Recombinant Human Albumin in Cell Culture: Evaluation of Growth-Promoting Potential for NRK and SCC-9 Cells In Vitro

Serum-derived albumin has for a long time been used in cell culture media, but the exact role of albumin and/or impurities bound to albumin has not been precisely defined. In this study, recombinant human albumin was evaluated for its growth-promoting activity on two cell lines, NRK and SCC-9. For NRK cells, the recombinant human albumin was found to exert an inhibitory effect. The fact that fatty acid free HSA was also inhibitory while HSA fraction V was stimulatory suggested a role for fatty acids or some other bound moieties in growth stimulation by HSA fraction V. Addition of oleic acid, cholesterol, phosphatidylcholine, phosphatidylserine or a combination of these lipids, however, did not significantly improve the growth stimulating activity of either fatty acid free HSA or the recombinant human albumin. For SCC-9 cells, both recombinant human albumin and fatty acid free HSA showed slight stimulation (although they were not as active as HSA fraction V), suggesting that in some cell systems, the albumin molecule per se may promote cell growth and survival. This revised version was published online in July 2006 with corrections to the Cover Date.     

Separation of growth-stimulating activity of BSA fraction V from the bulk of albumin using Heparin Sepharose Chromatography

Bovine serum albumin (BSA) is a potential source of biological contamination in cell culture medium. The aim of this work was to attempt to replace BSA in low serum and serum-free medium (SFM). BSA fraction V was subjected to a variety of processes in order to determine if the growth promoting activity observed for NRK cells could be extracted from the BSA molecule. These included solvent extractions, diafiltration, reverse phase HPLC and affinity chromatography using heparin sepharose. Solvent extraction and diafiltration failed to remove the activity from the BSA. Affinity chromatography using heparin sepharose indicated that all of the activity observed with BSA was retained in the 0.5 M NaCl fraction and was associated with less than 3% of the original protein. The major protein band in the 0.5 M NaCl fraction had the same apparent molecular weight as albumin (as seen by SDS-PAGE and analytical reverse phase HPLC). Unlike the untreated BSA, the 0.5 M NaCl fraction was partially susceptible to proteolytic digestion and to variations in pH.Abbreviations HS heparin sepharose - DHS donor horse serum - SFM serum free-medium     

Immunoassay for human serum albumin using capillary electrophoresis–semiconductor laser-induced fluorometry

Capillary electrophoresis combined with semiconductor laser-induced fluorometry was applied to an immunoassay of human serum albumin. Human serum albumin was labeled with a fluorescent molecule (Cy5), which has an absorption maximum at 649 nm. The labeled albumin was purified by ultrafiltration in order to reduce signals, which are unreacted labeling reagent, product, and fragment products derived therefrom. After the purification, no signal for unreacted labeling reagent and fragment products was detectable in the electropherogram of the labeled albumin. The labeled albumin was then reacted with anti-albumin to form an immunocomplex, which was separated from the excess free albumin. The competitive immunoassay was used in the determination of human serum albumin in a controlled serum sample, using the labeled albumin. The obtained value was found to be 0.21±0.02 mg/ml, which is in good agreement with other known values.     

Critical variables controlling cell proliferation in primary cultures of rat tracheal epithelial cells

Summary The purpose of our experiments was to examine variables affecting early events in the establishment of rat tracheal epithelial (RTE) cultures as well as factors regulating long-term RTE cell growth. The experiments showed that when RTE cells were seeded into complete serum-free medium between 13 and 30% of the seeded cells attached. Of the seeded cells, only ∼2% entered into DNA synthesis and underwent repeated cell divisions to form colonies containing >20 cells. Coating the dishes with extracellular matrix components had little effect on cell attachment or colony forming efficiency (CFE). However, coating the dishes with fetal bovine serum markedly increased CFE. The media components bovine serum albumin and bovine pituitary extract were shown to be important in promoting cell attachment as well as CFE. Cholera toxin on the other hand had no effect on cell attachment but significantly increased CFE. These and other studies showed that cell attachment and cell proliferation are independently regulated. Studies on long-term culture growth indicated that the number of progeny produced per colony forming unit (CFU) is inversely proportional to the number of CFUs seeded. Inasmuch as the cultures did not become confluent under any of the culture conditions tested and media obtained from high density cultures were shown to be growth inhibitory, these findings suggest that a diffusible growth restraining factor is being produced by the cultures limiting clonal expansion. Experiments showing growth inhibitory effects of media conditioned by high cell density cultures support this interpretation. The putative factor reaches critical concentrations earlier in cultures seeded with high numbers of CFU than in cultures seeded with low numbers of CFU. Because the cultures are known to produce transforming growth factor-beta, this growth regulator probably plays a role in controlling RTE cell proliferation. However, it is likely than other events, such as depletion of growth factors from the media, also are significant in regulating the growth of the cultures.     

