet halves, as shown in Fig. 2. The mean heat dissipation for revitalized leaflet havles is 22.03 microwatts, whereas that for control non-revitalized leaflet halves is 10.54 microwatts. Heat dissipation correlates directly with the metabolic energy flow of cells. Therefore, the metabolic energy flow, and hence the capacity of the tissue to function, is more than 2-times greater in revitalized leaflet halves than in control, nonrevitalized leaflet halves. This is in close agreement With the results seen when 2-deoxyglucose incorporation is used as a measurement of relative metabolic energy status and functional capacity (i.e., Example 2). Thus, by two independent measurements of tissue metabolic energy status and functional capacity it is determined that revitalized tissue is far superior to nonrevitalized tissue after thawing.
Whole hearts are procured and shipped to the laboratory as described in Example 1. The aortic and pulmonary valves are dissected out, placed in a tissue culture medium (e.g. DMEM with glucose) and incubated at 37 C for about 3 to about 12 hours. If desired, antibiotics may be included in the incubation solution. After the incubation and concomitant revitalization, the valves are cryopreserved via the protocol described in U.S. application serial no. 000,095 which is incorporated herein by reference. Upon transplantation, such valves are of superior quality to those that had not been revitalized prior to cryopreservation.
In summary, the revitalization of tissue for a~out 5 minutes to about 24 hours prior to cryopreservation results in approximately 2 to 3 fold greater cellular viability than that noted when tissue is given a pre-cryopreserva~ion incubation at 4C, even when compared to tissue which has been given a six to sixteen hour post-thaw incubation at 37C. ~ence, post-ischemic revitalization leads to recovery of the cells from transient, ischemia-induced metabolic lesions and depressed functional capacity. The improved metabolic state and functional capacity are maintained after cryopreservation, thawing, and incubation. Revitalization markedly improves the metabolic energy status, cellular function and hence, the overall quality of the cryopreserved and thawed tissue.
Furthermore, these results show that revitalized tissue would have a marked advantage over non-revitalized tissue upon transplant.
While the invention herein is described in detail, numerous alternative embodiments are possible and fall within the appended claims. Consequently, the scope of the invention is not to be limited thereby.
METHOD OF REVITALIZING CEL~S PRIOR TO CRYOPRESERVATION
TECHNICAL FIELD
The present invention relates to a method for revitalizing cells or tissues that are to be cryopreserved for storage at ultracold temperature.
Hence, the metabolic energy status and the tissue or cellular function are preserved, and maximized upon subsequent thawing and transplantation.
More particularly, the process described herein results in increased cell viability and functional capacity upon thawing. Tissue cryopreserved after this revitalization process is of much higher quality for transplantation than tissue cryopreserved without use of the revitalization process described herein.
BACKGROUND OF THE INVENTION
Current medical technology allows the use of several different types of tissue for transplantation to correct congenital, diseased-induced or degenerative failure of a recipient's tissue. Some examples include allograft human heart valves, veins, corneas, bone marrow, etc. Investigators have generally agreed that fresh tissue gives improved performance over old or dead tissue.
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Human tissue remains viable :in vitro for short periods of time, e.g., usually less than t~o to three days. Storage periods of this limited duration are usually inadequate for most tissue types due to the complexities in assuring the best match of donor to recipient (e.g. such factors as relative size of a graft, human leukocyte antigen and ABO blood group), as well as the time needed to test the tissue for pathogens. Consequently, much of the available donor tissue is unused due to the severe loss of cell viability over time. These shortcomings may be circumvented by the viable cryopreservation and ultracold storage of the tissue.
Ultracold storage of cells and tissues became possible after the discovery in 1949, by Polge, Smith and Par~s, that glycerol could be used to protect cells from injury due to freezing. With the advent of 1QW
temperature biology, workers in medical and biological fields have been seeking better ways to maintain the viability of frozen donor cells or tissues.
Several methods for freezing cells and cell aggregates have been repor~ed. For example, U.S. Patent No. 3,303,662 discloses a process for cell preservation that utilizes a cryoprotectant in the freezing process.
