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Hormonal requirements for neuronal survival in culture
Neuroscience Letters, 13 (1979) 225--230 O Elsevier/North-Holland Scientific Publishers Ltd.
225
HORMONAL REQUIREMENTS FOR NEURONAL SURVIVAL IN CULTURE E.Y. SNYDER and S.U. KIM
Dwision of Neuropathoiogy, Department of Pathology, University of Pennsylvania, School o f Med'w~me, Philadelphia, PA 19104 (U.S.A.) (Received March 22rid, 1979) (Revised version received April 26th, 1979) (Accepted April 26th, 1979)
SUMMARY
The inabilityto grow neurons in culture in the absence of complex and undefined biological fluids (e.g. serum) has proved a major obstacle to a rigorous formulation of the growth, nutritionaland hormonal requirements of the developing nervous system. W e have been successful in maintaining dissociated chick aorsal root ganglia neurons in a serum-free, defined medium composed of F12 synthetic medium and, substitutingfor sexum, a combination of hormones (insulin,PTH, triiodothyronine, TRH, somatomedin, hydrocortis0ne, testosterone) and other factors (tr~sferrin). Not only were these hormones found to be sufficientfor the maintenance of neurons in vitro,but, by the selectiveelimination of one component from the mixture, the role of specifichormones in neutral development could be grossly assessed. The elimination of insulin proved to be most inimical to neuronal survival.
Despite one unquantifled and uncorroborated report in the older literature [13], it is generally acknowledged that synthetic media alone (even when supplemented with Nerve Growth Factor (NGF)) fail to support the in vitro survival of adequate numbers of mature, differentiated neurons [14--16]. The inability to maintain neural tissue in culture in the absence of complex and poorly defined biological fluid supplements (e.g. sera, embryonic extracts) has proved a major obstacle to a rigorous investigation of the nutritional, hormonal and survival requirements of the developing nervous system. We now report, however, the successful maintenance of chick dorsal root ganglion (DRG) neurons in a serum-free, chemically defined medium composed of Ham's F12 synthetic medium [10] and a combination of hormones and other factors. Not only were these hormones fou~td to be sufficient for the z~taintenance of neurons in culture, but, by
226
the selective elimination of components from the mixture, the role o f specific hormones in neuronal sundval could be grossly assessed. The recen'~ report by Hayashi and Sato [ 11] that a p ' ~ tumor cell line could be supported in vitro by a specific combination of hormones in lieu of serum and the realization that different cell types most likely have different hormone requirements, had stimulated us to formulate our own hormone 'cocktail'. The hormones chosen were those that (a)have been implicated in the past as being of importance to neunfl development (e.g. triiodothyronine [8]), (b) have neural actions difficult to assess in vivo (e.g. hydrocortisone -- it is difficult to study the effects of adrenal corticosteroids in the neonate ~nce adrenalectomy stops all growth almost completely), (c) have been found to have an unexplained pv~ence in various loci of the nervous system (e.g. thyrotropin releasing hormone [20]) or (d) were found to be valuable to Hayashi and Sato [ 11] in growing pituitary tumor cells (e.g. somatomedins A and C, tran~errin). Lumbar and thoracic dorsal root ganglia were aseptically dissected from 9--11~1ay embryonic chicks and dissociated in 0.25% trypsin (in Ca 2 * Mg2*-free Hank's balanced salt solution (BSS)) at 37°C for 30 rain. Having washed the cells 3 times in Ham's F12 synthetic medium [ 10], with aadition on the last wash of a so/bean trypsin inhibitor (6.67 mg/ml, Sigma), cel~ were further dissociate~i by gentle pipetting and cen1~Yugation at 1000 rev./min for 5 min. Th~ pellet was resuspended in F12 and seeded onto collagen~oated glass coverslips [ 1] at a density of approx. 