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WO1998014058A1 - Cryoconservation de cellules pancreatiques de foetus et de personne adulte et de plaquettes humaines - Google Patents

Cryoconservation de cellules pancreatiques de foetus et de personne adulte et de plaquettes humaines Download PDF

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Publication number
WO1998014058A1
WO1998014058A1 PCT/US1997/017591 US9717591W WO9814058A1 WO 1998014058 A1 WO1998014058 A1 WO 1998014058A1 US 9717591 W US9717591 W US 9717591W WO 9814058 A1 WO9814058 A1 WO 9814058A1
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Prior art keywords
cells
trehalose
islets
accordance
platelets
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Inventor
Gillian M. Beattie
John H. Crowe
Fern Tablin
Alberto Hayek
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Priority to AU46596/97A priority Critical patent/AU4659697A/en
Publication of WO1998014058A1 publication Critical patent/WO1998014058A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • 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
    • A01N1/10—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes

Definitions

  • Field of the Invention lies in the field of cryopreservation of living cells and tissue, and of cryopreservation media.
  • Type I diabetes insulin-dependent diabetes is a widespread disease, arising from an autoimmune disorder in which insulin-secreting beta cells in the pancreas are destroyed, depriving the pancreas of its ability to secrete insulin. This impairs the body's ability to take up glucose from the blood, and the result is high glucose levels, which can lead to blindness, kidney disease, nerve damage, and ultimately death.
  • the therapy most commonly applied is the injection of insulin to compensate for the lack of beta cells, but blood sugar levels can still fluctuate widely. Methods of lessening the fluctuations have included the use of small, frequent doses of insulin and the use of mechanical pumps that mimic the action of the pancreas, but these require the rigors of continuous or periodic maintenance, and the results are often of limited success.
  • An alternative is a pancreatic transplant, but this requires major surgery with the attendant risk of complications, and the limited availability of donor pancreases makes this a less than viable option in general.
  • Islets of Langerhans are clusters of cells in the pancreas that include the insulin-secreting beta cells, and their transplantation entails considerably less risk than the transplantation of a pancreas.
  • Sources for islet transplantation include adult pancreatic tissue, fetal pancreatic tissue and islet-like cell clusters (ICCs).
  • ICCs islet-like cell clusters
  • Fetal tissue offers a greater content of islets in proportion to its mass, as well as a greater capacity for proliferation with its less mature cells.
  • Islet-like cell clusters are heterogeneous cell populations that include epithelial cells that differentiate after transplantation to form various types of cells including mature islets.
  • Islet tissue of the various types identified above that is available for transplantation is scarce, and islets must be banked and transported in order to obtain sufficient islets for a single recipient. This requires cryopreservation, and unfortunately a very low percentage of the cells are able to undergo freezing and thawing and retain their functionality. Cryoprotectants, of which dimethyl sulf oxide is the most prominently used, lessen the damage to the islets, but still entail some loss of islet functionality. This indicates a need for a cryopreservation medium that will promote the retention of a higher degree, if not all, of the islet tissue functionality upon thawing.
  • Platelets are a fraction of human blood which are important contributors to hemostasis. Platelets are generally oval to spherical in shape, with a diameter of 2-4 ⁇ m, and contain about 60% protein, 15% lipid, and 8.5% carbohydrate. Included in the chemical composition of platelets are serotonin, epinephrine, and norepinephrine, each of which aids in promoting the constriction of blood vessels at the site of injury. Platelets also contain platelet factors, including platelet thromboplastin, which is a cephalin-type phosphatide, and adenosine diphosphate, both of which are important in blood coagulation. The maintenance of functional platelets is important in preserving whole blood for storage in blood banks, and in preserving concentrated platelet fractions.
  • platelets raises certain problems, however. While the remaining fractions of blood can be preserved by cold storage for extended periods of time, platelets tend to become activated at low temperatures and thereby useless. To avoid activation, platelets are isolated and stored separately as concentrated fractions at 22-24 °C. This raises other risks, however, namely bacterial infection as well as metabolic and enzymatic reactions known collectively as "platelet storage lesion. " As a result, platelet storage is generally limited to five days. It would clearly be desirable to be able to freeze and store platelets in a manner that would result in the retention of their biological functions upon thawing.
  • Trehalose is a disaccharide found in high concentrations in a wide variety of organisms that are capable of surviving complete dehydration. Trehalose is known to stabilize membranes, proteins, and prokaryotic cells during freezing and drying in vitro.