The performance of a cryopreserved, transplantable tissue correlates directly with the viability of that tissue upon thawing. One parameter that provides an assessment of the cellular viability of tissue is the general metabolic energy status of the cells. In order for transplanted cells to perform their critical roles in the recipient, these cells must have sufficient metabolic capacity to carry out key energy-dependent processes. For example, one such process that is dependent on cellular metabolic energy is the biosynthesis of proteins. Furthermore, essentially any cellular, tissue or organ function is ultimately dependent on energy derived from cellular metabolism.
~ 3 ~ ~ v ~ r 7 Cells that are metabolically and functionally suppressed after thawing may not recover sufficiently to endure the shock of transplantation into a donor, and thus may not survive.
There are several steps in the handling of human tissue for cryopreservation that can decrease the metabolic energy status and depress the energy-dependent functions of the cells. The time between death and the harvest of the tissue (warm ischemia) and the time from harvest until cryopreservation (cold ischemia) are most influential. Prolonged warm and/or cold ischemia results in cells that are severely metabolically and functionally depressed.
Cryopreservation itself appears to reduce cellular energy and metabolic capacity, and to reduce energy-dependent functions at least minimally. Hence, there is a long-standing need for a method of maintaining tissue viability post-implant and for revitalizing cells in the tissue post-harvest, such that the cells essentially completely recover from the transient metabolic lesions and loss of function induced by warm and cold ischemia.
The invention therefore fulfills this long-term need for greatly improved viability, and maximizes the functional capacity of cryopreserved cells upon thawing and transplantation. In addition, revitalized cells are better able to withstand the rigors of cryopreservation.
Tissues are currently placed into solutions such as tissue culture media, Lactated Ringers, saline or Collins solution on wet ice for shipping. The concentration of compounds contained in these solutions, the time period during which the tissues are retained therein, and the temperature at which the tissues are shipped can vary widely. Due to the combined effects of these variables, and due to variations in the times of warm and cold ischemia, it is difficult to predict the degree of metabolic and functional depression for any given tissue. One important feature of the present 7 ~ ~
invention is that the method improves the metabolic status, and hence the capacity to function of tissues upon transplant, even with widely varying degrees of metabolic and functional suppression.
Accordingly, it is one object of the present invention to provide a method for revitalizing cells or tissues prior to cryopreservation.
It is another object of the present invention to provide a method of enhancing transplant cell viability and functional capacity upon thawing.
It is yet another object of the present invention to provide a method that improves the ability of a cryopreserved tissue or cell to survive and function upon thawing and transplantation.
It is yet another object of the present invention to provide a method for cell revitalization that can be used concomitantly with other procedures, such as antibiotic sterilization, which may be necessary in the preparation of transplantable tissue for cryopreservation.
These and other objects, features, a~d advantages of the present invention will become apparent after review of the following detailed description of the disclosed embodiments and the appended claims.
SUMMARY OF THE INVENTION
The present invention encompasses a method for optimizing cell revitalization comprising placing cells into a nutrient medium and incubating said cells prior to cryopreservation, at a temperature and for a period of time effective for optimizing cell revitalization.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 discloses the cellular energy and functional status of cells following preincubation, cryopreservation, thawing and post-thaw incubation. The Figure compares cellular energy and functional status of tissue that is held at 4C prior to cryopreservation to that of tissue that is revitalized (preincubated at 37C) in accordance with the preferred embodiment of the present invention. Time o for preincubation represents the hypothetical decrease of cellular metabolic and functional status to 50% of that seen for tissue in situ. This decrease is attributed to the combined effects of warm and cold ischemia, prior to preincubation.
Figure 2 shows continuous records of heat dissipation by heart valve leaflets. Revitalized leaflet halves ( ) were incubated at 37 C for six hours and then cryopreserved and thawed as described in Example 2. The control half leaflets were (+) held at 4 C for six hours and then cryopreserved and thawed as described in Example 2. Revitalized leaflets produce heat at a rate more than 2-times that of control, nonrevitalized leaflets.
