Cultural medium for maintaining neural cells in ambient atmosphere

The present invention provides a minimal essential medium for maintaining neural cell or tissue viability in an environment containing ambient levels of CO.sub.2. The medium contains less than about 2000 .mu.M bicarbonate, a buffer having a pKa of from about 6.9 to about 7.7, wherein the medium is free of ferrous sulfate, glutamate and aspartate, from 0 to about 3000 .mu.M CaCl.sub.2, from about 0.05 to about 0.8 .mu.M Fe(NO.sub.3).sub.3, from about 2500 to about 10000 .mu.M KCl, from 0 to about 4000 .mu.M MgCl.sub.2, from about 74000 to about 103000 .mu.M NaCl, from about 400 to about 2000 .mu.M NaHCO.sub.3, from about 250 to about 4000 .mu.M NaH.sub.2 PO.sub.4, from about 0.2 to about 2 .mu.M ZnSO.sub.4, from about 2500 to about 50000 .mu.M glucose and from about 23 to about 500 .mu.M sodium pyruvate. The present invention also provides a process of extending neural cell or tissue viability in an atmosphere having ambient levels of carbon dioxide whereby the neural cells or tissue are placed in such a medium. Preferably, the medium is supplemented with a growth-promoting medium that contains effective amounts of hormones, essential fatty acids and anti-oxidants for neural cells.

TECHNICAL FIELD OF THE INVENTION
 The field of this invention is cell culture media. More particularly, the
 present invention pertains to a medium for neural cells or tissue that
 maintains viability of those cells or tissue in an atmosphere having
 ambient levels of carbon dioxide.
 BACKGROUND OF THE INVENTION
 A major problem attendant to studies of central nervous system tissue is
 the maintenance of cell viability of such tissues. The inability to
 maintain central nervous system tissue viability in culture for prolonged
 periods of time and under various environmental conditions has impeded the
 development of effective therapeutic regimens for treating central nervous
 system disorders.
 A nutrient balanced salt solution (medium) for maintaining central nervous
 system tissue viability in a high-carbon dioxide atmosphere (5% CO.sub.2)
 has recently been developed. That medium, Neurobasal.TM. (Gibco/Life
 Technologies, Inc., Gaithersburg, Md.), is a bicarbonate buffered medium
 optimized for the growth of embryonic rat hippocampal neurons at a pH of
 7.3 in 5% CO.sub.2. Neurobasal.TM. is a derivative of Dulbecco's Modified
 Eagle's Medium (DMEM) and was formulated to optimize embryonic rat
 hippocampal cell survival. When compared to DMEM, Neurobasal.TM. has less
 NaCl and less NaHCO.sub.3, resulting in a lower osmolality, and lesser
 amounts of cysteine and glutamine, resulting in diminished glial growth.
 In addition, Neurobasal.TM. contains alanine, asparagine, proline and
 vitamin B12, all of which are absent from DMEM.
 Although neurons can be maintained in a 5% CO.sub.2 atmosphere in this high
 bicarbonate medium, when supplemented with B27 (a hormone and anti-oxidant
 supplement available from Life Technologies, Inc.), neurons undergo rapid
 death when transferred to ambient CO.sub.2 (0.2%) conditions. Death is
 associated with a rapid rise in medium pH to a value of 8.1.
 The preparation and study of neural tissue and cells frequently requires
 the use of ambient CO.sub.2 levels outside of an incubator. Existing
 methods for controlling the pH of cells outside of incubators include the
 use of weak buffers (e.g., as found in Dulbecco's modified Eagle's medium
 or L 15 medium) and the use of continuous gassing with 5-10% CO.sub.2 to
 maintain physiological pH.
 A simple test, however, shows that ambient CO.sub.2 causes the pH of DMEM
 to quickly rise to a value of 8.1 outside the incubator. The common
 practice of buffering with HEPES slows but does not prevent this
 substantial alkalinization. The practice of continuously gassing tissues
 to maintain high CO.sub.2 levels and physiological pH is cumbersome and
 expensive. There continues to be a need in the art, therefore, for a
 medium that can maintain physiological pH and neural cell viability in
 ambient CO.sub.2 conditions.