  • cryoprotectants trehalose and dimethyl sulfoxide as cryoprotectants. These materials are conveniently absorbed by the cells as the cells are chilled through the thermotropic lipid phase transition temperature prior to the actual freezing of the cells. The success of the cryoprotectant combination is evident by observation of the low rate of DNA synthesis and the higher rate of insulin concentration in the cells after freezing and thawing. The use of the trehalose-DMSO combination in a cryopreservation protocol thus leads to previously unobtainable survival rates of functional human endocrine tissue.
  • the trehalose-DMSO combination is useful in the cryopreservation of platelets, enabling them to be frozen and thawed while retaining their ability to be activated upon the appropriate stimuli. Platelets can thus be stored for extended periods of time at temperatures sufficiently low to control bacterial infection and platelet storage lesion, without the risk of premature activation.
  • trehalose can be incorporated into the interiors of eukaryotic cells in general whose cell walls contain a lipid bilayer, by suspending the cells in a liquid solution of trehalose and cooling or warming the suspension through the thermotropic lipid phase transition of the cells. This occurs independently of the presence or absence of DMSO.
  • FIGS, la and lb which do not represent data taken within the scope of this invention, are portions of the infrared spectra of islet cells taken at 0°C and 27 °C. Both temperatures are represented on each of the two spectra, the FIG. lb spectrum representing a portion of the FIG. la spectrum.
  • FIG. 2 which also does not represent data taken within the scope of this invention, is a plot of the frequency of the peak maximum for the CH 2 stretch from infrared spectra of islet cells as a function of temperature, showing the temperature being varied by both cooling and warming.
  • FIG. 3a which illustrates the present invention, is a plot of the frequency of the peak maximum for islet cells vs. temperature during a cooling transition as in FIG. 2, except that cooling was performed in the presence of trehalose. The plot is superimposed over plots of the amount of trehalose incorporated into the islets as a function of temperature during the same cooling transition.
  • FIG. 3b is a plot taken from that of FIG. 3a, showing the amount of trehalose incorporated as a function of the frequency of the peak maximum.
  • FIG. 4 which does not represent data taken within the scope of this invention, is a fluorescence-activated cell sorting (FACS) scan of human platelets, both stimulated with thrombin and non-stimulated.
  • FIG. 5 is a FACS scan illustrating the invention, showing corresponding data on platelets that have been frozen in the presence of a cryopreservation medium containing trehalose and DMSO.
  • FIG. 6 is a graph showing two plots superimposed — a plot of the frequency of the peak maximum of the CH 2 stretch in an infrared spectrum of human platelets vs. temperature as the platelets are being cooled, and a plot of the amount of trehalose incorporated into the platelets as a function of temperature during the same cooling transition.
  • Cryopreservation in accordance with this invention is performed in a liquid cryopreservation medium that contains preservation-enhancing amounts of trehalose and DMSO.
  • preservation-enhancing amount refers to any amount that will produce a detectable improvement in the retention of islet viability and functionality upon freezing and thawing relative to islets that have undergone freezing and thawing in the absence of a cryoprotectant.
  • islet will at times be used in this specification to include both adult and fetal islets, as well as ICCs.
  • the actual amounts of trehalose and DMSO can vary, although considerations of the economical use of materials and labor, and considerations of the cryopreservation protocol, i.e.
  • the choice of procedural steps used for cooling and thawing the islets together with the cooling and thawing rates may affect the selection of concentration ranges that will provide the most efficient and effective preservation.
  • concentration ranges that will provide the most efficient and effective preservation.
  • trehalose best results in most cases will be achieved at concentrations between about 10 uM and about 1,500 mM, preferably between about 100 mM and about 500 mM, in the cryopreservation medium.
  • DMSO best results in most cases will be achieved at concentrations between about 0.3 M and about 15 M, preferably between about 0.5 M and about 10 M, and most preferably between about 1 M and about 3 M.
  • the trehalose and DMSO are preferably dissolved in a liquid tissue culture medium, which includes any liquid solution that contains the appropriate solutes to render the solution capable of preserving living cells and tissue.
  • a liquid tissue culture medium which includes any liquid solution that contains the appropriate solutes to render the solution capable of preserving living cells and tissue.
  • Many types of mammalian tissue culture media are known in the literature and available from commercial suppliers, such as Sigma Chemical Company, St. Louis, Missouri, USA; Aldrich Chemical Company, Inc. , Milwaukee, Wisconsin, USA; and Gibco BRL Life Technologies, Inc. , Grand Island, New York, USA.
