CN1177905A - Cryosreservation solution - Google Patents
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- CN1177905A CN1177905A CN 96192425 CN96192425A CN1177905A CN 1177905 A CN1177905 A CN 1177905A CN 96192425 CN96192425 CN 96192425 CN 96192425 A CN96192425 A CN 96192425A CN 1177905 A CN1177905 A CN 1177905A
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Abstract
The invention is a simple, inexpensive, physiologically compatible, cry preservation solution which includes the innocuous components of (i) glycerol, (ii) an alkali metal chloride salt, (iii) a monosaccharide, and (iv) serum albumin.
Description
Technical field
The present invention relates to Cryosreservation solution.More particularly, the present invention is specially adapted to the various blood circulation cells of freezing preservation and bone marrow cell, for example Cryosreservation solution of candidate stem cell and CFU-GM.
The invention still further relates to freezing preservation cell and recovery method through the cell of freezing preservation.
Technical background
Blood
Have the blood circulation cell that to discern form and difference in functionality and comprise red blood cell, neutrophilia, acidophilia and basophilic granulocyte, bone-marrow-derived lymphocyte, T lymphocyte, non-bone-marrow-derived lymphocyte, non-T lymphocyte and blood platelet.These mature cells derive from the precursor that each pedigree has the division that can discern form, and when needs, can replace by them, these precursors for example have the erythroblast of erythron, granulocytic myeloblast, promyelocyte and myelocyte, and thrombocytic megacaryocyte.
Candidate stem cell and CFU-GM
Precursor is from the more original cell that can simply be divided into two subgroups: stem cell and CFU-GM.Definition to stem cell and CFU-GM is a manipulation type, depends on profile rather than its form standard.Some stem cell breaks up when needed, but some stem cell or its daughter cell generate other stem cell to keep the set of this valuable class cell.Like this, multipotential stem cell except the kind system that can keep oneself, can also be divided into several subbreed, have comparatively limited self ability or do not have the CFU-GM of self ability.Has the precursor that to discern form by final generation of these CFU-GM.CFU-GM can be bred and be broken up along one or more myelocyte sample differentiation pathway.
Account for very little percentage in stem cell and the CFU-GM karyocyte in marrow, spleen and blood.Be present in these cells in the spleen than lacking 10 times approximately in the marrow, in adult's blood even still less.For example, have that one thousandth is arranged in the bony nodule myelocyte approximately is CFU-GM; Stem cell still less.
Can build the blood system again by bone-marrow transplantation.Lorenz and colleague proof thereof can be protected the radiation (Lorenz, 20E is etc., 1951, National Cancer Institute magazine (J.Natl.Cancer Inst.) 12:197-201) of mouse opposing lethal dose by venoclysis marrow.Later studies show that, protective effect from cell the settling down in acceptor marrow of infusion (Lindsley, D.L., etc., 1955, experimental biology and account (Proc.Sco.Exp.Biol.Med.) 90:512-515 of medical association; Nowell, P.C., etc., 1956, (cancer research (CancerRes.) 16:258-261; Mitchison, N.A., 1956, Britain's experimental pathology magazine (Br.J.Exp.Pathol.) 37:239-247; Thomas, E.D. etc., 1957, New England Journal of Medicine (N.Engl.J.Med.) 257:491-496).Like this, the haemocyte of being responsible for protective immunity, tissue repair, blood coagulation and other blood function can be bred and replace to stem cell in the donor bone marrow and CFU-GM.In the bone-marrow transplantation of success, all rallied from the cell of donor in blood, marrow, spleen, thymus gland and other immune organ.
Bone-marrow transplantation more and more successfully is used to treat many diseases that cause death or disable, anaemia comprising some type, alpastic anemia (Thomas for example, E.D. etc., on February 5th, 1972, lancet (TheLancet) pp.284-289), anemia Fanconi's (Gluckman, E. etc., 1980, Britain's hematology magazine (Brit J.Haematol.) 45:557-564; Gluckman, E. etc., 1983, Britain's blood magazine (Brit J.Haematol.) 54:431-440; Gluckman, E. etc., 1984, hematology comment (Seminars inHematology): 21 (1): 20-26), immune deficiency (Good, R.A. etc., 1985, cellular immunology (CellularImmunol.) 82:36-54), cancer, for example lymphoma and leukemia (Cahn, J.Y. etc., 1986, Britain's hematology magazine (Brit J.Haematol.) 63:457-470; Blume, K.J. and Forman S.J., 1982, stechiology supplementary issue (J.Cell.Physiol.Supp.) 1:99-102; Cheever, M.A. etc., 1982, New England Journal of Medicine (N.Engl.J.Med.) 307 (8): 479-481), cancer (Blijham, G etc., 1981 European cancer magazines (Eur.J.Cancer) 17 (4): 433-441), various solid tumors (Ekert, H. etc., 1982, cancer (Cancer) 49:603-609; Spitzer, G. etc., 1980, cancer (Cancer) 45:3075-3085), and various hematopoiesis genetic disease.
