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Title:  Methods for storing neural cells

United States Patent:  6,821,779

Issued:  November 23, 2004

Inventors:  Koopmans; Jan (Groningen, NL); Jacoby; Douglas B. (Wellesley, MA); Dinsmore; Jonathan (Brookline, MA)

Assignee:  University Hospital Groningen, Inc. (Groningen, NL); Diacrin, Inc. (Charlestown, MA)

Appl. No.:  743242

Filed:  August 17, 2001

PCT Filed:  July 1, 1999

PCT NO:  PCT/US99/15115

PCT PUB.NO.:  WO00/01231

PCT PUB. Date:  January 13, 2000

Abstract

The instant methods pertain to improved methods for storing neural cells, preferably dissociated neural cells, prior to their use in transplantation and to the cells obtained using such methods. One embodiment pertains to methods for storing the neural cells in medium lacking added buffer or added protein, other embodiments feature neural cells which are maintained at 4oC. prior to cryopreservation and have comparable viability and/or functionality to freshly harvest cells. In addition, methods for storing and/or transplantation of porcine neural cells are described.

SUMMARY OF THE INVENTION

This invention solves the problems referred to above by providing methods for storing neural cells without significant decreases in cell viability and/or functionality. Such methods greatly enhance the availability of cells for in vitro analysis and/or transplantation in vivo. Such methods are also useful when pooling of cells is desired.

In one aspect. the invention pertains to a method for storing cells in a cryopreserved state in which fresh or cultured neural cells are suspended in a cryopreservation solution, the temperature of the cell suspension is decreased in a controlled manner to about -196oC. and the cells are maintained in a frozen state.

In another aspect, the invention pertains to a method for storing cells in hibernation in which fresh, cultured, or cryopreserved neural cells are suspended in a hibernation medium which is preferably free of added protein, free of a buffer, or free of added protein and a buffer, and the cell suspension is maintained at temperatures which are above freezing and sufficiently below normal body temperature such that normal physiological cell processes are decreased or halted.

In another aspect, the invention pertains to cultures of cells which have been stored according to a cryopreservation method and/or hibernation method of the invention. Such cells are useful for in vitro growth, development, and analysis as well as for transplantation in vivo.

In another aspect, the invention pertains to methods of implantation which utilize neural cells which have been stored according to the methods disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains, inter alia, to improved methods of storing neural cells, preferably dissociated neural cells, prior to their use in transplantation and to the cells obtained using such methods. This invention provides for long-term storage of neural cells without significant decreases in cell viability and/or function. Accordingly, the present invention represents a significant advance over the previous cell storage methods. The instant invention is based, in part, on the discovery that neural cells can be stored and/or frozen in a medium, which lacks any buffer or added protein. In addition, improved methods for storing porcine cells are provided. One aspect of the invention features methods which employ a hibernation step of as long as 3-5 days prior to and/or post freezing (or instead of freezing) while still recovering neural cells suitable or transplantation. The ability to store cells without loss of viability and/or function allows for the separation in time between cell preparation and implantation into a subject. The ability to store cells also enables the pooling of cells from multiple donors and adequate time for quality control assessment of cells and other in vitro analysis.

In one aspect, the invention pertains to a method for storing a population of fetal porcine neural cells suitable for transplantation comprising: a) contacting a population of porcine neural cells with a hibernation medium to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4oC. to thereby store a population of neural cells suitable for transplantation.

In another aspect the invention pertains to a method for cryopreserving a population of fetal porcine neural cells suitable for transplantation comprising: a) contacting a population of porcine neural cells with a cryopreservation solution to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC. to thereby cryopreserve a population of neural cells suitable for transplantation.

In yet another aspect, the invention pertains to a method of obtaining a population of fetal porcine neural cells suitable for transplantation comprising: a) contacting a population of porcine neural cells with a cryopreservation solution to thereby obtain a population of cells for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC. to obtain cryopreserved neural cells; c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation; and d) contacting the population of porcine neural cells suitable for transplantation with a hibernation medium and maintaining the cells at about 4oC. prior to transplantation.