  • Examples of media that are commercially available are Basal Medium Eagle, CRCM-30 Medium, CMRL Medium-1066, Dulbecco's Modified Eagle's Medium, Fischer's Medium, Glasgow Minimum Essential Medium, Ham's F-10 Medium, Ham's F-12 Medium, High Cell Density Medium, Iscove's Modified Dulbecco's Medium, Leibovitz's L-15 Medium, McCoy's 5A Medium (modified), Medium 199, Medium 199, Minimum Essential Medium Eagle, Alpha Minimum Essential Medium, Earle's Minimum Essential Medium, Medium NCTC 109, Medium NCTC 135, RPMI-1640 Medium, William's Medium E, Waymouth's MB 752/1 Medium, and Waymouth's MB 705/1 Medium.
  • the islets can be isolated from pancreas tissue by methods known to those skilled in the art.
  • the islets are then suspended in the cryopreservation medium ( . e. , the tissue culture medium plus the added trehalose and DMSO) .
  • the concentration of the islets in the medium that will provide optimal results can vary, and the concentration selected for use in any given procedure will be governed primarily by considerations of economy and efficiency. Effective results will generally be achieved with suspensions containing from 10 to 50,000 islets per milliliter of cryopreservation medium, preferably from 100 to 10,000 islets/mL, and most preferably from 300 to 3,000 islets/mL.
  • the protocol for cooling the islets past the thermotropic transition temperature can be any protocol known to those skilled in the art for freezing cells of this type while avoiding damage to the cells.
  • the islets are incubated while the temperature is lowered in stages and the DMSO and trehalose concentrations are likewise increased in stages. Adjustments to the medium are achieved by centrifugation and removal of the liquid followed by replacement of the liquid removed.
  • the final cryopreservation medium is preferably one that provides isotonicity with the frozen cells. During thawing, the medium is replaced with a medium that does not contain DMSO, since DMSO is toxic at physiologic temperature.
  • thermotropic phase transition that the islets pass through while being chilled toward the freezing temperature is a characteristic of lipid bilayers in their transition between the liquid crystalline (i.e. , fluid) and gel (i.e. , solid) phases.
  • the lipid molecules are loosely aligned according to their hydrophilic and lipophilic regions, with the lipophilic regions facing each other, away from the aqueous environment.
  • the loose alignment of the structure imparts flexibility to the bilayer, but the distance between the loosely aligned molecules is small enough that the permeability of the lipid bilayer is limited.
  • thermotropic phase transition a transitional phase known as the thermotropic phase transition
  • regions of a loosely packed liquid crystalline phase alternate with regions of a densely packed gel phase.
  • the two phases are not fully compatible, and at locations where the two phases are adjacent to each other, the bilayer molecules form packing irregularities or defects. These packing defects cause an increase in permeability, greater than that of either the liquid crystalline phase or the gel phase. It is this increase in permeability at the locations of the packing defects that permits trehalose to pass through the bilayer and into the cells.
  • trehalose is introduced into the cells by cooling the cells through the thermotropic phase transition while the cells are suspended in a liquid solution of trehalose.
  • the final temperature to which the islets will be cooled can vary, depending again on considerations of economy and practicality. Typical temperatures for effective storage and transport will be about 5°C or below, preferably about 0°C or below, and more preferably about -5°C or below. The most preferred storage temperature is between about -40°C and about -196°C (the temperature of liquid nitrogen).
  • transplantation is accomplished by conventional techniques known to those skilled in the art of tissue transplantation.
  • the platelets are first isolated and concentrated. This can be done according to conventional techniques such as those used to isolate platelets for platelet counting. According to one such technique, blood is collected in an anticoagulant, then centrifuged at a speed which is selected to produce a supernatant which is platelet- rich. Higher concentrations can be achieved by recovery of the supernatant followed by further centrifuging. Other methods are known to those skilled in the art.
  • the platelets are suspended in a solution containing the trehalose-DMSO combination.
  • the solvent can be a tissue culture medium such as those listed above for use with islets, or any other medium that will not cause damage to the platelets.
  • concentration of platelets in the suspension can vary. In most cases, appropriate concentrations for efficient and economical cryopreservation will be from about 10 6 to about 10 10 cells/mL, preferably from about 10 7 to about 10 9 cells/mL, and most preferably about 10 8 cells/mL.
  • the platelets can be maintained in suspension at 22 °C until ready for preservation. While the freezing and thawing protocols and the methods of incorporating trehalose can vary, a presently preferred method is to cool the platelet suspension at 1 °C per minute to a final temperature of -70 °C, then to transfer the cooled platelets to liquid nitrogen.
  • the platelets can be stored in liquid nitrogen for as much as 32 days or more.
  • thermotropic liquid phase transition As they are being cooled, the cells pass through the thermotropic liquid phase transition, which occurs between 15°C and 20°C. Passage through this phase transition will result in the passage of the trehalose through the platelet membrane and into the interior of the platelets due to the transitory increase in membrane permeability as described above.
