- Email: [email protected]
[19] Mammalian nerve cell culture
162
ANIMAL ORGAN AND CELL PREPARATIONS
[19]
tal, aryl hydrocarbons, and ingested toxins. 45-47 Therefore techniques for the isolation of villous and crypt cells may be of value in studying not only the development of drug metabolizing systems but also their mechanism(s) of induction in the gut. 4~ E. Heitanen, M. Laitinan, and U. Koivusaari, Enzyme 25, 153 (1980). ~ C. M. Schiller, Environ. Health Perspect. 33, 91 (1979). 47 S y m p o s i u m on "Target Organ Toxicity: I n t e s t i n e " (C. M. Schiller, g u e s t ed.), in Environ. Health Perspect. 33, 1-126 (1979).
[19]
Mammalian
Nerve Cell Culture
B y W. DIMPFEL
Tissue culture offers a unique possibility for combining the advantages of experimentally well-controlled in vitro conditions with the advantage of working with living cells. Nevertheless, a great number of methodological difficulties had to be overcome with neuronal tissue before the technique could advance from a mere morphological description of living tissue in vitro 1 to a model system that allows meaningful statements about the function of the nervous system. 2 The technical procedure for obtaining reproducible primary nerve cell cultures as described herein has been successfully applied in several laboratories for more than 6 years. It can be regarded as the further development of the successfully introduced, and already widely used, explant cultures. 3-5 Standard Procedure The close interrelations between different cell types within the central nervous system (CNS) and the extensive arborization of neurites in the adult account for the difficulty in dissociating tissue into a single cell suspension that could be plated in culture dishes. Therefore, embryonic tissue is best suited for culturing purposes. The embryos (12-14 days in utero for mice; 14-16 days for rats) are obtained under sterile conditions by cesarean section and placed under a stereo microscope at low magnification into prewarmed buffered saline, preferably Ca 2+ free. I R. G. Han'ison, Anat. Rec. 1, 116 (1906). 2 W. Dimpfel, Arch. Toxicol. 44, 55 (1980). a W. Hild and I. Tasaki, J. Neurophysiol. 25, 277 (1962). S. M. Crain and E. R. Peterson, J. Cell. Comp. Physiol. 64, 1 (1964). 5 R. P. Bunge, M. B. Bunge, and E. R. Peterson, J. Cell Biol. 24, 163 (1965).
METHODS IN ENZYMOLOGY,VOL. 77
Copyright © 1981 by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181977-9
[19]
NERVE CELLS
163
The brain or portions of the CNS are excised by means of iridectomy scissors and watchmaker forceps, placed into a small separate dish together with a drop of saline, and minced until the tissue pieces are smaller than 1 mm3. 6 Warm saline, 2 ml, containing 0.6 units per ml of Dispase (Boehringer, Mannheim, FRG) is added and the dish kept at 37° for 10 min. The tissue suspension is triturated cautiously at least 5 to 6 times by means of a Pasteur pipet and diluted further to give 8-10 ml of complete medium. The suspension is gently forced through a nylon mesh (gauze: 135/zm) by means of a glass rod. At this stage, a single cell suspension should have been attained, enabling a cell count. After further dilution of the single cell suspension, approximately 106 cells/ml are plated onto collagen-coated plastic dishes. Careful handling, without major shaking, prevents aggregation of the cells. The dishes are placed in an automatically controlled CO2 incubator. Either 5 or 10% CO~ is used, depending on the amount of bicarbonate buffer in the growth medium (usually Dulbecco's modification of Eagle medium). The cultures are fed three times per week by aspirating two-thirds of the fluid and replacing it with fresh medium. There are two reasons for retaining one-third of the fluid in the dish: the cells are conditioning the medium according to their needs; and, drying out of the cell layer is prevented, especially if 30 or 40 dishes are changed together--the usual practice needed to save time. The daily observation of one or two cultures from each dissection by phase-contrast microscopy is recommended in order to detect microbial contamination. Such contamination is always a possibility, especially if antibiotics are not used in the culture medium. Although working without antibiotics is practicable, it is difficult to achieve without contamination. On the other hand, because neurons are extremely sensitive to antibiotics, one should at least strive to use very low concentrations of antibiotics if they cannot be avoided, i.e., less than 10 units per ml of a penicillin--preferably carbenicillin because of its lower neurotoxicity. If glial cells tend to overgrow the neurons, a mitotic inhibitor such as fluorodeoxyuridine (2 ~g/ml) together with uridine (50 /zg/ml) may be given for a short period (2 days) after the initial week of culture. The cultures are generally grown for 2 to 3 weeks before experiments are performed with them; this interval appears to be needed for this system of cells to reach a certain degree of "maturity" in terms of synaptogenesis. The cultures are harvested for biochemical purposes by scraping with a rubber policeman following a washing procedure, which depends on the specific purpose for which the cells are to be used. E. W. Godfrey, P. G. Nelson, B. K. Schrier, A. C. Breuer, and B. R. Ransom, Brain Res. 90, I (1975).