500,000 cells/ml. A homogeneo~:s distribution was sought by gentle stirring of cel~ with a gl~ss rod befor, they settled. Each coverslip wm transferred to an individual 35-mm plastic petri dish, incubated at 37°C in a 5% CO2-alr atmosphere and fed, every 3--4 days, one of three media: (a) Har~'s F12 alone (F12 consisting of nothing more than a balanced salt solution, amino acids, glucose, vitamins) [ 10], (b) F12 plus 10~ fetal calf serum (FCS), or (c) F12 supplemented, not with serum, but with the following hormone 'cocktail': insulin (50/~g/ml), triidothyronine (T3) (3 • 10 -1° M), thyrotropin releasing hormone (TRH) (1 • 10 -9 M), somatomedin A and C preparation (50 mU/ml), hydrocortisone (2- 10 -~ M), testosterone ( 2 . 1 0 -9 M), parathyroid hormone (PTH) t0.5 ng/ml), transfenfin (5/~2/ml). Eight additional groups of sister cuitures were fed a 'cocktail' from which one component -- a different component for each group -- was selectively removed. A final control group was fed Hanks' BSS alone. All cultures were observed daily through an inverted phase microscope (at 100X ) (Fig. 1) and direct cell counts performed after 1 week on cultures fixed and stained by a modification of the Bodian-protargol method [12] (Table I). Following I week in vitro, serum-fed control cultures evinced, as expected, abundant round, phase bright and refractile neurons bearing processes which branched into extensive networks. The neurons were distributed upon a nearly confluent layer of non-neuronal cells (mostly fibroblasts) (Fig. 1A). In F12 alone, virtually no neurons and very few non-neuronal cells survived
r:
i
Fig. 1. Living dorsal root ganglion (DRG) neurons 7 days in vitro, viewed through the pJuwe contrast microscope (100X). Cultures were prepared as described in the text, different groups of cultures receiving the various experimental feeding media under investigation. Photomicrographs of representative cultures from some of these groups are presented here (see text for descriptions): (A)F12 + 10% fetal calf serum; (B)F12 alone; (C) F12 • hormone 'cocktail' (see text for ingredients); (D) F12 + 'cocktail' minus somatomedins A and C; (E)F12 + 'cocktail' minus triiodothyronine; (F) F12 + 'cocktail' minus insulin; n, neuron; f, filzroblag. (Fig. 1B). In F 1 2 plus the 'cocktail', however, neurons were seen which resembled tho~e o f serum-fed cultures in their abundance, healthy appearance and d e v e l o p m e n t of processes (Fig. 1C). The striking diff,erence between serum-fed and serum-free cultures was the conspicuously diminished n u m b e r
228 TABLE I NEURONAl, SURVIVAL IN VARIOUS CULTURE MEDIA-- DATA FROM DIRECT CELL COUNTS After I week in vitro, ~ltm'm were stained by • modifimtioa of the Bodim-tpmtml~ metimd [12] and direct counts of neurom perfm'm~ und~ 500x. ~ eemtts listed repr,~nt, the m~m vslum from 3--6 m p l k ~ eultm~ in ,meh ~ zmd/l~n from 2n of ~lm'Jm~ts. ~
sm~dwl
F12 + 109~,fetal calf serum FI2 ; fu~l'~ocktm3'
100~a ( + + + + ) b 9~r, ( + ÷ + + )
F12 + (eoektaU min,.~ triiodothyrmfiae ) F12 + (eoektail mim~tomatomedim Aand C)
1~ (+ + + + ) 93% (++++)
FI2 + (cocktail m;nm h y ~ o c f i s m w ) F12 + (eoektm'lminus'rRH) F12 + (eoek'..ail miaus PrH)
106% (++++) 99~ (++++) ~5~ (+ + + + )
F12 + ( e o e k t a i i m i ~ m ~ o n e ) F12 + (eoekudl mlmm t n m s f ~ ) F12 + (eoektafl minus ~ ) F12 ~)me e~SS,Jo.e
95% ( + + + + ) 105~, ( + + + + ) 9% (+) 6% (+) 0~ (0)
•
i
iiiii
[i[
J
i
ii
ii i
i
.
.
.
.
.
.
.
.
• To eompere data from multiple ezpedmmt8 sad to matrol for e ~ m 4 z t ~ h m a t vadatlou, the neuron eount in say i~vu cuitm~ is ezpnmed m petm atsp of that mesa total noted in s~mm-fed cultures of the same e x i t . 'l'n~, by d M ~ o n , ~ in ser,m eq~ds 100%. bBeeau~ cell counts cannot be compsrod dgorom~, ~ m ~ on t/ae b~Js of them eotmtl seeor~mg to the foUowlall seek: + + + + , 90--l10'g; ++ +, 60-BP~,; + + , 20--59%; +, 1--19%.