Recently, bone-marrow transplantation also is used to treat inheritance thesaurismosis (Hobbs, J.R., 1981, lancet (Lancet) 2:735-739), major thalaseemia (Thomas, E.D. etc., 1982, lancet (Lancet) 2:227-229), sickle cell disease (Johnson, F.J. etc., 1984, New England Journal of Medicine (N.Engl.J.Med.) 311:780-783), osteopetrosis (Coccia, P.F., Deng, 1980, New England Journal of Medicine (N.Engl.J.Med.) 302:701-708) (comprehensively argumentation can be referring to Storb, R. and Thomas, E.D., 1983, immunology comment (Immunol.Rev.) 71:77-102; O ' Reilly, R. etc., 1984, hematology comment (Sem.Hematol.) 21 (3): 188-221; 1969, the preservation of marrow, cultivation and transplanting, sub-committee's procceedings, Moscow (Bone-Marrow Conservation, Culture and Transplantation, Proceedings of aPanel, Moscow), July nineteen sixty-eight 22-26, International Atomic Energy Agency, Vienna; McGlave, P.B. etc., 1985, hematology latest developments (Recent Advances in Haematology), Hoffbrand, A.V. compiles, Churchill Livingstone, London, pp.171-197).
The use of bone-marrow transplantation now is subjected to strict restriction, because be difficult to find the donor of coupling (congruence in the heredity) fully, the bone marrow cell that only obtains identical twins or paracmasis patient is preserved with the freezing state of living.Even pollute the danger that causes at such malignant cell of in system, failing to discover, and patient must be enough strong so that carry out bone-marrow transplantation, still causes serious restriction.Except that such autoplastic transplantation, hereditary mismatch is to a certain degree arranged inevitably, this can cause serious is fatal complication sometimes.These complication are dual.At first, form immunosupress for preventing that patient from taking medicine usually in advance to immunological rejection (HVGR) patient of external bone marrow cell.Secondly, transplant successfully when (if with) donor bone marrow cell, they will attack (graft versus host disease(GVH disease)) as foreign matter with patient.The complication that causes can not cause heredity to go up bone-marrow transplantation causes between different individualities many death or disease yet even relatives' donor of matched, these parts match.
Also studied in addition source (Nothdurtt, W. etc., 1977, Scandinavia hematology magazine (Scand.J.Haematol.) 19:470-471 of peripheral blood as the stem cell that is used for building again the blood system; Sarpel, S.C. etc., 1979, experimental hematology (Exp.Hematol.) 7:113-120; Ragharachar, A. etc., 1983, cellular biochemistry supplementary issue (J.Cell.Biochem.Suppl.) 7A:78; Juttner, C.A. etc., 1985, Britain's hematology magazine (Brit.J.Haematol.) 61:739-745; Abrams, R.A. etc., 1983, cellular biochemistry supplementary issue (J.Cell.Biochem.Suppl.) 7A:53; Prummer.O. etc., 1985, experimental hematology (Exp.Hematol.) 13:891-898; ).In some research, for various leukaemics (Reiffers, J. etc., 1986, experimental hematology (Exp.Hematol.) 14:312-3l5 (using the cell of freezing preservation); Goldman, J.M. etc., 1980, Britain's hematology magazine (Br.J.Haematol.) 45:223-231; Tilly, H. etc., on July 19th, 1986, lancet (Lancet) pp.154-155; Simultaneously referring to To, L.B. and Juttner, C.A., 1987, Britain's hematology magazine (Brit.J.Haematol.) 66:285-288, and the list of references of quoting in this article) and lymphoma patient (Korbling, M. etc., 1986, blood (Blood) 67:529-532) obtained optimistic result.The ability that prompting is seen in some cancer patient, adult's autologous peripheral blood is built the blood system again reduces much more circulation CFU-GM relevant (the rebound phenomenon) (To of quantity that produces in the peripheral blood of back with the cell that strong chemotherapy and/or radiotherapy cause, L.B. and Juttner, C.A., 1987, the Britain lexical or textual analysis of hematology magazine (Annot., Brit.J.Haematol.) 66:285-288; Simultaneously referring to 1987, the document of quoting in Britain hematology magazine (Brit.J.Haematol.) 67:252-253 and this article).Other research of use peripheral blood is failed to produce and is built effect (Hershko, C. etc., 1979, lancet (The Lancet) 1:945-947 again; Ochs, H.D. etc., 1981, paediatrics research (Pediatr.Res.) 15 (4 part 2): 601).
