MXPA97006843A - Criopreservac solution - Google Patents

Abstract

The present invention relates to a simple, inexpensive and physiologically compatible cryopreservation solution, which includes the harmless components of glycerol, an alkali metal chloride salt, a monosaccharide and an albumin

Description

CRIOP SOLUTION ESERVñCIQN CFH1PO OF THE INVENTION This invention relates to cpopreservation solutions. Specifically, this invention is directed to cpopreservation solutions which are particularly suitable for use to cryopreserve various types of cells circulating in the blood or found in the bone marrow, such as stem cells and progenitor cells. . This invention also relates to m all for cprespressing and recovering cpreserved cells.

BACKGROUND OF THE INVENTION Blood Morphologically recognizable and functionally capable cells circulating in the blood include erythrocytes, neutrophilic granulocytes, eosinophils, and basophils, B lymphocytes, T lymphocytes, non-B lymphocytes, non-T lymphocytes, and platelets. These mature cells derive from and are replaced, with the demand, by morphologically recognizable dividing precursor cells for the respective lines, such as co-eri + roblaetos for the series of erythrocytes, ieloblasts, promiolocytes and ielocytes for the series of granulocytes and rnegacapocytes. for the plaque * as ..

Stem cells and hematopoietic progenitors The precursor cells are derived from more primitive cells that can be divided into two main groups: stem cells and progem cells + oras. The definitions of stem cells and progenitors are operational and depend on functional rather than morphological criteria. Stem cells have extensive ability to au + or renovate "Some of the stem cells differentiate according to need, but some stem cells or their fixed cells produce other stem cells to maintain * the valuable supply of these cells. In this way, in addition to maintaining its own class, pluripotent stem cells are capable of differentiation into several sub-lines of stem cells with a more limited capacity for self-renewal or no capacity for self-renewal. These progetarian cells finally give rise to the morphologically recognizable precursor cells. Progenitor cells are able to proliferate and differentiate along one, or more than one, of the myeloid differentiation trajectories. Stem cells and progenitors form a very small percentage of nucleated cells in the bone marrow, vessel and blood. Approximately 10 times less of these cells are present in the vessel in relation to the bone marrow, and even less present in the blood of an adult. As an example, one of the nucleated cells of the bone marrow is a progenitor cell; Stem cells occur at a lower frequency. The reconstitution of the hernatopoietic system has been made by bone marrow transplant. Lorene and coworkers showed that mice could be protected against lethal irradiation by intravenous infusion of bone marrow (Lorenz, 20 E., et al., 1951, 3, Nati. Cancer Inst. 12: 197-201). Subsequent investigation demonstrated that protection of the colonization of the recipient bone marrow by the infused cells resulted (Lindsley, DL, et al. 1995, Proc. Soc. Exp. Biol. Fed. 90: 512-515; No ell, PC and others, 1956, Cancer Res 15: 258-261, Mitchison, NA, 1956, Br. 3. Exp 'Pathol, 37: 239-247, Thomas, ED and others 1957, N. Engl., 1. 257: 491-496). In this way, stem cells and progeny in the donated bone marrow can multiply and replace the blood cells responsible for protective immunity, tissue repair, coagulation and other blood functions. In the bone marrow factorial transplant, blood, bone marrow, vessel, thymus and other immunity organs are repopulated with cells derived from the donor. Bone marrow has been used with increasing success to treat various fatal or deteriorating diseases, including certain types of anemia, such as aplastic anemia (Thomas, ED, and others Feb. 5, 1972, The Lancet, pp. 284-289), Fanconi anemia (Gluc man, E, and others 1980, Brit 3. Haematol, 45: 557-564; Gluckman, E, et al., 1983, Brit, 3. Haematol, 54: 431-440; Glickrnan, E "and others, 1984, Semina sm Hematology: 21 (1): 20-26), immune deficiencies (Good, RA, et al., 1985, Cellular Irnununol., 82: 36-54), cancers such as li fornas or leukemia (Cahn, 3. Y., and others, 19R6, Bpt 3. Haematol 63: 457-470, Blurne, K.3 and Forman S.3., 1982, 3. Cell Physiol. Supp. 1: 99-102; Cheever, Fl.ft., et al., 1982, N »Fngl 3. lied. 307 (8): 479-481), carcinomas (Blijharn, G., et al., 1981, Eur, 3. Cancer 17 (4): 433-441), several sun lumps Ldos (Ekert, H., et al., 1982, Cancer 49: 