The effect of human serum albumin on the extended storage of human oral keratinocyte viability under mild hypothermia

Isolated oral keratinocytes in suspension provide a number of advantages for use in maxillofacial surgery, however, the poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. The purpose of the present study was to evaluate whether human serum albumin (HSA) could serve as an effective constituent of a storage medium to enhance human oral keratinocyte (HOK) viability under conditions of mild hypothermia. Primary human oral keratinocytes were isolated from small pieces of the non-inflamed gingival tissues obtained during the extraction of the third molars of patients. HOK were cultured on collagen type I-coated culture dishes in keratinocyte growth medium (KGM). After the trypsinization of a culture dish (passage 2 or 3), freshly isolated HOK were stored for 24, 48, and 72 h at 4 degrees C or at room temperature in KGM, saline, Dulbecco's modified Eagle's medium (DMEM), saline supplemented with 10% HSA or DMEM supplemented with 10% (v/v) HSA under one atmosphere pressure. After storage, HOK cell survival was determined by dye exclusion using trypan blue and colony-forming assay and cell cycle change was obtained by flow cytometry. Highest cell viability was obtained in saline supplemented with 10% HSA and DMEM supplemented with 10% (v/v) HSA at 4 degrees C and at room temperature. Under these conditions no significant decline in keratinocyte viability was observed for at least 48 h. The cell cycle profiles of these cells were also maintained for at least 48 h at room temperature. These observations demonstrate that HSA might be better at preserving the viability of HOK stored under hypothermic and mild hypothermic conditions up to 48 h.     

Surface plasmon resonance analysis of ricin binding to plasma membranes isolated from NIH 3T3 cells

As the potential for bioterrorism has appeared to increase, the need for simple systems for identifying potential inhibitors of the binding of such biological agents to cell membranes has increased. In this work, surface plasmon resonance (SPR) was used to monitor binding of ricin, a ribosome-inactivating protein, to the plasma membranes of NIH 3T3 cells. Once conditions were established, efficacy of the system for monitoring effectiveness of compounds at inhibiting ricin binding was ascertained by determining the IC₅₀ values for asialofetuin (ASF) and for bovine serum albumin derivatized with an average of 34 lactosyl moieties (BSA-Lac₃₄). Results indicated that SPR is an efficient method for measuring adherence of a toxin to isolated cell plasma membranes. SPR can also indicate whether a compound that is an effective inhibitor of binding when a single ligand such as ASF is used will be as effective when used in studies with cells that may express multiple cell surface ligands for ricin and/or the inhibitor.     

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500?µg/L) and the half-life of IGF-1 in blood circulation is only 4.5?min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA–IGF-1 reached 100?mg/L. The fusion protein HSA–IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA–IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA–IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA–IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.     

Direct analysis of growth factor requirements for isolated human fetal hepatocytes

Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein (1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification and characterization of additional normal hepatocyte growth factors. This work was supported by NIH grant DK35310. Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes. In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate that the malignant transformation of these cells may involve the production of autocrine growth stimulators.     

Synthesis of F16 conjugated with 5‐fluorouracil and biophysical investigation of its interaction with bovine serum albumin by a spectroscopic and molecular modeling approach

