Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

Culture of epithelial cell lines in chemically defined media on microcarriers in biogenerators

A rat liver epithelial cell line has been propagated on microcarriers in 11 or 21 laboratory culture vessels for cell culture in suspension on microcarriers (biogenerators) with Ham F10 or DME as basal synthetic culture medium either serum-supplemented (SSM), or serum-free (SFM), or serum- and protein-free (SPFM). Without serum, the use of DME allows a cellular growth in the biogenerator at least equivalent to that obtained in culture dishes. For the cultivation on microcarriers in SFM in a biogenerator the use during the first day of culture of spent serum-free medium previously incubated (SFMI) in confluent culture dishes avoids the substratum treatment with serum. Results concerning the Vero cell line cultured in SPFM are shown.     

Culture media conditioned by wounded cells modify the fibronectin localization pattern of unwounded confluent PtK2 cells

A confluent PtK2 cell sheet was incised in a serum-free culture medium, at 15 min, 2 hr and 24 hr after wounding. The culture media were collected in the same way and used as conditioned media. Unwounded confluent cells were cultured in the conditioned medium for 24 hr. They showed a modification of fibronectin localization similar to that which we had previously observed in wounded confluent PtK2 cells: cells lost their normal fibronectin fibrils and were surrounded by fibronectin lace. This finding suggested that during wound healing, the cells released soluble chemical factors which could modify the fibronectin localization pattern of unwounded confluent cells. Subconfluent cells did not respond to conditioned media, showing that confluent cells and subconfluent cells had different susceptibilities.     

Changes in cell-surface fucose-containing glycopeptides and adhesion of cultured intestinal epithelial cells as a function of cell density.

Confluent cultured intestinal epithelial cells displayed greater adhesion to the substratum than did subconfluent cells. Subconfluent and confluent cells were labelled with [3H]fucose for 24h and the cell-surface components were released by mild Pronase treatment. After extensive Pronase digestion, cell-surface and cell-residue glycopeptides were fractionated on Bio-Gel P-6. The cell surface contained a higher proportion of lower-molecular-weight glycopeptides than the residue. No significant difference in elution pattern was found between total cell-surface glycopeptides of subconfluent and confluent cells. However, confluent cells contained almost twice as much [3H]-fucose-labelled glycopeptides that were bound to concanavalin A-Sepharose and were subsequently eluted with 20mM-methyl alpha-D-glucopyranoside as subconfluent cells. When the bound glycopeptides were chromatographed on Bio-Gel P-6, it was found that confluent cells contained a larger proportion of lower-molecular-weight glycopeptides than subconfluent cells. This difference in size was eliminated after treatment of glycopeptides with sialidase. When growth of subconfluent cells was inhibited with a non-toxic concentration of retinoic acid, no significant effect on the elution pattern of [3H]fucose-labelled glycopeptides was observed on either Bio-Gel P-6 or concanavalin A-Sepharose. No significant difference was found in the total [3H]fucose-labelled glycoproteins from subconfluent and confluent cells by two-dimensional gel electrophoresis. It is suggested that the differences in [3H]fucose-labelled glycopeptides between subconfluent and confluent cells are cell-density-dependent rather than growth-dependent, and that these differences are likely to result from some changes in glycosylation mechanism(s). Furthermore, the differences in cell-surface glycopeptides may be related to the changes in the adhesion of the cells to the substratum.     

Application of percent labeled mitoses (PLM) analysis to the investigation of melanoma cell responsiveness to MSH stimulation throughout the cell cycle

Dissociated embryonic chick dorsal root ganglionic cells were plated on collagen-coated tissue culture dishes in Eagle's basal medium containing 10% fetal calf serum (FCS). After 48 h, which allowed adequate cell attachment, the cultures were washed with serum-free medium and then received fresh medium supplemented with 10% FCS or serum-free defined medium (N1), which was supplemented with insulin, transferrin, progesterone, putrescine and selenium. In addition, both media required the addition of Nerve Growth Factor (NGF). N1 medium selectively maintained the neurons and did not support proliferation or even survival of almost all non-neuronal elements (fibroblasts and Schwann cells). Survival of neurons in N1 was initially as good and eventually better than in serum-containing medium. After 6 days in N1 the cultures consisted almost entirely of neurons (>95%), which had smaller cell bodies but more extensive process formation than in serum-supplemented medium. The omission of any one of the supplements resulted in a reduction of neuron survival. The ability to generate cultures of pure neurons in a serum-free defined medium may be useful for studying (i) the role of specific hormones and growth factors normally supplied by serum in the maintenance of neurons and (ii) biochemical parameters of neurons in the absence of the substantial background due to non-neuronal elements.     