603-609; Spi + zer, G., et al., 1980, Cancer 45: 3075-3085) and genetic disorders of hematopoiesis. Bone marrow transplantation has also recently been applied to the treatment of inherited storage diseases (Hobbs, 3.R., 1981, Lancet 2: 735-739), major thalasernia (Thomas, ED, et al., 1982, Lancet 2: 227-229), sickle cell anemia (3ohnson, F.3., Et al., 1984, N. Engl 3. ried 311: 780-783), and osteopet rosis (Coccia, PF, et al., 1980, N. Engl 3. Hex 302: 701-708) (for discussions, see Srotb, R. and Thomas, ED, 1983, Immunol Rev. 71: 77-102; O'Reilly, R. and others , 1984, Sern. Hermatol.21 (3): 188-221, 1969, Bone-Marrow Conservation, Culture and Transplantation, Proceedmgs of a Panel, Moscow, 22-26 July 1968, International Onatomic Energy flgency, Vienna; McGlave , RB, et al., 1985, Recent Developments in Haematology, Hoffbrand, OV, ed., Churchill Livmstone, London, pp. 171-197) The present use of bone marrow transplantation is severely restricted, since it is extremely rare to have perfectly matching donors (genetically identical), except in cases in which a twin is available or in which the bone marrow cells of the patient in remission are stored in a viable frozen state, flun in such autologous system, the danger due to No detectable concentration with malignant cells and the need to have a patient healthy enough to undergo bone marrow procurement, present certain limitations. Except in such autologous cases, there is an inevitable genetic inequality of some degree, which entails serious and sometimes lethal complications. These complications are double. In the first place, the patient is ordinarily incapacitated in unology in advance, in order to avoid immune rejection of foreign bone marrow cells (reaction of the host against the graft). Secondly, when the donated cells of the bone marrow are established, they can attack the patient (graft disease against the host), whom they recognize as foreign, with donors of the congenital family between ceracanes, these complications of partial inequality are the cause of substantial mortality and morbidity directly due to the bone marrow transplantation of a genetically different individual. Peripheral blood has also been investigated as a source of stem cells for hematopoietic reconstitution (Nothdurtt, U. et al., 1977, Scand., 3. Haematol, 19: 470-471, Sarpel, SC et al., 1979, Exp. Haematol 7: 113-120, Ragharachar, fi, et al., 1983, 3. Cell, Biochem, Suppl 70:78, 3uttner, C.fi., et al., 1985, Bpt. 3. Habernatol. 61: 739-745; ftbrarns, RP, et al., 1983, 3. Cell. Biochem., Suppl 70:53, Prurnmer, 0., et al., 1985, Exp. Hematol., 13: 891-898). In some studies, promising results have been obtained in patients with several leukaemias (Reiffers, 3. et al., 1986, Exp. Hematol.14: 312-315 (using cpoproserved cells), Goldman, 3. ti., And others. , 1980, Br. 3. Haematol 45: 223-231, Tilly, H. et al., 3ul 19, 1986, The Lancet, pp. 154-155, see also To, LB and 3uttner, CR, 1987 , Bpt. 3. Habernatol 65: 285-288, and references cited therein); and with lirnfoma (Korblmg, M. et al., 1986, Blood 67: 529-532). It has been suggested that the ability of adult autologous peripheral blood to reconstitute the hernatopoietic system, seen in some cancer patients, is associated with the much larger amounts of blood stem cells in penpency blood produced after cytoreduction, because to chemotherapy and / or intensive radiation (the rejection phenomenon) (To, LB and 3uttner, C.fi., 1987, Onnot, Bpt. 3. Haernatol., 66: 285-288; see also 1987, Brit. 3. Haematol. 67: 252-0253, and references cited therein). Other studies using peripheral blood have failed to effect reconstitution (Hershko, C, et al., 1979, The Lancet 1: 945-947, Ochs, HD et al., 1981, Pediatr. Res. 15 (4 Part 2) : 60l). Other studies have investigated the use of lethal liver cell transplantation (Cain, GR, et al., 1986, Transplantation 41 (l): 32-25, Ochs, HD, et al., 1981, Pediatr. Res. 15 (4 Part 2): 601; Paige, C.3., And others., 1981, 3. Exp., 153: 154-165; Tourame, 3.L., 1980, Excerpta Med. 514: 277 Touraine, 3.L., 1983, Birth Defects 19: 139, see also, R.fi., and others 1983, Celllular? Mrnmol 82: 44-45 and references cited therein) or cell transplantation neutral of the vessel (Yums, E.3., et al., 1974, Proc.Nati, fic.Sc. USfl.72: 4100) as sources of stem cells for hernatopoietic reconstitution. Neonatal thymus cells have also been transplanted in immune reconstitution experiments (Vickery, RC, et al., 1983, 3. Parasitol.69 (3): 478-4B5; Hiroka a, K », and others., 1982, Clin, [rnrnunol, I unopathol, 22: 297-304).

