Neurosvience Letters, 11 (1979) 137--141 © Elsevier/North-Holland Scientific Publishers Ltd.
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137
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TISSUECULTURE O F A D U L T H U M A N
N~IURONS
SEUNG U. KIM, KENNETH G. WARREN and MADHU KALIA
Division of Neurol~tholOgy, Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, and (M.K.) Department of Physiology, Hahnemann Medical College, Philadelphia, PA (U.S.A.) (Received October 31st, 1978) (Accepted November 2nd, 1978)
SUMMARY
Dissociated neurons from adult humml t~geminal and superior cervical ganglia were cultured in vitro for more than 2 months. Immediately after dissociation by incubation in 0.06% collagena3e for 15--18 h, the cultures consisted of singleneurons or clumps of neurons and degenerating fmgment~ of myelinated or non-myelinated axons. After 7--10 days, bipol~ Schwann cells,largeneurons and fine nerve fiberswere observed. Electron microscopic examination of these neurons revealed allthe ultrastructur~lfeatures o~ healthy adult neurons including those of lipofuscin pigments. By ~;'.ctrophysiological technique, extracellularrecording to action potentials generated by these neurons were obtained indicating the neurons were rUve and healthy. The availabilityof adult human neurons in culture should provide a model system for investigationrelated to the pathomechanism of lipofuscin formation and aging in general.
Since the first report of neural tissue culture by Harrison in 1907 [4] tissues from various parts o f thenervous system and various species have been successfully grown in vitro [.2,7,9,13] .However, most of these studies have used embryonic or early postnatal tissues and the relatively few studie:~ which have attempted ~ u s e adUlt neural tissue met with little success [ 1,3,5,,6,10]. One of the exceptions w a s e x p ~ t cultures of ~adulthuman sympathetic ganglia by Murray and ~ t ~!~!947 [8]~,Sincethen, no serious attezupt h ~ been made to culture a d ~ t ! ~ m ~ , n e u r 0 n s . R e c e n t l y , however, ~.ott h ~ described a technique by W h i ~ h e : ~ l a ~ d adult mouse dorsal root ganglianeurons and maintained them for up to 5 months [11]. W e ~now lmpo~:that adult human trigeminal and superior cervical ganglia neurons,;~en from human cadavers 4-'6 h post mortem, after di~socation by a modification of Scott's procedure, survive and regenerate in culture for more than 2 months.
138
Trigeminal and superior cervicalganglia were removed from 4 adult males of 19--46 years of age, 4--6 h afterdeath due to trauma. Individual ganglia were incubated in 10 ml of 0.06% collagenase (type CLS, Worthington Chemical) in Hank's balanced saltsolution (BSS) in 25 ml Erlenmeyer flasksfor 15--18 h
Fig.. 1. Living cultures of adult human neurons. A: three large neurons (N) isolated from the superior cervical ganglion are shown in the center of the field; axons (A) and Schwann cells (S) are noted outgrowing radially from the cell clump. 14 days in vitro, x 250. B: a large enuron from the trigeminal ganglion. Note an eccentric positioning of the nucleus and slightly granular cytoplasm. 26 days in vitro. × 850.
139
Fi ~ 2. Electron micrographs of cultured adult human neurons. A: the nucleus of the neuron occupies the lower right portion of the field, and embedded in the karyoplasm is a reticular nucleolus. In the cytoplasm are Golgi apparatus, mitochondria, rough endoplasmic reticulum and lipofuscin pigments. Trigeminai ganglion neuron 26 days in vitro. × 4000. B: higher magnification of neuronal cytoplasm. Several lipofuscin pigments (L), microtubules (M) and intermediate filaments (F) are indicated. Trigeminal ganglion neuron 26 days in vitro. x 25,000.
and neurons resulting from the incubation were centrifuged, washed in 2 changes of BSS, resuspended in nutrien~ medium and plated on polylysine or ~elatincoated Aclar coverslips (11 × 22 ram, Allied Chemical). Nutrient medium consisted of 10% horse serum, 10% fetal calf serum and 5 mg/ml glucose in Eagle's minimum essential medium. Cultures were refed every 3--4 days.