 BRIEF SUMMARY OF THE INVENTION
 In one aspect, the present invention provides a minimal essential medium
 for maintaining neural cell or tissue viability in an environment
 containing ambient levels of CO.sub.2. The medium contains less than about
 2000 .mu.M bicarbonate, has an osmolality of from about 230 mOsm to about
 300 mOsm, contains a buffer having a pKa of from about 6.9 to about 7.7,
 and is free of ferrous sulfate, glutamate and aspartate. A medium of the
 present invention is effective in maintaining viability of neural cells or
 tissue derived from embryonic tissues or adult tissues.
 Where the cells or tissue are of embryonic origin, the osmolality is from
 about 230 mOsm to about 250 mOsm. Such a medium comprises, in final
 concentration, 0 to about 3000 .mu.M CaCl.sub.2, 0.05 to about 0.8 .mu.M
 Fe(NO.sub.3).sub.3, 2500 to about 10000 .mu.M KCl, 0 to about 4000 .mu.M
 MgCl.sub.2, 74000 to about 81000 .mu.M NaCl, 400 to about 2000 .mu.M
 NaHCO.sub.3, 250 to about 4000 .mu.M NaH.sub.2 PO.sub.4, 0.2 to about 2
 .mu.M ZnSO.sub.4, 2500 to about 5000 .mu.M glucose, 0 to about 100 .mu.M
 phenol red, and 23 to about 500 .mu.M sodium pyruvate.
 Where the neural cells or tissue are of adult origin, the osmolality is
 from about 250 mOsm to about 300 mOsm. Such a medium comprises, in final
 concentration, 0 to about 3000 .mu.M CaCl.sub.2, 0.05 to about 0.8 .mu.M
 Fe(NO.sub.3).sub.3, 2500 to about 10000 .mu.M KCl, 0 to about 4000 .mu.M
 MgCl.sub.2, 86000 to about 103000 .mu.M NaCl, 400 to about 2000 .mu.M
 NaHCO.sub.3, 250 to about 4000 .mu.M NaH.sub.2 PO.sub.4, 0.2 to about 2
 .mu.M ZnSO.sub.4, 2500 to about 5000 .mu.M glucose, 0 to about 100 .mu.M
 phenol red, and 23 to about 500 .mu.M sodium pyruvate.
 A preferred buffer is 3-[N-morpholino]propane-sulfonic acid (MOPS). A
 medium of the present invention contains effective amo unts of at least
 ten essential amino acids. In one embodiment, the medium contains, in
 final concentration: a) from about 250 to about 2500 .mu.M each of
 L-isoleuoine, L-leucine, L-threonine and L-valine; b) from about 150 to
 about 1500 .mu.M L-glutamine; c) from about 120 to about 1200 .mu.M each
 of L-arginine, glycine, L-phenylalanine, L-serine and L-tyrosine; d) from
 about 60 to about 600 .mu.M each of L-histidine and L-methionine; e) from
 about 25 to about 250 .mu.M L-tryptophan; f) from about 25 to about 250
 .mu.M L-proline; g) from about 6 to about 60 .mu.M L-alanine; h) from
 about 3 to about 30 .mu.M L-cysteine; and i) from about 1.5 to about 15
 .mu.M each of L-asparagine and L-lysine.
 A medium of the present invention further includes vitamins in amounts
 effective to sustain neural cell or tissue viability. The medium contains,
 in final concentration, from about 12 to about 120 .mu.M i-inositol, from
 about 10 to about 100 .mu.M niacinamide, from about 9 to about 90 .mu.M
 choline chloride, from about 6 to about 60 .mu.M pyridoxal, from about 3
 to about 30 .mu.M thiamine, from about 2.5 to about 25 .mu.M each of folic
 acid and D-Ca pantothenate, from about 0.3 to about 3 .mu.M riboflavin,
 and from about 0.06 to about 0.6 .mu.M vitamin B12.
 In another aspect, the present invention provides a process of extending
 neural cell or tissue viability in an atmosphere having ambient levels of
 carbon dioxide. The process includes the steps of placing neural cells or
 tissue in a medium of the present invention and maintaining the cells or
 tissue in that medium under ambient CO.sub.2 conditions. In a preferred
 embodiment of a process of the present invention, the medium is
 supplemented with a serum-free growth promoting supplement that contains
 effective amounts of hormones, essential fatty acids and anti-oxidants for
 neural cells. A preferred growth-promoting supplement contains biotin,
 L-carnitine, corticosterone, ethanolamine, D(+)-galactose, reduced
 glutathione, linoleic acid, linolenic acid, progesterone, putrescine,
 retinyl acetate, selenium, triodo-1-thyronine, DL-.alpha. tocopherol,
 DL-.alpha. tocopherol acetate, bovine albumin, catalase, insulin,
 superoxide dismutase and transferrin.
