An improved method for dissociation and aggregate culture of human fetal brain cells in serum-free medium.
Moscona, in the early sixties [A.A. Moscona, Recombination of dissociated cells and the development of cell aggregates, in: B.M. Willmer (Ed.), Cells and Tissues in Culture, Academic Press, New York, 1965, pp. 489-529.] [16], discovered that aggregation of dissociated cells is a property of embryonal cells. Several features of the aggregate culture system are particularly attractive for the conduct of biochemical and molecular studies on the human fetal brain. (i) All the pertinent procedural parameters can be readily controlled and standardized, resulting in a consistently reproducible system suitable for quantitative analyses. (ii) Neuronal enriched aggregates can be readily obtained, with minimal neurotoxicity. (iii) Aggregates can be easily harvested for biochemical and molecular studies. Aggregate cultures, generated from rodent fetal brains, have been extensively utilized as a tool to study regulation of aminergic neurons [P. Honegger, E. Richelson, Biochemical differentiation of mechanically dissociated brain in aggregating cell culture, Brain Res. 109 (1976) 335-354; P. Honegger, E. Richelson, Biochemical differentiation of aggregating cell cultures of different fetal rat brain regions, Brain Res. 133 (1977) 329-339.] [11,12] and peptidergic neurons (neuropeptide Y (NPY) and somatostatin (SRIF) [A. Barnea, E. Anthony, G. Lu, G. Cho, Morphological differentiation of neuropeptide Y neurons in aggregate cultures of dissociated fetal cortical cells: a model system for glia-neuron paracrine interactions, Brain Res. 625 (1993) 313-322; A. Barnea, G. Cho, G. Lu, M. Mathis, Brain-derived neurotrophic factor induces functional expression and phenotypic differentiation of cultured fetal neuropeptide Y producing neurons, J. Neurosci. Res. 42 (1995) 638-647; A. Barnea, A. Hajibeigi, G. Cho, P. Magni, Regulated production and secretion of immunoreactive neuropeptide Y by aggregating fetal brain cells in culture, Neuroendocrinology 54 (1991) 7-13; P. Magni, A. Barnea, Forskolin and phorbol ester stimulation of neuropeptide Y (NPY) production and secretion by aggregating fetal brain cells in culture: evidence for regulation of NPY biosynthesis at transcriptional and posttranscriptional levels, Endocrinology 130 (1992) 976-984.]) [4-6,14]. However, very few studies have utilized this system to study regulatory processes of human fetal neurons/glia [M. McCarthy, L. Resnik, F. Taub, R.V. Stewart, R.D. Dix, Infection of human neural cell aggregate cultures with a clinical isolate of cytomegalovirus, J. Neuropathol. Exp. Neurol. 50 (1991) 441-450; L. Pulliam, M.E. Berens, M.L. Rosenblum, A normal human brain cell aggregate model for neurobiological studies, J. Neurosci. Res. 21 (1988) 521-530.] [15,17]. In a series of studies in our laboratory [N. Aguila-Mansilla, A. Barnea, Human fetal brain cells in aggregate culture: a model system to study regulatory processes of the developing human neuropeptide Y (NPY) producing neuron, Int. J. Dev. Neurosci. 14 (1996) 531-539; A. Barnea, N. Aguila-Mansilla, H.T. Chute, A.A. Welcher, Comparison of neurotrophin regulation of human and rat neuropeptide Y (NPY) neurons: induction of NPY production in aggregate cultures derived from rat but not from human fetal brains, Brain Res. 732 (1996) 52-60; A. Barnea, N. Aguila-Mansilla, G. Lu, R.H. Ho, Opposite effects of astrocyte-derived soluble factor(s) on the functional expression of fetal peptidergic neurons in aggregate cultures: enhancement of neuropeptide Y and suppression of somatostatin, J. Neurosci. Res. 50 (1997) 605-617; A. Barnea, J. Roberts, R.H. Ho, Evidence for a synergistic effect of the HIV-1 envelope protein gp120 and brain-derived neurotrophic factor (BDNF) leading to enhanced expression of somatostatin neurons in aggregate cultures derived from the human fetal cortex, Brain Res. 815 (1999) 349-357.] [1-3,7], we have established a human-derived aggregate culture system, maintained in serum-free medium for up to 28 days, in which expression
Barnea
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Department of Obstetrics and Gynecology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235-9032, USA. ayalla.barnea@email.swmed.edu
Roberts
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D001921:Brain / Q000166:cytology* / Q000196:embryology; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D003851:Deoxyribonucleases; D005333:Fetus / Q000166:cytology*; D006801:Humans; D009457:Neuroglia / Q000166:cytology; D009474:Neurons / Q000166:cytology; D009478:Neuropeptide Y / Q000096:biosynthesis; D013004:Somatostatin / Q000096:biosynthesis; D013048:Specimen Handling; D013314:Stress, Mechanical; D014357:Trypsin
D016895:Culture Media, Serum-Free; D009478:Neuropeptide Y; D013004:Somatostatin; D003851:Deoxyribonucleases; D014357:Trypsin