Cryopreservation Solutions and Techniques Freezing has been used for a long time to preserve living cells, such as blood cells, after they have been removed separately from a donor organism. The cpopreservacion and the recovery of alive cells, nevertheless, have turned out to be considerably complicated. The cells are subjected to relatively severe conditions during the freezing or co-freezing cycles involved in the cryopreservation of cells, resulting in a low proportion of over-capacity. Freezing is destructive to most living cells. As the external environment freezes, the cells try to maintain equilibrium and lose water, increasing the intracellular concentration of solute, so intracellular freezing occurs between -10 and -15 ° C. It is generally believed that both the intracellular effects of freezing and of solution are responsible for cellular damage *. For example, it has been proposed that the destruction by freezing of the extracellular ice * is essentially a damage to the plasma membrane that results from the osmotic dehydration of the cell. Considerable time and effort have been invested in an effort to maximize the viability of the thawed cells. Such efforts have generally focused on the development of cpoprotective agents and the establishment of optimal degrees of cooling. It is believed that cryoprotection by the addition of solute occurs through two potential mechanisms: (i) intracellular; reduction of the amount of ice formed inside the cell; and / or di) extracellular; decreasing the flow of water leaving the cell in response to a decreased vapor pressure caused by the formation of ice in the solute surrounding the cells. Different optimal degrees of cooling have been described for different cells. Several groups have observed the effect of the cooling rate and the cpoconservatives after the survival or effectiveness of transplantation of bone marrow cells and red blood cells (Lovelock, 3.E., and Bishop, fl.UH, 1959, Nature 183: 1394-1395; Flsh Ood-Smith, M.3., 1961, Nature 190: 1204-1205; Rows, UK). and Rinfret, fi.P., 1962, Blood 20: 636; Rowe, fl.U. and Fellmg, 3., 1962, Fed. Proc. 21: 157; Rowe, fl. )., 1966, Cryobiology 3 (l): 12-18; Lewis, 3.P., et al., 1967, Transfusion 7 (1): 17-32; Rapatr, G., et al., 1968, Cpobiology 5 (1): 18-25; ftaZur, p. , 1970, Science 168: 939-949; Nazur, P., 1977, Cpobiology 14: 251-272; Rowe, fi.U. and Lenny, L.L., 1983, Cpobiology 20: 717; Stj ff, P.3., And others., 1983, Cnobiology 20: 17-24; Gorin, N.C., 1986, Clinics in Hae atology 15 (1): 19-48). Generally, optimal results can be achieved for the cooling of herna progenitor and stem cells with a cooling rate of about 1-2 ° C per change with a final storage temperature of about -70 to about ~ 196 ° C with other blood cells usually within the same scales. The successful recovery of cells from human bone marrow after prolonged storage in liquid nitrogen has been described (198, American Type Culture Collection, Quarterly Newsletter 3 (4): 1). In addition, it was shown that the stem cells in the bone marrow were able to withstand cpopreservation and thawing (Fabi n, I., et al., 1982, Exp. Hematol, 10 (1): 119-122).