5‐Fluorouracil (5‐FU) has been widely used as a chemotherapy agent in the treatment of many types of solid tumors. Investigation of its antimetabolites led to the development of an entire class of fluorinated pyrimidines. However, the toxicity profile associated with 5‐FU is significant and includes diarrhea, mucositis, hand–foot syndrome and myelosuppression. In aiming at reducing of the side effects of 5‐FU, we have designed and synthesized delocalized lipophilic cations (DLCs) as a vehicle for the delivery of 5‐FU. DLCs accumulate selectively in the mitochondria of cancer cells because of the high mitochondrial transmembrane potential (ΔΨ_m). Many DLCs exhibited anti‐cancer efficacy and were explored as potential anti‐cancer drugs based on their selective accumulation in the mitochondria of cancer cells. F16, the DLC we used as a vehicle, is a small molecule that selectively inhibits tumor cell growth and dissipates mitochondrial membrane potential. The binding of the conjugate F16–5‐FU to bovine serum albumin (BSA) was investigated using spectroscopic and molecular modeling approaches. Fluorescence quenching constants were determined using the Stern–Volmer equation to provide a measure of the binding affinity between F16–5‐FU and BSA. The activation energy of the interaction between F16–5‐FU and BSA was calculated and the unusually high value was discussed in terms of the special structural block indicated by the molecular modeling approach. Molecular modeling showed that F16–5‐FU binds to human serum albumin in site II, which is consistent with the results of site‐competitive replacement experiments. It is suggested that hydrophobic and polar forces played important roles in the binding reaction, in accordance with the results of thermodynamic experiments. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of serum and serum-derived factors on the growth and production of α-fetoprotein and albumin by human hepatoma cell lines

A serum-free culture system of human hepatoma cell lines (HuH-6 and HuH-7) was used to investigate the activity of bovine serum (BS) and of serum-derived factors on the growth and production of -fetoprotein (AFP) and albumin. At higher concentrations, dialyzed BS was inhibitory to the growth of HuH-6 and caused reduction of the level of AFP production by the cells. AFP and albumin levels in HuH-6 and HuH-7 were reduced or unchanged by fetuin, bovine serum albumin (BSA) and transferin (TF), although no cytotoxicity was shown by any of them. Commercial preparations of platelet-derived growth factor exhibited cytotoxicity to HuH-6 and HuH-7 and induced a decrease of AFP and albumin levels in a dose-dependent manner. Transforming growth factor (TGF-) exhibited no cytotoxicity to HuH-6. AFP levels in HuH-6 were unchanged with 1000 pg/ml TGF-, but albumin levels were decreased. TGF-7 at a concentration of 1000 pg/ml was cytotoxic to HuH-7 and AFP levels were a little increased. Albumin levels, however, were unchanged. Following exposure to cycloheximide, AFP and albumin levels in HuH-6 were inhibited.Abbreviations AFP -fetoprotein - BS bovine serum - BSA bovine serum albumin - EDTA ethylenediaminetetraaceticacid - ELISA enzyme-linked immunosorbent assay - HBSS Hank's balanced salt solution - PBS phosphate buffered saline - PDGF platelet-derived growth factor - TF transferrin - TGF-\ transforming growth factor beta     

Enhancement of albumin expression in bone tissues with healing rat fractures

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.     

Effects of defined medium,fetal bovine serum,and human serum on growth and chemosensitivities of human breast cancer cells in primary culture: Inference for in vitro assays

Summary We compared the effects of defined medium, fetal bovine serum (FBS) and human serum (HuS) on the growth and responses to chemotherapeutic agents of human breast cancer cells in primary culture. Normal and tumor tissues were dissociated to small aggregates and single cells and seeded onto collagen-gel-coated wells in defined medium or medium supplemented with 5% FBS or 5% HuS. In all cases examined, defined medium and medium containing HuS were superior to medium containing FBS in supporting growth of both normal and tumor cell cultures. However, cultures in defined medium showed an initial cell loss. Cells from the same tumor cultured in different media varied in their responses to chemotherapeutic agents. In light of these results, medium supplemented with HuS, which promoted attachment of these cells in culture and stimulated their growth, should be the most appropriate nutrient environment for determining the effects of therapeutic agents on cells as it most closely resembles the in vivo situation. Because there were also variations in growth rates and chemosensitivities of tumor cells cultured in different human serum samples, we suggest that optimal conditions in which to culture these cells include the serum of the patient whose tumor is removed. This serum may provide host factors that influence cell growth and interact with exogenous factors. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia. J. T. Emerman is a research scholar of the National Cancer Institute of Canada.     