 The present invention also provides a composition comprising neural cells
 or tissue in a medium as set forth above. The medium containing the cells
 or tissue can optionally be supplemented with a growth-promoting
 supplement as described above.

DETAILED DESCRIPTION OF THE INVENTION
 I. Composition of Matter
 In one aspect, the present invention provides a medium for neural cells or
 tissue maintained in an environment containing ambient amounts of carbon
 dioxide (CO.sub.2). As is well known in the art, ambient air contains less
 than about 0.25% CO.sub.2 and, more typically, about 0.2% CO.sub.2. A
 medium of the present invention is designed and formulated to allow for
 prolonged survival and viability of neural cells or tissue exposed to such
 low CO.sub.2 levels.
 As is well known in the art, there are a variety of preparative steps to
 obtain primary neural cell cultures. By way of example, specific regions
 of the brain are typically dissected as a tissue and placed in a suitable
 medium. This neural tissue is further processed to obtain cells which are
 typically plated in a culture container and the medium is placed over the
 plated tissue. In both cases, the medium is changed as needed to maintain
 viability. As used herein, the terms "suspended" or "cultured" are used to
 refer both to suspensions of tissue, or individual cells and plated
 individual cells. As used herein, the phrase "neural cells" means either
 individual cells of the same or different type whether those cells exist
 as isolated cells or exist in the form of aggregates or collections.
 A medium of the present invention is a derivative of a basal medium
 previously described by the present inventor to provide prolonged
 viability of neural cells or tissue in a high-CO.sub.2 environment (e.g.,
 5% CO.sub.2) such as typically used in cell incubators (Brewer et al., J.
 Neuroscience Res., 35:567-576, 1993). The basal medium described in that
 publication was designated Neurobasal.TM..
 A medium of the present invention contains the same nutrients (e.g.,
 glucose, amino acids) and vitamins as Neurobasal.TM.. Differences between
 Neurobasal.TM. and a medium of the present invention are changes in the
 levels of bicarbonate and sodium chloride and the use of MOPS as the inert
 buffer (HEPES is used in Neurobasal.TM.).
 Like Neurobasal.TM., a medium of the present invention can be used with
 neural cells or tissue derived from any area of the central nervous system
 of a mammal, including humans. The cells or tissues can be derived or
 obtained from embryonic, neo-natal or adult central nervous system tissue.
 A medium of the present invention can also be used to maintain viability
 of neural cells or tissue obtained after death of the donor animal
 (post-mortem harvesting). Post-mortem tissue harvesting is particularly
 important where the donor animal is killed rather than anesthetized prior
 to tissue extraction.
 A medium of the present invention is characterized by the presence of very
 low levels of a bicarbonate buffer system. The pH value of the medium is
 controlled predominantly by non-bicarbonate buffers. Those buffers include
 sodium phosphate (e.g., NaH.sub.2 PO.sub.4) and inert buffers having a pKa
 between a pH value of about 6.9 and about 7.7. Such inert buffers are well
 known to those of skill in the art and readily available from commercial
 sources. A preferred such inert buffer is 3-[N-morpholino]
 propane-sulfonic acid (MOPS) having a pKa of about 7.2. An inert buffer
 used in a medium of the present invention must be non-toxic toward neural
 cells. Thus, for example, the use of HEPES, which has been shown to be
 phototoxic to certain neural cells should be avoided. The inert buffer is
 present at a final concentration of from about 5000 .mu.M to about 25000
 .mu.M and, more preferably from, about 8000 .mu.M to about 12000 .mu.M.
 A medium of the present invention is effective in maintaining viability of
 neural cells or tissue derived from embryonic tissues or adult tissues.
