Antibody production and growth of mouse hybridoma cells in Nutridoma media supplements

Traditionally, cell culturists have relied upon the addition of serum to culture medium for the growth and maintenance of cell lines. However, many aspects of the use of serum in tissue culture are problematic. Cell culture supplements that circumvent the need for serum are readily available and provide a consistent protein composition. This defined environment allows the antibody to be more easily purified from culture supernatants. Nutridoma media supplements were formulated to support the growth of lymphoblastoid cells in a defined culture environment. In this study, Nutridoma media supplements were tested in parallel with serum-containing cultures to determine if Nutridoma supplemented medium is effective in supporting hybridoma cell growth and antibody production in three hybridoma cell lines. Data, based on cell growth and antibody production, show the importance of basal media selection when serum is replaced with Nutridoma media supplements. SDS-PAGE results show that cell supernatants from Nutridoma supplemented cultures contain very few contaminating proteins.     

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.     

Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells

Background aimsEx vivo propagation of sparse populations of human mesenchymal stromal cells (hMSC) is critical for generating numbers sufficient for therapeutic applications. hMSC culture media have typically been supplemented with animal serum and, recently, human-sourced materials. However, these supplements are ill-defined and, thus, undesirable for clinical and research applications. Previously reported efforts to develop defined media for hMSC culture only resulted in slow or limited proliferation, and were unsuccessful in expanding these cells from primary cultures. Therefore a major step forward would be the identification of defined, serum-free culture conditions capable of supporting both the isolation and rapid expansion of hMSC.MethodsUsing classical approaches of medium development, we were able to identify a set of growth and attachment factors that allowed the serum-free isolation and expansion of hMSC from bone marrow.ResultsHeparin, selenium and platelet-derived growth factor (PDGF)-BB were found to be inhibitory for the growth of hMSC, whereas basic fibroblast growth factor (bFGF) was critical and worked synergistically with transforming growth factor (TGF)-β1 to allow significant cell expansion. Ascorbic acid, hydrocortisone and fetuin were also found to be important growth and attachment factors that, in conjunction with substrate-coating proteins, allowed the isolation of hMSC from primary culture and their subsequent expansion.ConclusionsWe report a defined medium formulation (PPRF-msc6), consisting of key recombinant and serum-derived components, for the rapid isolation and expansion of hMSC in the absence of serum. This work represents an important step forward for achieving an ideal, completely defined synthetic medium composition for the safe use of hMSC in clinical settings.     

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1 g L⁻¹ increased the maximal cell density by 70 %, while a mixture of YE (1 g L⁻¹) and YP.A (4 g L⁻¹) increased IgG production by 180 %. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20 %. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.     

Determinants and rate laws of growth and death of hybridoma cells in continuous culture

Experimental data from six hybridoma cell lines grown under diverse experimental conditions in both normal continuous and perfusion cultures are analyzed with respect to the significance of nutrients and products in determining the growth and death rates of cells and with respect to their mathematical modeling. It is shown that neither nutrients (glucose and glutamine) nor the common products lactic acid, ammonia, and monoclonal antibody can be generally assumed to be the clear-limiting or inhibiting factors for most of the cultures. Correspondingly, none of the unstructured models existing in the literature can be generally applied to describe the experimental data obtained over a relatively wide range of cultivation conditions as considered in this work. Surprisingly, for all cultures the specific growth rate (mu) almost linearly correlates with the ratio of the viable cell concentration (NV) to the dilution (perfusion) rate (D). Similarly, the specific death rate (kd) is a function of the ratio of the total cell concentration (Nt) to the dilution (perfusion) rate. These results strongly suggest the formation of not yet identified critical factors or autoinhibitors that determine both the growth and death rates of hybridoma cells. Based on these observations, simple kinetic models are developed for mu and kd which describe the experimental data satisfactorily. Analysis of the experimental data with the kinetic models reveals that under the current cultivation conditions the formation rate of the autoinhibitor(s) or the sensitivity of cell growth and death to the autoinhibitor(s) is mainly affected by the medium composition. Irrespective of the cell lines, cells grown on serum-containing media have almost the same model parameters, which are distinctively different from those of cells grown on serum-free media. Furthermore, in contrast to the prevailing view, kd is shown to positively correlate with mu if the effects of cell concentration and dilution (perfusion) rate are considered. Several important implications of these findings are discussed for the optimization and control of animal cell culture.     