Cell-density-dependent changes in cell-surface glycopeptides and in adhesion of cultured intestinal epithelial cells

We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-³H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-β-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [³H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.     

Human endometrial carcinoma cells release factors which inhibit the growth of normal epithelial cells in culture

Autocrine and paracrine interactions between cells are important homeostatic mediators in normal tissues. Alterations to growth factor signalling pathways are likely to play a role in multistep carcinogenesis. In this study normal human endometrial epithelial cells (NHEC) after 3 days in culture were treated with serum-free medium conditioned for 24 h by log phase or confluent cultures of established RL95-2, HEC1A, or AN3CA endometrial carcinoma (EC) cell lines. By day 4, NHEC treated with either log phase or confluent conditioned medium (CM) showed a significant decrease (50–90% of control) in [³H]thymidine ([³H]TdR) incorporation. DNA synthesis was inhibited more by confluent than by log phase CM. By day 7, NHEC treated with CM exhibited fewer colonies per culture, fewer cells per colony, and an increased percentage of single cells. Several growth-regulatory gene products found in the nucleus or at the cell membrane have been shown to be expressed differently in normal and transformed cells. We selected the p53 and c-Ha-ras p21 proteins to further investigate the mechanism of alteration of proliferation in cells treated with carcinoma CM. Thus, by day 7, the percentage of NHEC with nuclear localization of wild type p53 (wt p53) was elevated by treatment with CM. In contrast, CM-treated EC cells continued to proliferate, and showed a decrease in the percentage of cells expressing nuclear wt p53 and an increase in the cytoplasmic expression of c-Ha-ras p21. Our studies show that EC cell lines release factors which inhibit the proliferation of NHEC, thus favoring the proliferation of EC cells.Abbreviations CM conditioned medium - EC endometrial adenocarcinoma - NHEC normal human endometrial epithelial cells     

Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions

Summary We have developed a culture system for early bovine embryos in serum-free media conditioned by oviduct cell monolayers. A gentle mechanical procedure for oviduct cell isolation has been applied for this purpose avoiding the use of proteolytic enzymes. The aim of the present study was to identify the cell types present in the monolayers and to examine their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and biochemical analyses. The cell dissociation procedure yielded two cell populations: ciliary cells and secretory cells that gradually dedifferentiate during culture. These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplemented with 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and all the cells contained cytokeratin filaments as determined by immunocytochemistry. The overall histoarchitecture of the monolayer was preserved after washing and further culture for 7 d in serum-free medium. However, some degenerative signs indicate that the serum-free culture should not be extended for more than 7 d. Confluent oviduct cells also maintained their metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly increased as a function of time and numerous peaks were detected after separation of proteins by high performance ion exchange chromatography. Protein elution patterns were reproducible and most of the proteins present in the culture medium were neosynthesized as determined by the incorporation of radiolabeled amino acids into nondialyzable proteins.     

Extracellular matrix of cultivated, poorly differentiated human gastric adenocarcinoma cells promotes attachment and spreading of mesenchymal cells

To clarify interactions between carcinoma and mesenchymal cells, we examined the extracellular matrix-substance remaining on culture dishes after confluent growths of gastric carcinoma cells were removed with EDTA. The matrix synthesized by poorly differentiated adenocarcinoma cells (cell lines KATO-III and MKN-45) cultivated in serum-free medium has a fibroblast (cell line WI38)-attachment activity, which is not detected in the matrix synthesized by a well differentiated adenocarcinoma (cell line MKN-28). This activity was not observed in KATO-III-matrix extracted with 6 M urea, but could be detected in a 1% SDS extract from the remaining matrix on the culture dishes after 6 M urea extraction. The activity was abolished by treatment with pronase (16 micrograms/ml), trypsin (0.005%) or alkali, but was unaffected by collagenase (80 micrograms/ml, 4 h) or chondroitinase ABC (1 U ml, 1 h). It is conceivable that the fibroblast-attachment activity of the matrix produced by poorly differentiated adenocarcinoma cells is related to the proliferation of interstitial connective tissue in vivo.     