 Where the cells or tissue are of embryonic origin, the medium has an
 osmolality from about 230 mOsm to about 250 mOsm. Such a medium comprises,
 in final concentration, from 0 to about 3000 .mu.M CaCl.sub.2, from about
 0.05 to about 0.8 .mu.M Fe(NO.sub.3).sub.3, from about 2500 to about 10000
 .mu.M KCl, from 0 to about 4000 .mu.M MgCl.sub.2, from about 74000 to
 about 81000 .mu.M NaCl, from about 400 to about 2000 .mu.M NaHCO.sub.3,
 from about 250 to about 4000 .mu.M NaH.sub.2 PO.sub.4, from about 0.2 to
 about 2 .mu.M ZnSO.sub.4, from about 2500 to about 50000 .mu.M glucose,
 from 0 to about 100 .mu.M phenol red, and from about 23 to about 500 .mu.M
 sodium pyruvate. More preferably, such a medium comprises, in final
 concentration, from about 900 to about 2500 .mu.M CaCl.sub.2, from about
 0.1 to about 0.4 .mu.M Fe(NO.sub.3).sub.3, from about 4000 to about 7000
 .mu.M KCl, from 500 to about 1500 .mu.M MgCl.sub.2, from about 75000 to
 about 77000 .mu.M NaCl, from about 600 to about 1200 .mu.M NaHCO.sub.3,
 from about 600 to about 1200 .mu.M NaH.sub.2 PO.sub.4, from about 0.4 to
 about 1.2 .mu.M ZnSO.sub.4, from about 15000 to about 35000 .mu.M glucose,
 from about 15 to about 40 .mu.M phenol red, and from about 150 to about
 250 .mu.M sodium pyruvate.
 An especially preferred such medium comprises, in final concentration, 1800
 .mu.M CaCl.sub.2, 0.2 .mu.M Fe(NO.sub.3).sub.3, 5360 .mu.M KCl, 812 .mu.M
 MgCl.sub.2, 76000 .mu.M NaCl, 880 .mu.M NaHCO.sub.3, 900 .mu.M NaH.sub.2
 PO.sub.4, 0.67 .mu.M ZnSO.sub.4, 25000 .mu.M glucose, 23 .mu.M phenol red,
 and 230 .mu.M sodium pyruvate.
 Where the neural cells or tissue are of adult origin, the osmolality is is
 from about 250 mOsm to about 300 mOsm. Such a medium comprises, in final
 concentration, from 0 to about 3000 .mu.M CaCl.sub.2, from about 0.05 to
 about 0.8 .mu.M Fe(NO.sub.3).sub.3, from about 2500 to about 10000 .mu.M
 KCl, from 0 to about 4000 .mu.M MgCl.sub.2, from about 86000 to about
 103000 .mu.M NaCl, from about 400 to about 2000 .mu.M NaHCO.sub.3, from
 about 250 to about 4000 .mu.M NaH.sub.2 PO.sub.4, from about 0.2 to about
 2 .mu.M ZnSO.sub.4, from about 2500 to about 50000 .mu.M glucose, from 0
 to about 100 .mu.M phenol red, and from about 23 to about 500 .mu.M sodium
 pyruvate. More preferably, such a medium comprises, in final
 concentration, from about 900 to about 2500 .mu.M CaCl.sub.2, from about
 0.1 to about 0.4 .mu.M Fe(NO.sub.3).sub.3, from about 4000 to about 7000
 .mu.M KCl, from 500 to about 1500 .mu.M MgCl.sub.2, from about 83000 to
 about 95000 .mu.M NaCl, from about 600 to about 1200 .mu.M NaHCO.sub.3,
 from about 600 to about 1200 .mu.M NaH.sub.2 PO.sub.4, from about 0.4 to
 about 1.2 .mu.M ZnSO.sub.4, from about 15000 to about 35000 .mu.M glucose,
 from about 15 to about 40 .mu.M phenol red, and from about 150 to about
 250 .mu.M sodium pyruvate.
 An especially preferred such medium comprises, in final concentration, 1800
 .mu.M CaCl.sub.2, 0.2 .mu.M Fe(NO.sub.3).sub.3, 5360 .mu.M KCl, 812 .mu.M
 MgCl.sub.2, 89000 .mu.M NaCl, 880 .mu.M NaHCO.sub.3, 900 .mu.M NaH.sub.2
 PO.sub.4, and 0.67 .mu.M ZnSO.sub.4, 25000 .mu.M glucose, 23 .mu.M phenol
 red, and 230 .mu.M sodium pyruvate.