Also study the use fetal liver cells and transplanted (Cain, G.R. etc., 1986, transplanting (Transplantation) 41 (1): 32-25; Ochs, H.D. etc., 1981, paediatrics research (Pediatr.Res.) 15 (4 part 2): 601; Paige, C.J. etc., 1981, The Journal of Experimental Medicine (J.Exp.Med.) 153:154-165; Touraine, J.L., 1980, Excerpta Medica (Excerpta Med.) 514:277; Touraine, J.L., 1983, connatae defective (Birth Defects) 19:139; Simultaneously referring to Good, R.A. etc., 1983, list of references in cellular immunology (Cellular Immunol.) 82:44-45 and this article) or neonate's spleen cell transplantation (Yunis, E.J. etc., 1974, record (Proc.Natl.Acad.Sci.U.S.A.) 72:4100 of American Academy of Sciences) as the source of building the stem cell of blood system again.In the immunologic reconstitution test, also used neonate's thymocyte to transplant (Vickery, A.C. etc., 1983, parasitology magazine (J.Parasitol.) 69 (3): 478-485; Hirokawa, K. etc., 1982, clinical immunology and immunopathology (Clin.Immunol.Immunopathol.) 22:297-304).
Cryosreservation solution and technology
For a long time, freezing be used to preserve take from or separate living cells from the donor body, for example haemocyte.But the freezing preservation of living cells and recovery seem and very bother.In the freeze thawing circulation of the freezing preservation of pair cell, cell has stood suitable exacting terms, causes lower survival rate.
Freezing meeting causes damage to most living cells.When medium on every side freezed, cell was because of attempting to keep the balance dehydration, and the solute concentration in the born of the same parents that raise thus is until taking place to freeze in the born of the same parents when bearing 10-15 ℃ approximately.It has been generally acknowledged that, freeze in the born of the same parents all to cause cellular damage with the solution effect.For example, the someone proposes, and the freezing injury that the outer ice crystal of born of the same parents causes comes down to the injury of plasmalemmae that cell causes because of the infiltration dehydration.
Bet a large amount of time and efforts in order to improve the survival ability that melts cell as far as possible.These effort mainly lay particular emphasis on the freezing preservation reagent of exploitation and determine best cooling velocity.
Be considered to be undertaken by the freezing preservation of adding solute: (i) in the cell by two kinds of potential mechanisms; By reducing the ice crystal amount that forms in the cell; And/or (ii) extracellular; Cause vapour pressure to reduce the cell dehydration that causes then by the formation that reduces because of ice crystal in the cell peripheral solute.
Different cell theories is understood its different best cooling velocity.The cooling velocity preservative agent has all been studied to the survival of freezing bone marrow cell and red blood cell or the influence of transplanting efficient (Lovelock, J.E. and Bishop, M.W.H., 1959, nature (Nature) 183:1394-1395 by many seminar; Ashwood-Smith, M.J., 1961, nature (Nature) 190:1204-1205; Rowe, A.W. and Rinfret, A.P., 1962, blood (Blood) 20:636; Rowe, A.W. and Fellig, J., 1962, federal account (Fed.Proc.) 21:157; Rowe, A.W., 1966, cryobiology (Cryolbiology) 3 (1): 12-18; Lewis, J.P. etc., 1967, blood transfusion (Transfusion) 7 (1): 17-32; Rapatz, G. etc., 1968, cryobiology (Crylobiology) 5 (1): 18-25; MaZur, P., 1970, science (Science) 168:939-949; Mazur, P., 1977, cryobiology (Cryolbiology) 14:251-272; Rowe, A.W. and Lenny, L.L., 1983, cryobiology (Cryolbiology) 20:717; Stiff, P.J. etc., 1983, cryobiology (Cryolbiology) 20:17-24; Gorin, N.C., 1986, clinical (the Clinics in Haematology) 15 (1) of hematology: 19-48).Usually, use the cooling velocity of about 1-2 ℃ of per minute ,-70 ℃ to-196 ℃ final storage temperature can obtain optimum to candidate stem cell and CFU-GM approximately, and other haemocyte is usually also in this scope.