Long-term culture of human esophageal explants and cells

Human esophageal epithelium obtained from intermediate autopsies (<12 h) was maintained as cell and explant cultures. In order to develop a serum-free, defined media culture model, several medias and additives were evaluated. The viability and differentiation of the epithelial cells cultured with serum-free, Keratinocyte Growth Media (KGM, Clonetics Co., San Diego, CA) was improved over that of esophageal cells and explants cultured in either serum-supplemented CMRL 1066 (OCM), serum-free additive-supplemented CMRL 1066, or cimetidine-supplemented CMRL 1066. The KGM component EGF was determined to be trophic for esophagus cells on the basis of findings of increased ³H-TdR tabelling in KGM cultures when compared to control cells grown in KGM without EGF (KBM). The morphologic pattern of the cytoskeletal proteins actin, keratin, and vimentin were characterized in isolated cell populations. The intermediate filaments, keratin, and vimentin were co-expressed in these epithelial cells. Esophageal explant viability, differentiation, and outgrowth from 15 cases were also evaluated in dishes coated with basement membrane associated proteins. Explants cultured in these dishes were equally well-preserved and differentiated. There were no significant differences in the explant histology when there was protein coating of the culture dishes, although one case showed improved outgrowth with laminin coating. A main advantage for using this culture system is that the same medium (KGM) can be used for both the culture of explants and isolated epithelial cells. Future applications of this model include determining: (1) the effect different concentrations of EGF and calcium in the media will have on esophageal proliferation and differentiation, and (2) the role of different basement membrane associated proteins on the plating efficiency of either isolated or outgrowth epithelial esophageal cells.This is publication #2544 from the Pathobiology Laboratory.     

Effect of cyclodextrin-encapsulated beta-carotene on progesterone production by bovine luteal cells

Experiments were conducted to examine the effect of cyclodextrin-encapsulated beta-carotene on basal or cholesterol (cyclodextrin-encapsulated), LH and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production by bovine corpus luteum cells isolated from mid-luteal heifer ovaries by collagenase digestion. Cells were cultured with serum-free DMEM/Ham's F12 medium in serum pre-treated plastic culture dishes for periods of up to 11 days. Medium was replaced after 24h and thereafter every 48 h. Beta-carotene was added to cultures in a carrier molecule, dimethyl-beta-cyclodextrin, to facilitate dissolution. All treatments were started on day 3 of culture. Treatment of cells with 1 or 2 micromol/l beta-carotene resulted in sharp inhibition of progesterone production. On the contrary, treatment of cells with 0.1 micromol/l beta-carotene resulted in significant stimulation (P<0.05) of both basal and cholesterol-stimulated progesterone secretion. The effect of beta-carotene on LH or dbcAMP-stimulated progesterone production was also examined. Treatment of cells with LH or dbcAMP always resulted in stimulation of progesterone secretion (P<0.001). However, cells treated with LH plus beta-carotene or dbcAMP plus beta-carotene both produced significantly (P<0.01) less progesterone relative to those cells treated with LH or dbcAMP alone on days 7, 9 and 11 of culture. These results indicate that beta-carotene can enhance luteal steroidogenesis when present at low concentrations but is inhibitory at higher concentrations and that encapsulation of beta-carotene in cyclodextrin is an effective method of supplying it to cells in culture.     

Mammalian cell cultivation on serum-coated microcarriers

Summary The cell culture on serum-coated microcarriers yielded higher efficiency of cell attachment to microcarriers and more favorable initial cell distribution on microcarriers than on the conventional microcarriers. By employing serum-coated microcarriers, the maximum cell density was increased by 46% in low serum medium and by 30% in 10% (v/v) serum-supplemented medium. Serum coating of microcarriers could provide cell attachment factors and may replace costly attachment factors supplemented in low serum medium and serum-free medium.     

Indication of selective growth of human endometrial epithelial cells on extracellular matrix