164
ANIMAL ORGAN AND CELL PREPARATIONS
[19]
By use of Petriperm dishes (Haereus, GiSttingen, FRG), which have a semipermeable membrane as a bottom, cultures may be harvested simply by cutting out the bottom for further processing, i.e., for extraction or measurement of radioactivity. Variations There have been a number of different suggestions for improving the standard procedure for obtaining increased neuron survival. Coating the dishes with collagenr might be replaced by coating with polylysine.8 Enzymatic dissociation of the cells has also been attained by using trypsin instead of Dispase. 9 The pore size of the nylon mesh used for filtration may be smaller than 135/~m, but at the expense of excluding certain larger cell types. The amount and type of serum has been a matter of debate: 10% fetal calf serum and 10% horse serum have been used in most cases, but replacement of the horse serum by neonatal calf serum or pig serum works equally well. The newest trend consists of leaving out the serum completely, in order to obtain a chemically defined growth medium, l° Much hope is placed on the effect of insulin in combination with high glucose concentrations. Recent findings provide some optimism. 11 Addition of gangliosides to the medium is currently under trial, because these glycosphingolipids have been shown to induce neurite outgrowth in vivo and in tumor cell cultures, lz Applications There are no major problems in the experimental design of morphological techniques with these dissociated cell cultures. Feasibility and interpretation of the results depend largely on the identification of particular cell types. Two major lines of approach can be observed with regard to methodological aspects. The first consists of improvement of " m i x e d " cultures that contain neurons and the variety of glial cells, ependymal cells, and fibroblasts. The objective is to retain, as nearly as possible, the normal physiological interactions between the different cell types. Such studies aim at the use of a physiological system as a tool for the study of drug activity at the cellular level. The close similarity of these types of 7 M. B. Bornstein, Lab. Invest. 7, 134 (1958). s E. Yavin and Z. Yavin, J. Cell Biol. 62, 540 (1974). 9 E. L. Giller, Jr., B. K. Schrier, A. Shainberg, H. R. Fisk, and P. G. Nelson, Science 182, 588 (1973). lo p. Honnegger, D. Lenoir, and P. Favrod, Nature (London) 282, 305 (1979). 11 E. Y. Snyder and S. U. Kim, Brain Res. 196, 565 (1980). 12 W. Dimpfel, W. M611er, and U. Mengs, Arch. Pharmacol. 308, Suppl., K46 (1979).
[19]
NERVE CELLS
165
cultures to in vivo features has been described in a series of papers dealing with morphological, biochemical, and electrophysiological characteristics,13-1~ and the successful use of this approach in studying toxin or drug actions has emerged as a completely new area of pharmacology. Ir The second approach attempts to separate the different cell types before culturing in order to achieve pure (mostly neural or glial) cultures. An alternative method of achieving this goal is to kill specific cell types within a mixed culture (by use of specific toxins or antibodies). These studies may allow new insights into cell-to-cell interactions and into the trophic requirements. Interactions between nerve and muscle cell have already received attention, 9 particularly with respect to receptor sensitivity, is In addition, trophic or nutritional factors in the CNS have been studied with regard to myelination. ~9 Biochemical analysis is inherently difficult because of the low amount of tissue that can be obtained from such cultures. The usual 60-ramdiameter dish contains only about 1.5 mg of protein. A partial solution to the problem is the use of isotopes in order to increase sensitivity. Another serious limitation, which applies only to the mixed cultures, is cell heterogeneity. The only way of approaching this goal is to quantitate the particular cell types and to correlate the observed measurements to the relative amount of a specific cell type under study. The discovery of tetanus toxin as a highly selective marker for neurons is currently simplifying research in this direction. .'° Other cell types have been quantitatively identified by immunological methods, for which radioimmunoassays must be developed be.fore quantitation can be achieved. 21'22 The choice of the culture system thus depends on the type of question asked. The "window to the brain"--as the dissociated cultures have been called--also offer advantages to electrophysiologists. Impalement of morphologically distinguishable cells under complete visual control by mi~'~B. R. Ransom, E. Neale, M. Henkart, P. N. Bullock, and P. G. Nelson, J. Neurophysiol. 40, 1132 (1977). 44 B. R. Ransom, C. N. Christian, P. N. Bullock, and P. G. Nelson,J. Neurophysiol. 40, 1157 (1977). ~5 B. R. Ransom, P. N. Bullock, and P. G. Nelson, J. Neurophysiol. 40, 1163 (1977). ~6 p. G. Nelson, B. R. Ransom, M. Henkart, and P. N. Bullock, J. Neurophysiol. 40, 1178 (1977). ~z W. Dimpfel, Exp. Neurol. 65, 53 (1979). ~ G. D. Fischbach and S. A. Cohen, Dev. Biol. 31, 147 (1973). ~9 S. U. Kim and D. Pleasure, Brain Res. 145, 15 (1978). 2o W. Dimpfel, J. H. Neale, and E. Habermann, Naunyn-Schmiedeberg's Arch. Pharmacol. 290, 329 (1975). 2~ M. Schachner and M. Willinger, Prog. Brain Res. 51, 23 (1979). 22 M. C. Raft, J. P. Brockes, K. L. Fields, and R. Mirsky, Prog. Brain Res. 51, 17 (1979).