DETAILED DESCRIPTION OF T~E PREFERRED EMBODIMENT
The present method for revitalizing tissue or cells assures increased cellular viability and functional capacity upon thawing. The tissues, which are typically metabolically and functionally depressed by exposure to various durations of warm and cold ischemia, are treated in a manner that fosters recovery from these transient, ischemia-induced lesions. Since it is usually not practical to remove tissue immediately after donor death, nor is it usually possible to cryopreserve the tissue immediately after procurement, periods of warm and/or cold ischemia are unavoidable in the processing of human tissue for viable cryopreservation. By treating the tissue prior to cryopreservation, the method of the present invention assures that the metabolic energy status and functional capacity of the tissue are restored.
As used herein, the term "revitalization" means h ~' t i~ .J
restoration or optimization of cellular or tissue metabolic processes and energy-dependent functions after cryopreservation, accomplished by treating the cells or tissue prior to cryopreservation. Tissue and cellular function are ultimately dependent upon cellular metabolism. Hence, cell/tissue viability and functional capacity are optimized upon transplant to maximize the transplant success rate. Revitalization measurement as used herein is not limited to specified metabolic effects or pathways, and any measure of metabolic activity or tissue or cellular function can be used.
For example, protein synthesis, vascular constriction, nerve conductivity, muscle contractility, radioactive precursor uptake, radioactive or fluorescent metabolite production as well as other measures of metabolic activity or processes dependent on metabolic energy can be evaluated.
The methods described herein apply to cells and tissue as well as organs which are to be transplanted, and are not limited to specific forms of transplantable tissue. Examples include bone marrow cells, tendon, ligament, islet cells, fibroblasts, cornea, blood vessel, heart, liver, embryo, etc. The terms "cells,"
"tissue" and "organ" when referring to the transplantable material described herein are therefore used in the most general sense, and are used interchangeably to refer to cells, tissues, organs, etc.
Upon receipt in the laboratory, the tissue of choice is dissected away from any unwanted tissue and placed into a suitable tissue culture medium, in a container that allows for sufficient oxygenation of the medium. The tissue and medium are placed into an incubator, or a shaking water bath for an effective length of time and at an effective temperature to optimize or maximize cell or tissue viability after the sample has undergone cryopreservation and has thawed and ?., ~ J ~ ~J~l been prepared for transplant into a patient in need for such treatment.
A number of tissue and/or cell culture media can be used successfully in practicing the present invention.
Media, such as balanced tissue culture media or simple phosphate buffered saline (supplemented with a nutrient such as glucose), can be used for most tissue types. In addition, a protein suspension, such as blood serum or artificial serum may he present in the media.
Revitalization is conducted for an effective time period and at a temperature which is effective for revitalizing the tissue, there~y maximizing transplant effectiveness.
The treatment time required for revitalization may range from about 5 minutes to about 24 hours, with the preferred time being about 30 minutes to 9 hours. The most preferred time for revitalization is about 6 hours.
The temperature at which the tissue is treated ranges from about 27C to about 42C, with about 35C to about 40C being preferred. The optimal temperature for revitalization is 37C.
Following the revitalization procedure, the tissue is cryopreserved following the sLandard methods, in the chosen solutions, and optionally in the presence of the cryoprotectant(s) that have been shown to be optimal for each given tissue.
The frozen tissue is stored at ultracold temperatures (e.g. -196C in liquid nitrogen). The thawing and dilution steps typically are those which have heen shown to be optimal for the given tissue. The metabolic and functional advantage (relative to non-revitalized tissue) gained by revitalizing the tissue prior to cryopreservation will be maintained after the thawing and dilution steps.
One of thP advantages of the present invention is that regardless of the cryopreservation procedure followed, treating the tissue prior to freezing results i e ' i in improved tissue quality (relative to non-revitalized tissue) upon thawing.
The following specific examples will illustrate the invention as it applies to the revitalization of human heart valve tissue prior to cryopreservation, and the maintenance of this metabolic and functional advantage after thawing and dilution. However, as described above, it will be appreciated that these teachings apply to all transplantable tissues; various alternatives will be apparent to those of ordinary skill in the art from the teachings herein, and the invention is not limited to the specific illustrative examples.
