Effects Of Fetal Bovine Serum Research Articles

Effects of fetal bovine serum and serum-free conditions on white and dark axolotl neural crest explants

Neural crest cells from both white mutant and dark (wildtype) axolotls (Ambystoma mexicanum) were cultured in increasing concentrations of fetal bovine serum (FBS; 2 to 20%). For each explant, the total number of cells that migrated and the percent of differentiated melanophores were recorded. At concentrations of FBS above 2% melanophore differentiation was essentially equivalent (32 to 59%) for both the white and dark neural crest cultures, but subtle differences in cell behavior and differentiation were found between the two phenotypes. By contrast there was a significant difference in the percent melanization of cells in serum-free control cultures, wherein melanophore differentiation in dark neural crest cultures was, on average, 18% compared to 5% in white cultures. Thus, contrary to all previously published work, white and dark neural crest cells are not intrinsically equivalent. Our culture results are discussed with regard to the probable in vivo conditions that cause the white phenotype.

In vitro Effects of Fetal Bovine Serum and Glucose on Multiplication of Cryptobia salmositica

Cryptobia salmositica multiplied rapidly at 10 C in a minimum essential medium (containing 1.0 mg glucose/ml, Hanks' salts and L-glutamine) supplemented with heat-inactivated fetal bovine serum (FBS) and HEPES buffer (25 mM). The multiplication rate of C. salmositica was related to the amount of FBS; the peak number (approximately 9 x 10(6) parasites/ml) was attained in about 6 wk when the medium contained 25% final concentration of FBS. Glucose utilization was related to the number of parasites; the maximum utilization was reached before peak numbers. Formation of rosette colonies was correlated with multiplication rate and numbers of parasites in cultures. Degenerate round forms found in old cultures probably were caused by the accumulation of metabolic wastes in the medium.

Influence of culturing temperature and proteinase inhibitors on the spontaneously occurring changes in the organ culture of psoriatic skin

Skin explants from involved psoriatic lesions showed dissociation of keratinocytes, dermal-epidermal separation and degenerative changes such as cytoplasmic swelling of subcorneal prickled cells within 24 h after culture initiation at 37°C in the absence of fetal bovine serum (FBS). These histological changes developed time dependently, while normal skin explants did not exhibit such phenomena. Some of the uninvolved psoriatic skin explants showed only degerative change 48 to 72 h after culture initiation at 37°C. To determine the nature of these spontaneously occurring changes in psoriatic skin explants and then to approach the pathogenesis of psoriasis, the effects of FBS, various proteinase inhibitors (PIs) and culturing temperature (37, 31, 24°C) were examined in skin organ culture of normal and involved psoriatic skin. At 37°C, only serine PIs (5 or 10 mg/ml of soybean trypsin inhibitor (SBTI), 1000 KIU/ml of aprotinin, or 2 mg/ml of comostat mesilate in the medium) or FBS (20% in the medium) could suppress the occurrence of dissociation of keratinocytes and dermal-epidermal separation but not the degenerative change in involved psoriatic skin explants, while other types of PIs did not exhibit any such inhibition. When the culturing temperature was reduced from 37°C to 31 or 24°C, the formation of dissociation of keratinocytes and dermal-epidermal separation was almost non-existent and only moderate degenerative change was observed. The addition of FBS or serine PIs to the culture at 31°C revealed the formation of very weak degenerative change. Furthermore, the addition of two different serine PIs, 2 mg/ml of camostat mesilate and 5 mg/ml of SBTI, almost completely inhibited not only dissociation of keratinocytes and dermal-epidermal separation but also degenerative change in involved psoriatic skin explants cultured at a reduced temperature (31°C). These results indicate that there were elevated levels of serine proteinase(s) in psoriatic epidermis which might be involved in dissociation of keratinocytes and/or dermal-epidermal separation with a possible heat-activation system, and may furthermore be involved in degeneration at higher layers of the psoriatic epidermis.