  • Other means of incorporating trehalose into the platelets to achieve an equivalent result will be readily apparent to those skilled in the handling of platelets.
  • a preferred final temperature for storage purposes is within the range of about -70 °C to about -196°C.
  • the platelet pellet When platelets that have been chilled below their thermotropic phase transition temperature are needed for clinical use, the platelet pellet will be rapidly thawed at 37°C to an ice ball and progressively diluted until ready for use. It is preferred that the DMSO be removed from the platelets and the suspending medium before the platelets are returned to physiologic temperature. Dilution is preferably performed in two or more, preferably four or more, volumes of buffer per volume
  • trehalose into eukaryotic cells independently of cryopreservation, and independently of the presence or absence of DMSO, is achieved in accordance with this invention by suspending the cells in a liquid solution of trehalose and cooling or warming (preferably cooling) the suspension through the thermotropic phase transition. While this specification provides examples in which this discovery is applied to islets, ICCs and platelets, these examples lead to the conclusion that trehalose incorporation by this technique is applicable to any eukaryotic cells or cell-like structures that contain a lipid bilayer in the cell membrane. Any liquid solution of trehalose can be used, although the tissue culture media cited above are preferred for purposes of protecting the cells during the phase transition.
  • the concentration of trehalose in the suspending solution is not critical and can vary. Nevertheless, the preferred concentrations are those cited above. Cooling or warming is performed at a rate slow enough to permit the passage of the trehalose through the disrupted bilayer, although the actual cooling rate is not critical. Effective rates are readily determined by analyzing the cells for trehalose content, using methods well known in the art. Examples of such methods are those used in the examples below. For cooling, preferred rates are those cited above. When the temperature transition is a cooling transition, the retention of the trehalose in the cells upon rewarming of the cells above the phase transition temperature, and to physiologic temperature if desired, is readily achieved by rewarming at a rapid rate, i.e.
  • eukaryotic cell is used herein to mean any nucleated cell, i.e. , a cell that possesses a nucleus surrounded by a nuclear membrane, as well as any cell that is derived by terminal differentiation from a nucleated cell, even though the derived cell is not nucleated. Examples of the latter are terminally differentiated human red blood cells. Mammalian, and particularly human, eukaryotes are preferred.
  • Gestational age was determined by several criteria including bi-parietal diameter, femur length and fetal foot measurement.
  • pancreases were digested with collagenase and cultured as islet-like cell clusters (ICCs), as described by Beattie, G.M. , et al. ,
  • Lipid phase transitions were measured by changes in membrane CH 2 vibrational frequency, using a Perkin-Elmer Fourier transform infrared microscope coupled to a Perkin-Elmer 1620 FTIR optical bench and equipped with a temperature controller. Data manipulations were limited to baseline adjustment and absorbance expansion, using the flat and abex routines in Perkin-Elmer IRDM software. Samples were prepared by placing the islets between BaF 2 windows, with a 10-micron spacer supporting the windows, and placing the windows and islets in the temperature controller on the microscope stage. All curve fitting was done by multiple iterations of a least squares algorithm on a microcomputer.
  • the ICCs and islets were frozen in two groups each, one using DMSO as the sole cryopreservative and the other using both DMSO and trehalose as combined cryopreservatives.
  • the first medium consisted of RPMI-1640 plus 10% human serum containing 11.1 mM glucose (serving as a cell nutrient) and 2 M DMSO; this medium is referred to herein as "CDG.
  • the second medium consisted of RPMI-1640 plus 10% human serum containing 2 M DMSO and 300 mM trehalose, and is referred to herein as "CDT.
  • CDT mM trehalose
  • the freezing and thawing protocols were the same in each case, and were as follows.
  • ICCs Integrated Circuits
  • RPMI-1640 normal human serum containing saccharide (either 11.1 mM glucose or 300 mM trehalose) in each of a series of 100 mm reusable sterile Kimble glass tubes with phenolic caps.
  • RPMI-1640 normal human serum containing saccharide
  • To these ICCs or islets were then added 0.1 mL of RPMI-1640 plus 10% normal human serum containing 2 M DMSO and either 11.1 mM glucose or 300 mM trehalose, and the resulting mixture was incubated for 5 minutes at 24 °C. An additional 0.1 mL of the same solution was added, and incubation was continued for an additional 25 minutes at 24 °C.
  • the tubes were partially thawed rapidly at 37 °C to an ice ball and transferred to ice.
  • the cells were then quickly pelleted by centrifugation for one minute, whereupon the supernatant was carefully removed and 1 mL of a cold solution containing RPMI-1640 plus 10% human serum and the saccharide (either trehalose at 300 mM trehalose or sucrose at 750 mM) was added to the pellet.
  • the resulting mixture was kept on ice for thirty minutes.