The existing record of human bone marrow cell that successful recovery was preserved in the liquid nitrogen midium or long term (1983, U.S. microbial preservation center (American Type Culture Collection), season and communication (Quarterly Newsletter) 3 (4): 1).In addition, the stem cell in the marrow be proved to be able to tolerate freezing and thaw (Fabian, I. etc., 1982, pu tests hematology (Exp.Hematol.) 10 (1): 119-122).
Being used for the universally recognized industrial cryoprotector that freezing preservation comprises the most cells of whole blood, Cord blood, marrow, granulocyte, neutrophil leucocyte, blood platelet and candidate stem cell and CFU-GM is dimethyl sulfoxide (DMSO) (DMSO).This is normally because experience and knowledge and a kind of general notion of the relevant Cryosreservation solution of generally knowing altogether based on DMSO, and promptly DMSO can provide the good protective effect and the cell survival rate of maximum.Though DMSO can achieve the above object usually effectively, also known it have physiology toxicity, especially when high concentration, and hypertension, vomiting take place and feel sick in the patient who tends to stimulate the people who relates to freezing preservation operation thus and be introduced into the cell of freezing.The toxicity of DMSO also is considered to cause the cell of freezing lower in external survival rate.
So, be badly in need of another kind and can both provide the quite Cryosreservation solution of the simple tool physiological compatibility of the cell survival rate of level with external in vivo.
Summary of the invention
This paper has developed a kind of simple, inexpensive, Cryosreservation solution that physiology is compatible, wherein comprises nontoxic composition (i) glycerine, (ii) alkali metal chloride, (iii) monose and (iv) seralbumin.
The Main Ingredients and Appearance of Cryosreservation solution is freezing preservative agent glycerine.Alkali metal chloride improves the survival rate of cell in the freeze thawing circulation.The effect of monose is the trophism carbon source as cell.Sero-abluminous effect is as protein source, in order to the cell membrane of parcel target cell, protects cell membrane in the preservation process of freeze thawing circulation and frozen cell.Seralbumin also is considered to have the function of removing oxygen radical, the known oxygen free radical is unfavorable for the survival of multiple target cell, for example above-mentioned red blood cell, neutrophilia, acidophilia and basophilic granulocyte, bone-marrow-derived lymphocyte, T lymphocyte, non-bone-marrow-derived lymphocyte, non-T lymphocyte and platelet cell.Sero-abluminous source is best to be complementary with the kind that will introduce the curee of protected target cell.
Cryosreservation solution can be the form that concentrates or fully dilute.Can be by in solution, adding thinner easily, for example suitably treated water is diluted to working concentration with concentrated type.Thinner is the physiological equilibrium salting liquid preferably, for example physiological saline.
To target cell; for example whole blood, Cord blood, marrow, granulocyte, neutrophil cell, blood platelet and comprise the candidate stem cell of CD34+ cell and the preservation of CFU-GM and later treatment thereof adopt Cryosreservation solution may further comprise the steps in using: (i) Cryosreservation solution that concentrates of dilution as required; (ii) target cell is added in the weak solution; (iii) freezing protected target cell; frozen soln is thawed to environmental condition, and (target cell after v) will thawing is introduced the curee who needs these target cells.The target cell that preferably will thaw before the introducing curee and the frozen soln that accompanies are warmed to body temperature (promptly about 37 ℃).The target cell that the physiological compatibility of cryoprotection solution allows to thaw is introduced into the curee and must separate with Cryosreservation solution by target cell.
Comprise the detailed description of preferred forms
This paper has developed a kind of simple, inexpensive, Cryosreservation solution that physiology is compatible, wherein mainly comprises nontoxic composition (i) glycerine, (ii) alkali metal chloride, (iii) monose and (iv) seralbumin.
This paper comprises when the component of " weight % " and Cryosreservation solution in claims is relevant, is based on the gross weight of the Cryosreservation solution that concentrates, unless otherwise mentioned.