Growth and Secretory Responses of Enriched Populations of Corticotropes*

The purpose of this study was to learn whether enriched populations of corticotropes could be grown without the other pituitary cell types. Corticotropes populations were enriched to 80-90% by counterflow centrifugation in an elutriator with the Sanderson chamber. After initial separation into small, medium, and large fractions, the cells were stimulated for 3 h with 0.5 nM corticotropin-releasing hormone (CRH) and then re-eluted to remove the enlarged corticotropes. More ACTH (6- to 10-fold) was released in media with 10% fetal bovine serum (FBS) than was released in media with no serum. The effects of FBS could not be mimicked by 0.3% BSA. Corticotropes grew in serum-containing media as long as they were plated at a density of at least 2500 cells per well. The corticotropes expanded in size and assumed two major morphological subtypes. Both stored ACTH and beta-endorphin. One subtype was flattened and pleomorphic. The other subtype was stellate with multiple processes. Cell counts showed a 2.5- to 3.8-fold increase in the number of labeled corticotropes during the first 21 days of culture. Then the numbers of cells declined rapidly. Basal secretion of ACTH rose 1.6-fold during the first week, plateaued after 14 days and then declined to less than 30% of first week levels. CRH stimulation produced dose-dependent increases in media ACTH. In 7 day cultures, both basal and stimulated levels of ACTH were similar to those in 7 day cultures of mixed pituitary cells (containing equivalent numbers of corticotropes). Stimulatory effects of CRH were evident for up to 42 days of culture. Arginine vasopressin enhanced CRH-mediated secretion in most cultures in the first week. Pretreatment with glucocorticoids (100 nM corticosterone) for 15 h blocked CRH-mediated secretion in all cultures. The studies showed that corticotropes do not need the other pituitary cell types for basic plating and basal and CRH-mediated secretory responses. Further tests of specific growth factors are needed to learn whether they will maintain function for longer periods.

Generation of natural killer cells and lymphokine-activated killer cells in human AB serum or fetal bovine serum

Interleukin 2 (IL-2) can induce or enhance the cytotoxic activity of natural killer (NK) cells and lymphokine-activated killer (LAK) cells. The effects of fetal bovine serum (FBS) and human AB serum (HABS) on the IL-2-induced NK cell and LAK cell activities of large granular lymphoctyes (LGL) were measured, respectively, against the NK-sensitive cell line K562 and the LAK-sensitive cell line Daudi. FBS and HABS were essentially equivalent in their effects on IL-2-dependent NK activity with prolonged culture. However, with prolonged incubation from 1 to 5 days of PBMC in the presence of IL-2, there was considerably less generation of LAK cell activity in FBS (Day 3: 5.3 ± 3.4 LU 10 7 cells ) compared to HABS (Day 3: 44.6 ± 4.2 LU 10 7 cells ) ( P < 0.05). These differences in IL-2-dependent LAK cell generation did not appear to be due to the lot of the FBS or to activating factors present in individual samples of HABS. Similarly, the suppressive effects of FBS could not be reversed with increasing concentrations of IL-2 ranging from 10 to 100 U/ml. Importantly, the presence of FBS in the cultures resulted in more cell death (15.9 ± 5.6%) at 4 days of culture compared to HABS (1.8 ± 1.0%) ( P < 0.05). These results suggest that FBS may inhibit generation of LAK effector cells, but not NK cells in cultures containing IL-2 and that the use of HABS as a culture supplement is preferable to FBS in studies of human LAK cell function.

Quantitative characterization of domes in primary mouse mammary epithelial tumor cell cultures.

Dome populations from primary cultures of mouse mammary tumor cells were quantitatively studied in regard to size, distribution, density and the area occupied by light-diffusion photography and image analysis. The effects of fetal bovine serum, insulin and hydrocortisone were analyzed. Quantitative characterization documented dome diameter (mode diameter 0.26 to 0.52 mm), dome area occupied (average 23%, maximum 38.7%), and density (average 78 domes per cm2, maximum 117 domes per cm2) for standard culture conditions. Insulin and hydrocortisone had a primary effect on dome density whereas 15% fetal bovine serum had a minimal effect. However, insulin and hydrocortisone had a synergistic optimal effect on dome population. Time-lapse cinematography revealed that the dome population is not static, but a very dynamic one. Domes underwent irregular pulsations of expansion and contraction. Dome enlargement was either by a series of expansions and contractions, by lateral involvement of other cells, or by coalescence of two or more domes. Domes have been considered to be the in vitro counterpart of the in vivo acinus of the mouse mammary gland. However, quantitative dome population characterization has not been available. Dome analysis by light-diffraction photography and image analysis lends itself towards correlative studies of domes and their differentiative products.