In one embodiment of the invention, the porcine neural cells are ventral mesencephalic cells. In a preferred embodiment, the ventral mesencephalic cells are porcine cells obtained between about days 25 and 33 or days 25 and 28 of gestation. In another embodiment, the porcine cells are spinal cord cells. In another embodiment, the porcine neural cells are striatal cells. In a preferred embodiment, the striatal cells are obtained from a lateral ganglionic eminence of porcine striatum. In another embodiment, the porcine neural cells are cortical cells.

In another embodiment, the invention pertains to a population of porcine neural cells for transplantation prepared according to the instant methods.

In yet another embodiment, the invention pertains to a method for treating a neurological disorder or dysfunction comprising transplanting the population of porcine neural cells obtained using the instant methods into a subject.

In another aspect, the invention pertains to a method for storing a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium which medium is free of added protein to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4oC. to thereby store a population of neural cells suitable for transplantation.

In another aspect, the invention pertains to a method for storing a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium which medium is free of a buffer to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4oC. to thereby store a population of neural cells suitable for transplantation.

In another aspect, the invention pertains to a method for storing a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium which medium is free of added protein and free of a buffer to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4oC. to thereby store a population of neural cells suitable for transplantation.

In another aspect, the invention pertains to a method for storing a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium which medium consists of glucose and sodium chloride to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4oC. to thereby store a population of neural cells suitable for transplantation.

In yet another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of added protein and which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC. to thereby cryopreserve a population of neural cells suitable for transplantation.

In another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of a buffer and which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells to about -196oC. to thereby cryopreserve a population of neural cells suitable for transplantation.

In another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of added protein and free of a buffer and which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC. to thereby cryopreserve a population of neural cells suitable for transplantation.

In another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution consisting of glucose, sodium chloride, and a cryopreservative to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC. to thereby cryopreserve a population of neural cells suitable for transplantation

In another aspect, the invention pertains to a method of obtaining a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of added protein which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC. to obtain cryopreserved neural cells; and c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation.

In another aspect, the invention pertains to a method of obtaining a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of a buffer and which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC. to obtain cryopreserved neural cells; and c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation.

In yet another aspect, the invention pertains to a method of obtaining a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of added protein and free of a buffer and which comprises a cryopreservative to thereby produce a population of neural cells suitable for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC. to obtain cryopreserved neural cells; and c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation.

In still another aspect, the invention pertains to a method of obtaining a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution consisting of: glucose, sodium chloride, and a cryopreservative to thereby obtain a population of cells for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC. to obtain cryopreserved neural cells; and c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation.

In certain embodiments of the invention, the neural cells are fetal human cells. In preferred embodiments, the neural cells are human neural stem or neural progenitor cells that have been induced to differentiate in vitro prior to storage using the instant methods.

In other embodiments of the invention, the neural cells are porcine cells. In still other embodiments, the porcine neural cells are ventral mesencephalic cells. In yet other embodiments, the porcine neural cells are porcine spinal cord cells. In still other embodiments, the porcine neural cells are striatal cells. In still other embodiments, the porcine neural cells are cortical cells. In still further embodiments, tie porcine cells are porcine neural stem cells or neural progenitor cells. In certain embodiments, the porcine neural stem cells or progenitor cells have been induced to differentiate in vitro prior to storage using the instant methods.

In other embodiments of the invention, the invention pertains to a population of human or porcine neural cells for suitable for transplantation prepared according to the instant methods.

In still other embodiments of the invention, the invention pertains to a method for treating a neurological disorder or dysfunction comprising transplanting a population of human or porcine neural cells stored according to the claimed methods into a subject.

In another aspect, the invention pertains to a method for storing a population of porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium to thereby produce a cell suspension; b) maintaining the cell suspension for at least about 24 hours at about 4oC. in hibernation medium to thereby store a population of neural cells suitable transplantation.

In another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium to thereby produce a cell suspension; b) maintaining the cell suspension for at least about 24 hours at about 4oC. in hibernation medium to thereby produce an adapted cell suspension; c) contacting the adapted cell suspension with a cryopreservation solution to thereby obtain a population of cells for cryopreservation; and d) decreasing the temperature of the population of neural cells suitable for cryopreservation to about -196oC. to thereby cryopreserve a population of neural cells suitable for transplantation.