o f fibrobksts. Cult~re~ in which one o f the 'cocktail' i n p e d k m t e w u withheld were euent/afiy indistingukhable under I/ght microscopic ~ y n o t only from the total 'cocktail', not only from esch other, b u t from N m m / t ~ e l f , (e4~. see Figs. I D and 1E), with one dramatic except/on: t h e ~ l y poor survival of neurons in culture deprived o f insulin b u t s u p p l k d the .nmmh~ler o f the hormones (Fig. 1F). Here, neurons failed to send pt~z~emm and d e S ~ erated. A second, more subtle difference one may note between seru_m-fed and serum-free cultures is the [p~ater degree o f clumping o f neurons seen in the latter. In the face o f fewer fibroblssts, upon which the neurons would ordinarily settle, the neurons tend to cling more to each other. 8inee single n e u r o m can neverthelees be essily dist/niplished in B o d i a n 4 t s / n ~ material under h i ~ p o w e r (X 500), cell counts were n o t affected by this phenomenon. That a combination of essential hormones can substitute so satisfactoriaUy for serum leads us to conclude, as did Hayashi and Sato [11], that the putpose o f serum in tissue culture med/a is to provide hormones, and that t h e serum requirement o f cells in culture is in reality a h o r m o n e requirement, which is met b y serum. Bam~ solely on an exam/nat/on of m o r p h o l o ~ c a l detail - - and t h a t at t h e light microscopic level -- it is d/fficult to draw conclusions concerning the relative importance of the various components o f the
229
'cocktail'. More extensive investigations employing biochemical measures and morphometric analyses of electron-microscopic data are indicated to detect nuances, if they exist, among the effects of the various hormonal deficiencies. At our level of,~solution, one can merely screen for the necessity and sufficiency of substances for the survival and differentiation of neurons. Cast in these fairly a l l , r - n o n e term a, there appears to be an absolute necessity for insulin in serum-free media. Cultures without insulin, even when fed the other hormones, failed to survive. The effect of insulin deprivation, while not entirely surprising (in view of previous studies which report a higher proportion of well-maintained cultures in insulin-supplemented serum media [2,9,18] ) is so dramatic as to demand further, rigorous investigation. Insulin's growth-promoting properties have been recognized in many non-neuronal cell types [7,19]. Further, the presence of factors in serum with insulin-like activity is well documented [6]. Also, the known insulin-like action and structural homology with insulin of NGF [2,5] and other growth factors [ 3] makesone suspect that the overlap between the substances is far from coincidental; insulin may have very important growth promoting effects in the early stages of neurogenesis in pezipheral and pezhaps even in central, nervous system (PNS, CNS) Perhaps insulinis one of the yet-to-be~liscovered nerve survival factors. Future investigations will seek to define the absolute minimal supplementation necessary for neuronal survival. The absence of detectable impairment in T3-free cultures supports previous in vivo studies in which the effect of thyroid hormone deficiency in the PNS has been ambiguous or minimal [4,17]. One might expect a greater deficit in dissociated CNS tissue in view of the documented relationship between mental retardation and hypothyroidism and reports in vivo or impaired neurite and synapse formation in hypothyroid animals [.8]. It would not be surprising to find differences between the hormone requirements of the PNS and the CNS.
An u n e x p e c t ~ finding in light of the dramatic effect reported by Hayashi and Sato [ 11] was the relatively unremarkable effect of somatomedin deprivation in neural cultures. This finding, however, serves to highlight the fact that different tiuue (and, indeed, the same tissues of different species) may well be found to have different hormone requirements, though som~ requirements may be common to all cells. 8upp~ fibroblMt and non.neuronal proliferation in the face of adequate neuronal survival would seem to indicate that the hormone mixture described is selective for neurons. While not dependent solely on serum for their survival, f i b r o b l a ~ apparently require mitogenic factors supplied by serum for their continued proliferation. That fibroblasts were most likely not secreting a neuron-promoting factor in our 'cocktail'-supported cultures is evident from the fact that the presence of fibroblasts was not sufficient Jn F12 alone to promote the survival of neurons. ACKNOWLEDGEMENTS We thank Dr. K.O. Uthne for generously providing us with the sornato-
230
merlin A and C p i t o n . This work was supported by gr~nts GM-02046, NS.10648, and KO4 N8-00151 from USPHS. REFER~ 1 Bomstein, M.B., Reeonsl~uted mt tadl collagen mind m m ~ for tisme eulturu on eovendips in Maximo~ tildes and roller tubes, Lab. Iuvmt., 7 (1958) 134--137. 2 Burnlmm, P.A., BiI~,J.A. and V~on,8., Ansbolkrespolmmof e m h r y ~ ~,4,J~miroot p n g l k to nerve growth faet~, immlin, eonemurral~ A or serum in vitro, J. Neuro¢Jmn., 2S (19V4) ~S~--695. 3 Choehinov, R.H. msd r_~m4hsdsy, WAL, Currtmt ~ of soms/omedin and other
b ~ y
rekt~i ~
f~,
~ ,
25 (I~76) 994--1007.