140 Immediately after the dissociation, the cultures consisted of single neurons or clumps of neurons and degenerating fragments of myelinated or nonmyelinated axons. After 1--3 days, Schwann cells started to extend their processes and assumed their typical bipolar morphology (Fig. la). Neurons, on the other hand, were slow to regenerate, and usually took 7--10 days to extend axonal processes. Some of surviving neurons attached onto the other cell elements, using fibrobiasts and failed to send axons (Fig. lb). Electron microscopic examination of these neurons revealed all the ultrastructural features of healthy adult neurons. Neuronal cytoplasmic organelles including Golgi apparatus, mitocho~.dria, rough endoplasmic reticulum as well as microtubules and intermediate filaments showed normal configuration (Fig. 2a,b). Lipofuscin granules were also found in the cytoplasm. These lipofuscin granules were bound by a single membrane and were larger than lysosomes, being 1.5--2.5 #m in diameter (Fig. 2b). An important feature of lipofuscin granules is that they have one or two peripherally located vacuoles or electron lucent areas, the rest of the granules being filled with a heterogenous mixture of electron dense particles [ 12]. This is the first time to our knowledge that lipofuscin granule~ have been demonstrated in cultured mammalian neurons including those of human. Lipofuscin has been observed in human and animal neurons usually concomitant with advancing age. The availability of adult human neuron culture thus would provide a model system for investigation of questions related to the pathomechanism of lipofuscin formation and aging in general.
Fig. 3' Extracellulaz ~recording .of action potentials from adulthuman ganglion neurons cultured 42 days in vitro.
141
Adult human neurons grown on coverslips were placed on an inverted microscope for electrophysiological study. Microelectrodes (platinum black electrode with tip diameter of 1--2 "tm) were positioned with a micromanipulator under direct visual contlol and extracellular re~.ordings were made with AC-differential preamplifier ~WP instruments) and 4~hanne~ storage oscilloscope (Tektronics). By positioning the tip of the microelectrode within a few ~m of the neurons, spontaneous extracellular action potentials were recorded from a majority of neurons tested indicating ~hose cultured adult human neurons were alive and firing actively (Fig. 3). It is surprising to see t ~ survival in culture of human adult neurons taken from individuals as long as 4 - 6 h after death. The ability of these adult neurons to sprout axons when transferred to adequate culture conditions might well be viewed as instances of regeneration, Cultures of human adult neurons should provide a promis-ng model system for further investigating this regenerative capaci~ of ati~lt mammaliar~ neurons. ACKNOWLEDGEMENT
We thank Myong Kim and Anna Stieber for their technical assistance. This work was supported by the USPHS Grant NS-10648, NS-05572 and the Multiple Sclerosis Society of Canada. S.U. Kim and M. Kalia are recipients of the Research Cancer Development Award from the USPHS (NS-00151 and HL-00103). REFERENCES 1 Costero, I. and Pomerat, C.M., Cultivation of neurons from the adult human cerebral and cerebellar cortex, Amer. J. Anat., 89 (1951) 405--467. 2 Crain, S., Neurophysiologic Studies in Tissue Culture, Rsvo.n Press, New York, 1976. 3 Geiger, R., Subcultures of adult mammalian cortex in vitro, Exp. Cell Res., 14 (1958) 541--566. 4 Harrison, R.G., Observations on living developing nerve fibers, Proc. Soc. Exp. Biol. Mad., 4 (1907) 140--143. 5 Hogue, M., A study of adult human brain cells in tissue culture, Amer. J. Anat., 93 (1953) 397--427. 6 Kiernan, J. and Pettit, D., Organ culture of central nervous system of the adult rat, Exp. Neurol., 32 (1971) 111--120. 7 Murray, M.R., Nervous tissue in vitro. In E.N. Willmer (Ed.), Cells and Tissues iu Culture, Vol. 2, Academic Press, New York, 1965, pp. 373.-455. 8 Murray, M.R. an~ Stout, A., Adult human sympathetic ganglion cells cultivated in vitro, Amer. J. Anat., ,~}0(1947) 225--273. 9 Nelson, P.G., Nerve and muscle cells in culture, Physiol. Ray., 55 (1975) 1--61. 10 Rorke~ L., Gil,~en, D., Wroblewska, Z. and Santoli, D., Human brain in tissue culture, 4. Morphological characteristics, J. comp. Neurol., 161 (1975) 329--339. 11 Scott, B.S., Adult mouse dorsal root ganglia neurons in cell culture, J. Neurobiol., 8 (1977) 417--427. 12 Sekhon, S. and Maxwell, D., Ultrastructural changes in neurons of the spinal anterior horn of ageing r.~ice with particular reference to the accumulation of lipofuscin pi~,ment,
J. Neurocytol., 3 (1974) 59--72. 13 Varon, S., Neurons and s~;4 in neural cultures, Exp. Neurol., 48 (1975) 93--134.
:
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•
137
.