In another aspect the invention pertains to a method of obtaining a population of human or porcine neural cells for transplantation comprising: a) contacting a population of neural cells with a hibernation medium to thereby produce a cell suspension; b) maintaining the cell suspension for at least about 24 hours at about 4oC. in hibernation medium to thereby produce an adapted cell suspension; c) contacting the adapted cell suspension with a cryopreservation solution to thereby obtain a population of cells for cryopreservation; d) decreasing the temperature of the population of neural cells for cryopreservation to about -196oC.; and e) increasing the temperature of the neural cells to thereby obtain population of neural cells suitable for transplantation.

In certain embodiments the cell suspension is maintained at about 4oC. for about 72 hours. In preferred embodiments the cell suspension is maintained at about 4oC. for at least about 40-48 hours. In more preferred embodiments, the cell suspension is maintained at about 4oC. for at least about 44 hours.

The invention is further described in the following subsections:

Neural Cells

Neural cells useful in the methods of this invention can be fresh cells or may be obtained from in vitro culture.

Preferably, the cells of the invention are of mammalian origin, i.e., are obtained from mammalian subjects (e.g., humans, pigs, or cows). In one embodiment, the cells are bovine. Preferred cells for use in the instant methods are porcine. Other preferred cells are human.

Neural cells can be derived or obtained from a variety of tissues which are selected based, at least in part, on the intended use for the cells, e.g., the particular function to be assessed or clinical indication to be addressed. For example, if the cells are to be used to treat paralysis, it may be desirable to obtain them from the spinal cord of a donor subject. When the cells are intended for implantation into humans with Parkinson's disease, they are preferably derived from a region of the brain giving rise to dopamine-producing cells.

Similarly, the neural cells useful in the methods of this invention may be obtained during various stages of development of the donor subject, including, fetal, juvenile, and adult cells. In general, the particular stage of development is selected based upon the intended use of the cells subsequent to storage and the species of animal from which the cells are derived.

In one embodiment, the cells for use in the present invention are fetal cells. Preferably, the cells are derived from the fetal central nervous system. In another embodiment, the fetal cells are spinal cord cells. In preferred embodiments the fetal cells are ventral mesencephalic cells. In still other preferred embodiments the fetal cells are striatal cells. In yet other preferred embodiments the striatal cells are obtained from a lateral ganglionic eminence of the striatum. In other embodiments, the fetal cells are cortical cells.

Preferably, cells for use in the instant methods are used after they have undergone their final maturation, but before they have sent out projections, e.g., axons. For example, in one embodiment, the fetal human cells are obtained from fetuses ranging in age from 7 to 18 weeks of gestation. In preferred embodiments, fetal human cells are obtained at between 7 and 11 weeks gestation. Fetal human cells for use in the claimed methods are obtained using methods known in the art and as required under the guidelines for use of human tissue (see e.g., DHEW publication OS 1975). In embodiments in which fetal porcine cells are used, preferably the cells are obtained between about days 20 and 115 of gestation, depending on the cell type to be isolated. For example, in certain embodiments, e.g., when the cells are porcine ventral mesencephalic cells, the cells are obtained between about days 25 and 33 or days 25 and 28 of gestation. Preferably the porcine VM cells are used between about days 26 and 27 of gestation. More preferably, the porcine VM cells are used at about 27 days of gestation. In the case of fetal porcine striatal cells, preferably the cells are obtained from a fetus at between about days 30 and 50 of gestation. In more preferred embodiments, the porcine striatal cells are obtained from a fetus between about days 31 and 38 of gestation. In particularly preferred embodiments, the porcine striatal cells are obtained from a fetus between about days 34 and 36 of gestation. In the case of porcine cortical cells, the cells are preferably obtained from a fetus between about days 20 and 30 of gestation. In particularly preferred embodiments, the porcine cortical cells are obtained from a fetus between about days 24 and 28 of gestation.

In certain embodiments, the cells for use in the instant methods are neural stem cells which have been induced to differentiate. In other embodiments, the cells are neural progenitor cells which have been induced to differentiate. Tissue containing stem or progenitor cells can be obtained from mammalian fetuses, juveniles, or from an adult organ donor. In preferred embodiments, stem cells to be used in the instant methods are porcine cells. In other preferred embodiments, stem cells to be used in the instant methods are human cells. In certain embodiments, autologous stem cells from the donor may be obtained, differentiated and cryopreserved using the instant methods.