Summary The culturing of human endometrium in conventional plastic dishes and media is only partially successful, mainly because a growth of a heterogeneous population of cells is achieved. Naturally produced extracellular matrix closely resembles the subepithelial basement membrane and seems to affect both growth and differentiation of cells. These qualities of the extracellular matrix (ECM) were applied for obtaining endometrial epithelial cultures. Endometrial tissue specimens were plated after slicing on ECM-coated dishes and kept for up to 8 d. The growth of a confluent homogeneous tissue composed of polygonal epithelial-like cells was demonstrated. To further characterize these cells, cultures were examined by scanning electron microscopy and transmission electron microscopy. Scanning electron microscopy revealed flattened polygonal cells covered with microvilli, among which ciliated cells were observed. By transmission electron microscopy the cells were seen as a monolayer, with some cells overlapping, closely adherent to the matrix. Microvilli, as well as intracellular vacuoles and glycogen granules were observed. Cell type specific cytoskeletal markers were demonstrated by antibodies to intermediate filament proteins (keratin and epithelial membrane antigen). Taken together, the morphologic and immunohistochemical studies indicate that a selective growth of the epithelial component of endometrial tissue was obtained after plating unprocessed endometrial tissue fragments on ECM-coated culture dishes. This work was supported by PHS grant no. CA 30289 to J.V.     

Primary culture and cryopreservation of mouse astrocytes under serumfree conditions

The methods of primary culture and cryopreservation of mouse astrocytes under serum-free conditions were examined. Cerebra from newborn C3H/He mice were employed as the source of astrocytes. The cultured cells were able to grow in a serum-free, chemically defined medium containing transferrin, hydrocortisone, biotin, sodium selenite, insulin, fibroblast growth factor and epidermal growth factor. After the culture was maintained in the medium for 3 weeks, purity was assessed using immunofluorescence staining. The great majority of the cells (>98%) contained glial fibrillary acidic protein and S-100 protein which are cell markers of astrocytes. To cryopreserve the enriched astrocytes under serum-free conditions, various cryoprotectants were examined. The combination of 10% dimethylsulfoxide and 0.1% methylcellulose gave the highest survival rate. These methods of primary culture and cryopreservation will be useful in physiological and biochemical studies which require mouse astrocytes.     

Serum-free culture of stromal and functionally polarized epithelial cells of guinea-pig endometrium: a potential model for the study of epithelial-stromal paracrine interactions.

Stromal and glandular epithelial (GE) cells were isolated from guinea-pig endometrium and growth to near confluency (6-8 days) in primary culture on plastic surfaces in a serum-supplemented medium (SSM). The stromal cells were subcultured on plastic dishes and maintained for 72 h in SSM. Then SSM was replaced by a chemically defined medium (CDM) and the stromal cells grown to confluency (5-7 days). The GE cells were subcultured in CDM, on a basement membrane matrix (Matrigel) applied to permeable Millicell-PC filters, and grown to confluency (5 days). Homogeneity of the subcultured endometrial cell populations was ascertained immunocytochemically. The filter-cultured GE monolayers were polarized morphologically, and displayed epithelial-specific specialized structures. These monolayers had functional tight junctions as verified by a measurable transepithelial resistance. The subcultured cell populations were distinguished by an analysis of their cellular and secretory proteins after labelling with [35S]-methionine and analysis by polyacrylamide gel electrophoresis. The filter-cultured GE monolayers allowed identification of the proteins released vectorially in the apical or the basal secretory compartment, thus demonstrating the functional polarization of GE cells in this bicameral culture system. Within the defined conditions of this culture system, the paracrine factors released by the two endometrial cell populations as well as the interplay of stromal-epithelial interactions and ovarian hormones could be investigated.     

A serum-free defined culture system which maintains follicle-stimulating hormone responsiveness and differentiation of porcine granulosa cells

A serum-free defined culture system has been developed that maintains follicle-stimulating hormone (FSH)-dependent differentiation of porcine granulosa cells from small follicles for up to six days in culture. Confluent monolayers of epithelioid cells were established after culture on fibronectin-coated culture dishes (FBN, 2 micrograms/cm2) in nutrient medium supplemented with human low-density lipoprotein (LDL, 10 micrograms/ml), insulin (I, 1 microgram/ml), and thrombin (TH, 1 NIH U/ml). Each of these factors was necessary to maintain the epithelioid morphology of the monolayers that attained 70% of the protein content and 71% of the cell number of replicate cultures maintained in nutrient medium supplemented with 10% fetal calf serum and insulin. Addition of FSH to the FBN/LDL/I/TH-supplemented cultures resulted in dose-dependent increases in progesterone secretion and [125I]-iodo-human chorionic gonadotropin (hCG) binding comparable to those obtained in the cultures containing serum. These results indicate that the attachment, epithelioid morphology, and differentiated function of porcine granulosa cells (GCs) can be maintained in defined culture conditions. This culture system will facilitate study of the effects of growth promoters and differentiative agents on GC function in the absence of poorly defined serum supplements.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

Effects of sex steroids and antihormones on growth, adhesiveness and receptors of L-929 cells cultured in serum-containing and serum-free media.