Serial cultivation of normal human keratinocytes: A defined system for studying the regulation of growth and differentiation

Summary We have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into a morphologically differentiated epidermis.     

Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells

Using the monoclonal antibody R26.4, we have previously identified a approximately 225-kD peripheral membrane protein, named ZO-1, that is uniquely associated with the tight junction (zonula occludens) in a variety of epithelia including the Madin-Darby canine kidney (MDCK) epithelial cell line (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). In this study we have analyzed the effects of cell-cell contact and extracellular calcium on the localization and the solubility of ZO-1. In confluent monolayers under normal calcium conditions, ZO-1 immunoreactivity is found exclusively at the plasma membrane in the region of the junctional complex. If MDCK cells are maintained in spinner culture under low calcium conditions, ZO-1 is diffusely organized within the cytoplasm. After the plating of suspension cells at high cell density in medium with normal calcium concentrations, ZO-1 becomes localized to the plasma membrane at sites of cell-cell contact within 5 h in a process that is independent of de novo protein synthesis. However, if suspension cells are plated at high density in low calcium medium or if suspension cells are plated at low cell density in normal calcium growth medium, ZO-1 remains diffusely organized. ZO-1 localization also becomes diffuse in monolayers that have been established in normal calcium medium and then subsequently switched into low calcium medium. These results suggest that both extracellular calcium and cell-cell contact are necessary for normal localization of ZO-1 to the plasma membrane. An analysis of the solubility properties of ZO-1 from suspension cells and monolayers revealed that high salt, nonionic detergent, and a buffer containing chelators were somewhat more effective at solubilizing ZO-1 from suspension cells than from monolayers.     

In vitro development of Cryptosporidium parvum in serum-free media

AIM: To compare commercially available serum-free media with common, standard, growth medium for their ability to support growth of Cryptosporidium parvum in HCT-8 cell cultures. METHODS AND RESULTS: Twelve serum-free media formulations with or without additional supplements were tested against a standard growth medium containing 2% FBS in HCT-8 cell cultures. After a 48-h incubation period, the level of parasite development was determined by ELISA. The extent of development in the serum-free media was determined as a percentage of infections compared with those obtained using a standard growth medium. CONCLUSIONS: Several of the serum-free media formulations, which included MDCK, UltraMDCK, PC-1, UltraCHO and UltraCulture, compared favourably with a traditional, standard growth medium. Moreover, increasing FBS concentrations to 10% actually resulted in an overall decrease in development in many cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Several serum-free medium formulations are available which allow development of C. parvumin vitro at levels comparable with standard media employing FBS. These serum-free media are particularly useful for applications, which may require a more defined medium without the presence of FBS. Moreover, the elimination of FBS as a variable allows investigators the ability to more closely regulate their experimental systems when growing C. parvum in cell cultures.     

Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture

Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPLBeta-Sf21-AE) and Trichoplusia ni (Tn 5Beta-1-4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150-160 rpm were necessary for maximum growth of suspended Tn 5Beta-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 x 10(5) cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for beta-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Beta-1-4 cells are 2-4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of beta-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5Beta-1-4 cells produced 2.6-4.4 and 2.7-3 times more beta-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the beta-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of beta-galactosidase by Tn 5Beta-1-4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production.     

Metabolite profiling of CHO cells with different growth characteristics

Mammalian cell cultures are the predominant system for the production of recombinant proteins requiring post-translational modifications. As protein yields are a function of growth performance (among others), and performance varies greatly between culture medium (e.g., different growth rates and peak cell densities), an understanding of the biological mechanisms underpinning this variability would facilitate rational medium and process optimization, increasing product yields, and reducing costs. We employed a metabolomics approach to analyze differences in metabolite concentrations of CHO cells cultivated in three different media exhibiting different growth rates and maximum viable cell densities. Analysis of intra- and extracellular metabolite concentrations over the course of the cultures using a combination of HPLC and GC-MS, readily detected medium specific and time dependent changes. Using multivariate data analysis, we identified a range of metabolites correlating with growth rate, illustrating how metabolomics can be used to relate gross phenotypic changes to the fine details of cellular metabolism.     