Neural stem or progenitor cells can be obtained from any area of the central nervous system, including the cerebral cortex, cerebellum, midbrain, brainstem, spinal cord, ventricular tissue, or from areas of the peripheral nervous system, including the carotid body and the adrenal medulla. Methods of obtaining neural progenitor or stem cells are known in the art (see e.g., U.S. Pat. No. 5,753,506; WO97/44442; WO96/04368; WO94/10292; WO94/02593; Gage et al. 1995 Ann. Rev. Neurosci. 18:159).

To expand a population of neural cells, (e.g., stem or progenitor cells) the cells can be grown in the presence of trophic factors, such as nerve growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, platelet-derived growth factor, thyrotropin releasing hormone, epidermal growth factor, amphiregulin, transforming growth factor, transforming growth factor .beta., insulin-like growth factor, or other growth factors using methods known in the art (see, e.g., U.S. Pat. Nos. 5,753,506, 5,612,211, 5,512,661, WO93/01275; Mehler and Kessler. 1995 Crit. Rev. Neurobiol 9:419).

Neuronal or glial cells can be differentiated from an expanded stem or progenitor cell population by treating the cells by any method known in the art which promotes differentiation of the cells, for example, phorbol esters or various growth factors (See, e.g., U.S. Pat. Nos. 5,750,376; 5,753,506; WO 96/15226; WO 94/02593; WO 96/15224). Alternatively, a surface such as poly-L-lysine can be used to induce differentiation (WO 93/01275). Such differentiated cells can be stored using the instant methods.

In certain embodiments, pieces of tissue are left intact, or stored as fragments, e.g., are not dissociated into individual cells prior to used in the instant methods. In preferred embodiments, the cells to be used in the claimed methods are dissociated, e.g., into individual cells to make a suspension prior to their use in the instant methods. For example, tissue can be dissociated using gentle trituration. Methods of dissociating tissue are known in the art and include the use of flame-polished pasteur pipets of diminishing orifice diameters or a succession of Angio-catheters of diminishing orifice diameters.

Hibernation Conditions

Numerous types of media can be used as hibernation media in conjunction with the instant methods. In preferred embodiments, hibernation media is free of added Ca++. In certain embodiments, medium for hibernating cells is free of added protein and/or free of a buffer. A preferred hibernation medium includes or consists of glucose in a saline solution, e.g., between about 0.21%-0.9% glucose in saline. In preferred embodiments, the hibernation medium includes or consists of about 0.35-0.9% glucose and 0.9% NaCl. In other preferred embodiments, the medium includes or consists of about 0.6% glucose and 0.9% NaCl. In certain embodiments, more complex media can be used, e.g., Hank's balanced salt solution, Dulbecco's minimal essential medium (see e.g., Nikkah et al. 1995 Brain Research 687:22), or Eagle's modified minimal essential medium. Another suitable hibernation medium has been described by Kawamoto and Barrett (1986 Brain Research 384:84). In certain embodiments it may be desirable to supplement the chosen hibernation medium with additives, for example, added protein (e.g., mammalian serum protein or whole serum (preferably heat inactivated)) buffers (e.g., phosphate buffers, HEPES, or the like) antioxidants, growth factors, KCl (e.g., at about 30 mM), lactate (e.g., at about 20 mM), pyruvate, MgCl2 (e.g., at about 2-3 mM), sorbitol (e.g., at about 300 mM) or other additives as are well known in the art (e.g., as taught by Kawamoto and Barrett supra).

In certain embodiments, the cells of the invention are hibernated at about 0-37oC., preferably about 4oC. In certain embodiments, cells are maintained at about 4oC. in hibernation medium prior to freezing or use. In other embodiments the cells of the invention are maintained at about 4oC. in hibernation medium post freezing. In still other embodiments, the cells of the invention are maintained at about 4oC. in hibernation medium without freezing. In certain embodiments, the cells of the invention are maintained in hibernation medium at about 4oC. for at least about 1 hour to 5 days prior to freezing, post freezing or prior to use in transplantation. In other embodiments, the cells of the invention are maintained in hibernation medium at about 4oC. for at least about 12-72 hours prior to freezing, post freezing or prior to use in transplantation. In certain embodiments the cells are maintained at 4oC. in hibernation medium for at least about 24 hours prior to freezing; post freezing or prior to use in transplantation. In a more preferred embodiment, the cells are maintained in hibernation medium from at least about 40-48 hours at about 4oC. prior to freezing, post freezing or prior to use. In a particularly preferred embodiment, the cells are maintained in hibernation medium for at least about 44 hours at about 4oC. prior to freezing, post freezing or prior to use.

