Serial passage of embryonic human astrocytes in serum‐free, hormone‐supplemented medium

Abstract

We applied serum-free cell culture methods that allow extended proliferation of mouse astrocyte precursor cells to the multipassage culture of embryonic human brain cells. Cells were cultured in nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibroblast growth factor, heparin, high-density lipoprotein, and fibronectin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized glial fibrillary acidic protein, an astrocyte marker, and expression of this protein was increased by incubation of the cells with transforming growth factor beta or serum. These results identify extracellular factors important for proliferation and differentiation of embryonic human astrocytes and provide a controlled system for multipassage culture.

Secretion of ribonucleases by normal and immortalized cells grown in serum-free culture conditions

“…HDLs and growth factors have been reported to support the long-term growth of several cell types in serum-free conditions while maintaining their morphological and biological characteristics (Gospodarowicz, 1984;Loo et al, 1989Loo et al, , 1991Sakai et al, 1990). In the present work, using a basal medium containing HDLs, FGF-2, insulin, and transfen'in, we demonstrated that serum could be omitted in CCL39, bSM, and EA.hy926 cell cultures.…”

Human neurocytoma cells in culture show characteristics of astroglial differentiation

“…Especially the long-term maintenance of the cells in EGF, which has to be added immediately to the primary cultures, appears to be a mandatory experiment according to our own experiment and the experience of others (Loo et al, 1987). In contrast to experiments with rodent embryonal brain cells (Loo et al, 1991), however, maintenance in serum free conditions alone failed to sustain proliferation or expand in vitro passages. Clinically it is known that neurocytomas are best treated by removal and subsequent irradiation; they are not known to produce other than local recurrencies (Nishio et al, 1988;Yasargil et al, 1992;Kim et al, 1992).…”

“…Especially the long-term maintenance of the cells in EGF, which has to be added immediately to the primary cultures, appears to be a mandatory experiment according to our own experiment and the experience of others (Loo et al, 1987). In contrast to experiments with rodent embryonal brain cells (Loo et al, 1991), however, maintenance in serum free conditions alone failed to sustain proliferation or expand in vitro passages.…”

Morphological differentiation and proteoglycan synthesis regulate Alzheimer amyloid precursor protein processing in PC‐12 and human astrocyte cultures

“…A4, the principal component of these deposits, corresponds to an APP sequence extending from the middle of the transmembrane domain to the proximal extracellular region. APP is normally cleaved in some neural and nonneural cells within the extracellular portion of A4 (Anderson et Rapidly dividing (passage 20) and slowly dividing (passage 55) human HBIB astrocytes were maintained in serum-free media supplemented with EGF and FGF as previously described (Loo et al, 1991). Neuroblastomas N-GP, N-LF, N-MB (obtained from Dr. G. Brodeur), IMR-32, and SK-SH-5Y and control or F-AD fibroblasts (AG04402A) obtained from the ATTC were maintained in Waymouth's MB media with 10% fetal calf serum.…”

“…Rapidly dividing (passage 20) and slowly dividing (passage 55) human HBIB astrocytes were maintained in serum-free media supplemented with EGF and FGF as previously described (Loo et al, 1991). Neuroblastomas N-GP, N-LF, N-MB (obtained from Dr. G. Brodeur), IMR-32, and SK-SH-5Y and control or F-AD fibroblasts (AG04402A) obtained from the ATTC were maintained in Waymouth's MB media with 10% fetal calf serum.…”