Cultural medium for maintaining neural cells in ambient...

Details Cultural medium for maintaining neural cells in ambient... Cultural medium for maintaining neural cells in ambient...

1999-06-14

2001-01-30

Tate, Christopher (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Culture medium, per se

C435S325000, C435S366000, C435S368000, C435S374000, C435S404000, C435S406000, C424S093700

Reexamination Certificate

active

06180404

ABSTRACT:

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
2
) has recently been developed. That medium, Neurobasal™ (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
2
. Neurobasal™ 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™ has less NaCl and less NaHCO
3
, resulting in a lower osmolality, and lesser amounts of cysteine and glutamine, resulting in diminished glial growth. In addition, Neurobasal™ contains alanine, asparagine, proline and vitamin B12, all of which are absent from DMEM.
Although neurons can be maintained in a 5% CO
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
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
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
2
to maintain physiological pH.
A simple test, however, shows that ambient CO
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
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
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
2
. The medium contains less than about 2000 &mgr;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 &mgr;M CaCl
2
, 0.05 to about 0.8 &mgr;M Fe(NO
3
)
3
, 2500 to about 10000 &mgr;M KCl, 0 to about 4000 &mgr;M MgCl
2
, 74000 to about 81000 &mgr;M NaCl, 400 to about 2000 &mgr;M NaHCO
3
, 250 to about 4000 &mgr;M NaH
2
PO
4
, 0.2 to about 2 &mgr;M ZnSO
4
, 2500 to about 5000 &mgr;M glucose, 0 to about 100 &mgr;M phenol red, and 23 to about 500 &mgr;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 &mgr;M CaCl
2
, 0.05 to about 0.8 &mgr;M Fe(NO
3
)
3
, 2500 to about 10000 &mgr;M KCl, 0 to about 4000 &mgr;M MgCl
2
, 86000 to about 103000 &mgr;M NaCl, 400 to about 2000 &mgr;M NaHCO
3
, 250 to about 4000 &mgr;M NaH
2
PO
4
, 0.2 to about 2 &mgr;M ZnSO
4
, 2500 to about 5000 &mgr;M glucose, 0 to about 100 &mgr;M phenol red, and 23 to about 500 &mgr;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 &mgr;M each of L-isoleuoine, L-leucine, L-threonine and L-valine; b) from about 150 to about 1500 &mgr;M L-glutamine; c) from about 120 to about 1200 &mgr;M each of L-arginine, glycine, L-phenylalanine, L-serine and L-tyrosine; d) from about 60 to about 600 &mgr;M each of L-histidine and L-methionine; e) from about 25 to about 250 &mgr;M L-tryptophan; f) from about 25 to about 250 &mgr;M L-proline; g) from about 6 to about 60 &mgr;M L-alanine; h) from about 3 to about 30 &mgr;M L-cysteine; and i) from about 1.5 to about 15 &mgr;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 &mgr;M i-inositol, from about 10 to about 100 &mgr;M niacinamide, from about 9 to about 90 &mgr;M choline chloride, from about 6 to about 60 &mgr;M pyridoxal, from about 3 to about 30 &mgr;M thiamine, from about 2.5 to about 25 &mgr;M each of folic acid and D-Ca pantothenate, from about 0.3 to about 3 &mgr;M riboflavin, and from about 0.06 to about 0.6 &mgr;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
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-&agr; tocopherol, DL-&agr; 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.


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patent: 4873230 (1989-10-01), Belzer et al.
patent: 4879283 (1989-11-01), Belzer et al.
patent: 5316938 (1994-05-01), Keen et al.
patent: 5716847 (1998-02-01), Simmons et al.
G.J. Brewer, Serum-Free B27/Neurobasal Medium Supports Differentiated Growth of Neurons From the Striatum, Substantia Nigra, Septum, Cerebral Cortex, Cerebellum, and Dentate Gyrus, Journal of Neuroscience Research 42;674-683 (1995).
G.T. Spierenburg, G.T.J.J. Oerlemans, J.P.R.M. Van Laarhoven, and C.H.M.M. De Bruyn, Phototoxicity of N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic Acid-buffered Culture Media for Human Leukemic Cell Lines, Cancer Research, May 1984, 44, 2253-2254.
Jerrolynn C. Kawamoto and John N. Barrett, Cryopreservation of Primary Neurons for Tissue Culture, Brain Research, 1986, 384 (1986) 84-93.
Albert Leibovitz, The Growth And Maintenance Of Tissue-Cell Cultures In Free Gas Exchange With The Atmosphere, Am.J.Hyg. 1963, vol. 78; 173-180.
C. Ward Kischer, A. Leibovitz and J. Pind

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