Freezing Conditions

Any cryopreservative known in the art can be used in a cryopreservative solution of the instant invention. In preferred embodiments, cryopreservation solutions of the present invention include intracellular cryopreservatives e.g., dimethylsulfoxide (DMSO), various diols and triols (e.g., ethylene glycol, propylene glycol, butanediol and triol and glycerol), as well as various amides (e.g., formamide and acetamide). However, extracellular cryopreservatives e.g., phosphomono and phosphodiester catabolites of phosphoglycerides (EP 0 354 474), polyvinylpyrrolidone (Fang and Zhong 1992 Cryobiology 29:267), or methylcellulose (e.g., at 0.1%, see e.g., Sautter et al. 1996 J. of Neuroscience Methods. 64:173) can also be used alone or in conjunction with an intracellular cryopreservative.

In preferred embodiments, DMSO is used as the cryopreservative. DMSO can be used at a wide range of concentrations (see e.g., Silani et al. 1988 Brain Research 473:169). In preferred embodiments, DMSO is used at a final concentration of about 4% to about 10%. In more preferred embodiments the concentration of DMSO ranges from about 7% to about 10%. In particularly preferred embodiments the concentration of DMSO is about 7%.

In certain embodiments, the cryopreservative is added to the cells in a stepwise manner in order to gradually increase the concentration of the cryopreservative until the desired final concentration of cryopreservative is achieved. In preferred embodiments, the cells are contacted with a cryopreservation solution containing the cryopreservative at the desired final concentration or the cryopreservative is added directly to the base medium without a gradual increase in concentration.

The cryopreservation solution includes the cryopreservative in an appropriate base medium. Any type of media can be used for this purpose. For example, any of the hibernation media listed above can be used as the base medium for a cryopreservation solution. In preferred embodiments, the base medium to which the cryopreservative is added is free of added Ca++. In certain embodiments the medium to which the cryopreservative is added is free of added protein and/or free of a buffer. In other embodiments, the base medium to which the cryopreservative is added includes or consists of about 0.2-0.9% glucose and about 0.9% NaCl. In preferred embodiments, the base medium to which the cryopreservative is added includes or consists of about 0.35-0.9% glucose and about 0.9% NaCl. In another preferred embodiment, the base medium to which the cryopreservative is added includes or consists of about 0.6% glucose and about 0.9% NaCl. Alternative media can also be used, see e.g., that described by Gage et al. (1985 Neuroscience Letters 60:133).

In certain embodiments the cryopreservation solution can also contain added protein, for example, serum, e.g., fetal calf serum or human serum, or a serum protein, e.g., albumin. In other embodiments, the cryopreservative can also contain other additives, such as those described above for inclusion in hibernation media, for example, antioxidants, growth factors, KCl (e.g., at about 30 mM), lactate (e.g., at about 20 mM), pyruvate, MgCl2 (e.g., at about 2-3 mM), sorbitol (e.g., to an osmolarity of about 300 mM) or other additives as are well known in the art (e.g., as taught by Kawamoto and Barrett supra).

Once the cells are suspended in cryopreservation solution, the temperature of the cells is reduced in a controlled manner. In cooling the cells to below freezing, preferably the reduction in temperature occurs slowly to allow the cells to establish an equilibrium between the intracellular and extracellular concentration of cryopreservative such that intracellular ice crystal formation is inhibited. On the other hand, the rate of cooling is preferably fast enough to protect the cells from excess water loss and the toxic effects of cryopreservatives. Controlled freezing may be accomplished with the aid of commercially available electronically controlled fire equipment, e.g., a Cryomed or Planer Biomed controlled rate freezer. In an exemplary freezing program a cell sample and the freezing chamber are brought to about 1oC. to 9oC., preferably about 4oC. The cells are then cooled at a rate of about 1oC./min to 7oC./min, preferably about 2oC./min, until the cells reach about -6oC. to +6oC., preferably about -1oC. The chamber is brought to about -6oC. to +6oC., preferably about -1oC., and the sample is held at this temperature for about 9-19 minutes, preferably about 14 minutes. The sample is cooled at a rate of about 1oC./minute to 6oC./minute, preferably about 1.2o /minute until the sample reaches about -16oC. to about -6oC., preferably about -11oC. The freezing chamber is subsequently cooled at a rate of about 83 to about 92oC./minute, preferably about 88o/minute until the sample reaches about -22oC. to -32oC., preferably about -27oC. The chamber is warmed to about -38 to about -28oC., preferably about -33oC. and the sample is held for about 5-15 minutes, preferably about 10 minutes. The cells are then cooled at a rate of about 1-5o/minute, preferably about 1o/minute until the sample reaches about -40 to about -50oC., preferably about -45oC. Next, the cells are cooled at about -1-7o/minute, preferably about 2o/minute until the cells reach about -55 to about -65oC., preferably about -60oC. The sample is then cooled at about 1o to about 10o/minute, preferably about 5o/minute until it reaches -90oC.