4 Clos, L sad L ~ , J., ~ of thyrmd def~,eney sad u n d ~ oQ growth and n~yellnatkm of the sciatic nerve fibers in the young rat. An electron ~ ~ N : d c study, BraJn P.m., 22 (19'70) 285--297. 5 Frazisr, W.A., Anf~ett, i, R.IL and ]~-Kbhlw, R.A., Nerve ~ htct~ and insu-n: stru~..~r~ ~ indicate an evo'm~ionsry r e h ~ ~ reflected by physiologic~ sctio'~, P~%enee, 176 (1972) 482--488. 6 F r o t ~ h , S.R., Burgi, I-L, Muller W., Humbel, R.E., Jakob, A. and Labhort, A., Nonsupp~euive insulin4ike activity of human serum: purification, physiochemical and biological properties and it8 relation to total Nrum ILA, ~ Progr. Horm. Res., 23 (]967) 568--616. 7 Go" p o d ~ , D. and Moran, J.8., Growth factors in mammalian cell culture, Ann. _R.e: Bic~b._..-n'...,45 (1976) 531~558. 8 Cra-;e, G.D., (Ed.), Thyroid Hormones and Brain Development, Raven Press, New York, 1977. 9 (;uIIlery, R.W. 8obkowlez, H. and Scott, G., Light and electron microscopical observation of the ventral horn and ventral root in long-term cultures of the spinal cord of the fetal m u s e , J. comp. Neurol., 134 (1968) 433--476. 10 Ham, R.G., Clonal growth of mammalian cells in a ¢hemte~ly &fined, synthetic medium, Pro¢. Nat. Acad. ,%;. (USA), 53 (1965) 288--293. 11 Hay~hl, I. and Sato, G.H., Replacement of serum by hormones permita growth of cells in a defined rm~ium, Nature (Load.), 259 (1976) 132--134. 12 Kim, S.U., Oboervation8 on cerebellqr granule cells in vitro. A silver and electron microscopic study, Z. Zellforsch., 107 (1970) 454-465. 13 Levi-Montalcini, R. and Angeletti, P.U., Ementlal role of the nerve growth factor in the survival and maintmmm~ of dkeociatsd ~ and sympathetic ~mbryonic nerve cells m vitro, l~velop. Biol., 7 (1963) 663--659. 14 Ludetwa, M.A., The growth of spinal [pmgiion mmrons in umun-free n~dium, D~elop. B,ol., 33 (1973) 470--476. 15 Murray, M.R., Nervous tissue in vitro. In E.N. WiIImer (Ed.), Celk and '.rksues in Cub ture, Vol. 2, Academic Press, New York, 1965, pp. 373--455. 16 Peterson, E.R. and Murray, M.R., Modification of development in isolated dorsal root ganglia by nutritional and physical factors, Develop. Biol., 2 (1960} 461-476. 17 Reier, PJ. and Hughes, A.F., An effect of neonatsl radiothyroidectomy upon nonm y e l i n a ~ axons and auo¢/ated Schwann calls during matt, ration of the mouse sciatic nerve, Brain R~., 41 (1972) 263--282. 18 Silberberg, D., Phenylketonuria metabolites in cerebellum culture morphology, Arch. Neurol., 17 (1967) 524--529. 19 Waymouth, C., The nutrition of animal cells, Int. Rev. Cytol., 3 (1954) 1--68. 20 Winokur, A. and Utiger, R., Thyrotropin-releasing hormone: regional distribution in rat brain, Science, 18r~ (1974) 265--267.