166
ANIMAL ORGAN AND CELL PREPARATIONS
[19]
croelectrodes with tip diameters far less than 1 /zm are reported by a number of laboratories. For these purposes, the culture dish is mounted on the fixed stage of an inverted phase contrast microscope and kept at 37°. Microelectrodes are held by micromanipulators and are guided into the cells either by free hand movement or by the aid of automatic microdrives. The electronic equipment is conventional and has been used widely, z3 Because neurons usually sit on top of a monolayer consisting of several other cell types, they are easily accessible although extremely fragile. This means that electrodes of at least 30 to 50 megohms (3 M KC1) have to be drawn in order to give a very fine tip. With experience it is even possible to insert two electrodes into the same cell. As neurons in these cultures mature to the stage at which synaptic connections are formed, it is also possible to record synaptic potentials, either occurring spontaneously or by stimulation of a different cell by a second electrode. One of the most exciting techniques currently applied is the use of iontophoresis of drugs or transmitters in order to determine the sensitivity of a specific neuron. 23 The possibilities provided by these cultures should facilitate examination of drugs acting at the neuronal membrane or at synapses.
z3 j. L. Barker and B. R. Ransom, J. Physiol. (London) 280, 331 (1978).
ANIMAL ORGAN AND CELL PREPARATIONS
[19]
tal, aryl hydrocarbons, and ingested toxins. 45-47 Therefore techniques for the isolation of villous and crypt cells may be of value in studying not only the development of drug metabolizing systems but also their mechanism(s) of induction in the gut. 4~ E. Heitanen, M. Laitinan, and U. Koivusaari, Enzyme 25, 153 (1980). ~ C. M. Schiller, Environ. Health Perspect. 33, 91 (1979). 47 S y m p o s i u m on "Target Organ Toxicity: I n t e s t i n e " (C. M. Schiller, g u e s t ed.), in Environ. Health Perspect. 33, 1-126 (1979).
[19]
Mammalian
Nerve Cell Culture
B y W. DIMPFEL
Tissue culture offers a unique possibility for combining the advantages of experimentally well-controlled in vitro conditions with the advantage of working with living cells. Nevertheless, a great number of methodological difficulties had to be overcome with neuronal tissue before the technique could advance from a mere morphological description of living tissue in vitro 1 to a model system that allows meaningful statements about the function of the nervous system. 2 The technical procedure for obtaining reproducible primary nerve cell cultures as described herein has been successfully applied in several laboratories for more than 6 years. It can be regarded as the further development of the successfully introduced, and already widely used, explant cultures. 3-5 Standard Procedure The close interrelations between different cell types within the central nervous system (CNS) and the extensive arborization of neurites in the adult account for the difficulty in dissociating tissue into a single cell suspension that could be plated in culture dishes. Therefore, embryonic tissue is best suited for culturing purposes. The embryos (12-14 days in utero for mice; 14-16 days for rats) are obtained under sterile conditions by cesarean section and placed under a stereo microscope at low magnification into prewarmed buffered saline, preferably Ca 2+ free. I R. G. Han'ison, Anat. Rec. 1, 116 (1906). 2 W. Dimpfel, Arch. Toxicol. 44, 55 (1980). a W. Hild and I. Tasaki, J. Neurophysiol. 25, 277 (1962). S. M. Crain and E. R. Peterson, J. Cell. Comp. Physiol. 64, 1 (1964). 5 R. P. Bunge, M. B. Bunge, and E. R. Peterson, J. Cell Biol. 24, 163 (1965).