Glycerine (C
3H
8O
3) can be available from how tame supplier, it is the commercially available polyalcohol of U.S.P. level and the food-grade of J.T.Baker.
Comprise about 60 weight % in the Cryosreservation solution that concentrates to about 80 weight % glycerine.The glycerol concentration that is lower than about 60 weight % can reduce cell survival rate, be higher than about 80 weight % can make solution have too high permeability (promptly be higher than about 2,000mOsm/kg).
Be applicable to that alkali metal chloride of the present invention comprises sodium chloride and potassium chloride.Alkali metal chloride is considered to be adjusted in about 500 to 2 by the permeability with Cryosreservation solution, (this value utilizes Wescor5500 type vapor pressure osmometer to record according to the method in instrument explanation and the service manual) improved cell survival rate between the 000mOsm/kg in the freeze thawing circulation.As a reference, the physiology permeability of blood is generally between about 260 to 320mOsm/kg.Best permeability in this general range depends on Several Factors, comprises concrete target cell, the concrete composition of Cryosreservation solution and freeze thawing speed.For example, about 700 to about 1,700 permeability is normally comparatively desirable concerning the freezing preservation of candidate stem cell and CFU-GM.
Concentrated frozen is preserved solution and is comprised and 5 to 10 weight % alkali metal chlorides.Alkali metal chloride concentration is lower than about 5 weight % and is higher than about 10 weight % and can reduce cell survival rate in the freeze thawing circulation, and concentration is higher than 10 weight % and also is subject to crystalline fracture.
Monose, for example glucose is used as the trophism carbon source of cell.Monose is by guaranteeing have sufficient nutrient to have to come the preservation that promotes cell in the preservation process of freeze thawing circulation and frozen cell in the cell.
Sero-abluminous effect is as the source of parcel target cell membrane with the protein of protection cell membrane in freeze thawing circulation and frozen cell preservation process.Seralbumin also is considered to have the function of removing oxygen radical, the known oxygen free radical is unfavorable for the survival of multiple target cell, for example above-mentioned red blood cell, neutrophilia, acidophilia and basophilic granulocyte, bone-marrow-derived lymphocyte, T lymphocyte, non-bone-marrow-derived lymphocyte, non-T lymphocyte and platelet cell.Sero-abluminous source is best to be complementary with the kind that will introduce the curee of protected target cell.
Adding about 0.2 to about 1 weight % monose can reach valid density in concentrated frozen preservation solution.Concentration is lower than about 0.2 weight % and causes concentration to reduce the survival rate of cell in the freeze thawing circulation inadequately, and concentration is higher than about 1 weight % and does not improve cell survival rate with being directly proportional, and the concentration of other component is had harmful effect.
Sero-abluminous effect is the source as protein, in order to form integument around target cell membrane, protects cell membrane then in freeze thawing circulation and frozen cell preservation process.
Add about 20 and can reach valid density to about 30 weight % seralbumins.Seralbumin concentration is lower than about 20 weight % to make that concentration is not enough and reduces cell survival rate, and concentration is higher than raising cell survival rate of about 30 weight % with being directly proportional, and it is about 2 to tend to make the permeability of solution to exceed, the above-mentioned desirable upper limit of 000mOsm/kg.
Can preserve about 1 to 10 part of solution by every part of concentrated frozen, be generally 1 to 2 part ratio and add suitable diluent (for example salt solution, water for injection and the autologous plasma of the 0.9 weight % for preparing with ultrafiltration water) concentrated frozen is preserved solution dilution to working concentration.Usually, Cryosreservation solution after the dilution mainly comprises (i) about 20 to about 40 weight % glycerine by the total restatement of Cryosreservation solution after diluting, (ii) about 1.5 to about 5 weight % alkali metal chlorides, (iii) about 0.1 to about 0.5 weight % monose and (iv) about 6 to about 15 weight % seralbumins.Its surplus is a thinner.
Cryosreservation solution must be mixed with pH between about 7 to 7.5.