225
HORMONAL REQUIREMENTS FOR NEURONAL SURVIVAL IN CULTURE E.Y. SNYDER and S.U. KIM
Dwision of Neuropathoiogy, Department of Pathology, University of Pennsylvania, School o f Med'w~me, Philadelphia, PA 19104 (U.S.A.) (Received March 22rid, 1979) (Revised version received April 26th, 1979) (Accepted April 26th, 1979)
SUMMARY
The inabilityto grow neurons in culture in the absence of complex and undefined biological fluids (e.g. serum) has proved a major obstacle to a rigorous formulation of the growth, nutritionaland hormonal requirements of the developing nervous system. W e have been successful in maintaining dissociated chick aorsal root ganglia neurons in a serum-free, defined medium composed of F12 synthetic medium and, substitutingfor sexum, a combination of hormones (insulin,PTH, triiodothyronine, TRH, somatomedin, hydrocortis0ne, testosterone) and other factors (tr~sferrin). Not only were these hormones found to be sufficientfor the maintenance of neurons in vitro,but, by the selectiveelimination of one component from the mixture, the role of specifichormones in neutral development could be grossly assessed. The elimination of insulin proved to be most inimical to neuronal survival.
Despite one unquantifled and uncorroborated report in the older literature [13], it is generally acknowledged that synthetic media alone (even when supplemented with Nerve Growth Factor (NGF)) fail to support the in vitro survival of adequate numbers of mature, differentiated neurons [14--16]. The inability to maintain neural tissue in culture in the absence of complex and poorly defined biological fluid supplements (e.g. sera, embryonic extracts) has proved a major obstacle to a rigorous investigation of the nutritional, hormonal and survival requirements of the developing nervous system. We now report, however, the successful maintenance of chick dorsal root ganglion (DRG) neurons in a serum-free, chemically defined medium composed of Ham's F12 synthetic medium [10] and a combination of hormones and other factors. Not only were these hormones fou~td to be sufficient for the z~taintenance of neurons in culture, but, by
226
the selective elimination of components from the mixture, the role o f specific hormones in neuronal sundval could be grossly assessed. The recen'~ report by Hayashi and Sato [ 11] that a p ' ~ tumor cell line could be supported in vitro by a specific combination of hormones in lieu of serum and the realization that different cell types most likely have different hormone requirements, had stimulated us to formulate our own hormone 'cocktail'. The hormones chosen were those that (a)have been implicated in the past as being of importance to neunfl development (e.g. triiodothyronine [8]), (b) have neural actions difficult to assess in vivo (e.g. hydrocortisone -- it is difficult to study the effects of adrenal corticosteroids in the neonate ~nce adrenalectomy stops all growth almost completely), (c) have been found to have an unexplained pv~ence in various loci of the nervous system (e.g. thyrotropin releasing hormone [20]) or (d) were found to be valuable to Hayashi and Sato [ 11] in growing pituitary tumor cells (e.g. somatomedins A and C, tran~errin). Lumbar and thoracic dorsal root ganglia were aseptically dissected from 9--11~1ay embryonic chicks and dissociated in 0.25% trypsin (in Ca 2 * Mg2*-free Hank's balanced salt solution (BSS)) at 37°C for 30 rain. Having washed the cells 3 times in Ham's F12 synthetic medium [ 10], with aadition on the last wash of a so/bean trypsin inhibitor (6.67 mg/ml, Sigma), cel~ were further dissociate~i by gentle pipetting and cen1~Yugation at 1000 rev./min for 5 min. Th~ pellet was resuspended in F12 and seeded onto collagen~oated glass coverslips [ 1] at a density of approx. 