Hearts were procured in toto and shipped to the laboratory. In preparation for transport, each heart was placed into a sterile intestinal bag with about 350 ml of Lactated Ringers, saline or Collins solution. The bag was secured with a plastic band or umbilical tape and was placed into a second intestinal bag, which was likewise secured. The heart, which is thus double bagged, was placed in a plastic container and the lid secured. The container was then put into a third sterile intestinal bag and put into a styrofoam shipping container with wet ice. Upon receipt, the aortic and/or pulmonary valves were dissected and placed in the original shipping solution.
The valves were stored at 4~C for 4 to 72 hours.
Following this storage period, the valve leaflets were dissected out, and each leaflet was cut into two equal parts. The valve leaflet pieces were placed into Dulbecco's Modified Eagle's Medium ("DMEM") (low glucose, with 10% fetal calf serum) in sterile tissue culture tubes and stored on ice. One half of each leaflet was left in this solution on ice. The other half of the leaflet was transferred into a sterile tissue culture tube, which contained the same solution, P1 3 r,~
but which had been warmed to 37C. The sterile tubes containing these half leaflets were placed into a 37~C
incubator. After six hours all leaflet halves were assayed for cell viability and functionality.
The assay measured the incorporation of 3H 2-deoxyglucose into 2-deoxyglucose 6-phosphate by leaflet cells. This assay determines the integrity of the cell membrane, the functional capacity of the transmembrane glucose transport proteins, the integrity of the hexokinase enzymes and the general energy status of the cellO The last parameter is important because ATP is needed for the 2-deoxyglucose to be phosphorylated.
The half leaflets were placed into approximately 2 ml of sterile Hanks solution at room temperature for 3-5 minutes. They were then transferred into 0.5 ml of Hanks (at 37C in a heating block) containing 10 uCi/ml H 2-deoxyglucose. After a 30 minute incubation, the half leaflets were immediately transferred to approximately 10 mls of ice cold Hanks solution. The solution was aspirated off with a pippette and another 10 mls of Hanks added. This washing procedure, which removes any extracellular 2-deoxyglucose and washes out any intracellular 2-deoxyglucose that is not phosphorylated, was repeated 4 more times. The half leaflets were then placed in 0.5 ml of 1 M NaOH and incubated at 60~C for 30 minutes. The tissue was then homogenized by sonication, and the resulting homogenate was centrifuged for 10 minutes in a table top Eppendorf centrifuge. The disintegrations per minute ("3H DPM") in the resulting supernatant were determined by liquid scintillation counting. All values for 3H DPM were normalized for the amount of protein present in the supernatant. The results are given in Table A.
TABLE A
Comparison of 2-deoxyglucose incorporation (DPM / mg protein) by half leaflets given a six hour incubation at f?, ~JJ ~ 2 ~ 7 37C to the incorporation by half leaflets held at 4C
for six hours.
DPM Values for Tissue Preincubated at: % 37C vs. 4C
4C 37C (37C/4C x 100) 138,431 318,742 230 132,568 364,123 274 261,695 302,192 115 258,983 430,819 166 197,876 451,193 228 202,518 318,179 157 116,452 144,760 124 109,274 307,184 281 131,514 367,644 280 x + SE = 206 + 21 %
The data demonstrates that there is an approximately 2-fold improvement in cell viability and functional capacity when the tissue is revitalized by incubation at 37C relative to tissue incubated at 4C.
This difference is presented diagrammatically at point A. Such results show that post-ischemic revitalization at 37C leads to recovery of the cells from transient, ischemia-induced metabolic lesions and from the concomitant depression of cellular function. Thus, the data indicate that revitalization at 37C markedly improves the metabolic energy status and function of the cells, and hence the overall quality of the fresh human heart valve leaflets.
Heart valve half leaflets were prepared and incubated at 4C or 37C as described in Example 1.