METHODS IN ENZYMOLOGY,VOL. 77
Copyright © 1981 by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181977-9
[19]
NERVE CELLS
163
The brain or portions of the CNS are excised by means of iridectomy scissors and watchmaker forceps, placed into a small separate dish together with a drop of saline, and minced until the tissue pieces are smaller than 1 mm3. 6 Warm saline, 2 ml, containing 0.6 units per ml of Dispase (Boehringer, Mannheim, FRG) is added and the dish kept at 37° for 10 min. The tissue suspension is triturated cautiously at least 5 to 6 times by means of a Pasteur pipet and diluted further to give 8-10 ml of complete medium. The suspension is gently forced through a nylon mesh (gauze: 135/zm) by means of a glass rod. At this stage, a single cell suspension should have been attained, enabling a cell count. After further dilution of the single cell suspension, approximately 106 cells/ml are plated onto collagen-coated plastic dishes. Careful handling, without major shaking, prevents aggregation of the cells. The dishes are placed in an automatically controlled CO2 incubator. Either 5 or 10% CO~ is used, depending on the amount of bicarbonate buffer in the growth medium (usually Dulbecco's modification of Eagle medium). The cultures are fed three times per week by aspirating two-thirds of the fluid and replacing it with fresh medium. There are two reasons for retaining one-third of the fluid in the dish: the cells are conditioning the medium according to their needs; and, drying out of the cell layer is prevented, especially if 30 or 40 dishes are changed together--the usual practice needed to save time. The daily observation of one or two cultures from each dissection by phase-contrast microscopy is recommended in order to detect microbial contamination. Such contamination is always a possibility, especially if antibiotics are not used in the culture medium. Although working without antibiotics is practicable, it is difficult to achieve without contamination. On the other hand, because neurons are extremely sensitive to antibiotics, one should at least strive to use very low concentrations of antibiotics if they cannot be avoided, i.e., less than 10 units per ml of a penicillin--preferably carbenicillin because of its lower neurotoxicity. If glial cells tend to overgrow the neurons, a mitotic inhibitor such as fluorodeoxyuridine (2 ~g/ml) together with uridine (50 /zg/ml) may be given for a short period (2 days) after the initial week of culture. The cultures are generally grown for 2 to 3 weeks before experiments are performed with them; this interval appears to be needed for this system of cells to reach a certain degree of "maturity" in terms of synaptogenesis. The cultures are harvested for biochemical purposes by scraping with a rubber policeman following a washing procedure, which depends on the specific purpose for which the cells are to be used. E. W. Godfrey, P. G. Nelson, B. K. Schrier, A. C. Breuer, and B. R. Ransom, Brain Res. 90, I (1975).
164
ANIMAL ORGAN AND CELL PREPARATIONS
[19]
By use of Petriperm dishes (Haereus, GiSttingen, FRG), which have a semipermeable membrane as a bottom, cultures may be harvested simply by cutting out the bottom for further processing, i.e., for extraction or measurement of radioactivity. Variations There have been a number of different suggestions for improving the standard procedure for obtaining increased neuron survival. Coating the dishes with collagenr might be replaced by coating with polylysine.8 Enzymatic dissociation of the cells has also been attained by using trypsin instead of Dispase. 9 The pore size of the nylon mesh used for filtration may be smaller than 135/~m, but at the expense of excluding certain larger cell types. The amount and type of serum has been a matter of debate: 10% fetal calf serum and 10% horse serum have been used in most cases, but replacement of the horse serum by neonatal calf serum or pig serum works equally well. The newest trend consists of leaving out the serum completely, in order to obtain a chemically defined growth medium, l° Much hope is placed on the effect of insulin in combination with high glucose concentrations. Recent findings provide some optimism. 11 Addition of gangliosides to the medium is currently under trial, because these glycosphingolipids have been shown to induce neurite outgrowth in vivo and in tumor cell cultures, lz Applications There are no major problems in the experimental design of morphological techniques with these dissociated cell cultures. Feasibility and interpretation of the results depend largely on the identification of particular cell types. Two major lines of approach can be observed with regard to methodological aspects. The first consists of improvement of " m i x e d " cultures that contain neurons and the variety of glial cells, ependymal cells, and fibroblasts. The objective is to retain, as nearly as possible, the normal physiological interactions between the different cell types. Such studies aim at the use of a physiological system as a tool for the study of drug activity at the cellular level. The close similarity of these types of 7 M. B. Bornstein, Lab. Invest. 7, 134 (1958). s E. Yavin and Z. Yavin, J. Cell Biol. 62, 540 (1974). 9 E. L. Giller, Jr., B. K. Schrier, A. Shainberg, H. R. Fisk, and P. G. Nelson, Science 182, 588 (1973). lo p. Honnegger, D. Lenoir, and P. Favrod, Nature (London) 282, 305 (1979). 11 E. Y. Snyder and S. U. Kim, Brain Res. 196, 565 (1980). 12 W. Dimpfel, W. M611er, and U. Mengs, Arch. Pharmacol. 308, Suppl., K46 (1979).