500,000 cells/ml. A homogeneo~:s distribution was sought by gentle stirring of cel~ with a gl~ss rod befor, they settled. Each coverslip wm transferred to an individual 35-mm plastic petri dish, incubated at 37°C in a 5% CO2-alr atmosphere and fed, every 3--4 days, one of three media: (a) Har~'s F12 alone (F12 consisting of nothing more than a balanced salt solution, amino acids, glucose, vitamins) [ 10], (b) F12 plus 10~ fetal calf serum (FCS), or (c) F12 supplemented, not with serum, but with the following hormone 'cocktail': insulin (50/~g/ml), triidothyronine (T3) (3 • 10 -1° M), thyrotropin releasing hormone (TRH) (1 • 10 -9 M), somatomedin A and C preparation (50 mU/ml), hydrocortisone (2- 10 -~ M), testosterone ( 2 . 1 0 -9 M), parathyroid hormone (PTH) t0.5 ng/ml), transfenfin (5/~2/ml). Eight additional groups of sister cuitures were fed a 'cocktail' from which one component -- a different component for each group -- was selectively removed. A final control group was fed Hanks' BSS alone. All cultures were observed daily through an inverted phase microscope (at 100X ) (Fig. 1) and direct cell counts performed after 1 week on cultures fixed and stained by a modification of the Bodian-protargol method [12] (Table I). Following I week in vitro, serum-fed control cultures evinced, as expected, abundant round, phase bright and refractile neurons bearing processes which branched into extensive networks. The neurons were distributed upon a nearly confluent layer of non-neuronal cells (mostly fibroblasts) (Fig. 1A). In F12 alone, virtually no neurons and very few non-neuronal cells survived
r:
i
Fig. 1. Living dorsal root ganglion (DRG) neurons 7 days in vitro, viewed through the pJuwe contrast microscope (100X). Cultures were prepared as described in the text, different groups of cultures receiving the various experimental feeding media under investigation. Photomicrographs of representative cultures from some of these groups are presented here (see text for descriptions): (A)F12 + 10% fetal calf serum; (B)F12 alone; (C) F12 • hormone 'cocktail' (see text for ingredients); (D) F12 + 'cocktail' minus somatomedins A and C; (E)F12 + 'cocktail' minus triiodothyronine; (F) F12 + 'cocktail' minus insulin; n, neuron; f, filzroblag. (Fig. 1B). In F 1 2 plus the 'cocktail', however, neurons were seen which resembled tho~e o f serum-fed cultures in their abundance, healthy appearance and d e v e l o p m e n t of processes (Fig. 1C). The striking diff,erence between serum-fed and serum-free cultures was the conspicuously diminished n u m b e r
228 TABLE I NEURONAl, SURVIVAL IN VARIOUS CULTURE MEDIA-- DATA FROM DIRECT CELL COUNTS After I week in vitro, ~ltm'm were stained by • modifimtioa of the Bodim-tpmtml~ metimd [12] and direct counts of neurom perfm'm~ und~ 500x. ~ eemtts listed repr,~nt, the m~m vslum from 3--6 m p l k ~ eultm~ in ,meh ~ zmd/l~n from 2n of ~lm'Jm~ts. ~
sm~dwl
F12 + 109~,fetal calf serum FI2 ; fu~l'~ocktm3'
100~a ( + + + + ) b 9~r, ( + ÷ + + )
F12 + (eoektaU min,.~ triiodothyrmfiae ) F12 + (eoektail mim~tomatomedim Aand C)
1~ (+ + + + ) 93% (++++)
FI2 + (cocktail m;nm h y ~ o c f i s m w ) F12 + (eoektm'lminus'rRH) F12 + (eoek'..ail miaus PrH)
106% (++++) 99~ (++++) ~5~ (+ + + + )
F12 + ( e o e k t a i i m i ~ m ~ o n e ) F12 + (eoekudl mlmm t n m s f ~ ) F12 + (eoektafl minus ~ ) F12 ~)me e~SS,Jo.e
95% ( + + + + ) 105~, ( + + + + ) 9% (+) 6% (+) 0~ (0)
•
i
iiiii
[i[
J
i
ii
ii i
i
.
.
.
.
.
.
.
.
• To eompere data from multiple ezpedmmt8 sad to matrol for e ~ m 4 z t ~ h m a t vadatlou, the neuron eount in say i~vu cuitm~ is ezpnmed m petm atsp of that mesa total noted in s~mm-fed cultures of the same e x i t . 'l'n~, by d M ~ o n , ~ in ser,m eq~ds 100%. bBeeau~ cell counts cannot be compsrod dgorom~, ~ m ~ on t/ae b~Js of them eotmtl seeor~mg to the foUowlall seek: + + + + , 90--l10'g; ++ +, 60-BP~,; + + , 20--59%; +, 1--19%.