These half leaflets were then cryopreserved, essentially via the method described in U.S. application serial no.
000,095. The frozen leaflets were stored at -196'C for at least 16 hours. The leaflets were thawed, and the cryoprotectant was diluted as described in the aforementioned patent application. Immediately after thawing and dilution, the half leaflets were assayed for 2-deoxyglucose incorporation as described in Example 1.
The results are shown in Table B.
TABLE B
Comparison of 2-deoxyglucose incorporation (DPM / mg protein) by half leaflets given a six hour incubation at 37C vs. half leaflets given a six hour incubation at 4C. After the incubations all leaflets were cryopreserved, thawed, diluted and immediately assayed.
?J ~ '~r ~3 ~
DPM Values for T.issue Preincubated at- % 37 C vs. 4C
4~C 37C (37 C/4 C x 100) 27,281 70,372 258 34,219 118,682 347 33,944 86,439 255 132,447 185,887 140 82,604 320,953 389 162,623 201,013 124 3949 17,215 436 3990 14,366 360 13,975 28,888 207 14,960 38,016 254 16,052 17,130 107 5193 18,951 365 17,320 29,482 170 2489 20,533 825 x + SE = 304 + 44 %
The data demonstrates that the revitalization of tissue by incubation at 37C for six hours, prior to cryopreservation, results in approximately a 3-fold greater cellular viability and functional capacity than that noted when tissue is given a pre-cryopreservation incubation at 4C. This difference is presented diagrammatically at point B. The revitalization at 37C
r, leads to recovery of the cells from transient, ischemia-induced metaboli~ lesions and functional depression, and the improved metabolic state and functional capacity are maintained after cryopreservation and thawing. Thus, revitalization at 37 C markedly improves the metabolic energy status and function, and hence the overall quality of the cryopreserved and thawed tissue.
Human heart valve leaflets were treated as described in Example 2, except that after thawing and dilution, all of the leaflet halves were placed in DMEM
(low glucose with 10% fetal calf serum) and given a 6 hour post-thaw incubation at 37C. After this incubation period, the half leaflets were assayed for 2-deoxyglucose incorporation as described in Example 1.
The results are shown in Table C.
2 ~
~14-TABLE C
Comparison of 2-deoxyglucose incorporation (DPM /
mg protein) by half leaflets given a six hour incubation at 37C vs. half leaflets given a six hour incubation at 4OC. After the incubations, all leaflets were cryopreserved, thawed, diluted and then given a 6 hour post-thaw incubation at 37C. After this incubation, the half leaflets were assayed for 2-deoxyglucose incorporation.
DPM Values for Tissue Preincubated at: % 37C vs. 4C
43C37C (37C/4C x 100) 109,009 171,963 158 79,676 103,058 129 148,937 176,034 118 127,787 459,873 360 150,302 239,906 160 119,150 224,751 189 x + SE = 186 ~ 33 %
The data shows that the revitalization of tissue by incubation at 37C prior to cryopreservation, results in approximately a two fold greater cellular viability than that noted when tissue is given a pre-cryopreservation incubation at 4~C, even after the tissue has heen given a six hour post-thaw incubation at 37C. This J~
difference is presented diagrammatically at point C.
Such results show that post-ischemic revitalization at 37C leads to recovery of the cells from transient, ischemia-induced metabolic lesions and functional depression, and that the improved metabolic state and functional capacity are maintained after cryopreservation, thawing, and incubation. Thus, the data indicates that revitalization at 37C markedly improves the metabolic energy status and functional capacity, and hence the overall quality of the cryopreserved and thawed tissue. Furthermore, these results show that revitalized tissue would have a marked advantage over non-revitalized tissue during the first six hours after transplantation.
Human heart valve leaflets were treated as described in Example 2, except that after thawing and dilution, all of the leaflet halves were placed in DMEM
(low glucose with 10% fetal calf serum) and given a 16 hour post-thaw incubation at 37C. After this incubation period, the half leaflets were assayed for 2-deoxyglucose incorporation as described in Example 1.
The results are shown below in Table D.