  • ICCs and islets that had undergone freezing and thawing in the presence of the two cryopreservation media (i.e. , those containing DMSO and saccharide) were compared with the functionalities of ICCs and islets that had been cultured in RPMI-1640 plus 10% human serum (i. e. , those containing neither DMSO nor saccharide), and had not undergone freezing and thawing.
  • the latter are referred to in the examples as "fresh" ICCs and islets.
  • Freezing and thawing was also performed using a medium that was identical to CDT except that it did not contain DMSO, i.e. , its composition was RPMI-1640 plus 10% human serum containing 300 mM trehalose. The protocol in this case was identical to that used for CDT.
  • Immunoreactive insulin was assayed in the medium and in acid ethanol extracts of cell sonicates using a solid phase radioimmunoassay. Incorporation of [ 3 H]-thymidine (64 Ci/mmol) into cellular DNA was determined by measuring trichloroacetic acid precipitable counts in a scintillation counter.
  • mice obtained through the NIH-Grantee Reimbursement Program from the Charles River Breeding Laboratories (Charles River, Massachusetts, USA), and were housed in microisolater cages in a semi-sterile room where they were maintained according to NIH guidelines.
  • the animals were sacrificed three months after transplantation, and the kidneys removed. The kidneys were then fixed in 4% paraformaldehyde, and 5- micron sequential sections were stained using hematoxylin and eosin and the immunoalkaline phosphatase technique.
  • the primary antibody used was guinea pig anti-porcine insulin. Normal rabbit serum was used as the control serum.
  • the grafted human tissue was carefully peeled away from the kidney under direct vision, then minced finely, homogenized in distilled water and sonicated. Aliquots of the sonicates were extracted with acid ethanol for insulin radioimmunoassay and dried for DNA quantitation as described above for the in vitro assays.
  • FIGS, la and lb Typical infrared spectra in the hydrocarbon chain stretching region in islet cells both before and after a phase transition are shown in FIGS, la and lb, where FIG. lb is an expansion of a portion of the horizontal scale of FIG. la. Spectra were taken both above the transition temperature (i.e. , at 27 °C, represented by the solid line in both Figures) and below the transition temperature (i.e. , at 0°C, represented by the dashed line in both Figures), with no cryopreservation agent having been used.
  • the CH 2 stretch near 2850 cm “1 is conveniently used to measure phase transitions in biological membranes, hence the expansion of the portion surrounding this band in FIG. lb.
  • the frequency of the maximum for this band was determined as a function of temperature, first during a cooling transition and then during a re-warming transition.
  • the results are plotted in FIG. 2, where the filled circles (•) represent the cooling transition and the open circles (O) represent the warming transition.
  • the plot shows that during cooling the frequency of this band falls from about 2853 cm "1 to about 2851 cm 1 , with a midpoint at about 5°C.
  • the re- warming transition has the same end frequencies, but the midpoint of the curve shifts to about 9°C, indicating a hysteresis of about 4°C.
  • the cooling and re- warming were then repeated, and the same shift in curve midpoint was observed.
  • EXAMPLE 2 This example shows the correlation between the passage of trehalose into cells and the location of the cells on the absorption peak wave number vs. temperature curve.
  • the cells used in this test included both islets from adults and
  • the cells were suspended in CDT and the suspension was gradually cooled below the phase transition of the cells.
  • the phase transition of the cells was monitored by recording the wave number of the band maximum for the CH 2 stretch, and the amount of trehalose entering the cells was determined by liquid scintillation counting.
  • the results are plotted in FIG. 3a, where the filled circles (•) represent the wave number and hence the phase transition, the open circles (O) represent the amount of trehalose in the adult islets, and the open squares (D) the amount of trehalose in the fetal islets.
  • the plot shows that the entry of trehalose coincides with the phase transition, and the trehalose reaches maximal values as the transition is completed.
  • phase transition i.e. , at temperatures above about 15 °C, only minimal amounts of trehalose had entered the cells.
  • This example presents in vitro test results on adult islets, with a comparison between results obtained using a cryopreservation medium containing trehalose in accordance with this invention (CDT), and those obtained with a medium containing glucose rather than trehalose (CDG). Freezing and thawing of adult islets in both media were performed as described above. Three days after thawing, the islets were assayed for insulin and DNA content, and for insulin release following stimulation with 16.7 mM glucose, as compared to a basal glucose concentration of 1.6 mM. Assays were also performed on islets that were not treated with either medium and had not undergone freezing and thawing. The results are shown in Table I below. TABLE I
  • Insulin content 43.7 + 2.4 46.6 ⁇ 6.5 56.5 ⁇ 7.9 (pmoles/ ⁇ g DNA)
  • Insulin release (pmoles/ ⁇ g DNA): 1.6 mM Glucose 1.2 ⁇ 0.1 2.3 ⁇ 0.2 1.3 + 0.2 16.7 mM Glucose 2.5 + 0.2 5.7 ⁇ 0.6 3.2 + 0.6
  • This example presents in vitro test results on fetal ICCs, again comparing results obtained using a cryopreservation medium containing trehalose (CDT) with those obtained with a medium containing glucose rather than trehalose (CDG).