Regulation of satellite cells during skeletal muscle growth and development

Satellite cells are myogenic cells attributed with the role of postnatal growth and regeneration in skeletal muscle. Following proliferation and subsequent differentiation, these cells will fuse with one another or with the adjacent muscle fiber, thereby increasing myonuclei numbers for fiber growth and repair. The potential factors which could regulate this process are many, including exercise, trauma, passive stretch, innervation, and soluble growth factors. Three classes of growth factors in particular (fibroblast growth factor, insulin-like growth factor, and transforming growth factor-beta) have been studied extensively with respect to their effects on satellite cell proliferation and differentiation in culture. Fibroblast growth factor has been shown to stimulate proliferation but depress differentiation. Insulin-like growth factor stimulates both proliferation and differentiation, although the latter to a much greater degree. Transforming growth factor-beta slightly depresses proliferation but inhibits differentiation. When administered in combination, these factors can induce satellite cell activities in culture which mimic those typical of satellite cells found in vivo in growing, regenerating, or healthy mature muscle. Alterations in the concentrations of these growth factors in the muscle environment as well as alterations in the cell's sensitivity or responsiveness to these factors represent potential mechanisms for regulating satellite cell activity in situ.     

Serum suppresses myeloid progenitor apoptosis by regulating iron homeostasis

The growth and survival of committed hematopoietic progenitors is dependent upon cytokine signaling. However, serum is also required for optimal growth of these progenitors in culture ex vivo. Here we report that serum withdrawal leads to myeloid progenitor cell apoptosis. Although serum deprivation-induced cell death has many hallmarks typical of apoptosis, these cell deaths were not inhibited by hemopoietins, survival factors such as IGF-I, or treatment with a broad-spectrum caspase inhibitor. Rather, apoptosis due to serum withdrawal was associated with damage to mitochondria. Surprisingly the serum factor required for myeloid cell survival was identified as iron, and loss of iron led to marked reductions in ATP production. Furthermore, supplementing serum-deprived myeloid cells with bound or free iron promoted cell survival and prevented mitochondrial damage. Therefore, serum suppresses hematopoietic cell apoptosis by providing an obligate source of iron and iron homeostasis is critical for proper myeloid cell metabolism and survival.     

Ligand binding strategies of human serum albumin: How can the cargo be utilized?

Human serum albumin (HSA), being the most abundant carrier protein in blood and a modern day clinical tool for drug delivery, attracts high attention among biologists. Hence, its unfolding/refolding strategies and exogenous/endogenous ligand binding preference are of immense use in therapeutics and clinical biochemistry. Among its fellow proteins albumin is known to carry almost every small molecule. Thus, it is a potential contender for being a molecular cargo/or nanovehicle for clinical, biophysical and industrial purposes. Nonetheless, its structure and function are largely regulated by various chemical and physical factors to accommodate HSA to its functional purpose. This multifunctional protein also possesses enzymatic properties which may be used to convert prodrugs to active therapeutics. This review aims to highlight current overview on the binding strategies of protein to various ligands that may be expected to lead to significant clinical applications. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Hepatocyte culture on biodegradable polymeric substrates

The interactions of primary rat liver cells with biodegradable polymeric substrates were investigated in vitro to assess the suitability of the polymer materials for use in cell transplantation devices. The kinetics of cell adhesion to, and the growth and biochemical function of cells maintained on, films formed from poly (D,L-lactic-co-glycolic acid, 88: 12) (PLGA) or from a 50/50 (w/w) blend of PLGA and poly (L-lactic acid) (PLLA) were evaluated in comparison to two control substrates, matrigel coated or collagen-coated polystyrene petri dishes. The rate of cell adhesion to both types of polymeric substrates was similar to the rate of adhesion to the collagen control substrate, but of the two polymers, only the blend was suitable for extended culture. Hepatocytes maintained on the polymer blend films showed retention of differentiated cell function as measured by the rate of albumin secretion-the rate of albumin secretion by cells on the films was the same as the rate for cells on matrigel and reached a level in the range of reported in vivo levels (140-160 mug/10(6) cells/24 h). In contrast, albumin secretion by hepatocytes maintained on collagen-coated polystyrene culture dishes declined over five days to a level one third that of the initial level and one fifth that of cells maintained on the polymer blend films on day five. Such retention of differentiated cell function by hepatocytes in culture has previously been observed only when hepatocytes were cultured in the presence of exogenous extracellular matrix proteins or were cocultured with another cell type. In addition to retention of differentiated function, the cells maintained on the polymer blend films also displayed rates of DNA synthesis similar to controls maintained on collagen-coated polystyrene, a substrate optimal for DNA synthesis.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.     