TABLE D
Comparison of 2-deoxyglucose incorporation (DPM / mg protein) by half leaflets given a six hour incubation at 37C vs~ half leaflets given a six hour incubation at 4C. After the incubations all leaflets were cryopreserved, thawed, diluted and then given a 16 hour post-thaw incubation at 37C. After this incubation, the half leaflets were assayed for 2-deoxyglucose incorporation.
DPM Values for Tissue Preincubated at: % 37C vs. 4C
4OC 37C (37C/4C x 100) 35,988 112,025 311 72,313 88,043 122 295,436 479,791 162 x + SE = 198 + 47 %
These results indicate that revitalized heart valve leaflets maintain their functional advantage over nontreated tissue even after 16 hours, post-thaw, at 37C. This difference is presented diagrammatically at point D.
Heart valve leaflets were processed as described in Example 2, except that pre-cryopreservation incubation of three hours duration at 37C was compared to a three hour incubation at 4*C. The results are shown in Table E.
2 ~ .J
TABLE E
Comparison of 2-deoxyglucose incorporation (DPM /
mg protein) by half leaflets given a three hour incubation at 37C vs. half leaflets given a three hour incubation at 4C. After the incubations all leaflets were cryopreserved, thawed, diluted and immediately assayed.
DPM Values for Tissue Preincubated at: % 37C vs. 4C
4C 37C (37C/4C x 100) 10,488 26,062 248 19,839 34,172 172 18,873 33,169 176 44,872 54,130 121 58,496 56,509 97 57,270 61,499 107 x + SE = 154 + 21 %
The data show that the revitalization of tissue by incubation at 37C for three hours, prior to cryopreservation, results in approximately 1.5-fold greater cellular viability than that noted when tissue is given a pre-cryopreservation incubation at 4C. Such results show that post-ischemic revitalization at 37C, which can be accomplished in as little as three hours, leads to recovery of the cells from transient ischemia-induced metabolic lesions and functional depression, and that the improved metabolic state and functional capacity are maintained after cryopreservation and thawing. Thus, revitalization at 37C for three hours markedly improves the metabolic energy status and functional capacity, and hence the overall quality of the cryopreserved and thawed tissue.
Human heart valve leaflets were treated as described in Example 4, except that after thawing and dilution, all of the leaflet halves were placed in DMEM (low glucose with 10%
fetal calf serum) and given a 6 hour post-thaw incubation at 37C. After this incubation period, the half leaflets were assayed for 2-deoxyglucose incorporation as described in Example 1. The results are shown in Table F.
TABLE F
Comparison of 2-deoxyglucose incorporation (DPM / mg protein) by half leaflets given a three hour incubation at 37c vs. half leaflets given a three hour incubation at 4C. After the incubations all leaflets were cryopreserved, thawed, diluted and then given a 6 hour post-thaw incubation at 37C.
After this incubation, the half leaflets were assayed for 2-deoxyglucose incorporation.
2~ 757 DPM Values for Tissue Preincubated at: % 37C vs. 4_ 4C 37C (37C/4C x 100) 13,196 20,852 158 11,364 17,760 156 7320 25,237 345 201,917 297,080 147 186,455 280,580 150 185,035 226,723 123 x + SE = 180 + 31 %
The data shows that the revitalization of tissue by incubation at 37 C for three hours prior to cryopreservation results in approximately 2-fold greater cellular viability than that noted when tissue is given a pre-cryopreservation incubation at 4 C, even a~ter the tissue has been given a six hour post-thaw incubation at 37 C. Such results show ~hat post-ischemic revitalization at 37~ C leads to recovery of the cells from transient, ischemia-induced metabolic lesions and functional depression, and that the improved metabolic state, and functional capacity are maintained after cryopreservation thawing, and incubation at physiological temperature. Thus, revitalization at 37 C markedly improves the metabolic energy status and functional capacity and hence, the overall quality of the cryopreserved and thawed human heart valve leaflets.
Furthermore, these results show that revitalized tissue would have a marked advantage over non-revitalized tissue within the t~J
first six hours after transplantation.