 A medium of the present invention contains effective amounts of at least
 ten essential amino acids. In one embodiment, the medium contains, in
 final concentration: a) from about 250 to about 2500 .mu.M each of
 L-isoleucine, L-leucine, L-threonine and L-valine; b) from about 150 to
 about 1500 .mu.M L-glutamine; c) from about 120 to about 1200 .mu.M each
 of L-arginine, glycine, L-phenylalanine, L-serine and L-tyrosine; d) from
 about 60 to about 600 .mu.M each of L-histidine and L-methionine; e) from
 about 25 to about 250 .mu.M L-tryptophan; f) from about 25 to about 250
 .mu.M L-proline; g) from about 6 to about 60 .mu.M L-alanine; h) from
 about 3 to about 30 .mu.M L-cysteine; and i) from about 1.5 to about 15
 .mu.M each of L-asparagine and L-lysine. More preferably, the medium
 contains, in final concentration: a) from about 500 to about 1200 .mu.M
 each of L-isoleucine, L-leucine, L-threonine and L-valine; b) from about
 250 to about 750 .mu.M L-glutamine; c) from about 200 to about 600 .mu.M
 each of L-arginine, glycine, L-phenylalanine, L-serine and L-tyrosine; d)
 from about 100 to about 300 .mu.M each of L-histidine and L-methionine; e)
 from about 50 to about 125 .mu.M L-tryptophan; f) from about 55 to about
 90 .mu.M L-proline; g) from about 15 to about 30 .mu.M L-alanine; h) from
 about 7 to about 15 .mu.M L-cysteine; and i) from about 2.5 to about 7.5
 .mu.M each of L-asparagine and L-lysine.
 In a most preferred embodiment, the medium contains, in final
 concentration: a) 800 .mu.M each of L-isoleucine, L-leucine, L-threonine
 and L-valine; b) 500 .mu.M L-glutamine; c) 400 .mu.M each of L-arginine,
 glycine, L-phenylalanine, L-serine and L-tyrosine; d) 200 .mu.M each of
 L-histidine and L-methionine; e) 80 .mu.M L-tryptophan; f) 67 .mu.M
 L-proline; g) 20 .mu.M L-alanine; h) 10 .mu.M L-cysteine; and i) 5 .mu.M
 each of L-asparagine and L-lysine.
 A medium of the present invention further includes vitamins in amounts
 effective to sustain neural cell or tissue viability. The medium contains,
 in final concentration, from about 12 to about 120 .mu.M i-inositol, from
 about 10 to about 100 .mu.M niacinamide, from about 9 to about 90 .mu.M
 choline chloride, from about 6 to about 60 .mu.M pyridoxal, from about 3
 to about 30 .mu.M thiamine, from about 2.5 to about 25 .mu.M each of folic
 acid and D-Ca pantothenate, from about 0.3 to about 3 .mu.M riboflavin,
 and from about 0.06 to about 0.6 .mu.M vitamin B12. More preferably, the
 final concentration of vitamins is from about 20 to about 60 .mu.M
 i-inositol, from about 15 to about 50 .mu.M niacinamide, from about 20 to
 about 40 .mu.M choline chloride, from about 10 to about 30 .mu.M
 pyridoxal, from about 5 to about 15 .mu.M thiamine, from about 5 to about
 12 .mu.M each of folic acid and D-Ca pantothenate, from about 0.5 to about
 1.5 .mu.M riboflavin, and from about 0.1 to about 0.3 .mu.M vitamin B12.
 In an especially preferred embodiment, the medium contains, in final
 concentration, 40 .mu.M i-inositol, 30 .mu.M niacinamide, 28 .mu.M choline
 chloride, 20 .mu.M pyridoxal, 10 .mu.M thiamine, 8 .mu.M each of folic
 acid and D-Ca pantothenate, 1 .mu.M riboflavin, and 0.2 .mu.M vitamin B12.
 II. Process of Maintaining Neural Cell Viability
 A medium of the present invention has many uses, all of which are related
 to the ability of that medium to prolong survival of neural cells or
 tissues in a low CO.sub.2 environment.
 In one embodiment, the medium can be used to store neural cells or tissue.
 The cells, once obtained from any brain region, are placed in a medium and
 maintained under ambient CO.sub.2 conditions. By way of example, tissue
 from the central nervous system can be placed in a medium of the present
 invention and stored at reduced temperature (e.g., 2.degree. C. to
 15.degree. C.) The storage of neural cells or tissue is useful both in
 preparing such cells for experimental procedures and for maintaining cells
 prior to transplantation or implantation for therapeutic purposes.
 Exemplary such uses include ex vivo gene therapy, repair of damaged
 circuits, clonal expression and proliferating autologous or heterologous
 cells for transplant.