The common cpoprotector widely accepted for use in the cpreservation of most cells, including whole blood, umbilical cord blood, bone marrow, large neutrophil cells, platelets and hematopoietic stem and progenitor cells, is the sulfoxide of dimethyl (DMSO). This can be attributed generally to the extensive experience and knowledge of DMSO-based preservation solutions and a general perception that DMSO provides superior protection and maximum cell viability. While DMSO is generally effective for these purposes, it is also known that it is physiologically pericious, particularly at higher concentrations and thus tends to irritate both those involved in the cryopreservation process and the patient to whom the cryopreserved cells are introduced with hypertension, vomiting and nausea. The pernicious nature of DMSO is also thought to result in a lower in vitro viability of thepreserved cells. Accordingly, there is a considerable need for a simple, physiologically compatible alternative cryopreservation solution capable of providing a comparable level of cellular viability both in vivo co or in vitro.

BRIEF DESCRIPTION OF THE INVENTION A simple, economical, physiologically compatible cpopreservation solution has been developed which includes the harmless components of (i) glycerol, (ii) an alkali metal chloride salt, (iii) a monosaccharide and (iv) serum albumin. The main constituent of the cryopreservation solution is the glycerol cryopreservation agent (also called glycerin). The alkali metal chloride promotes the viability of the freeze-thaw cycle. The rnonosacápdo works like a nutpcional source of carbon for the cells. Serum albumin functions as a protein source that provides a coating around the membrane of the target cell in order to protect the membrane during the freeze-thaw cycle and the freezing of frozen cells. It is also believed that serum albumin functions as a scavenger of oxygen free radicals that is known to adversely affect the viability of several targets, such as erythrocytes, neutrophilic, eosinophilic and basophilic granulocytes, B lymphocytes, T lymphocytes, lymphocytes that are not B, lymphocytes that are not T and platelet cells mentioned above. The source of serum albumin is prebly matched to the species of the selected individual in which the protected target cells will be introduced. The cryopreeervation solution can be provided in I? Form either concentrated or completely diluted. The concentrated form can be diluted to use strength by simply adding a diluent, such as water that has been adequately treated for injection, to the solution. The diluent is prebly a physiologically balanced salt solution, such as saline. The use of the cpreservation solution in the preservation and subsequent therapeutic use of target cells, such as whole blood, umbilical cord blood, bone marrow, granulocytes, neutrophils, platelets and stem cells and hematopoietic progenitors including those defined as cells CD 34 +, include the steps of (i) diluting the concentrated cryopreservation solution as needed, di) introducing the target cells to the diluted solution, (iii) freezing the protected target cells, (iv) thawing the frozen solution at ambient conditions and (v) introducing * the thawed target cells to an individual in need of such target cells. Prebly, the frozen target cells and the cryopreservation solution are heated, which echoes the body temperature (ie, approximately 37 ° C) before introduction to the individual. The physiological compatibility of the cnoprotection solution allows the defrosted target cells to be introduced to the individual without separation of the target cells and the cryopreservation solution.