  • CDT cryopreservation medium containing trehalose
  • CDG medium containing glucose rather than trehalose
  • Freezing and thawing of fetal ICCs in both media were performed as described above. Three days after thawing, the ICCs were assayed for insulin and DNA content, and for [ 3 H]-thymidine incorporation and insulin release following stimulation with 16.7 mM glucose and 10 mM theophylline, and with a basal glucose concentration of 1.6 mM. Assays were also performed on islets that were not treated with either medium and had not undergone freezing and thawing. The results are shown in Table II below.
  • Insulin content 2.2 + 0.3 0.4 + 0.1 2.3 + 0.4 (pmoles/ ⁇ g DNA)
  • Insulin release (fmoles/ ⁇ g DNA): 1.6 mM Glucose 69. ⁇ 24 49. + 13 39. + 8 16.7 mM Glucose + 10 mM Theophylline 134. ⁇ 37 126. + 16 139. + 37
  • This example is a repeat of the experiment reported in Example 4, showing the synergistic effect of DMSO and trehalose.
  • In vitro tests on fetal ICCs were performed, in one group of tests using CDT as defined above, in another using the same medium minus the DMSO (i.e. , containing trehalose as the only cryoprotectant), and in a third using the CDG as defined above (i.e. , in which DMSO was the only cryoprotectant).
  • Total DNA was assayed three days after thawing, as in Example 4, and the results were as follows:
  • This example presents in vivo test results obtained by transplanting adult islets and fetal ICCs into mice, followed by measurements of the insulin content of the grafts.
  • Cells cryopreserved in CDT were compared with those cryopreserved in CDG and with fresh cells (that had not undergone freezing).
  • Equivalent numbers of fresh and cryopreserved and thawed adult islets and fetal ICCs were transplanted under the kidney capsules of nude mice. Three months after transplantation, the grafts were removed. Those preserved with CDT were observed to be much larger than those preserved with CDG. Sections were immunostained by the alkaline phosphatase method with guinea pig anti-porcine insulin as the primary antibody.
  • This example investigates thrombin- induced activation of human platelets, and illustrates the ability of the DMSO-trehalose combination to preserve the ability of the platelets to respond to thrombin after having been frozen and thawed.
  • Human platelets were isolated from whole blood by low-speed differential centrifugation. The platelets were washed free of plasma proteins, suspended in the CDT cryopreservation medium at a concentration of 10 8 cells/mL, cooled at a rate of 1 °C/min to -70°C, held overnight, then transferred to liquid nitrogen where they were held for 32 days. The platelets were then thawed according to the protocols described above. The platelets were then treated with thrombin (a physiological platelet agonist), and analyzed for P-selectin (GMP-140) on the platelet surface. P-Selectin is an alpha granule membrane protein that is present on the platelet surface only upon activation of the platelets, and that therefore serves as an activation marker. Analysis for P-selectin was performed by fluorescence-activated cell scanning (FACS).
  • FACS fluorescence-activated cell scanning
  • FIGS. 4 and 5 represent fresh washed platelets that have not undergone freezing and thawing
  • FIG. 5 represents washed platelets that have been frozen and thawed in the presence of CDT medium.
  • the curve labeled "Isotypic" shows the level of non-specific background fluorescence.
  • the remaining two curves represent the platelets that have not been stimulated by thrombin (labeled "Unstimulated") and those that have been stimulated by thrombin (labeled "Thrombin Stimulated”).
  • the thrombin stimulation causes an increase in the number of cells that express P-selectin.
  • the thrombin stimulation still causes an increase in the number of cells expressing P-selectin, although the increase is less than that observed in the fresh cells.
  • stimulation after a freeze-thaw cycle produced no platelet activation at all.
  • EXAMPLE 8 This example illustrates the incorporation of trehalose into human platelets as the platelets are cooled through the thermotropic phase transition.
  • Human platelets were washed as in Example 7, then suspended at room temperature in a cryopreservation medium consisting of RPMI-1640 plus 10% human serum and 300 mM trehalose (DMSO was not present), the trehalose including [ 14 C] -trehalose as a tracer.