The cells can then be cryopreserved at a temperature of between -20oC. and about -250oC. Preferably, the cells are stored below -90oC. to minimize the risk of ice recrystalization. In particularly preferred embodiments, the cells are cryopreserved in liquid nitrogen at about -196oC.

Thawing Conditions

After cryopreservation, the cells are preferably thawed rapidly, e.g., by quick immersion in liquid at 37oC. Once the cells are thawed, dilution of the cryopreservative is accomplished by gradual addition of a dilution media. Preferably, the cryopreservative is gradually diluted by a slow multi-step addition of media For example, cells can be diluted slowly by adding a dilution medium (e.g., a 1:1 dilution) and allowed to sit for 5 minutes at room temperature. The 1:1 dilution can be repeated twice more by slowly adding dilution medium, waiting 5 minutes, and then adding more dilution medium. In other embodiments, a one-step dilution procedure can be used.

Any media can be used for diluting the cryopreservation solution which is in contact with the thawed cells. For example, any of the media listed above for use in hibernating cells can be used for diluting the cryopreservation solution. Other media are also appropriate, for example, Hank's balanced salt solution (preferably without Ca++) containing glucose (about 0.6%) can be used. Additives, e.g., as listed above for inclusion in hibernation or freezing media can also be used in media for dilution. Exemplary additives include, for example, buffers (e.g., phosphate buffers, HEPES, or the like) antioxidants, growth factors, KCl (e.g., at about 30 mM), lactate (e.g., at about 20 mM), pyruvate, MgCl2 (e.g., at about 2-3 mM), sorbitol (e.g., to an osmolarity of about 300 mM) or others additives as are well known in the art (e.g., as taught by Kawamoto and Barrett supra). Another suitable additive includes DNase (e.g., commercially available from Genentech, Incorporated as PULMOZYME.RTM.). The medium which is used for diluting the cryopreservation solution can, optionally, contain added protein, e.g., added protein (e.g., mammalian serum (preferably heat inactivated) or a serum protein such as albumin (e.g., commercially available from Alpha Therapeutic Corporation)). In other embodiments, the medium contains no added protein and/or no added buffer.

When cells have been frozen as pieces of tissue, the thawed tissue can be dissociated, if desired, into individual cells, using methods known in the art and described supra.

After dilution of the cryopreservative, the cells can then be allowed to settle or a pellet of cells can be formed under centrifugal force in order to remove as much of the cryopreservation solution from the cells as possible. The cells can then be washed in medium which does not contain a cryopreservative. It is preferable for the cells to remain at room temperature after the addition of the wash media and prior to letting the cells settle or form a pellet under centrifugal force. In preferred embodiments, the cells remain at room temperature for at least 15 minutes prior to the second centrifugation. Any medium known in the art can be used to wash the cells, for example, any of the hibernation or dilution media set forth above can be used.

For use in transplantation, cells should be suspended in a final medium which is suitable for administration to a subject. In certain embodiments, the cells are resuspended in a solution including or consisting of glucose (e.g., about 0.2-0.9%) and sodium chloride (e.g., about 0.9%). In preferred embodiments, the cells are resuspended in a solution including or consisting of glucose (e.g., about 0.3-0.6%) and sodium chloride (e.g., about 0.9%). In particularly preferred embodiments, the cells are resuspended in a final solution including or consisting of about 0.35% glucose and about 0.9% saline

In addition, the thawed cells may be maintained in hibernation medium as described above at between 0 and 37oC., preferably about 4oC. for up to 3-5 days prior to use in transplantation without a statistically significant loss in viability.