DETAILED DESCRIPTION OF THE INVENTION INCLUDING THE ME3QR MODALITY A simple, economical, physiologically compatible cryopreservation solution has been discovered, consisting essentially of (i) glycerol, (n) an alkali metal chloride salt, (iii) a rnonosacchar and (v) serum albumin. As used herein, excluding the claims, "% by weight", when used in connection with the constituents of the preservation solution, is based on the total weight of the concentrated cryopreservation solution unless it is expressed in another way. Glycerol (C3H8O3) is a polyol that is commercially available from the United States Pharmacopoeia and food classes from a number of distributors including 3.T. Baker. The concentrated cryopreservation solution includes from about 60 to about 80% by weight of the glycerol. Concentrations of less than about 60% by weight of glycerol result in reduced cell viability while solutions containing more than approximately 80% by weight of glycerol result in a solution with unacceptably high oxygen (i.e., greater than approximately 2,000 mOsrn / kg). The alkali metal chloride salts suitable for use in this invention include sodium chloride and potassium chloride. It is believed that the alkali metal chloride salt promotes cell viability through the freeze / thaw cycle by adjusting the osmolality of the cpreservation solution between approximately 500 and 2,000 rnOsrn / kg measured with a vapor pressure oscilloscope * model Uescor 5500 in accordance with the protocol provided in the instruction / service manual for the instrument. By way of reference, the physiological osmolality of the blood is generally within the range of about 250 to 320 rnOsrn / kg. Optimum osrnolality within this general scale depends on several factors including the particular target cells, the specific constitution of the buffering solution, and the velocity of freezing-de-icing. As an example, a value of approximately 700 to approximately between 1,700 is generally desired for the preservation of the hematopoietic stem and progenitor cells. The concentrated cryopreeervation solution includes from about 5 to about 10% by weight of the alkali metal chloride salt. Concentrations of less than about 5% by weight and more than about 10% by weight of alkali metal chloride salt result in reduced cell viability through the freeze-thaw cycle, with concentrations greater than 10% in weight also susceptible to damage crystallization. E) Onosaccharide, such as glucose, functions as a source of carbon for the cells. The availability of monosacap promotes the conservation of the cell through the freeze-thaw cycle and the storage of frozen cells, ensuring the presence of sufficient nutrients within the cell. Serum albumin functions as a source of protein that provides a coating around the membrane of the target cell to protect the membrane during the freeze-thaw cycle and the storage of frozen cells. It is also believed that serum albumin functions as a scavenger of oxygen free radicals that are known to adversely affect the viability of several target cells such as erythrocyte, neutrophilic, eosmophiliac, and basophilic granulocytes, B-cell lymphocytes. ? nfoc? ts ~ T, non-B lymphocytes, non-T lymphocytes and platelet cell mentioned above. The source of serum albumin is made to coincide * preferably with the species of the subject into which the protected target cells will be introduced. Effective concentrations can be obtained by incorporating approximately 0.2 to approximately 1% by weight of monosaccharide in the concentrated cryopreservation solution. A load of less than about 0.2% provides insufficient concentration and reduces the viability of the cell through the freeze-thaw cycle, while concentrations of more than about 1% by weight do not provide a proportional increase in the viability of the cell. and they adversely affect the concentrations of the constituent parts. Serum albumin functions as a source of protein that provides a coating around the membrane of the target cell to protect the membrane during the freeze-thaw cycle and the storage of frozen cells. Effective concentrations can be obtained by incorporating about 20 to about 30% by weight of serum albumin. A concentration of less than a? R * ox? 202% in addition provides insufficient concentration and reduces the viability of the cell, while ace concentrations of approximately 30% by weight do not provide a proportional increase in cell viability and tend to increase the osnolality of the solution above the