  • the suspension was then cooled at a rate of l°C/minute from room temperature to 4°C. During the chilling, aliquots of the suspension were removed and rapidly filtered on Millipore filters, and the filtered platelets were transferred to scintillation vials. Scintillation cocktail (a mixture of organic solvents) was added to each sample, and the radioactivity in each sample was determined by liquid scintillation counting.
  • infrared spectra of human platelets were taken at various points during an identical cooling protocol, and the wave number of the band maximum for the CH 2 stretch was recorded as a function of temperature.
  • the radioactivity of the filtered cells (representing the amount of trehalose incorporated into the cells) and the wave number of the band maximum, both taken during the cooling procedure, are plotted as a function of temperature in FIG. 6, where the filled circles represent the radioactivity in counts per minute per milligram of platelets (dry weight) and the open circles represent the wave number.
  • the plot shows that the transition points of the two curves occur approximately at the same temperature, and that trehalose is in fact incorporated in the cell interiors as an result of the phase transition.

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Abstract

La combinaison de tréhalose et de diméthylsulfoxyde donne un agent exceptionnellement efficace de cryoprotection d'îlots et de groupes de cellules de type îlot, ainsi que de plaquettes. Ces îlots et groupes de cellules de type îlot conservent leur fonctionnalité lorsqu'on les réfrigère par le biais d'une transition de phase thermotropique et en présence de ladite combinaison d'agents de traitement, et qu'on les laisse ensuite revenir à leur température physiologique. De même, on peut réfrigérer et réchauffer des plaquettes sans que celles-ci ne subissent une activation prématurée. En général, on peut incorporer le tréhalose dans les cellules eucaryotes, en suspendant les cellules dans une solution de tréhalose et soit en réfrigérant, soit en réchauffant la solution par le biais d'une transition thermotropique des cellules.
PCT/US1997/017591 1996-10-03 1997-09-29 Cryoconservation de cellules pancreatiques de foetus et de personne adulte et de plaquettes humaines Ceased WO1998014058A1 (fr)

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Cited By (15)

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WO2000015032A1 (fr) * 1998-09-11 2000-03-23 The General Hospital Corporation Doing Business As Massachusetts General Hospital Poration controlee reversible pour la conservation de matieres vivantes
US6176089B1 (en) 1998-10-27 2001-01-23 Modex Th{acute over (e)}rapeutics Methods and compositions for cryopreservation of cells and tissues
US6878543B1 (en) 1999-10-25 2005-04-12 Nsgene Sa Cultures of GFAP+ nestin+ cells that differentiate to neurons
EP1427811A4 (fr) * 2001-08-09 2006-11-02 Univ California Cellules erythrocytaires et methode de preservation de cellules
EP1430067A4 (fr) * 2001-08-09 2006-11-02 Univ California Cellules eucaryotes et procede de conservation associe
US8034635B2 (en) 2002-05-16 2011-10-11 Absorber Ab Methods of donor specific crossmatching
WO2012031162A1 (fr) 2010-09-01 2012-03-08 Doris Taylor Procédé de recellularisation d'un tissu ou d'un organe pour une transplantabilité améliorée
WO2016154447A1 (fr) 2015-03-26 2016-09-29 Miromatrix Medical Inc. Matrice extracellulaire décellularisée remplie de gaz
US10016463B2 (en) 2012-08-01 2018-07-10 United Therapeutics Corporation Treatment of pulmonary arterial hypertension with prostacyclin-treated endothelial progenitor cells
US10071123B2 (en) 2012-08-01 2018-09-11 United Therapeutics Corporation Treatment of pulmonary arterial hypertension with mesenchymal stem cells
US10080730B2 (en) 2013-01-09 2018-09-25 United Therapeutics Corporation Treatment of vasculopathy with prostacyclin and mesenchymal stem cells
IT202000026467A1 (it) * 2020-11-05 2022-05-05 Lipobank S R L Metodo per la crioconservazione di campioni biologici
US11571444B2 (en) 2016-10-24 2023-02-07 United Therapeutics Corporation Enhancement of MSC immunomodulatory properties by treprostinil
US12426594B2 (en) 2020-09-24 2025-09-30 Everest Medical Innovation GmbH Cryoprotective compositions and methods for protection of a surgical site during cryosurgery
US12453805B2 (en) 2020-09-24 2025-10-28 Everest Medical Innovation GmbH Cryoprotective compositions, surgical kits, and methods for protection of a surgical site during cryosurgery