o f fibrobksts. Cult~re~ in which one o f the 'cocktail' i n p e d k m t e w u withheld were euent/afiy indistingukhable under I/ght microscopic ~ y n o t only from the total 'cocktail', not only from esch other, b u t from N m m / t ~ e l f , (e4~. see Figs. I D and 1E), with one dramatic except/on: t h e ~ l y poor survival of neurons in culture deprived o f insulin b u t s u p p l k d the .nmmh~ler o f the hormones (Fig. 1F). Here, neurons failed to send pt~z~emm and d e S ~ erated. A second, more subtle difference one may note between seru_m-fed and serum-free cultures is the [p~ater degree o f clumping o f neurons seen in the latter. In the face o f fewer fibroblssts, upon which the neurons would ordinarily settle, the neurons tend to cling more to each other. 8inee single n e u r o m can neverthelees be essily dist/niplished in B o d i a n 4 t s / n ~ material under h i ~ p o w e r (X 500), cell counts were n o t affected by this phenomenon. That a combination of essential hormones can substitute so satisfactoriaUy for serum leads us to conclude, as did Hayashi and Sato [11], that the putpose o f serum in tissue culture med/a is to provide hormones, and that t h e serum requirement o f cells in culture is in reality a h o r m o n e requirement, which is met b y serum. Bam~ solely on an exam/nat/on of m o r p h o l o ~ c a l detail - - and t h a t at t h e light microscopic level -- it is d/fficult to draw conclusions concerning the relative importance of the various components o f the
229
'cocktail'. More extensive investigations employing biochemical measures and morphometric analyses of electron-microscopic data are indicated to detect nuances, if they exist, among the effects of the various hormonal deficiencies. At our level of,~solution, one can merely screen for the necessity and sufficiency of substances for the survival and differentiation of neurons. Cast in these fairly a l l , r - n o n e term a, there appears to be an absolute necessity for insulin in serum-free media. Cultures without insulin, even when fed the other hormones, failed to survive. The effect of insulin deprivation, while not entirely surprising (in view of previous studies which report a higher proportion of well-maintained cultures in insulin-supplemented serum media [2,9,18] ) is so dramatic as to demand further, rigorous investigation. Insulin's growth-promoting properties have been recognized in many non-neuronal cell types [7,19]. Further, the presence of factors in serum with insulin-like activity is well documented [6]. Also, the known insulin-like action and structural homology with insulin of NGF [2,5] and other growth factors [ 3] makesone suspect that the overlap between the substances is far from coincidental; insulin may have very important growth promoting effects in the early stages of neurogenesis in pezipheral and pezhaps even in central, nervous system (PNS, CNS) Perhaps insulinis one of the yet-to-be~liscovered nerve survival factors. Future investigations will seek to define the absolute minimal supplementation necessary for neuronal survival. The absence of detectable impairment in T3-free cultures supports previous in vivo studies in which the effect of thyroid hormone deficiency in the PNS has been ambiguous or minimal [4,17]. One might expect a greater deficit in dissociated CNS tissue in view of the documented relationship between mental retardation and hypothyroidism and reports in vivo or impaired neurite and synapse formation in hypothyroid animals [.8]. It would not be surprising to find differences between the hormone requirements of the PNS and the CNS.
An u n e x p e c t ~ finding in light of the dramatic effect reported by Hayashi and Sato [ 11] was the relatively unremarkable effect of somatomedin deprivation in neural cultures. This finding, however, serves to highlight the fact that different tiuue (and, indeed, the same tissues of different species) may well be found to have different hormone requirements, though som~ requirements may be common to all cells. 8upp~ fibroblMt and non.neuronal proliferation in the face of adequate neuronal survival would seem to indicate that the hormone mixture described is selective for neurons. While not dependent solely on serum for their survival, f i b r o b l a ~ apparently require mitogenic factors supplied by serum for their continued proliferation. That fibroblasts were most likely not secreting a neuron-promoting factor in our 'cocktail'-supported cultures is evident from the fact that the presence of fibroblasts was not sufficient Jn F12 alone to promote the survival of neurons. ACKNOWLEDGEMENTS We thank Dr. K.O. Uthne for generously providing us with the sornato-
230
merlin A and C p i t o n . This work was supported by gr~nts GM-02046, NS.10648, and KO4 N8-00151 from USPHS. REFER~ 1 Bomstein, M.B., Reeonsl~uted mt tadl collagen mind m m ~ for tisme eulturu on eovendips in Maximo~ tildes and roller tubes, Lab. Iuvmt., 7 (1958) 134--137. 2 Burnlmm, P.A., BiI~,J.A. and V~on,8., Ansbolkrespolmmof e m h r y ~ ~,4,J~miroot p n g l k to nerve growth faet~, immlin, eonemurral~ A or serum in vitro, J. Neuro¢Jmn., 2S (19V4) ~S~--695. 3 Choehinov, R.H. msd r_~m4hsdsy, WAL, Currtmt ~ of soms/omedin and other
b ~ y
rekt~i ~
f~,
~ ,
25 (I~76) 994--1007.
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