Use the Cryosreservation solution after the dilution can freezing easily preservations target cell, only need (i) that target cell is added in the Cryosreservation solution after diluting and (ii) freezing or low temperature is preserved protected target cell.The volume that adds rare Cryosreservation solution of target cell depends on the ratio of the volume ratio that contains target cell solution and Cryosreservation solution and target cell and Cryosreservation solution simultaneously.The volume ratio that contains the solution of target cell and Cryosreservation solution usually should be between about 1: 1 to 1: 10, and the ratio that makes target cell and Cryosreservation solution simultaneously is about 1 * 10
5Individual cell/ml is to about 1: 1 * 10
9Individual cell/ml is more preferably usually about 2 * 10
8Individual cell/ml is to about 3 * 10
8Individual cell/ml.Being higher than aforementioned proportion can reduce viable cell concentrations because of Cryosreservation solution is not enough, is lower than the availability that aforementioned proportion then obviously can reduce Cryosreservation solution, and can introduces excessive Cryosreservation solution in order to introduce the target cell of effective dose to the curee.Usually the target cell that can be used for preserving can only be that (it is about 1 * 10 that the stem cell of for example, concentrating from peripheral blood contains concentration usually for dilution form as a kind of component in certain biofluid
6Individual cell/ml is to about 3 * 10
8The stem cell of individual cell/ml, and all the other components that comprise other blood components such as blood plasma and blood platelet).The sample of these biological dilutions must mix with Cryosreservation solution with about 2: 1 to 1: 4 volume ratio (certainly usually, this depends on multiple factor, concrete composition comprising rare Cryosreservation solution, target cell concentration in the biological dilute sample, target cell type etc.) to obtain the best target cell and the ratio of Cryosreservation solution.The solution that contains target cell at last contains have an appointment 4 to 12 weight % glycerine and about 2 to 10 weight % albumin usually, considers some applicable cases, and concentration can be outside above-mentioned general range.
Before being introduced the curee, target cell the target cell of freezing preservation protection must be thawed.Give the credit to the physiological compatibility of Cryosreservation solution, the target cell after thawing can directly be introduced the curee and need not target cell is separated with Cryosreservation solution.Target cell after thawing can at room temperature be kept a few hours and not serious loss cytoactive.For with the permeability of solution from desirable freezing Save Range 500 to 2,000mOsm/kg reduces to physiological range 260 to 320mOsm/kg, can utilize the solution after the slow dilution of salt solution or other suitable solion is thawed, to reduce the impact that permeability flip-flop cell membrane causes.
There is the cooling velocity slowly of control can obtain best cytoactive usually effectively.Generally believe, dissimilar cells have different best cooling velocities (referring to, for example, Rowe, A.W. and Rinfret, A.P., 1962, blood (Blood) 20:636; Rowe, A.W., 1966, cryobiology (Cryo Biology) 3 (1): 12-18; Lewis, J.P. etc., 1967, blood transfusion (Transfusion) 7 (1): 17-32; With Mazur P., 1970, science (Science) 168:939-949, relevant cooling velocity to marrow in the survival of cell and to the influence of its transfer ability).
Before the use, Cryosreservation solution can be kept at room temperature, and wherein the target cell of Hun Heing can be before freezing beginning be thirty minutes long in room temperature preservation and the significantly sacrificing of cytoactive do not occur.This is opposite with poisonous cryoprotector DMSO, the latter must before adding target cell, be cooled to about 4 ℃ accelerating refrigerating process, and require after DMSO is added cell, to get started refrigerating process in order to reduce the cell death that DMSO causes as far as possible.
Can the freezing of control be arranged by using programmable refrigerating plant.Programmable freezing equipment can be determined best cooling velocity and carry out standardized reproducible cooling easily.
The container that holds cell must keep stable under deep cooling low temperature, and can carry out the Rapid Thermal transmission so that effectively regulate and control freezing and course of defrosting.The plastic jar (for example Nunc and Wheaton cryules) or the glass ampule of sealing can be used for multiple a small amount of (1 to 2ml), and 100 to 200ml larger volume can be at the polyolefin bag that is fixed between the metallic plate, and be for example freezing in the bag available from Fenwal.
Frozen cell can be transferred to long-term low temperature then and preserve in the container,, can be kept in liquid nitrogen (196 ℃) or the liquid nitrogen steam (105 ℃) sample is freezing one preferably in the embodiment.Efficient liquid nitrogen storage case greatly facilitates such preservation.
When needed, preferably the quick-thawing frozen cell also is maintained at about 37 ℃ before use.Because Cryosreservation solution is a physiological compatibility, so need before introducing the curee, not isolate cell.
Can be with freezing preservation and umbilical cord cell, blood platelet and candidate stem cell after thawing and CFU-GM therapeutic be applied in reconstitute hematopoiesis system in the suitable patient body.Can utilize any known method introducing cell in this area, usually preferred whole body infusion.