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WO2000015032A1 (fr) * 1998-09-11 2000-03-23 The General Hospital Corporation Doing Business As Massachusetts General Hospital Poration controlee reversible pour la conservation de matieres vivantes
US6176089B1 (en) 1998-10-27 2001-01-23 Modex Th{acute over (e)}rapeutics Methods and compositions for cryopreservation of cells and tissues
US8501467B2 (en) 1999-10-25 2013-08-06 Stemcells California, Inc. Cultures of GFAP+ nestin+ cells that differentiate to neurons
US6878543B1 (en) 1999-10-25 2005-04-12 Nsgene Sa Cultures of GFAP+ nestin+ cells that differentiate to neurons
US7303912B2 (en) 1999-10-25 2007-12-04 Nsgene A/S Cultures of GFAP nestin cells that differentiate to neurons
US7651853B2 (en) 1999-10-25 2010-01-26 Nsgene A/S Cultures of GFAP+ nestin+ cells that differentiate to neurons
EP1427811A4 (fr) * 2001-08-09 2006-11-02 Univ California Cellules erythrocytaires et methode de preservation de cellules
EP1430067A4 (fr) * 2001-08-09 2006-11-02 Univ California Cellules eucaryotes et procede de conservation associe
US8034635B2 (en) 2002-05-16 2011-10-11 Absorber Ab Methods of donor specific crossmatching
US8173372B2 (en) 2002-05-16 2012-05-08 Absorber Ab Methods of donor specific crossmatching
WO2012031162A1 (fr) 2010-09-01 2012-03-08 Doris Taylor Procédé de recellularisation d'un tissu ou d'un organe pour une transplantabilité améliorée
US12310992B2 (en) 2012-08-01 2025-05-27 United Therapeutics Corporation Treatment of pulmonary arterial hypertension with prostacyclin-treated endothelial progenitor cells
US10016463B2 (en) 2012-08-01 2018-07-10 United Therapeutics Corporation Treatment of pulmonary arterial hypertension with prostacyclin-treated endothelial progenitor cells
US10071123B2 (en) 2012-08-01 2018-09-11 United Therapeutics Corporation Treatment of pulmonary arterial hypertension with mesenchymal stem cells
US11666602B2 (en) 2012-08-01 2023-06-06 United Therapeutics Corporation Treatment of pulmonary arterial hypertension with mesenchymal stem cells
US11839631B2 (en) 2012-08-01 2023-12-12 United Therapeutics Corporation Treatment of pulmonary arterial hypertension with prostacyclin-treated endothelial progenitor cells
EP3492084A1 (fr) 2012-08-01 2019-06-05 United Therapeutics Corporation Traitement de l'hypertension artérielle pulmonaire avec des cellules progénitrices endothéliales traitées à la prostacycline
US10842823B2 (en) 2012-08-01 2020-11-24 United Therapeutics Corporation Treatment of pulmonary arterial hypertension with prostacyclin-treated endothelial progenitor cells
US10080730B2 (en) 2013-01-09 2018-09-25 United Therapeutics Corporation Treatment of vasculopathy with prostacyclin and mesenchymal stem cells
US11141393B2 (en) 2013-01-09 2021-10-12 United Therapeutics Corporation Treatment of vasculopathy with prostacyclin and mesenchymal stem cells
EP3878452A1 (fr) 2013-01-09 2021-09-15 United Therapeutics Corporation Traitement de la vasculopathie au moyen de la prostacycline et de cellules souches mésenchymateuses
EP3415150A1 (fr) 2013-01-09 2018-12-19 United Therapeutics Corporation Traitement de la vasculopathie au moyen de prostacycline et de cellules souches mésenchymateuses
US11839596B2 (en) 2013-01-09 2023-12-12 United Therapeutics Corporation Treatment of vasculopathy with prostacyclin and mesenchymal stem cells
US12274684B2 (en) 2013-01-09 2025-04-15 United Therapeutics Corporation Treatment of vasculopathy with prostacyclin and mesenchymal stem cells
WO2016154447A1 (fr) 2015-03-26 2016-09-29 Miromatrix Medical Inc. Matrice extracellulaire décellularisée remplie de gaz
US11571444B2 (en) 2016-10-24 2023-02-07 United Therapeutics Corporation Enhancement of MSC immunomodulatory properties by treprostinil
US12426594B2 (en) 2020-09-24 2025-09-30 Everest Medical Innovation GmbH Cryoprotective compositions and methods for protection of a surgical site during cryosurgery
US12453805B2 (en) 2020-09-24 2025-10-28 Everest Medical Innovation GmbH Cryoprotective compositions, surgical kits, and methods for protection of a surgical site during cryosurgery
IT202000026467A1 (it) * 2020-11-05 2022-05-05 Lipobank S R L Metodo per la crioconservazione di campioni biologici
EP3994984A1 (fr) * 2020-11-05 2022-05-11 Lipobank S.r.l. Procedé de cryoconservation de tissu adipeux

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