upper limit desired of approximate 2, 000 rnOsrn / kg "The concentrated cpopreservation solution can be diluted to use concentration by editing a suitable diluent (e.g., 0.9% by weight of saline from ultra-illed water, water for injection and autologous plasma) in a ratio of from about 1 to 10 parts, typically from 1 to 2 parts, diluent to 1 part of cpopreservation solution concent reda. Generally, the diluted cryopreservation solution will consist essentially of (i) about 20 to about 40% by weight of glycerol, (11) about 1.5 to about 5% by weight of alkali metal chloride salt, (m) about 0.1 to about 0.5% by weight of rnonosacchar and (iv) about 6 to about 15% by weight of serum albumin, based on the diluted coppreservation solution. The balance is the dilutent. The cpreservation solution should be formulated to provide a pH of between approximately 7 and 7.5. The target cells can be conveniently cpreserved using the diluted cryopreservation solution by simply (i) int oducing the target cells in the diluted cpreservation solution and (n) freezing or cpopreservating the target cells. The volume of diluted cryopreservation solution to be added to the target cells depends both on the volumetric ratio of the solution containing the target cell to the cpreservation solution and the ratio of the target cell to the cnopreservation solution. The volumetric relegation of the solution containing the target cell to the cryopreservation solution should generally be from about 1: 1 to 1:10, the ratio of the target cells to the cpreservation solution being from about 1x10 * cells / ml to approximately l: lxl09 cells / ml, with a generally preferred ratio of between approximately 2x108 cells / ml to 3x108 cells / rnl. Prior relements of those established above tend to result in a decrease in the concentration of viable cells due to an insufficient cryopreservation solution, whereas lower ratios than those previously established simply result in an inefficient use of the storage and storage solution. need to introduce excessive cpopreservation solution in a subject to introduce effective amounts of the target cells. The target cells are available for preservation only in diluted form or a constituent in a biological fluid (e.g., medre concentrated cells from peripheral blood commonly contain stem cells at a concentration of approximately 1 × 10 6 cells / ml to an appropriate amount). 3x108 cells / ml, the balance comprising other sengre components such as plasma and platelets). These biologically diluted samples should generally be combined with the cpreservation solution in a volumetric ratio of approximately 2: 1 to approximately 1: 4 (depending, of course, on a number of factors including the specific formulation of the diluted cpopreservation solution, the concentration of target cells in the biologically diluted sample, the type of target cells, etc.) to obtain the optimal ratio of target cells to cpopreservation solution. The solution containing final target cell should generally contain approximately 4 to 12% by weight of glycerol and approximately 2 to 10% by weight of albumin, with variations outside this general scale being contemplated for certain applications. The protected cpopreserved target cells must be thawed before the introduction of the target cells into a subject. Thanks to the physiological compatibility of the cryopreservation solution, the thawed target cells can also be introduced into the subject without separating the target cells and the co-preservation solution. the thawed target cells can be maintained during vanas at ambient temperature without appreciable loss in cell viability. The thawed solution can be slowly diluted with saline solution or other appropriate isotonic solution to reduce the osnolality of the solution from the desired cpopreservation scale of 500 to 2., 000 rnOsrn / kg up to the physiological scale of 260 to 320 rnOsm / kg to reduce the shock to the cell walls caused by sudden changes in the osrnolality. A controlled slow approach speed can usually be effective to obtain an optimal cell viailided. It is generally believed that different types of cells have different optimal cooling velocities (see, for example, Rowe, R.U. and Rinfret, R.P., 1962, Blood 20: 636; RoUe, R.U., 1966, Cryo Biology 3 (1): 12-18; Lewis, 3.P. and others., 1967, transfusion 7 (1), -17-32; and Mazur, P., 1970 Science 168: 939-949 for cooling velocity effects on the survival of marrow stem cells and their transplantation potential).