The medical treatment that is reconstituted in of hemopoietic system (or immune system) is very valuable to numerous disease and imbalance upward.Be used for building again the candidate stem cell of freezing preservation of blood system and the infusion of CFU-GM and can perform well in treating known disease of curing now with autologous bone marrow transplantation.
The imbalance that available stem cell infusion is treated includes but not limited to five big classes.The first kind is the disease (being alpastic anemia and low hyperplasia stem cell disease) that normal plasma cell generates and ripe defunctionalization or obstacle cause.Second class comprises the malignancy disease (for example leukemia and lymphoma) in the blood forming organ.The 3rd class comprises the patient in the no hematopoiesis source of suffering from multiple malignant solid tumor.Stem cell infusion in these patients plays the marrow rescue, and infusion has been accepted to carry out after possible fatal malignant tumor chemotherapy or the radiotherapy these patients.The 4th class disease comprises autoimmune state, and stem cell plays the abnormal immune system therein and replaces the source.The 5th class disease comprises the many genetic diseases that can correct by the candidate stem cell infusion of homology preferably, and stem cell has been accepted gene therapy before transplanting.
Claims (32)
1. a concentrated frozen is preserved solution, mainly comprises:
(a) glycerine;
(b) concerning improving cell survival rate, be the alkali metal chloride of effective dose;
(c) but concerning the keeping of cell under environmental condition, be the monose of the biological metabolism of effective dose;
(d) concerning the protection membrane integrity, be the seralbumin of effective dose.
2. Cryosreservation solution according to claim 1 wherein also comprises thinner.
3. Cryosreservation solution according to claim 1, the pH of solution is between about 7 to 7.5.
4. Cryosreservation solution according to claim 1, concentrate wherein mainly comprises (a) about 60 to about 80 weight % glycerine, (b) about 5 to about 10 weight % alkali metal chlorides, and (c) about 0.2 to about 1 weight % monose with (d) about 20 to about 30 weight % seralbumins.
5. Cryosreservation solution according to claim 2, solution wherein mainly comprises (a) about 20 to about 40 weight % glycerine, (b) about 1.5 to about 5 weight % alkali metal chlorides, (c) about 0.1 to about 0.5 weight % monose, and (d) about 6 to the about 15 weight % seralbumins and (e) thinner of surplus.
6. concentrated frozen according to claim 1 is preserved solution, and alkali metal chloride wherein is a sodium chloride.
7. Cryosreservation solution according to claim 2, alkali metal chloride wherein is a sodium chloride.
8. concentrated frozen according to claim 1 is preserved solution, and monose wherein is glucose.
9. Cryosreservation solution according to claim 2, monose wherein is glucose.
10. concentrated frozen according to claim 1 is preserved solution, and seralbumin wherein is a human serum albumins.
11. Cryosreservation solution according to claim 2, seralbumin wherein is a human serum albumins.
12. a concentrated frozen is preserved solution, mainly comprises:
(a) about 60 to 80 weight % glycerine;
(b) about 5 to 10 weight % alkali metal chlorides;
(c) about 0.2 to 1 weight % glucose;
(d) about 20 to 30 weight % seralbumins.
13. a Cryosreservation solution mainly comprises:
(a) about 20 to 40 weight % glycerine;
(b) about 1.5 to 5 weight % alkali metal chlorides;
(c) about 0.1 to 0.5 weight % glucose;
(d) about 6 to 15 weight % seralbumins;
(e) thinner of surplus.
14. the method for a freezing preservation cell comprises that (i) adds the protected target cell of the described Cryosreservation solution (ii) freezing preservation of neutralization of claim 2 with target cell.
15. method according to claim 14, the volume ratio that wherein contains the solution of target cell and Cryosreservation solution are between about 1: 1 to 1: 10, the ratio of target cell and Cryosreservation solution is about 1 * 10
5Individual cell/ml is to about 1 * 10
9Between individual cell/ml.
16. method according to claim 14, target cell wherein are candidate stem cell or CFU-GM.
17. method according to claim 14, target cell wherein is a platelet cell.
18. method according to claim 14, target cell wherein are the CD34+ cells.
19. method according to claim 14, target cell wherein is from Cord blood.
20. the method for a freezing preservation cell comprises that (i) adds the protected target cell of the described Cryosreservation solution (ii) freezing preservation of neutralization of claim 13 with target cell.
