Fibroblasts serum-free medium

Vitamins and lipids are often required for animal cells to grow in serum-free medium. Phosphoethanolamine and ethanolamine are key additives that facilitate the growth of the mammary tumor cell line 64024 (Kano-Sueoka and Errick, 1981). In addition, ethanolamine promotes the growth of human lymphocytes and mouse hybridoma cells. Short-term cultures of human diploid lung and foreskin fibroblasts grow in medium that includes among its supplements soybean lecithin, cholesterol, sphingomyelin, and vitamin E. 

Fig. 2. Effect of serum concentration on the attachment and spreading of BHK-21 cells onto TCP2 surface. BHK-21 cells were seeded in media containing the indicated concentrations of intact serum (open squares), Fn-depleted serum (triangles). Vn-depleted serum (circles), or serum-free medium alone (the single closed square) and the attachment panel (A) and spreading panel (B) of the cells were determined after 90 min culture on TCP (panel (A, B)) Mean SEM. (Reproduced from J. Biomed. Mater. Res. [Ref. 11 Role of serum vitronection and fibronectin in adhesion of fibroblasts following seeding onto tissue culture polystyrene] through the courtesy of John Wiley Sons, Inc.) BHK-21 Fibroblast cell lines from Baby Hamster Kidney 2 Similar results on Primaria are also presented in [Ref 11]...

Deposition from serum-free medium culturing in serum-containing medium Fluorinated polymer Spatially separated coculture of human alveolar epithelial cells, human hepatoma cells, and mouse 3T3 fibroblast Photocatalytic removal of fluorinated polymer (mask lithography) 2009 [164]... 

The second category includes mesenchymal cells, such as fibroblasts (BALB/c 3T3, Swiss 3T3), adipocytes, endothelial cells, smooth thin muscle cells, and neuroectodermic cells (such as glia cells). Most of these cells need maintenance factors. Some cells, such as the NIH-3T3, can grow in a serum-free medium containing minimal medium supplemented with transferrin (25 pg/ml), insulin (10 pg/ml), EGF (100 ng/ml), bFGF (100 ng/ml), and PDGF (0.5 U/ml). 

Because of the low protein content of some serum-free media the cells may be more fragile and fibroblasts and epithelial cells may attach only poorly to the substratum. In such cases it is important to adapt cells to growth in serum-free medium by growing cells in steadily decreasing concentrations of serum. The following protocol is adapted from information supplied by Boehringer Company Limited for adaption of cells for growth on 1% Nutridoma ... 

Temin (1970) and Todaro et al. (1965) showed similar effects for chicken fibroblasts and 3T3 mouse fibroblasts. The low level of serum is important for survival as well as for the subsequent stimulation of DNA synthesis (Cherrington, 1984). A kinetic analysis using time lapse cinematography (Zetterberg and Larsson, 1985) showed that Swiss 3T3 cells were only susceptible to cell cycle arrest in a short period (3-4 h) following mitosis. Even a 1-h exposure to serum-free medium during this time forced the cells into GO from which they required 8 h to return to Gl. The length of the postmitotic sensitive phase was very constant at between 3 and 4 h but considerable intercellular variability existed in the duration of the pre S-phase Gl period consistent with a transition probability event ( 10.4). 

Fig. 2.2. Positive and negative adhesion of fibroblasts on fibronectin and tenascin coating of a tissue culture dish, after saturation with BSA. After I h of plating in serum-free medium, cells appear loosely attached on BSA, well spread on fibronectin and not attached on tenascin. Crystal violet staining. (Photograph courtesy of Ruth Chiquet, Friedrich Miescher Institute.)...

L929 mouse fibroblast cells in this device changed their morphology due to the forces exerted on them. A gradient was created by introducing a standard culture medium with fetal bovine serum (FBS) at one inlet and a serum-free medium at the other. FBS contains proteins, such as serum albumin, that are needed for cell survival, division and growth. The cell density and attachment depended on a combination of sheer stress and FBS concentration [28]. 