The cryopreservation solution can be maintained at room temperature before use and the mixed target cells maintained at room temperature for about thirty minutes before freezing without appreciable loss in cell viability. This is in contrast to the toxic cpoprotector DMSO that must be cooled to approximately 4 ° C before the addition of target cells to make the freezing procedure faster and requires that the freezing procedure begin immediately after the DMSO is added. to the cells to minimize cell death induced by the presence of DMSO. Controlled freezing can be carried out by using a programmable freezing device. Programmable freezing devices allow to determine optimal cooling speeds and facilitate normal reproducible cooling. The container containing the cells should be stable at cryogenic temperatures and allow rapid heat transfer - for effective control of both freezing and thawing. Sealed plastic bottles (eg, Nunc and Uheeton crioles) or glass ampules can be used for multiple small amounts (2 ml) while larger volumes of 100-200 ml can be frozen in polyolefin bags, such as or those available from Fenwal, entenides between metal plecas.

The frozen cells can then be transferred to a long-term cryogenic storage vessel. In a preferred embodiment, samples can be stored cryogenically in liquid nitrogen (-196 ° C) or liquid nitrogen vapor (-105 ° C). Such is easily facilitated by the availability of highly efficient liquid nitrogen refrigerators. When necessary, the frozen cells are preferably thawed rapidly and kept at approximately 37 ° C until used. Since the cpoprotective solution is physiologically compatible, it does not need to be withdrawn before its introduction into the subject. Cryopreserved and thawed cells, platelets and stem cells and hernetopoietic progenitors of the umbilical cord can be used therapeutically for the reconstitution of the atopoietic system in a suitable patient. The cells can be introduced by any method known in the art, the systematic infusion being generally preferred. The reconstitution of the hematopoietic system (or in unological system) can be therapeutically valuable for a large number of diseases and disorders. The infusion of stem cells and progeitoras hematopoietic cryopreservations for hematopoietic reconstitution can be beneficially used in the treatment of those diseases that are known to be cured by autogenous bone marrow transplantation. The branches that can be treated with the mfusion of stem cells include, but are not limited to, five broad categories. First there are the diseases that result from an insufficiency or dysfunction in the production and normal maturation of blood cells (ie, aplastic anemia and disorders of the mother cell, oproliferative). The second group includes neoplastic malignancies in the hernatopoietic organs (e.g., leukemia and lympholas). The third group of disorders comprises those patients with a broad spectrum of malignant solid tumors of non-hernatopoietic origin. The infusion of stem cells in these patients serves as a bone marrow rescue procedure, which is provided to a patient after another lethal chemotherapy or irradiation of the malignant tumor. The large group of diseases consists of autoimmune conditions, in which the stem cells serve as a source of replacement for an abnormal immune system. The fifth group of diseases comprises a number of genetic disorders that can be corrected by the infusion of hernatopoietic stem cells, preferably signene, which prior to transplantation have undergone gene therapy.

Claims (26)

NOVELTY OF THE INVENTION CLAIMS

1. - A concentrated cpopreservation solution, consisting essentially of: (a) glycerol; (b) an alkali metal chloride salt in an amount effective to promote cell viability; (c) a biologically metabolizable rnonosaccharide in an amount effective to support * cellular maintenance under ambient conditions; and (d) a serum albumin in an amount effective to support the integrity of the plasma membrane.

2. The cpopreservation solution according to claim 1, further comprising a diluent.

3. The cpopreservation solution according to claim 1, further characterized in that the pH of the solution is between about 7 and 7.5.

4. The cpreservation solution according to claim 1, further lacking in that the concentrate consists essentially of (a) about 60 to about 80% by weight of glycerol, (b) about 5 to about 10% by weight. weight of alkali metal chloride salt, (c) from about 0.2 to about 1% by weight of monosaccase, and (d) from about 20 to about 30% by weight of serum albumin.

5. - The cryopreservation solution according to claim 2, further characterized in that the solution consists essentially of (a) about 20 to about 40% by weight of glycerol, (b) from about 1.5 to about 5% by weight of chloride salt of alkali metal, (c) from about 0.1 to about 1.5% by weight of the rnonosaccharide, (d) from about 6 to about 15% by weight of serum albumin, and (e) the remaining diluent ..

6.- The solution cPenpressed concentrate according to claim 1, further characterized in that the alkali metal chloride salt is sodium chloride.

7. The cryopreservation solution according to claim 2, further characterized in that the alkali metal chloride salt is sodium chloride.

8. The concentrated cpopreservation solution according to claim 1, further characterized in that the onosaccharide is glucose.