TISSUECULTURE O F A D U L T H U M A N
N~IURONS
SEUNG U. KIM, KENNETH G. WARREN and MADHU KALIA
Division of Neurol~tholOgy, Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, and (M.K.) Department of Physiology, Hahnemann Medical College, Philadelphia, PA (U.S.A.) (Received October 31st, 1978) (Accepted November 2nd, 1978)
SUMMARY
Dissociated neurons from adult humml t~geminal and superior cervical ganglia were cultured in vitro for more than 2 months. Immediately after dissociation by incubation in 0.06% collagena3e for 15--18 h, the cultures consisted of singleneurons or clumps of neurons and degenerating fmgment~ of myelinated or non-myelinated axons. After 7--10 days, bipol~ Schwann cells,largeneurons and fine nerve fiberswere observed. Electron microscopic examination of these neurons revealed allthe ultrastructur~lfeatures o~ healthy adult neurons including those of lipofuscin pigments. By ~;'.ctrophysiological technique, extracellularrecording to action potentials generated by these neurons were obtained indicating the neurons were rUve and healthy. The availabilityof adult human neurons in culture should provide a model system for investigationrelated to the pathomechanism of lipofuscin formation and aging in general.
Since the first report of neural tissue culture by Harrison in 1907 [4] tissues from various parts o f thenervous system and various species have been successfully grown in vitro [.2,7,9,13] .However, most of these studies have used embryonic or early postnatal tissues and the relatively few studie:~ which have attempted ~ u s e adUlt neural tissue met with little success [ 1,3,5,,6,10]. One of the exceptions w a s e x p ~ t cultures of ~adulthuman sympathetic ganglia by Murray and ~ t ~!~!947 [8]~,Sincethen, no serious attezupt h ~ been made to culture a d ~ t ! ~ m ~ , n e u r 0 n s . R e c e n t l y , however, ~.ott h ~ described a technique by W h i ~ h e : ~ l a ~ d adult mouse dorsal root ganglianeurons and maintained them for up to 5 months [11]. W e ~now lmpo~:that adult human trigeminal and superior cervical ganglia neurons,;~en from human cadavers 4-'6 h post mortem, after di~socation by a modification of Scott's procedure, survive and regenerate in culture for more than 2 months.
138
Trigeminal and superior cervicalganglia were removed from 4 adult males of 19--46 years of age, 4--6 h afterdeath due to trauma. Individual ganglia were incubated in 10 ml of 0.06% collagenase (type CLS, Worthington Chemical) in Hank's balanced saltsolution (BSS) in 25 ml Erlenmeyer flasksfor 15--18 h
Fig.. 1. Living cultures of adult human neurons. A: three large neurons (N) isolated from the superior cervical ganglion are shown in the center of the field; axons (A) and Schwann cells (S) are noted outgrowing radially from the cell clump. 14 days in vitro, x 250. B: a large enuron from the trigeminal ganglion. Note an eccentric positioning of the nucleus and slightly granular cytoplasm. 26 days in vitro. × 850.
139
Fi ~ 2. Electron micrographs of cultured adult human neurons. A: the nucleus of the neuron occupies the lower right portion of the field, and embedded in the karyoplasm is a reticular nucleolus. In the cytoplasm are Golgi apparatus, mitochondria, rough endoplasmic reticulum and lipofuscin pigments. Trigeminai ganglion neuron 26 days in vitro. × 4000. B: higher magnification of neuronal cytoplasm. Several lipofuscin pigments (L), microtubules (M) and intermediate filaments (F) are indicated. Trigeminal ganglion neuron 26 days in vitro. x 25,000.
and neurons resulting from the incubation were centrifuged, washed in 2 changes of BSS, resuspended in nutrien~ medium and plated on polylysine or ~elatincoated Aclar coverslips (11 × 22 ram, Allied Chemical). Nutrient medium consisted of 10% horse serum, 10% fetal calf serum and 5 mg/ml glucose in Eagle's minimum essential medium. Cultures were refed every 3--4 days.