 A medium of the present invention can also be used to maintain the
 viability of plated neural cells that are undergoing study or
 manipulation. Thus, for example, cells can be maintained under normal
 ambient conditions during electrophysiological examination. In a similar
 fashion, neural cells or tissue placed in a medium of the present
 invention can be transfected or transformed with expression vectors
 carrying gene inserts. Once transformed, those cells can be implanted into
 various regions of the brain as part of a gene therapy regimen. A medium
 of the present invention can also be used as a perfusate or irrigant for
 central nervous system tissue during central nervous system operations.
 Use of a medium of the present invention has the advantage that neural
 cells or tissue can be studied or manipulated under normal, ambient
 conditions without exposing those cells or tissue to the deleterious
 effects of high CO.sub.2 levels and changes in pH. Still further, there is
 no need to continuously gas a medium of the present invention. Thus, use
 of a medium of the present invention results in substantial time and cost
 savings.
 When used to sustain or prolong neural cell or tissue viability, a medium
 of the present invention is preferably supplemented with a
 growth-promoting supplement that contains essential fatty acids, hormones
 and anti-oxidants needed for growth of the particular neural cells being
 cultured. Such growth-promoting media are well known in the art. By way of
 example, the present inventor has formulated a growth-promoting media for
 use with embryonic rat hippocampal cells. That medium is designated B27
 and is commercially available from GIBCO/Life Technologies, Inc.,
 Gaithersburg, Md. The growth promoting supplement, B27, contains essential
 fatty acids, hormones and anti-oxidants in amounts that optimize embryonic
 hippocampal neuron growth. The fatty acids, hormones and anti-oxidants in
 B27 are biotin, L-carnitine, corticosterone, ethanolamine, D(+)-galactose,
 reduced glutathione, linoleic acid, linolenic acid, progesterone,
 putrescine, retinyl acetate, selenium, triodo-1-thyronine, DL-.alpha.
 tocopherol, DL-.alpha. tocopherol acetate, bovine albumin, catalase,
 insulin, superoxide dismutase and transferrin.
 The present invention also provides a composition comprising neural cells
 or tissue placed in a medium as set forth above. The culture can further
 comprise a growth-promoting supplement as set forth above.
 The following Example illustrates preferred embodiments of the present
 invention and is not limiting of the specification and claims in any way.
 EXAMPLE 1
 Two basic types of experiments were conducted. In the first, rat embryonic
 day 18 hippocampal neurons were isolated and cultured as previously
 described at 160 cells/mm.sup.2 in B27-supplemented Neurobasal.TM. (Life
 Technologies, Inc., Gaithersburg, Md.) on 24-well plastic substrates
 coated with polylysine. After 4 days in culture at 37.degree. C. in 9%
 O.sub.2, 5% CO.sub.2, the entire medium was changed to a pre-warmed
 CO.sub.2 independent medium supplemented with B27 as indicated. The
 composition of that CO.sub.2 independent medium is set forth below in
 Table 1.
 TABLE 1
 Component Concentration in .mu.M
 inorganic salts
 CaCl.sub.2 (anhydrous) 1800
 Fe(NO.sub.3).sub.3 9H.sub.2 O 0.2
 KCl 5360
 MgCl.sub.2 (anhydrous) 812
 NaCl 76000
 NaHCO.sub.3 880
 NaH.sub.2 PO.sub.4 H.sub.2 O 900
 ZnSO.sub.4 7H.sub.2 O 0.67
 other components
 D-glucose 25000
 phenol red 23
 MOPS 10000
 sodium pyruvate 230
 amino acids
 L-alanine 20
 L-arginine HCl 400
 L-asparagine H.sub.2 O 5
 L-cysteine 10
 L-glutamine 500
 glycine 400
 L-histidine HCl H.sub.2 O 200
 L-isoleucine 800
 L-leucine 800
 L-lysine HCl 5
 L-methionine 200
 L-phenylalanine 400
 L-proline 67
 L-serine 400
 L-threonine 800
 L-tryptophan 80
 L-tyrosine 400
 L-valine 800
 vitamins
 D-Ca pantothenate 8
 choline chloride 28
 folic acid 8
 i-inositol 40
 niacinamide 30
 pyridoxal HCl 20
 riboflavin 1
 thiamine HCl 10
 vitamin B12 0.2
 Culture was continued at 37.degree. C. in a bacteriologic incubator at
 ambient O.sub.2 and CO.sub.2. The plates were placed in a Plexiglas
 plastic box adjacent to open dishes of water for hydration. At various
 times, plates were removed for counting live cells by staining with
 fluorescein diacetate and dead cells with propidium iodide. Survival was
 calculated as the number of live cells divided by the total cells
 (live+dead).