9. The cryopreservation solution according to claim 2, further characterized in that the monosaccharide is glucose.

10. The concentrated cryopreservation solution according to claim 1, further characterized in that serum albumin is human serum albumen.

11. The cryopreservation solution according to claim 2, further characterized in that the serum albumin is human serum albumin.

12. A concentrated solution of co-preservation, consisting essentially of: (a) from about 60 to about 80% by weight of glycerol; (b) from about 5 to about 10% by weight of the alkali metal chloride salt; (c) from about 0.2 to eproximally 1% by weight of glucose; and (d) from about 20 to about 30% by weight of serum albumin.

13. A cpreservation solution consisting essentially of: (a) from approximately 20 to about 40% by weight of glycerol; (b) from about 1.5 to about 5% by weight of alkali metal chloride salt; (c) from about 0.1 to about 0.5% by weight of glucose; (d) from about 6 to about 15% by weight of serum albumin; and (e) is a remaining diluent.

14. A method of cryopreservating cells, comprising (i) introducing target cells into the cryopreservation solution according to claim 2, and (ii) cryopreserving the protected target cells.

15.- The method according to the claim 14, further characterized in that the volume ratio of the solution containing target cells to the cryopreservation solution is between about 1: 1 and 1:10 and the ratio of the target cells to the cnopreservation solution is between 1 x 10s cells / ml to approximately 1 x 10 cells / nl.

16. - The method according to claim 14, further characterized in that the target cells are stem cells or progemtoras cells.

17. The method according to claim 14, further characterized in that the target cells are platelets.

18. The method according to claim 14, characterized in that the target cells are cells identified as CD 34 * cells. 19.- The method of compliance with the rei indication 14, further characterized in that the target cells are obtained from umbilical cord blood. 20. A cpopreservative cell method, comprising (i) introducing target cells into the cryopreserve solution according to claim 13, and (n) cprespressing the target protean cells. 21. The method according to claim 20, further characterized in that the volumetric return of the target cell containing solution to the cryopreservation solution is between 1: 1 to 1:10 and the ratio of target cells to solution. of cryopreservation is approximately between 1 x 10 5 cells / ml to approximately 1 x 10 9 cells / rnl. 22. The method according to claim 20, further characterized in that the protected target cells are cooled at a rate of 1-2 ° C per minute at a temperature between about -70 and about -196 ° C. 23. A method for cprespressing and recovering viable cells, comprising (1) introducing target cells into the cryopreservation solution according to claim 7, (ii) cprespressing the protected target cells, and (m) dissolving the protected target cells . 24. The method according to claim 23, further characterized in that the volume ratio of the target cell containing solution to the cryopreservation solution is between about 1: 1 to 1:10 and the ratio of target cells to cryopreservation solution it is approximately between 1 x 10 5 cells / ml to approximately 1 x 10 9 cells / ml. 25.- The method of compliance with the claim 23, further characterized in that the target cells are stem cells or progenitor cells. 26.- A method for the cryopreservation, recovery and therapeutic use of cells, comprising (i) introducing target cells to the cryopreservation solution according to claim 2, (ii) cryopreservating the protected target cells, (in) diluting the protected target cells, and (iv) introducing the target proteins protected in a subject having the need for said target cells without seperation of the target cells and the cryopreservation solution. 2 .- The method of compliance with claim 26, further characterized in that the volumetric ratio of the target cell containing solution to the cryopreservation solution is between about 1: 1 to 1:10 and the ratio of target cells to solution of The preservation is approximately between 1 x 10 cells / ml to approximately 1 x 10 9 cells / ml. 28. The method according to claim 26, characterized in that the target cells are stem cells or progenitor cells. 29. The method according to claim 26, further characterized in that the target cells are platelets. 30. The method according to claim 26, characterized in that the target cells are cells identified as CD 34 + cells. 31. The method according to claim 26, characterized in that the target cells are obtained from blood of the umbilical cord. 32.- The method of compliance with the claim 26, further characterized because the subject is human.