140 Immediately after the dissociation, the cultures consisted of single neurons or clumps of neurons and degenerating fragments of myelinated or nonmyelinated axons. After 1--3 days, Schwann cells started to extend their processes and assumed their typical bipolar morphology (Fig. la). Neurons, on the other hand, were slow to regenerate, and usually took 7--10 days to extend axonal processes. Some of surviving neurons attached onto the other cell elements, using fibrobiasts and failed to send axons (Fig. lb). Electron microscopic examination of these neurons revealed all the ultrastructural features of healthy adult neurons. Neuronal cytoplasmic organelles including Golgi apparatus, mitocho~.dria, rough endoplasmic reticulum as well as microtubules and intermediate filaments showed normal configuration (Fig. 2a,b). Lipofuscin granules were also found in the cytoplasm. These lipofuscin granules were bound by a single membrane and were larger than lysosomes, being 1.5--2.5 #m in diameter (Fig. 2b). An important feature of lipofuscin granules is that they have one or two peripherally located vacuoles or electron lucent areas, the rest of the granules being filled with a heterogenous mixture of electron dense particles [ 12]. This is the first time to our knowledge that lipofuscin granule~ have been demonstrated in cultured mammalian neurons including those of human. Lipofuscin has been observed in human and animal neurons usually concomitant with advancing age. The availability of adult human neuron culture thus would provide a model system for investigation of questions related to the pathomechanism of lipofuscin formation and aging in general.
Fig. 3' Extracellulaz ~recording .of action potentials from adulthuman ganglion neurons cultured 42 days in vitro.
141
Adult human neurons grown on coverslips were placed on an inverted microscope for electrophysiological study. Microelectrodes (platinum black electrode with tip diameter of 1--2 "tm) were positioned with a micromanipulator under direct visual contlol and extracellular re~.ordings were made with AC-differential preamplifier ~WP instruments) and 4~hanne~ storage oscilloscope (Tektronics). By positioning the tip of the microelectrode within a few ~m of the neurons, spontaneous extracellular action potentials were recorded from a majority of neurons tested indicating ~hose cultured adult human neurons were alive and firing actively (Fig. 3). It is surprising to see t ~ survival in culture of human adult neurons taken from individuals as long as 4 - 6 h after death. The ability of these adult neurons to sprout axons when transferred to adequate culture conditions might well be viewed as instances of regeneration, Cultures of human adult neurons should provide a promis-ng model system for further investigating this regenerative capaci~ of ati~lt mammaliar~ neurons. ACKNOWLEDGEMENT
We thank Myong Kim and Anna Stieber for their technical assistance. This work was supported by the USPHS Grant NS-10648, NS-05572 and the Multiple Sclerosis Society of Canada. S.U. Kim and M. Kalia are recipients of the Research Cancer Development Award from the USPHS (NS-00151 and HL-00103). REFERENCES 1 Costero, I. and Pomerat, C.M., Cultivation of neurons from the adult human cerebral and cerebellar cortex, Amer. J. Anat., 89 (1951) 405--467. 2 Crain, S., Neurophysiologic Studies in Tissue Culture, Rsvo.n Press, New York, 1976. 3 Geiger, R., Subcultures of adult mammalian cortex in vitro, Exp. Cell Res., 14 (1958) 541--566. 4 Harrison, R.G., Observations on living developing nerve fibers, Proc. Soc. Exp. Biol. Mad., 4 (1907) 140--143. 5 Hogue, M., A study of adult human brain cells in tissue culture, Amer. J. Anat., 93 (1953) 397--427. 6 Kiernan, J. and Pettit, D., Organ culture of central nervous system of the adult rat, Exp. Neurol., 32 (1971) 111--120. 7 Murray, M.R., Nervous tissue in vitro. In E.N. Willmer (Ed.), Cells and Tissues iu Culture, Vol. 2, Academic Press, New York, 1965, pp. 373.-455. 8 Murray, M.R. an~ Stout, A., Adult human sympathetic ganglion cells cultivated in vitro, Amer. J. Anat., ,~}0(1947) 225--273. 9 Nelson, P.G., Nerve and muscle cells in culture, Physiol. Ray., 55 (1975) 1--61. 10 Rorke~ L., Gil,~en, D., Wroblewska, Z. and Santoli, D., Human brain in tissue culture, 4. Morphological characteristics, J. comp. Neurol., 161 (1975) 329--339. 11 Scott, B.S., Adult mouse dorsal root ganglia neurons in cell culture, J. Neurobiol., 8 (1977) 417--427. 12 Sekhon, S. and Maxwell, D., Ultrastructural changes in neurons of the spinal anterior horn of ageing r.~ice with particular reference to the accumulation of lipofuscin pi~,ment,
J. Neurocytol., 3 (1974) 59--72. 13 Varon, S., Neurons and s~;4 in neural cultures, Exp. Neurol., 48 (1975) 93--134.