21. method according to claim 20, the volume ratio that wherein contains the solution of target cell and Cryosreservation solution are between about 1: 1 to 1: 10, the ratio of target cell and Cryosreservation solution is about 1 * 10
5Individual cell/ml is to about 1 * 10
9Between individual cell/ml.
22. method according to claim 20 wherein is cooled to-70 to-196 ℃ approximately with 1-2 ℃/minute speed with protected target cell.
23. a freezing preservation and recover the method for living cells comprises that (i) adds target cell in the described Cryosreservation solution of claim 2, the protected target cell of (ii) freezing preservation and (iii) make protected cell thawing.
24. method according to claim 23, the volume ratio that wherein contains the solution of target cell and Cryosreservation solution are between about 1: 1 to 1: 10, the ratio of target cell and Cryosreservation solution is about 1 * 10
5Individual cell/ml is to about 1 * 10
9Between individual cell/ml.
25. method according to claim 23, target cell wherein are candidate stem cell or CFU-GM.
26. the method for a freezing preservation, recovery and therapeutic application cell; comprise that (i) adds target cell in the described Cryosreservation solution of claim 2; the protected target cell of (ii) freezing preservation; protected target cell introducing after (iii) making protected target cell thaw and (iv) will thaw needs to need not target cell is separated with Cryosreservation solution in curee's body of these target cells.
27. method according to claim 26, the volume ratio that wherein contains the solution of target cell and Cryosreservation solution are between about 1: 1 to 1: 10, the ratio of target cell and Cryosreservation solution is about 1 * 10
5Individual cell/ml is to about 1 * 10
9Between individual cell/ml.
28. method according to claim 26, target cell wherein are candidate stem cell or CFU-GM.
29. method according to claim 26, target cell wherein is a platelet cell.
30. method according to claim 26, target cell wherein are the CD34+ cells.
31. method according to claim 26, target cell wherein is from Cord blood.
32. method according to claim 26, curee wherein is the people.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 96192425 CN1177905A (en) | 1995-03-08 | 1996-03-07 | Cryosreservation solution |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/399,077 | 1995-03-08 | ||
| CN 96192425 CN1177905A (en) | 1995-03-08 | 1996-03-07 | Cryosreservation solution |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1177905A true CN1177905A (en) | 1998-04-01 |
Family
ID=5128265
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 96192425 Pending CN1177905A (en) | 1995-03-08 | 1996-03-07 | Cryosreservation solution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1177905A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101939417B (en) * | 2007-12-04 | 2014-02-05 | 蛋白生物动力私人有限公司 | Protection of progenitor cells and regulation of their differentiation |
| CN105076116A (en) * | 2015-09-17 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryopreservation liquid, application thereof and cryopreservation method of megakaryocyte progenitor cells |
| CN105325402A (en) * | 2015-11-13 | 2016-02-17 | 黄林海 | Cryopreservation protective agent for vitrification cryopreservation of marrow mesenchymal stem cells |
| CN107372469A (en) * | 2017-09-06 | 2017-11-24 | 广州赛莱拉干细胞科技股份有限公司 | The frozen stock solution and cryopreservation methods of a kind of endothelial progenitor cells |
| CN108184817A (en) * | 2018-01-11 | 2018-06-22 | 南京三生生物技术股份有限公司 | A kind of umbilical cord blood hematopoietic stem cell frozen stock solution and cryopreservation methods |
-
1996
- 1996-03-07 CN CN 96192425 patent/CN1177905A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101939417B (en) * | 2007-12-04 | 2014-02-05 | 蛋白生物动力私人有限公司 | Protection of progenitor cells and regulation of their differentiation |
| CN105076116A (en) * | 2015-09-17 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryopreservation liquid, application thereof and cryopreservation method of megakaryocyte progenitor cells |
| CN105325402A (en) * | 2015-11-13 | 2016-02-17 | 黄林海 | Cryopreservation protective agent for vitrification cryopreservation of marrow mesenchymal stem cells |
| CN107372469A (en) * | 2017-09-06 | 2017-11-24 | 广州赛莱拉干细胞科技股份有限公司 | The frozen stock solution and cryopreservation methods of a kind of endothelial progenitor cells |
| CN108184817A (en) * | 2018-01-11 | 2018-06-22 | 南京三生生物技术股份有限公司 | A kind of umbilical cord blood hematopoietic stem cell frozen stock solution and cryopreservation methods |
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