Serum-free Primary Culture System Research Articles

Effects of Fatty Acids on Proliferation of Cultured Wild-type and FABP5-KO Thymic Epithelial Cells.

Lipids including fatty acids (FAs), which are water-insoluble molecules, are not only a cellular energy source but also signaling molecules that induce and modulate the expression of various cellular functions. Fatty acid-binding proteins (FABP) bind FAs in the cytoplasm, and are thought to determine the cellular localization of FAs. In a previous observation, FABP5 was expressed in thymic epithelial cells (TEC) in the thymus and was influenced by FAs. Fatty acids have mostly inhibitory effects on various cell types, including cancer cells, but their effects on TEC have not been well investigated. In this study, we investigated the effects of long-chain FAs (LCFAs) and the involvement of FABP5 in cell proliferation using a serum-free primary culture system. The results showed that saturated fatty acids did not affect proliferation, but n-3 long-chain polyunsaturated FA (LCPUFA) reduced, n-6 LCPUFA increased, and retinoic acid strongly reduced the percentage of proliferating wild-type TEC. The proliferation of FABP5-KO TEC was more significantly affected by LCPUFA, suggesting that FABP5 is an important modulator of FA-mediated TEC proliferation. These observations may provide a basis for exploring the properties of TEC.

Rolipram potentiates bevacizumab-induced cell death in human glioblastoma stem-like cells

AimsGlioblastoma cancer stem-like cells (GCSCs) promote themselves proliferation by secreting the vascular endothelial growth factor A (VEGFA) in an autocrine manner, positively regulated by phosphodiesterase IV (PDE4). In the current study, we investigated the putative cytotoxic effect of bevacizumab, a VEGFA blocker, alone and in combination with a specific inhibitor of PDE4 called rolipram on GCSCs isolated from human surgical tumor specimen with a focus on PI3K/AKT pathway. Main methodsCD133+/CD15+ GCSCs were characterized by flow cytometry and expanded in a serum-free primary culture system. The cell survival, apoptosis, and protein expression values were measured using MTT assay, TUNEL staining and western blot, successively. Intracellular cAMP and free secreted VEGFA levels were assessed by cAMP enzyme immunoassay and ELISA, respectively. Key findingsBevacizumab suppressed GCSCs survival with IC50~6.5μg/ml and enhanced the levels of apoptosis, p53 and cleaved-caspase3 along with a decrease in free VEGFA levels and ERKs activation. However, there was no significant modulation of AKT phosphorylation on serine 473, the intracellular PDE4A, VEGFA and cAMP levels. More cytotoxicity in co-treated cells coupled with a more substantial decline in the free VEGFA levels and a greater increase in the quantities of p53 and cleaved-caspase3 compared to those treated with bevacizumab alone. Co-treatment reduced phospho-AKT, endogenous VEGFA and PDE4A values but elevated cAMP levels. SignificanceThis study highlighted a booster cytotoxic effect of combined rolipram and bevacizumab treatment on the GCSCs primary culture, suggesting that this approach is warranted in treatment of GBMs overexpressing VEGFA and PDE4A.

L-tyrosine induces melanocyte differentiation in novel pink-eyed dilution castaneus mouse mutant showing age-related pigmentation

BackgroundThe mouse pink-eyed dilution (oculocutaneous albinism II; p/Oca2p) locus is known to control tyrosinase activity, melanin content, and melanosome development in melanocytes. Pink-eyed dilution castaneus (pcas/Oca2p−cas) is a novel mutant allele on mouse chromosome 7 that arose spontaneously in Indonesian wild mice, Mus musculus castaneus. Mice homozygous for Oca2p−cas usually exhibit pink eyes and beige-colored coat on nonagouti C57BL/6 (B6) background. Recently, a novel spontaneous mutation occurred in the progeny between this mutant and B6 mice. The eyes of this novel mutant progressively become black from pink and the coat becomes dark gray from beige with aging. ObjectiveThe aim of this study is to clarify whatever differences exist in melanocyte proliferation and differentiation between the ordinary (pink-eyed) and novel (black-eyed) mutant using serum-free primary culture system. MethodsThe characteristics of melanocyte proliferation and differentiation were investigated by serum-free primary culture system using melanocyte-proliferation medium (MDMD). ResultsThe proliferation of melanoblasts in MDMD did not differ between the two mice. However, when the epidermal cell suspensions were cultured with MDMD supplemented with l-tyrosine (Tyr), the differentiation of black-eyed melanocytes was greatly induced in a concentration-dependent manner compared with pink-eyed melanocytes. Immunocytochemistry demonstrated that the expression of tyrosinase and tyrosinase-related protein-1 (Tyrp1) was greatly induced or stimulated both in pink-eyed and black-eyed melanocytes, whereas the expression of microphthalmia-associated transcription factor (Mitf) was stimulated only in black-eyed melanocytes. ConclusionThese results suggest that the age-related coat darkening in black-eyed mutant may be caused by the increased ability of melanocyte differentiation dependent on l-Tyr through the upregulation of tyrosinase, Tyrp1, and Mitf. This mutant mouse may be useful for animal model to clarify the mechanisms of age-related pigmentation in human skin, such as melasma and solar lentigines.

Co-Mitogenic Effect of Pancreatic Polypeptide on Adult Rat Hepatocytes in Primary Culture

Proliferation of hepatocytes in liver regeneration is regulated by humoral and neuronal factors. Humoral factors include hormones and growth factors (mitogens). The neuronal factors, however, have not yet been identified. Factors that amplify the effect of complete mitogens (epidermal growth factor, transforming growth factor a and hepatocyte growth factor) are termed co-mitogens, which include vasopressin, angiotensin II, noradrenaline, neurotensin and neuropeptide-y. We have surveyed various other neuropeptides and gastrointestinal hormones that may have a co-mitogenic effect on rat hepatocytes using a complete serum-free primary culture system. Pancreatic polypeptide at 10 - 9 to 10 - 6 M amplified DNA synthesis stimulated by a low concentration of EGF (1 ng/mL) by 4- to 5-fold. Neuropeptide Y or peptide YY that have a structural similarity to pancreatic polypeptide did not amplify DNA synthesis. A similar amplification was found for secretin (10 - 1 0 to 10 - 7 M). These results indicate that pancreatic polypeptide and secretin can be regarded as novel co-mitogens, and suggest that these peptides and other co-mitogenic neuropeptides, most of them are found also in neurons, are candidate neuronal factors in hepatocyte proliferation in vivo, i.e. liver regeneration.

Positive Modulation of the Adipoconversion of Adipoblasts Derived from Genetically Obese Rats by Sodium Butyrate

Using a new serum-free primary culture system, we have previously reported genotypic differences between adipoblasts derived from the epididymal adipose deposit of lean and obese 8-week-old Zucker and Wistar Diabetic Fatty (WDF) rats (15). In these strictly controlled culture conditions, obese-derived adipoblasts expressed low levels of the late markers of differentiation (lipid accumulation, GPDH). In order to further characterize obese-derived adipoblasts and analyze the critical relationship between growth and differentiation, growth arrest was induced in lean- and obese-derived cultures using sodium butyrate treatment. Addition of 2.5 mM sodium butyrate to the serum-free medium from day 1 reduced markedly the growth of lean as well as obese-derived cells. Adipoconversion of lean-derived adipoblasts was not altered, similar levels of LPL and GPDH activities being obtained in control and butyrate-treated groups. By contrast, a marked increase in both activities was observed in obese-derived cultures, restoring the level of both markers of differentiation to the lean level. Similar results were obtained with adipoblasts derived from subcutaneous inguinal (ING) fat pad of obese Zucker as well as adipoblasts derived from ING and EPI fat deposits from obese WDF rats. Taken together, these results suggest that adipose deposits of these genetically obese rats contain a specific adipoblast population which differs from lean-derived adipoblasts in respect to its adipoconversion capacity and/or its stage of commitment to differentiation.

Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture.

It has been a subject of controversy whether fibroblastic cells are necessary for the proliferation of intestinal epithelial cells in primary culture. To answer this question, we have developed a serum-free primary culture system which allows reproducible and quantitative assays of proliferation and differentiation of fetal rat intestinal epithelial cells in the absence of fibroblastic cells. Pure intestinal epithelial tissues were obtained from 16.5-day fetal rats without contamination of mesenchymal cells, and were successfully cultured on a collagen gel in a medium consisting of Ham's F12, bovine serum albumin, epidermal growth factor (EGF), insulin, cholera toxin, transferrin and hydrocortisone. The epithelial nature of the cultured cells was confirmed by the presence of cytokeratin in the cells. Under optimal culture conditions, intestinal epithelial cells readily attached to the substratum in a day, and proliferated rapidly in vitro, increasing their number about 10 times in the first 5 days. EGF, insulin, cholera toxin, transferrin and hydrocortisone synergistically induced the epithelial proliferation, and lack of any one of them resulted in a significant reduction of the proliferation. In contrast, fetal bovine or horse serums, which have been widely used to supplement culture media, severely inhibited the epithelial proliferation. Histological examination showed that the epithelial cells formed simple cuboidal epithelia with basally-located nuclei when cultured on collagen gels. The intestinal epithelial nature of the cells was affirmed by the presence of villin on their luminal surface. Ultrastructurally, cells were connected by tight junctions and desmosomes at the subluminal region, and microvilli were projecting on the luminal surface, indicating that the cells in primary culture retained some characteristics of absorptive epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation and differentiation of mouse uterine epithelial cells in primary serum-free culture: Estradiol-17β suppresses uterine epithelial proliferation cultured on a basement membrane-like substratum