The subdomain structure of human serum albumin in solution under different pH conditions studied by small angle X-ray scattering

Small-angle X-ray scattering (SAXS) was used to study structural characteristics of human serum albumin (HSA) in solution under different pH conditions. Guinier analysis of SAXS results yielded values of the molecular radius of gyration ranging from 26.7 Å to 34.5 Å for pH varying from 2.5 to 7.0. This suggests the existence of significant differences in the overall shape of the molecule at different pH. Molecular models based on subdomains with different spatial configurations were proposed. The distance distribution functions associated with these models were calculated and compared with those determined from the experimental SAXS intensity functions. The conclusion of this SAXS study is that the arrangement of molecular subdomains is clearly pH dependent; the molecule adopting more or less compact configuration for different pH conditions. The conclusions of this systematic study on the modification in molecular shape of HSA as a response to pH changes is consistent with those of previous investigations performed for particular pH conditions. Correspondence to: J. R. Olivieri     

Effects of serum and serum-derived factors on growth and differentiation of mouse keratinocytes

Summary Mouse epidermal keratinocytes (MK cells) were grown as replicating subcultures at clonal density, in a serum-free, low calcium basal medium supplemented with seven different growth factors (Bertolero et al., Exp. Cell. Res. 155:64–80, 1984). This serum-free system was used to investigate the activity of cells. bovine serum (FBS) and of serum-derived factors on the growth and differentiation of MK cells. Unfractionated, whole FBS inhibited growth and induced terminal differentiation of normal MK cells. The growth inhibitory activity was considerably reduced by passing whole FBS over a resin (Chelex) to remove Ca²⁺ and other di- and trivalent cations. It is not known whether this treatment removed other factors. Addition of individual serum components either stimulated or inhibited cell-growth and differentiation. Fetuin, a major α-globulin of FBS, and high density lipoprotein strongly inhibited the colony forming efficiency (CFE) of MK cells, whereas bovine serum albumin increased the CFE 4.5-fold and stimulated the growth rate as well. The addition of impure commercial preparations of platelet-derived growth factor inhibited the CFE and induced the morphological features of squamous terminally-differentiating keratinocytes. As reported in other systems, transforming growth factor beta (TGF-β) inhibited the growth of secondary keratinocytes in a dose-dependent manner. Thus, at least three factors present in FBS inhibited growth whereas others were stimulatory. These observations explain the difficulties in obtaining replicating subcultures of mouse keratinocytes in serum-supplemented media and emphasize the importance of a serum-free system for studies on growth control and carcinogenesis in keratinocytes. Editor’s Statement This report contributes significantly to our knowledge of keratinocyte cell biology in two ways. First, a serum-free medium has been developed that can now be used by many investigators to define growth versus differentiation factors for these cells. This is important since several impure or relatively crude preparations of factors are known to influence these cells. Second, the finding that TGF-Beta is an inhibitor of keratinocyte growth opens new avenues to investigate the biochemical events leading to differentiation. David A. Sirbasku     

Continuous culture of rat C6 glioma in serum-free medium

In this communication we describe serum-free culture conditions for the serial propagation of the C6 glioma cell line. The growth rate, saturation density, and morphology of these cells are equivalent to those of their serum-grown counterparts when cultured in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's medium F-12 supplemented with trace elements, insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and a serum-spreading factor (SSF) partially purified from human plasma. The requirement for SSF in the medium can be satisfied by preincubating the tissue culture dishes with SSF. Tissue culture dishes sequentially pretreated with poly-D-lysine and purified cold insouluble globulin will also substitute for this requirement. The fatty acid-free bovine serum albumin/linoleic acid complex increases the growth rate of these cells but has no appreciable effect on their morphology, saturation density, or ability to grow with repeated subculture. The growth stimulation caused by this complex appears to be dependent on the fatty acid, as the fatty acid-free bovine serum albumin alone has no effect on the growth rate. Linoleic acid is cytotoxic in the absence of bovine serum albumin, and the fatty acid-free bovine serum albumin prevents this toxicity. Other fatty acids including oleic, arachidonic, and palmitic only partially substitute for the growth-promoting effect of linoleic acid.