L-929 cells contain distinct steroid hormone receptors for glucocorticosteroids, for androgens and for estrogens. We studied the effects of different hormones at physiological concentrations on androgen and estrogen receptor protein accumulation and on cell multiplication. The cells were cultured in steroid-free serum-containing medium, either in Petri dishes or in suspension cultures, and in serum-free medium in Petri dishes. The presence of androstanolone (30 nM) in suspension cultures decreased the concentration of estradiol receptor-binding sites in the cytoplasmic fraction. This decrease was progressive following 3, 5 or 10 days of suspension culture in the presence of the androgen; simultaneously a parallel increase in cell multiplication and DNA was observed. The estradiol receptor decrease was approx. 50% after 10 days of treatment and was unaltered after a further 5 days. It was verified that the low androstanolone concentration in the medium did not provoke the translocation of the estradiol receptor into the nucleus. Progesterone 50 nM also decreased the cytoplasmic estradiol binding sites but had no influence on cell growth and no cytoplasmic progesterone receptor could be found. Diethylstilbestrol (30 nM) did not decrease the concentration of androgen receptor.Cell multiplication was stimulated after several days of suspension culture in the presence of either diethylstilbestrol, estradiol or androstanolone at a concentration of 10–30 nM. The specific anti-hormones, tamoxifen and cyproterone acetate, inhibited selectively the growth effects of estrogens and androgen, respectively. L-929 cells could be cultured for a long period of time in serum-free medium in Petri dishes. Cell adhesiveness was increased in the presence of 40 nM androstanolone or 40 nM estradiol, as well as cell multiplication. Dexamethasone had a negative effect on cell adhesiveness and cell growth. The experimental data suggest that at low concentrations the different steroids operated each through its own receptor and were active on cell growth even in serum-free medium.     

Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF

A simple dissociation procedure and the collagen gel culture system have been utilized to determine the effects of mammogenic hormones and epidermal growth factor (EGF) on the proliferation of normal rat mammary epithelial (RME) cells in serum-free culture. Epithelial fragments, isolated from normal virgin F344 rat mammary glands by enzyme digestion followed by Percoll density gradient centrifugation, were embedded within a rat tail collagen matrix. A three- to four-fold increase in cell number was observed when ovine prolactin (PRL) and progesterone (P) were present in the basal medium during 7 days of culture. Mouse EGF stimulated one cell doubling during the same culture period. Isolated mammary organoids produced a 'stellate' type colony when PRL + P were present in the culture medium. These colonies were composed of small, tightly packed cuboidal cells. The addition of EGF to the basal medium produced a diffuse 'basket' type colony which was composed of large, elongate cells. When the complete hormonal and growth factor combination (PRL + P + EGF) was present, a 'mixed' type colony was observed which contained both the large and small epithelial cell types. Immunocytochemical analysis revealed that both the cuboidal and elongate cells present in the two colony types stained with antibodies to keratin indicating that these cells were epithelial in nature. The small cuboidal cells also expressed thioesterase II and alpha-lactalbumin, both specific for secretory mammary epithelial cells. The large, elongate cell type, however, was positive for actin but did not stain for either secretory epithelial specific marker. The results reported here suggest that normal rat mammary tissue may contain two epithelial populations, one which responds to PRL + P and the other which responds to EGF.     

Fibronectin and polylysine requirement for proliferation of neuroblastoma cells in defined medium

We have demonstrated in this study that we could eliminate the requirement of a serum preincubation for proliferation of B104 neuroblastoma cells in defined medium. When cells were plated directly into serum-free defined medium after trypsin or EGTA detachment, they had no difficulty in adhering or remaining attached to the plastic substratum but were incapable of cell division. However, the addition of human plasma fibronectin to serum-free defined medium and precoating the tissue culture dishes with polylysine at each subculture permitted cell division to occur. Fibronectin was only required at the time of subculture and did not need to be replenished at each medium change. In addition, we have shown that clonal growth and serial subculture are possible in serum-free defined medium provided that the cell inoculum encounters the appropriate substratum. These findings are consistent with a role for fibronectin and a positively charged substratum in the growth regulation of B104 neuroblastoma cells. This completely defined culture system will be of great benefit in studying the growth regulation and differentiation of these neuronal cells.