Low serum and serum-free culture of multipotential human adipose stem cells

BACKGROUND: Adipose tissue provides an easily accessible and abundant source of putative stem cells for translational clinical research. Currently prevalent culture techniques include the use of FBS, a highly variable and undefined component, which brings with it the potential for adverse patient reactions. In an effort to eliminate the use of animal products in human adipose stem cell (ASC) cultures, we have developed two new culture methods, a very low human serum expansion medium and a completely serum-free medium. METHODS: Through serial testing, a highly enriched medium formulation was developed for use with and without the addition of 0.5% human serum, an amount easily obtainable from autologous blood draws. RESULTS: Very low-serum culture yielded population-doubling times averaging 1.86 days in early passage, while the serum-free formulation was associated with less robust cell growth, with doubling times averaging 5.79 days. ASC in both conditions maintained its ability to differentiate into adipo-, chondro- and osteogenic lineages in vitro, despite lower expression of CD34 in early passage. Expression of ALDH, HLA, CD133, CD184, and CD31 was comparable with that seen in cells cultured in 10% FBS. DISCUSSION: These newly developed culture conditions provide a unique environment within which to study ASCs without the interference of animal serum, and allow for the rapid expansion of autologous ASCs in culture in an animal product-free environment for use in human clinical trials.     

Transforming growth factor beta 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis.

Transforming growth factor-beta 1 (TGF-beta 1) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-beta 1; however, in three-dimensional (3-D) collagen gels TGF-beta 1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-beta 1 treated cultures. In 2-D cultures TGF-beta 1 is known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-beta 1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3-D cultures there is increased synthesis and secretion of type V collagen in both control and TGF-beta 1 treated cultures over 2-D cultures. Even though an equal amount of type V collagen is seen in both 3-D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF-beta 1 treated cultures. EM morphological analyses on 3-D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-beta 1 treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-beta 1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the ZO-1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endothelial cells (RFC) monolayer lysates probed with anti-ZO-1 label a 220 kd band which co-migrates with the bonafide ZO-1 protein. These data confirm and support the hypothesis that TGF-beta 1 is angiogenic in vitro, eliciting microvascular endothelial cells to form tube-like structures with apparent tight junctions and abluminal basal lamina deposition in three-dimensional cultures.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses

The development of media free of serum and animal or human proteins is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. In order to reduce the risk of contamination, we have modified the serum free medium MDSS2, a very efficient serum free medium for the production of various biologicals including experimental vaccines using different cell lines (Merten et al., 1994), by replacing the animal derived products by plant extracts. The new serum and animal protein free medium (MDSS2N) can be efficiently used for biomass production of various cell lines. These cells grow equally well or better in this new serum-free medium than in the old formulation (MDSS2): • BHK-21/BRS cells, adapted to MDSS2N, showed an overall specific growth rate of 0.0197 h-1 (μ_max = 0.0510±0.0058 h-1), whereas those cultivated in MDSS2 grew with an average specific growth rate of 0.0179 h-1 (μ_max = 0.0305±0.0177 h-1). • Vero cells grew with an average specific growth rate of 0.0159 h-1 and 0.0153 h-1 in MDSS2 and MDSS2N, respectively. Very similar growth rates were obtained in microcarrier cultures in stirred tank reactors: the specific growth rates were 0.0161 h-1 and 0.0166 h-1 for MDSS2 and MDSS2N cultures, respectively. • For MDCK cells, when cultured on microcarriers in bioreactors, a higher average specific growth rate was observed in MDSS2N than in MDSS2; values of 0.0248 h-1 and 0.0168 h-1, respectively, were obtained. The capacity of MDSS2N to support the production of different viruses was equally evaluated and it could be established that for certain viruses there are no or insignificant differences between MDSS2N and MDSS2 (influenza and polio virus), whereas, the production of rabies virus is somewhat reduced in MDSS2N when compared to MDSS2. The use of MDSS2N for cell culture and the production of various viruses is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of the distribution and phosphorylation state of ZO-1 in MDCK and nonepithelial cells