~ eart valve leaflets are treated as described in Example 2, except that after thawing and dilution, two revitalized leaflet halves (preincubated at 37 C for six hours prior to cryopreservation), from the same valve are rinsed briefly with Hanks and placed into an open-flow microcalorimeter and perfused with Hanks at a flow rate of 15 ml/minute. The tissue chamber is maintained at 37 C, and heat dissipation by the tissue is recorded continuously for a 2 hour period. The two control leaflet halves (preincubated at 4 C
prior to cryopreservation) are thawed, diluted and treated in an identical manner.
The rate of heat dissipation by the revitalized leaflet halves upon thawing is more than ~-times the rate seen with the control, nonrevitalized leaflet halves, as shown in Fig. 2. The mean heat dissipation for revitalized leaflet havles is 22.03 microwatts, whereas that for control non-revitalized leaflet halves is 10.54 microwatts. Heat dissipation correlates directly with the metabolic energy flow of cells. Therefore, the metabolic energy flow, and hence the capacity of the tissue to function, is more than 2-times greater in revitalized leaflet halves than in control, nonrevitalized leaflet halves. This is in close agreement With the results seen when 2-deoxyglucose incorporation is used as a measurement of relative metabolic energy status and functional capacity (i.e., Example 2). Thus, by two independent measurements of tissue metabolic energy status and functional capacity it is determined that revitalized tissue is far superior to nonrevitalized tissue after thawing.
Whole hearts are procured and shipped to the laboratory as described in Example 1. The aortic and pulmonary valves are dissected out, placed in a tissue culture medium (e.g. DMEM with glucose) and incubated at 37 C for about 3 to about 12 hours. If desired, antibiotics may be included in the incubation solution. After the incubation and concomitant revitalization, the valves are cryopreserved via the protocol described in U.S. application serial no. 000,095 which is incorporated herein by reference. Upon transplantation, such valves are of superior quality to those that had not been revitalized prior to cryopreservation.
In summary, the revitalization of tissue for a~out 5 minutes to about 24 hours prior to cryopreservation results in approximately 2 to 3 fold greater cellular viability than that noted when tissue is given a pre-cryopreserva~ion incubation at 4C, even when compared to tissue which has been given a six to sixteen hour post-thaw incubation at 37C. ~ence, post-ischemic revitalization leads to recovery of the cells from transient, ischemia-induced metabolic lesions and depressed functional capacity. The improved metabolic state and functional capacity are maintained after cryopreservation, thawing, and incubation. Revitalization markedly improves the metabolic energy status, cellular function and hence, the overall quality of the cryopreserved and thawed tissue.
Furthermore, these results show that revitalized tissue would have a marked advantage over non-revitalized tissue upon transplant.
While the invention herein is described in detail, numerous alternative embodiments are possible and fall within the appended claims. Consequently, the scope of the invention is not to be limited thereby.
Claims (16)
1. A method for optimizing cell revitalization comprising placing cells into a nutrient medium and incubating said cells at a temperature and for a period of time effective for optimizing cell revitalization.
2. The method of claim 1 wherein the cells constitute a transplantable tissue.
3. A method of maximizing transplantable tissue vitality after cryopreservation comprising placing transplant tissue into a nutrient medium prior to cryopreservation, and incubating said tissue at a temperature and for a period of time effective for maximizing tissue vitality upon transplant into a patient in need of such treatment.
4. The method of claim 3 wherein said transplantable tissue is incubated at a temperature ranging from about 27°C to about 42°C.
5. The method of claim 4 wherein the said transplantable tissue is incubated for a period ranging from about five minutes to about twenty four hours.
6. The method of claim 5 wherein said transplantable tissue in incubated for about three to about nine hours.
7. The method of claim 6 wherein said transplantable tissue is incubated for about 6 hours.
8. The method of claim 4 wherein said transplantable tissue is incubated at 37°C.
9. An optimally revitalized transplantable tissue comprising cells suitable for administration to a patient in need of such treatment, said cells being incubated prior to cryopreservation for a time period and at a temperature effective for optimally revitalizing said cells after cryopreservation and thawing.