[19]
NERVE CELLS
165
cultures to in vivo features has been described in a series of papers dealing with morphological, biochemical, and electrophysiological characteristics,13-1~ and the successful use of this approach in studying toxin or drug actions has emerged as a completely new area of pharmacology. Ir The second approach attempts to separate the different cell types before culturing in order to achieve pure (mostly neural or glial) cultures. An alternative method of achieving this goal is to kill specific cell types within a mixed culture (by use of specific toxins or antibodies). These studies may allow new insights into cell-to-cell interactions and into the trophic requirements. Interactions between nerve and muscle cell have already received attention, 9 particularly with respect to receptor sensitivity, is In addition, trophic or nutritional factors in the CNS have been studied with regard to myelination. ~9 Biochemical analysis is inherently difficult because of the low amount of tissue that can be obtained from such cultures. The usual 60-ramdiameter dish contains only about 1.5 mg of protein. A partial solution to the problem is the use of isotopes in order to increase sensitivity. Another serious limitation, which applies only to the mixed cultures, is cell heterogeneity. The only way of approaching this goal is to quantitate the particular cell types and to correlate the observed measurements to the relative amount of a specific cell type under study. The discovery of tetanus toxin as a highly selective marker for neurons is currently simplifying research in this direction. .'° Other cell types have been quantitatively identified by immunological methods, for which radioimmunoassays must be developed be.fore quantitation can be achieved. 21'22 The choice of the culture system thus depends on the type of question asked. The "window to the brain"--as the dissociated cultures have been called--also offer advantages to electrophysiologists. Impalement of morphologically distinguishable cells under complete visual control by mi~'~B. R. Ransom, E. Neale, M. Henkart, P. N. Bullock, and P. G. Nelson, J. Neurophysiol. 40, 1132 (1977). 44 B. R. Ransom, C. N. Christian, P. N. Bullock, and P. G. Nelson,J. Neurophysiol. 40, 1157 (1977). ~5 B. R. Ransom, P. N. Bullock, and P. G. Nelson, J. Neurophysiol. 40, 1163 (1977). ~6 p. G. Nelson, B. R. Ransom, M. Henkart, and P. N. Bullock, J. Neurophysiol. 40, 1178 (1977). ~z W. Dimpfel, Exp. Neurol. 65, 53 (1979). ~ G. D. Fischbach and S. A. Cohen, Dev. Biol. 31, 147 (1973). ~9 S. U. Kim and D. Pleasure, Brain Res. 145, 15 (1978). 2o W. Dimpfel, J. H. Neale, and E. Habermann, Naunyn-Schmiedeberg's Arch. Pharmacol. 290, 329 (1975). 2~ M. Schachner and M. Willinger, Prog. Brain Res. 51, 23 (1979). 22 M. C. Raft, J. P. Brockes, K. L. Fields, and R. Mirsky, Prog. Brain Res. 51, 17 (1979).
166
ANIMAL ORGAN AND CELL PREPARATIONS
[19]
croelectrodes with tip diameters far less than 1 /zm are reported by a number of laboratories. For these purposes, the culture dish is mounted on the fixed stage of an inverted phase contrast microscope and kept at 37°. Microelectrodes are held by micromanipulators and are guided into the cells either by free hand movement or by the aid of automatic microdrives. The electronic equipment is conventional and has been used widely, z3 Because neurons usually sit on top of a monolayer consisting of several other cell types, they are easily accessible although extremely fragile. This means that electrodes of at least 30 to 50 megohms (3 M KC1) have to be drawn in order to give a very fine tip. With experience it is even possible to insert two electrodes into the same cell. As neurons in these cultures mature to the stage at which synaptic connections are formed, it is also possible to record synaptic potentials, either occurring spontaneously or by stimulation of a different cell by a second electrode. One of the most exciting techniques currently applied is the use of iontophoresis of drugs or transmitters in order to determine the sensitivity of a specific neuron. 23 The possibilities provided by these cultures should facilitate examination of drugs acting at the neuronal membrane or at synapses.
z3 j. L. Barker and B. R. Ransom, J. Physiol. (London) 280, 331 (1978).