 The data in FIG. 1 show that HEPES, bicarbonate buffered Neurobasal.TM.
 supplemented with B27 produced rapid loss of neuron viability in ambient
 CO.sub.2, with an LD.sub.50 of less than 6 hr. When this medium was more
 appropriately buffered and the bicarbonate concentration reduced, much
 better survival was obtained. This new formulation (See Table 1) extended
 the LD.sub.50 beyond 24 hr. Slightly inferior results were obtained with a
 simple balanced salt solution (Hank's, Life Technologies, Inc. #14060-016)
 supplemented with 0.1% glucose, 1 .mu.M sodium bicarbonate, 1 .mu.M
 pyruvate, 10 .mu.M HEPES, pH 7.3. Further improvement in viability was
 obtained with the use of anti-oxidants, essential fatty acids, hormones
 and other ingredients as in the B27 supplement. With the B27 supplement,
 full viability was maintained for at least 2 days in culture in ambient
 CO.sub.2. The LDSO was extended beyond 3 days. Developed axons and
 dendrites showed little evidence of neurodegeneration such as retracted or
 beaded neurites or swollen somae. These neurons after 3 days in ambient
 CO.sub.2 were comparable to neurons cultured in 5% C.sub.2.
 Comparisons of viability and sprouting were also made for ambient and 5%
 CO.sub.2 conditions after one and four days in culture. After one day,
 viable cells in cultures started in 5% CO.sub.2 in B27-supplemented
 Neurobasal.TM. were 63.+-.10% of those plated (mean.+-.S.D., n=12) and
 54.+-.9% of these had neurite sprouts greater than one cell diameter. In
 ambient CO.sub.2 and the B27-supplemented medium of Table 1, viability on
 day 1 was reduced to 50.+-.7% (p&lt;0.05) and sprouting was reduced to
 22.+-.12% (p&lt;0.001). After 4 days, all cells were dead in the
 B27-supplemented medium of Table 1 in ambient CO.sub.2 while normal
 culture in B27-supplemented Neurobasal.TM. in 5% CO.sub.2 produced 71%
 viability. These data show that hippocampal neurons need a start in 5%
 CO.sub.2 to remain healthy.
 In the second type of experiment, hippocampi were dissected as usual, but
 placed in 2 ml of the medium of Table 1 supplemented with B27 in a 15 ml
 centrifuge tube (Corning, Oneonta, N.Y.). The tube was placed in a
 refrigerator at 8.degree. C. At various times, the tube was removed to a
 laminar flow hood at room temperature. One ml of the medium was removed
 and saved. The hippocampus in 1 ml medium was triturated by mild suction
 and expulsion 10 times (or until most pieces of tissue are dispersed)
 through the plastic tip of a 1 ml pipettor (Gilson, Middleton, Wis.). The
 volume was returned to 2 ml with the original medium. After letting
 undispersed pieces settle for 3 min., the supernatant was transferred and
 centrifuged for 1 min. at 200.times.G. The supernatant was discarded. To
 loosen the pellet, the tube was agitated by hand before resuspension in 1
 ml of B27-supplemented Neurobasal.TM. medium. After counting cells that
 excluded 0.2% trypan blue, cells were plated and cultured as above.
 The data in FIG. 2 show that hippocampal tissue can be stored for at least
 one month in the refrigerator before losing half of the surviving cells,
 measured after 4 days in normal culture at 5% CO.sub.2. These results are
 superior to prior studies reporting good survival after one week of
 storage, but little, if any, survival after 4 weeks in a low sodium, low
 calcium medium (Kawamoto et al, Brain Res., 384:84-93, 1986). Another
 medium sometimes used for trituration and storage is Leibovitz's L-15, a
 medium with high concentrations of amino acids (Leibovitz, Amr. J. Hyg.,
 78:173-180, 1963). The data show that the use of a medium of the present
 invention is superior to L-15 for refrigerated viable storage of
 hippocampal tissue. The yield of viable cells after 1 week of tissue
 hibernation in B27-supplemented medium of Table 1 was 1.3.+-.0.3 million
 cells/hippocampus (mean.+-.S.E., n=6). This is comparable to cell yields
 from fresh tissue.