Eccleston, P.A., Gunton, D.J. and Silberberg, D.H. (1985) Requirements for brain cell attachment, survival and growth in serum-free medium effects of extracellular matrix, epidermal growth factor and fibroblast growth factor. Dev. Neurosci. 7 308-322. 

The steroid derivative 1,25-dihydroxy vitamin D (vit D3), a metabolically active form of vitamin D, was shown to act as an inducer of the NGF gene in murine fibroblasts cultured in a serum-free medium (Wion et al., 1991). This effect could be detected as early as 3 h after the addition of vit D3 and resulted in an increase in both cellular mRNA and secreted NGF protein. Further experiments in the same culture system (Jehan et al.,... 

Figure 3. Focal adhesions in (A,B) embryonic fibroblasts and (C,D) tumor cells removed from MMTY-PyMT mice. Cells were plated on fibronectin in serum-free medium. Fibroblasts were stained with rhodamine-phalloidin, FITC conjugated anti-paxillin antibodies, and Hoechst 33258 stain. Tumor cells were stained with rhodamine-phalloidin and FITC conjugated anti-vinculin antibodies. Fluorescence images of the cells were obtained using a decovolution microscope. Paxillin and vinculin (green) are localized to ends of microfilaments (red) in focal adhesions. Mgat5 (A) fibroblasts and (C) tumor cell show focal adhesions but these structures are absent in MgatS (B) fibroblasts and (D) tumor cells. (E), T cell receptor dependent stimulation measured by H-thymi-dine incorporation in response to anti-CD3 antibodies at 48h (F) Model of responses to variable substratum adhesions for Mgat5 and Mgat5 cells.

Figure 10. Primary cultures of mouse kidney cells. Primary cultures of kidney epithelial cells derived from 10-day-old mice were grown either in hormonally defined medium with five supplements (5 pg/ml insulin, 5 pg/ml transferrin, 25 ng/ml PCE, 5 X10" M hydrocortisone, and 5 x 10" M Tj), or in medium supplemented with 10% fetal calf serum. After 10 days, primary cultures still were epithelial in morphology serum free (a) but were overgrown with fibroblasts with serum (b). (Taub et al., 1979 with permission.)...

Feeder cells for fusion cultures (see Note 6) essential for fusions employing rat myelomas. Quickly thaw irradiated rat fibroblasts, prepared as described in Note 6, just before commencing the cell fusion. Add the cells to 10 mL of serum-free DMEM, centrifuge, and wash once in serum-free DMEM Resuspend feeders in HAT medium just before addition of the fusion mixture. Alternatively, use thymocytes from spleen donors (mouse). 

Fibroblasts are cultured under standard conditions in F10+ medium with 15% fetal calf serum. The level of free NeuAc is greatly influenced by culture conditions. Do not use Chang medium (leads to very high control values). The final culture is done in a 175-cm2 culture flask for 7 days, the last 3 days with 5% fetal calf serum. Medium is removed, cells are washed with phosphate-buffered saline (PBS), trypsinized, centrifuged at 100 xg, and washed with PBS. The resultant pellets are frozen before use, in 1.5-ml sample vials at -80°C. 

Radiolabeled [l- C]18 3n-3 was purchased from Perkin-Elmer Life Sciences (Boston, MA). The free acid form of 24 5n-3 and 24 6n-3 were generous gifts from A. Spector and H. Sprecher. Human skin fibroblasts from normal controls and patients with peroxisomal or mitochondrial disorders were received from the Mental Retardation Research Center of the Kennedy Krieger Institute. Cells at 90% confluence were incubated with 0.05 pCi albumin-bound [1- C] 18 3n-3 in Dul-becco s modified Eagle s minimum essential medium supplemented with 10% fetal bovine serum for 3 d. At harvest, cells were removed and washed with Hanks solution. An aliquot of cells was removed for protein determination, and the remainder was used for analysis of fatty acids after conversion to their methyl esters (17). Radiolabeled fatty acid methyl esters were separated by reversed-phase high performance liquid chromatography (18), and collected by a fraction collector. The radioactivity was counted by liquid scintillation counting. 

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