Fibroblasts serum-supplemented medium

It is also unlikely that the increased GSL in these cells is related to the uptake of GSL from serum supplemented medium, since the medium has previously been shown not to be the primary source of cellular GSL in cultured fibroblasts (29) (see also above). The effect of serum and lipoproteins on GSL biosynthesis will be the subject of future studies in this laboratory. 

Fig. 5.2. Primary baby mouse kidney cultures were established at about 103 cells/cm2 in medium based on a 50 50 mixture of DMEM F12 supplemented with 10% FBS (a) or a mixture of 5 hormones (PGE1, hydrocortisone, triodothyronine, insulin and transferrin (b). Although over 99% of the attached cells were epithelial at day 1, by the time the photograph was taken (day 11), fibroblasts had completely overgrown the epithelial cells in the serum-supplemented medium. Only epithelial cells are present in the hormone-supplemented culture. (Reproduced from Taub et al., 1979, with thanks.)...

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. 

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). 

Both multiple carboxylase deficiencies are characterized by deficient activities of the three mitochondrial carboxylases in peripheral blood leukocytes prior to biotin treatment. The carboxylase activities increase to near normal or normal after treatment with pharmacological doses of biotin. Patients with biotin holocarboxylase synthetase deficiency have deficient activities of the three mitochondrial carboxylases in fibroblasts incubated in medium with low biotin concentrations (containing only the biotin contributed by fetal calf serum added to the medium for cell growth), whereas patients with biotinidase deficiency have normal carboxylase activities under these conditions. The activities of the carboxylases in biotin holocarboxylase synthetase deficiency become near normal to normal when cultured in medium supplemented with high concentrations of biotin. 

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.)...

Primary fibroblasts from skin biopsy samples are grown at 37°C in the presence of 5% C02 in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% foetal calf serum (FCS) and 1% penicillin/streptomycin. For passaging, confluent cells are washed with phosphate-buffered saline (PBS) and incubated for 5-10 min at 37°C in the presence of trypsin. 

As an alternative, primary skin fibroblasts or lymphoblasts of patients suspected to be affected with a cholesterol biosynthesis defect can be cultured for 3-7 days in medium supplemented with fetal calf serum depleted of lipoproteins to induce cholesterol biosynthesis, whereupon the specific defect can be determined by sterol analysis using GC-MS as described above. This procedure will readily identify patients affected with Smith-Lemli-Opitz syndrome, desmosterolosis, lathosterolosis, hydrops-ectopic calcification-motheaten (HEM) skeletal dysplasia and most patients with Conradi-Hunermann syndrome (CDPX2). Patients with congenital hemidys-plasia with ichthyosiform nevus and limb defects (CHILD) syndrome may not be identified with this assay, but they can be readily diagnosed on the basis of their typical clinical presentation. 

Endothelial cells are maintained in a basal culture medium, such as DMEM or a proprietary medium supplied with the cells, which is supplemented with hydrocortisone (lug/mL). epidermal growth factor (EGF 100 ug/mL) bovine fibroblast growth factor (FGF 1 ng/mL), antibiotics (gentamicin and amphotericin, at 50 ug/mL) and fetal calf serum (2-10%). Alternatively, bovine brain extract (3 ug/mL) can be used instead of EGF and FGF. The cells are cultured in either standard tissue culture flasks directly on plastic or on flasks coated with collagen. The cells are grown to confluence and subcultured and reseeded in a ratio of 1 3. For all experiments primary endothelial cells should be used between passages 3 and 12. 

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. 

Normal, malignant, and virus-transformed diploid fibroblast -like derivatives exhibit differing morphological features and proliferating capacities when cultured with growth medium supplemented with a single serum type. For example, medium containing only fetal bovine serum (10-15%) provides more than adequate nutrition for virus-transformed cells and the more advanced normal diploid and aneuploid cell types. However, this same medium will prove to be detrimental for the routine initiation of cultures... 

Fig. IB. The effect of SAB on the repair kinetics of potentially lethal damage in normal human fibroblasts, Ewing s sarcoma, and human lung adenocarcinoma. No inhibitor (open circles) 8SAB (closed circles). Data points are means 1 standard deviation. Both normal fibroblast and human tumor cell cultures were grown and maintained in Eagle s MEM supplemented with 10% (VA ) fetal bovine serum, streptomycin and penicillin, at S7°C in a humidified chamber of 95% air and 5% CO2. Stock cultures in exponential growth were detached by trypsinization and appropriate cell numbers plated in 25 cm flasks and incubated to permit cell attachment. Inhibitors of poly(ADP-ribose) synthetase (SAB or BZ) were added to the appropriate cultures 2 hr prior to irradiation in air at room temperature. Following irradiation, the cells were exposed to the inhibitors for 24 hr, washed, and replenished with fresh medium. Refeeding was performed every S days and after 10-14 days, cells were fixed and the colonies stained with Giemsa. Only colonies of 50 cells or more were scored as survivors. All experiments were performed in triplicate and the survival data expressed as the mean S.E. of three separate survival experiments.

HeLa cells and 3T3 Fibroblasts were routinely grown in 75 cm cell culture flasks at 37 °C with 5% CO2 in a humidified atmosphere. Dulbecco s Modified Eagle s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin was used as culture medium for HeLa cell line and fibroblasts. After reaching the 80-90% confluence, the cells were washed with Dulbecco s Phosphate Buffered Saline (PBS) and harvested through trypsinization. 

Swiss 3T3 fibroblast morphology 48 h postseeding on PNIPAM brushes of thickness (a) 3 nm, (b) 5 nm, and (c) 37 nm. Swiss 3T3 cells were seeded at 1(P cells/cm and cultured in Dulbecco s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2% L-glutamine, 1% penicillin (100 U/ml), and 1% streptomycin (100 U/ml) for 48 h. Cells were observed at 37°C using a Leitz Orthoplan microscope in bright field. Scale bar = 100 pm. 

The culture of Hela cells was carried out with Eagle s minimal essential medium, and the fibroblast cell line derived from human kidney was cultured with RPMI medium 1640. Both were supplemented with calf serum to a concentration of 10% and with penicillin and streptomycin sulfate to concentrations of 100 units per ml. Before cells were cultured, several slides were immersed in the cell culture bottle. While covered with cultured cells, they were taken out and put in the bottles containing Hank s solution (3 ml) and AaHI (30-80 pg/ml). These bottles were incubated at 37°C and the slides were examined microscopically at various time intervals. 

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