10. The tissue of claim 9 wherein the cells constitute heart tissue.
11. The tissue of claim 10 wherein said tissue is incubated at a temperature ranging from about 27°C to about 47°C prior to cryopreservation.
12. The tissue of claim 11 wherein the tissue is incubated at a temperature ranging from about 35°C to about 42°C prior to cryopreservation.
13. The tissue of claim 12 wherein the tissue is incubated at 37°C prior to cryopreservation.
14. The tissue of claim 11 wherein the tissue is incubated for from about five minutes to about 24 hours prior to cryopreservation.
15. The tissue of claim 14 wherein the tissue is incubated for about three to nine hours.
16. The tissue of claim 15 wherein the tissue is incubated for six hours.
Applications Claiming Priority (2)
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|---|---|---|---|
| US07/344,013 US5171660A (en) | 1989-04-26 | 1989-04-26 | Process of revitalizing cells and tissue prior to cryopreservation |
| US07/344,013 | 1989-04-26 |
Publications (1)
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|---|---|
| CA2012757A1 true CA2012757A1 (en) | 1990-10-26 |
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| CA002012757A Abandoned CA2012757A1 (en) | 1989-04-26 | 1990-03-22 | Method of revitalizing cells prior to cryopreservation |
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| DE (1) | DE69024260T2 (en) |
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| US4559298A (en) * | 1982-11-23 | 1985-12-17 | American National Red Cross | Cryopreservation of biological materials in a non-frozen or vitreous state |
| FR2589165B1 (en) * | 1985-10-24 | 1989-01-20 | Inst Nat Sante Rech Med | PROCESS FOR OBTAINING A HUMAN CULTURE EPITHELIUM, EPITHELIAL FABRIC OBTAINED AND ITS APPLICATION TO ALLOGRAFTS |
| US4678003A (en) * | 1986-10-10 | 1987-07-07 | Griffin Beacher C | Safety cap for valve on high-pressure cylinder |
| IT1207525B (en) * | 1987-06-23 | 1989-05-25 | Ist Naz Ric Sul Cancro | METHOD FOR THE PRESERVATION OF TRANSPLANTABLE SHEETS OF EPITELIUM CULTIVATED IN VITRO VITRO. |
| US5145769A (en) * | 1987-08-21 | 1992-09-08 | Cryolife Inc. | Method for cryopreserving blood vessels |
-
1989
- 1989-04-26 US US07/344,013 patent/US5171660A/en not_active Expired - Lifetime
-
1990
- 1990-03-22 CA CA002012757A patent/CA2012757A1/en not_active Abandoned
- 1990-04-12 EP EP90304038A patent/EP0399647B1/en not_active Expired - Lifetime
- 1990-04-12 AT AT90304038T patent/ATE131686T1/en not_active IP Right Cessation
- 1990-04-12 ES ES90304038T patent/ES2081927T3/en not_active Expired - Lifetime
- 1990-04-12 DE DE69024260T patent/DE69024260T2/en not_active Expired - Fee Related
- 1990-04-24 JP JP2106660A patent/JP2859925B2/en not_active Expired - Fee Related
-
1992
- 1992-08-10 US US07/927,768 patent/US5424207A/en not_active Expired - Lifetime
-
1996
- 1996-02-01 GR GR960400270T patent/GR3018876T3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0399647A1 (en) | 1990-11-28 |
| DE69024260T2 (en) | 1996-06-27 |
| ATE131686T1 (en) | 1996-01-15 |
| JPH0368501A (en) | 1991-03-25 |
| ES2081927T3 (en) | 1996-03-16 |
| US5424207A (en) | 1995-06-13 |
| DE69024260D1 (en) | 1996-02-01 |
| JP2859925B2 (en) | 1999-02-24 |
| US5171660A (en) | 1992-12-15 |
| GR3018876T3 (en) | 1996-05-31 |
| EP0399647B1 (en) | 1995-12-20 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |