Goat serum: an alternative to fetal bovine serum in biomedical research

Serum is frequently added to the defined basal medium as a source of certain nutritional and macromolecular growth factors essential for cell growth. Although a number of synthetic media have been prepared serum continues to be used in cell culture by many investigators. The best supplementation to a basal medium is fetal bovine serum (FBS) that is most frequently used for all types of cell cultures. During last four decades National Institute of Virology, Pune, has been working on isolation and identification of viruses from clinical specimens, employing tissue culture. Initially FBS was used for this purpose. However, due to its prohibitive cost and uncertain supply an alternative was sought. Commercially available sera from newborn calf, sheep, horse, human and serum obtained from goat blood (available from local abattoir) were tried. Goat serum (GS) was found to be suitable for most of the cell lines and primary cultures. Primary cultures from guinea pig embryo, monkey kidney, chick embryo, mouse peritoneal macrophages, and established cell lines were prepared and grown in growth media supplemented with GS. These cultures were studied for their morphology and growth in comparison with cultures grown in FBS containing media, and were used for mass cultivation of cells, quantitation and susceptibility of various virus strains, studies on effects of different nutrients and natural substances on cellular metabolism and virus replication, epitope analysis of various strains of Japanese encephalitis (JE) virus, strain differentiation studies, studies on antibody dependent plaque enhancement, assay of murine migration inhibition factor. Monoclonal antibodies against JE virus adapted to GS were characterised for their retention of functionalities. The results were comparable to those of cell cultures grown in FBS containing media. Similar results on chromosome studies were obtained from patient's whole blood cultures prepared in GS and FBS containing growth media. Organ cultures from mammalian, reptile and avian hosts; successfully grown in GS supplemented growth media, were used for different virological studies. Growth media supplemented with GS were used for in vitro cultivation of malarial parasites. Thus since the last three decades many scientists are using GS in place of FBS, in various fields of biomedical research. The present article reviews an account of the same.     

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.     

Effects of serum reduction and VEGF supplementation on angiogenic potential of human adipose stromal cells in vitro

Objectives

This study investigated effects of reduced serum condition and vascular endothelial growth factor (VEGF) on angiogenic potential of adipose stromal cells (ASCs) in vitro.

Materials and methods

Adipose stromal cells were cultured in three different types of medium: (i) F12/DMEM (FD) supplemented with 10% FBS from passage 0 (P0) to P6; (ii) FD supplemented with 2% FBS at P6; and (iii) FD supplemented with 2% FBS plus 50 ng/ml of VEGF at P6. Morphological changes and growth rate of ASCs were recorded. Changes in stemness, angiogenic and endogenic genes’ expressions were analysed using Real‐Time PCR.

Results

Adipose stromal cells changed from fibroblast‐like shape when cultured in 10% FBS medium to polygonal when cultured in 2% FBS plus VEGF‐supplemented medium. Their growth rate was lower in 2% FBS medium, but increased with addition of VEGF. Real‐Time PCR showed that ASCs maintained most of their stemness and angiogenic genes’ expression in 10% FBS at P1, P5 and P6, but this increased significantly in 2% FBS at P6. Endogenic genes expression such as PECAM‐1, VE chaderin and VEGFR‐2 decreased after serial passage in 10% FBS, but increased significantly at P6 in 2% FBS. Addition of VEGF did not cause any significant change in gene expression level.

Conclusion

Adipose stromal cells had greater angiogenic potential when cultured in reduced serum conditions. VEGF did not enhance their angiogenic potential in 2% FBS‐supplemented medium.

     

The effects of bovine serum albumin and fetal bovine serum on the development of pre- and postcleavage-stage bovine embryos cultured in modified CR2 and M199 media

The effects of protein supplementation on bovine embryo development in vitro was evaluated using a 4 × 2 factorial arrangement with ten replications. A total of 6438 oocytes collected from abattoir ovaries were used. Bovine serum albumin (BSA) and fetal bovine serum (FBS) were added in various combinations to simple (modified CR2) and complex (M199) media during culture of precleavage-stage IVM/IVF-derived ova from 18 h after insemination to 72 h and postcleavage-stage embryos after 72 h of culture. Cleavage rates did not differ (p > 0.05) between media supplemented with FBS or with BSA. However, the postcleavage development to the blastocyst stage of in vitro-derived bovine embryos is better in media supplemented with FBS than BSA. A greater (p < 0.05) proportion of cleaved occytes developed to blastocysts and hatched blastocysts in media supplemented with FBS during postcleavage culture. The percentage of embryos that stopped development at the morula stage was significantly (p < 0.05) greater in media supplemented with BSA during postcleavage culture. Viability of blastocysts produced in CR2 and M199 supplemented with FBS were further assessed by transfer to recipients. In CR2, 25 transferred blastocysts resulted in seven pregnancies and the birth of three normal calves. In M199, 24 transferred blastocysts resulted in five pregnancies and the birth of two normal calves. There was no difference (p > 0.05) in rate of embryo development between CR2 and M199.     

Glycogen synthase kinase-3 regulates microglial migration,inflammation, and inflammation-induced neurotoxicity

Microglia play a prominent role in the brain's inflammatory response to injury or infection by migrating to affected locations, secreting inflammatory molecules, and phagocytosing damaged tissue. However, because severe or chronic neuroinflammation exacerbates many neurological conditions, controlling microglia actions may provide therapeutic benefits in a diverse array of diseases. Since glycogen synthase kinase-3 (GSK3) promotes inflammatory responses in peripheral immune cells, we investigated if inhibitors of GSK3 attenuated microglia responses to inflammatory stimuli. Treatment of BV-2 microglia with GSK3 inhibitors greatly reduced the migration of microglia in both a scratch assay and in a transwell migration assay. Treatment of BV-2 microglia with lipopolysaccharide (LPS) stimulated the production of interleukin-6 and increased the expression of inducible nitric oxide synthase (iNOS) and NO production. Each of these microglia responses to inflammatory stimulation were greatly attenuated by GSK3 inhibitors. However, GSK3 inhibitors did not cause a general impairment of microglia functions, as the LPS-induced stimulated expression of cylcooxygenase-2 was unaltered. Regulation of microglia functions were also evident in cultured mouse hippocampal slices where GSK3 inhibitors reduced cytokine production and microglial migration, and provided protection from inflammation-induced neuronal toxicity. These findings demonstrate that GSK3 promotes microglial responses to inflammation and that the utilization of GSK3 inhibitors provides a means to limit the inflammatory actions of microglia.     

Study of the Chemotactic Response of Multicellular Spheroids in a Microfluidic Device

We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it.     

Removal of Transmissible Spongiform Encephalopathy Prion from Large Volumes of Cell Culture Media Supplemented with Fetal Bovine Serum by Using Hollow Fiber Anion-Exchange Membrane Chromatography

Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrP^Sc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrP^Sc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrP^Sc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrP^Sc from up to 1000 mL of Dulbecco’s modified Eagle’s medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrP^Sc in the initial 200mL flow-through was reduced by 2.5 to > 3 log₁₀, compared with that of the starting material. These results indicate that QSD filtration removes PrP^Sc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth media supplemented with FBS.     

Effects of serum concentration on some growth characteristics of cultured murine parietal yolk sac carcinoma cells

Murine parietal yolk sac carcinoma cells were examined by scanning and transmission electron microscopy to determine the ultrastructural changes resulting from growth, in vitro, in media containing different serum concentrations. Cells grown in medium supplemented with 10% fetal bovine serum (FBS) formed spherical bodies, were generally oval with numerous surface microvilli, well-organized microtubules, abundant free polysomes and a well-developed Golgi apparatus. By contrast, cells grown in 1% FBS failed to form multicellular spheres, were generally flattened over the growth surface and lacked the surface and intracellular features demonstrated when cells were grown in 10% serum. These differences could explain the alterations in the glycosylation of secreted glycoprotein associated with culture in the presence of low serum.     

Serum-free hybridoma culture: ethical, scientific and safety considerations

Despite considerable progress in the development of cell culture techniques, including the development of the serum- and protein-free media that now routinely support hybridoma and mammalian cell growth, fetal bovine serum (FBS) supplemented media are still commonly used: a practice that raises ethical, scientific and safety concerns. The use of FBS in hybridoma culture media is examined here, with regards to the development and production of monoclonal antibodies (mAbs), and it is our recommendation that researchers adopt serum-free cell culture methods to reduce animal use in this area.     

Fibroblast growth factor-stimulated growth of porcine aortic endothelial cells depends on hypoxanthine in fetal bovine serum in culture media

The rate of proliferation of porcine aortic endothelial cells (PAEC) in response to stimulation of fibroblast growth factors (FGFs) was largely retarded in media supplemented with 10% dialyzed fetal bovine serum (FBS) in place of nondialyzed FBS. This inhibition was overcome by supplement of dialyzable fraction, and hypoxanthine was purified from the dialyzable fraction as the active compound which stimulated the basal and FGF-dependent growth rates of dialyzed FBS-treated PAEC. Addition of hypoxanthine (5 microM) to media with 10% dialyzed FBS containing FGFs (10 ng/ml) markedly increased the rate of both cell proliferation and DNA synthesis of PAEC, and their maximal levels were comparable to those attained by cells in media with 10% nondialyzed FBS. Hypoxanthine changed the spindle-like morphology of dialyzed FBS-treated PAEC even in the presence of FGFs into the cobblestone-like morphology of regular PAEC in media with 10% FBS.     

Endogenous thymidine and hypoxanthine are a source of error in evaluating methotrexate cytotoxicity by clonogenic assays using undialyzed fetal bovine serum

None of 13 fresh human tumor samples of various histology cloned in a two-layer agar culture system with 20% undialyzed fetal bovine serum (FBS) showed sensitivity to three antifolates, methotrexate (MTX), trimetrexate and 5,8-dideazaisofolic acid (IAHQ), even after continuous exposure to the highest concentrations (100 microM) for 21 days. In order to investigate this lack of antifolate drug effect, we compared the toxicity of continuous MTX exposure in the human colon carcinoma cell line HCT-8, cloned in a thymidineless medium (RPMI 1640) supplemented with 10% horse serum (HS), 10% fetal bovine serum (FBS), 20% FBS or 20% dialyzed FBS. In the presence of native FBS, when the minimum clone size was set at 30 cells/colony, the survival of HCT-8 cells reached a plateau at approximately 60% of untreated control after exposure to MTX concentrations between 0.1 microM and 100 microM. Only when the minimum clone size was set at 2 X 10(3) cells/colony was the sensitivity of HCT-8 cells to the antimetabolite comparable to that obtained in HS or dialyzed FBS (ED50 values in the range of 0.01 microM). MTX protection experiments indicated that even very small concentrations of thymidine and hypoxanthine together were sufficient to reproduce the pattern of sensitivity to MTX observed under culture conditions with undialyzed FBS. We conclude that for a proper evaluation of MTX cytotoxicity in clonogenic assays, dialyzed FBS and thymidine-less media should be employed; if native FBS is an absolute requirement for growth, only very large colonies (at least 10 cell divisions) should be scored.     

Application of a new medium supplement for propagation and storage of human cytomegalovirus

A new medium supplement, NU-SERUM, was evaluated for cultivation of human embryonic lung fibroblasts (HEL) and for propagation and storage of human cytomegalovirus (HCMV). NU-SERUM was comparable to fetal bovine serum (FBS) in promoting rapid growth of HEL if they were seeded at a sufficient density. HCMV replicated quite satisfactorily in HEL cultured with media supplemented with NU-SERUM as well as FBS. Inactivation of HCMV at 37 C occurred similarly when the medium contained FBS or NU-SERUM. However, at -70 C, HCMV was less stable in NU-SERUM-containing medium than in FBS-containing medium. Sorbitol added to the NU-SERUM-containing medium improved the unstableness of HCMV at -70 C, and HCMV was storable with such medium. Thus, NU-SERUM is useful as an alternative to FBS not only for growth of HEL but also for propagation and storage of HCMV.     

Albumin inhibits cytotoxic activity of lysophosphatidylcholine by direct binding

Fetal bovine serum (FBS) was found to protect Jurkat T cells from LPC-induced cytotoxicity. Twenty micromolar LPC-induced cytotoxicity of 80-90% of the cells in media without FBS for 3 h, whereas 50-70% in media with 0.5% FBS. However, LPC-induced cytotoxicity was not observed in the presence of 5% FBS in media. The cytotoxicity was specific for LPC among lysophospholipids tested and significantly observed with palmitoyl (C16:0) LPC, stearoyl (C18:0) LPC, and oleoyl (C18:1) LPC among 11 synthetic LPCs. Furthermore, the cytoprotective effect of FBS was observed only when it was added before the treatment, but not after the treatment of LPC, and premixing of FBS and LPC before addition to the cells ameliorated LPC-induced cytotoxicity. Finally, albumin, a major constituent of FBS, prevented completely LPC-induced cytotoxicity even at as low as 3 microM concentration. We also found that five molecules of LPC could sequentially bind to one BSA using isothermal titration calorimetry. The above results suggest that the cytotoxic activity of LPC could be attenuated by albumin in blood. Finally, it should be cautioned that, when experiments are conducted with LPC dissolved in assay buffers containing albumin, the albumin in the buffer could influence the results.     

Decreasing variability in your cell culture

Culturing mammalian cells has not significantly changed in almost 50 years. Typically, a synthetic basal medium is chosen to meet the environmental and nutritional requirements of a given cell line. Components, such as amino acids, vitamins, inorganic salts, and a carbon source such as glucose are commonly found in the classical basal media formulation. These basal formulations normally will not support cell growth alone, but must be further supplemented with animal serum, usually fetal bovine serum (FBS) at a concentration of 5-20%. Recent advances in serum-free and chemically defined media formulations have provided cell culturists with options. When considering FDA regulations and potential risks to human health when manufacturing biologics or considering cell therapies, eliminating serum is of paramount concern. For a large majority of researchers however, using classical media with serum builds on previous generations of research and makes cell culture easier to perform.     

Toward improving selectivity in affinity chromatography with PEGylated affinity ligands: The performance of PEGylated protein A

Chemical modification of macromolecular affinity chromatography ligands with polyethylene glycol chains or “PEGylation” can potentially improve selectivity by sterically suppressing non‐specific binding interactions without sacrificing binding capacity. For a commercial protein A affinity media and with yeast extract (YE) and fetal bovine serum (FBS) serving as mock contaminants, we found that the ligand accounted for more than 90% of the media‐associated non‐specific binding, demonstrating an opportunity for improvement. The IgG static binding affinity of protein A mono‐PEGylated with 5.0 and 20.7 kDa poly(ethylene glycol) chains was found to be preserved using a biomolecular interaction screening platform. Similar in situ PEGylations of the commercial protein A media were conducted and the modified media was functionally characterized with IgG solutions spiked with YE and FBS. Ligand PEGylation reduced the mass of media‐associated contaminants by a factor of two to three or more. Curiously, we also found an increase of up to 15% in the average recovery of IgG on elution after PEGylation. Combined, these effects produced an order of magnitude increase in the IgG selectivity on average when spiked with YE and a two‐ to three‐fold increase when spiked with FBS relative to the commercial media. Dynamic binding capacity and mass‐transfer resistance measurements revealed a reduction in dynamic capacity attributed to a decrease in IgG effective pore diffusivity and possibly slower IgG association kinetics for the PEGylated protein A ligands. Ligand PEGylation is a viable approach to improving selectivity in affinity chromatography with macromolecular ligands. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1364–1379, 2014     

Analyses of basal media and serum for in vitro expansion of suspension peripheral blood mononucleated stem cell

Transplantation of stem cells requires a huge amount of cells, deeming the expansion of the cells in vitro necessary. The aim of this study is to define the optimal combination of basal medium and serum for the expansion of suspension peripheral blood mononucleated stem cells (PBMNSCs) without resulting in loss in the differentiation potential. Mononucleated cells were isolated from both mice and human peripheral blood samples through gradient centrifugation and expanded in α-MEM, RPMI, MEM or DMEM supplemented with either NBCS or FBS. The suspension cells were then differentiated to osteoblast. Our data suggested that α-MEM supplemented with 10 % (v/v) NBCS gives the highest fold increase after 14 days of culture for both mice and human PBMNSCs, which were ~1.51 and ~2.01 times, respectively. The suspension PBMNSCs in the respective medium were also able to maintain osteoblast differentiation potential as supported by the significant increase in ALP specific activity. The cells are also viable during the differentiated states when using this media. All these data strongly suggested that α-MEM supplemented with 10 % NBCS is the best media for the expansion of both mouse and human suspension PBMNSCs.     

Serum‐free media for the production of human mesenchymal stromal cells: a review

The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large‐scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)‐supplement for optimal cell proliferation. FBS‐based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum‐use to serum‐free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large‐scale production of MSCs.     

TGFβ (transforming growth factor β) and keratocyte motility in 24 h zebrafish explant cultures

Fish keratocytes are used as a model system for the study of the mechanics of cell motility because of their characteristic rapid, smooth gliding motion, but little work has been done on the regulation of fish keratocyte movement. As TGFβ (transforming growth factor β) plays multiple roles in primary human keratinocyte cell migration, we investigated the possible involvement of TGFβ in fish keratocyte migration. Studying the involvement of TGFβ1 in 24 h keratocyte explant allows the examination of the cells before alterations in cellular physiology occur due to extended culture times. During this initial period, TGFβ levels increase 6.2‐fold in SFM (serum‐free medium) and 2.4‐fold in SFM+2% FBS (fetal bovine serum), while TGFβ1 and TGFβRII (TGFβ receptor II) mRNA levels increase ～3‐ and ～5‐fold respectively in each culture condition. Two measures of motility, cell sheet area and migration distance, vary with the amount of exogenous TGFβ1 and culture media. The addition of 100 ng/ml exogenous TGFβ1 in SFM increases both measures [3.3‐fold (P=4.5 × 10^?5) and 26% (P=2.1 × 10^?2) respectively]. In contrast, 100 ng/ml of exogenous TGFβ1 in medium containing 2% FBS decreases migration distance by 2.1‐fold (P=1.7 × 10^?7), but does not affect sheet area. TGFβ1 (10 ng/ml) has little effect on cell sheet area in SFM cultures, but leads to a 1.8‐fold increase (P=1.5 × 10^?2) with 2% FBS. The variable response to TGFβ1 may be, at least in part, explained by the effect of 2% FBS on cell morphology, mode of motility and expression of endogenous TGFβ1 and TGFβRII. Together, these results suggest that expression of TGFβ and its receptor are up‐regulated during zebrafish keratocyte explant culture and that TGFβ promotes fish keratocyte migration.     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Effect of phenol red and steroid hormones on cystic fibrosis transmembrane conductance regulator in mouse endometrial epithelial cells

Previous studies have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-mediated Cl(-)channel found in most epithelia including reproductive tract, could be regulated by various culture conditions. The present study further investigated the effect of phenol red, a pH indicator widely used in growth medium, and steroid hormones, present in the supplement fetal bovine serum (FBS), on primary cultured endometrial epithelial cells by monitoring ion channel activities using the short-circuit current technique. When compared to the results obtained with normal medium supplemented with regular FBS, the forskolin-stimulated I(SC), presumably mediated by CFTR, obtained in phenol red-free medium was significantly reduced, from 16.95+/-1.53 microA/cm(2)(control) to 9.72+/-0.89 microA/cm(2)(medium without phenol red, P< 0.05). The forskolin-activated I(SC)was further attenuated to 5.29+/-0.46 microA/cm(2)in the phenol red-free medium when supplemented with charcoal/ dextran-treated FBS where steroid hormones were removed. Our data suggest that phenol red and steroid hormones present in culture medium and FBS supplement, respectively, may somehow upregulate CFTR expression in vitro. Our study demonstrates the need for carefully choosing the culture media and supplements due to the effect of steroid hormones.