Methods of Determining Viability and/or Functionality of Recovered Cells

After storage, it may be desirable to assay the viability and/or functionality of the cells prior to transplantation to confirm their suitability for use, e.g., in transplantation. This can be accomplished using a variety of methods known in the art. For example, the cells can be stained using vital stains, such as, e.g., trypan blue or ethidium bromide or acridine orange. In certain embodiments, a population of cells suitable for transplantation is at least between about 75-100% viable. In preferred embodiments, a population of cells suitable for transplantation is at least about 80% viable. In particularly preferred embodiments, such a population of cells is at least about 85% viable.

In other embodiments, the morphometric characteristics of the cells can be determined as a measure of the suitability of cells for use in transplantation (see e.g., Petite and Calvet. 1997 Brain Research 769:1). In preferred embodiments, the morphology of cells which have been stored using the instant methods and are suitable for transplantation does not differ (e.g., statistically significant) from that of fresh cells. The morphology of the cells and their ability to integrate into the host nervous system can also be tested post-transplantation (Nikkhah et al. 1995 Brain Research 687:22). In preferred embodiments, the in vivo morphology of cells which have been stored using the instant methods and are suitable for transplantation does not differ (e.g., statistically significant) from that of fresh cells. Graft volume of transplanted cells can also be measured (Sauer and Brundin. 1991 Restorative Neurology and Neuroscience 2:123). In preferred embodiments, the graft volume of cells which have been stored using the instant methods and are suitable for transplantation does not differ (e.g., statistically significant) from that of fresh cells.

Cells which have been stored can also be assayed for the presence of neural cell markers to determine if they are suitable for use in transplantation. For example, methods and reagents useful in detecting the expression of glial fibrillary acidic protein, gamma amino butyric acid (GABA), neuron-specific enolase, tyrosine hydroxylase (TH), norepinephrine, serotonin, 3,4,-dihydroxyphenylacetic acid (DOPAC), homovanillic acid, 5-hydroxyindole acetic acid, acetyl cholinesterase, or other markers are available (see, e.g.,. Petite and Calvet 1995 Brain Research 669:263; Collier et al. 1987 Brain Research 436:363; Petite and Calvet 1997 747:279). In preferred embodiments, cells suitable for transplantation display an immunoreactivity pattern, e.g., TH activity, which is not lower (e.g., statistically significant) than that demonstrated in a fresh population of cells.

Additionally, or alternatively, the cells can be tested for their functionality. For example, the ventral mesencephalon cells could be transplanted into 6-OHDA lesioned rats (Kamo et al. 1986 Brain Research 397:372). The ability of the cells to reduce pregraft ipsilateral amphetamine-induced motor asymmetry can be tested. In preferred embodiments, a population of cells suitable for transplantation compensates (e.g., statistically significant) for a neural defect in such an in vivo animal model. For example, a population of cells obtained by the instant methods compensates for a 6-OHDA lesion as well as or better than (e.g., statistically significant) a fresh population of cells.

Treatment of Disease

Cells which have been cryopreserved using the instant methods can be used to treat a variety of neurodegenerative diseases or dysfunctions. For example, Parkinson's disease, Huntington's disease, Lou Gehrig's disease or amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer's disease, have all been linked to the degeneration of neurons in specific locations in the brain or spinal cord. In addition, damage to the nervous system, caused by, for example, stroke, epilepsy, cerebral palsy, spinal cord injury, or chronic pain all result in neuronal loss and can be treated to restore neuronal physiology using cells obtained by the instant methods.

Claim 1 of 25 Claims

What is claimed:

1. A method for treating a neurological disorder or dysfunction comprising:

a) contacting a population of human or porcine neural cells with a hibernation medium free of Ca++ and free of protein and free of buffer to thereby produce a cell suspension;

b) maintaining the cell suspension at about 4oC. for between at least about 12-72 hours to thereby store a population of neural cells suitable for transplantation, wherein the population of neural cells is between at least about 75-100% viable;

c) transplanting the population of neural cells to a subject with a neurodegenerative disorder or dysfunction, such that the neurological disorder or dysfunction is treated.




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