The effects of different substrata and estradiol-17 beta (E2) on proliferation and differentiation of mouse uterine epithelial cells was examined in a serum-free primary culture system. When cultured on rat-tail collagen gels, the epithelial cells rapidly increased in number to form a simple squamous cell layer that exhibited a relatively undifferentiated state (a few short microvilli, no secretory granules, and poorly developed endoplasmic reticulum). Addition of E2 into the culture medium did not affect the proliferation of epithelial cells on collagen gel. Uterine epithelial cells grown on a reconstituted basement membrane-like substratum (Matrigel) formed a simple columnar/cuboidal cell layer exhibiting fully developed characteristics (many long microvilli, many secretory granules, and fully developed endoplasmic reticulum). Examination of epithelial proliferation by counting substratum-attached cell number revealed only a slow increase in cell growth on Matrigel, and E2 did not significantly affect it. However, measurement of proliferating cells by labeling cells with 5-bromo-2'-deoxyuridine revealed that cells on Matrigel were replicating and that E2 (10(-7) to 10(-11) M) actually significantly suppressed epithelial proliferation. However, there was not an effect of E2 on total cell number, indicating that the cells in control medium replicate faster and detach more readily from the substratum than those in E2-supplemented medium on Matrigel. Thus, it is probable that E2 significantly reduces the rate of cell detachment from the substratum, which may mimic the in vivo condition where significant decrease in apoptosis or cell death is induced by E2.

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

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 produce 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.

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors

A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mammary glands of immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbecco's modified Eagle's medium-F12 culture medium containing 10 micrograms/ml insulin, 1 microgram/ml prolactin, 1 microgram/ml progesterone, 1 microgram/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 micrograms/ml transferrin, and 5 microM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like projections, stellate colonies, and two- and three-dimensional web units. Optimal proliferation required insulin, prolactin, progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane. In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this model should prove to be an important tool to study the hormonal regulation of the growth and development of rat mammary cells.

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, beta-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated.

Induction of Mineral Deposition by Primary Cultures of Chicken Growth Plate Chondrocytes in Ascorbate-containing Media: Evidence of an association between matrix vesicles and collagen

A serum-free primary culture system for chicken growth plate chondrocytes has been developed which consistently undergoes mineral deposition. Upon attainment of confluency, the chondrocytes develop locally into multilayer cellular nodules leading to matrix calcification. Mineralization first occurs in matrix vesicles (MV) that are abundant in the extraterritorial matrix between the hypertrophic cells. Studies with 45Ca reveal that significant accumulation of Ca2+ occurs as early as day 12, continuing progressively throughout the culture period. By day 24, the nodules become densely calcified. Fourier transform infrared spectroscopy reveals the mineral to be similar to apatite, with features essentially identical to those of mineral formed by MV in vitro. The presence of ascorbate is critical to the culture system; in its absence, calcification is rarely observed. Ascorbate stimulates MV formation and synthesis of cellular protein, alkaline phosphatase, and especially types II and X collagens. In addition, there is strong evidence that the types II and X collagens are associated with MV. 1) Electron microscopy reveals MV embedded in a type II collagenous network; 2) Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MV using monospecific antibodies to types X and II collagen indicate that both collagens are present in specific MV fractions; 3) sucrose gradient purification of MV does not remove associated collagens; 4) graded salt extraction selectively releases type II collagen from MV; and 5) incubation of radiolabeled types II and X collagens with MV leads to their cosedimentation upon subsequent centrifugation. Taken together, the data suggest that coordinated synthesis of the collagens, alkaline phosphatase, MV formation, and Ca2+ accumulation by the cultures combine to induce mineral deposition in the multilayer nodules.

A serum-free primary culture system for studying cell-substrate interactions during newt epidermal cell migration

To study the interaction of migrating newt epidermal cells with purified extracellular matrix (ECM) molecules we have developed an in vitro migration assay using pieces of newt skin explanted onto culture dishes coated with various ECM molecules and cultured for 18 h in defined serum-free medium. Newt epidermal cells migrate out from explants placed on dishes coated with either collagen, vitronectin, fibronectin, or fibrinogen but not on albumin-coated or uncoated dishes. Explant outgrowth on collagen was best in CEM 2000 medium diluted to 60% of mammalian osmolarity. Other media such as RPMI 1640 or Ex-Cell 300, diluted similarly, may also be used although in our hands CEM 2000 always allowed more migration. We found no migration on collagen when skin explants were incubated in Holtfreter's solution (an amphibian saline solution that we have previously shown allows reepithelialization on amputated newt limbs). Supplementation of Holtfreter's solution with glucose did not improve its ability to support migration. By testing various supplement combinations in conjunction with CEM 2000 and RPMI 1640 we found that neither serum, insulin, selenium, transferrin, nor L-glutamine is required for explant outgrowth. Of the additives tested, outgrowth was stimulated only by insulin. Epidermal cell outgrowth on collagen was inhibited by both puromycin and cycloheximide, indicating the necessity for protein synthesis in this system. Whether the effects of these protein synthesis inhibitors are specifically on migration-related events or on general metabolic requirements is not clear. Inasmuch as there was no correlation (r = -0.227) between DNA synthesis (measured by incorporation of tritiated thymidine) and the amount of outgrowth, we believe that our assay is a measure of cell migration alone rather than a combination of mitosis and migration. This explant outgrowth system represents a new and relatively simple assay that can be used in the study of cell-substrate interactions during newt epidermal cell migration over extracellular matrix molecules in a defined serum-free environment.