We have examined the distribution and extent of phosphorylation of the tight junction-associated protein ZO-1 in the epithelial MDCK cell line, and in three cell types that do not form tight junctions: S180 (sarcoma) cells, S180 cells transfected with E-cadherin (S180L), and primary cultures of astrocytes. In shortterm calcium chelation experiments on MDCK cells, removal of extracellular calcium caused cells to pull apart. However, ZO-1 remained concentrated at the plasma membrane and no change in ZO-1 phosphorylation was observed. Maintenance of MDCK cells in low calcium medium, conditions where no tight junctions are found, resulted in altered ZO-1 distribution and lower total phosphorylation of the protein. In S180 cells, ZO-1 was diffusely distributed along the entire cell surface, with concentration of the antigen in motile regions of the cell. Cell-cell contact was not a prerequisite for ZO-1 localization at the plasma membrane in this cell type, and the phosphate content of ZO-1 was found to be lower in S180 cells relative to MDCK cells. Expression of Ecadherin in S180L cells did not alter either the distribution or phosphorylation of ZO-1. In contrast to S180 cells, ZO-1 in primary cultures of astrocytes was concentrated at sites of cell-cell contact, and the phosphorylation state was the same as that in control MDCK cells. Comparison of one-dimensional proteolytic digests of ³²P-labeled ZO-1 revealed the phosphorylation of two peptides in control MDCK cells that was absent in both MDCK cells grown in low calcium and in S180 cells.We would like to thank Cheryl Richards for her help with the cell culture and immunohistochemistry; David Begg, Gary Firestone, Vik Maraj, Manijeh Pasdar and Colin Rasmussen for helpful discussions; Jaclyn Peebles and Greg Morrison for help with graphics and photography; and Grace Martin and Bob Campenot for rat tail collagen. We are grateful to all the members of our laboratories for their friendship, advice and support. This work was supported by an Establishment Award to B.R.S. from the Alberta Heritage Foundation for Medical Research and grants to B.R.S. from the Kidney Foundation of Canada and the Medical Research Council of Canada. A.H. is funded by a Studentship from the AHFMR. K.L.S. was supported by a grant from the National Institutes of Health (DK-42799) to Gary L. Firestone. B.R.S. is a Medical Research Council of Canada and AHFMR Scholar.     

Rapid Growth of Acanthamoeba In Defined Media; Induction of Encystment By Glucose-Acetate Starvation

Defined media are described that support 14-20 h generation times for Acanthamoeba castellanii and A. rhysodes in monolayer cultures. the media differ in minor ways from previously described media, but the growth rates are greatly improved over previously reported values. Maximum growth rates were observed for A, castellanii in a complex medium containing 21 amino acids, but near-maximum rates could be achieved in relatively simple media containing 9 amino acids. Growth occurred with 6 amino acids, as reported by others, but generation times exceeded 30 h. Amitosis was a common problem during early subcultures in defined media, but became infrequent after repeated transfers. Synchronous encystment resulting in 70-80% cyst formation could be induced in the defined media by glucose and acetate starvation. the rate of encystment varied with cell density at the time of starvation and was optimal at initial densities of 400-800 amebae/mm2.     

Formulation and production of a blood-free and chemically defined virus production media for VERO cells

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.     

A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies

In order to promote the uniform and reproducible differentiation of human embryonic stem cells (HESCs) in response to exogenously added growth factors, we have developed a method (spin embryoid bodies (EBs)) that uses a recombinant protein-based, animal product-free medium in which HESCs are aggregated by centrifugation to form EBs. In this protocol we describe the formulation of this medium, denoted APEL (Albumin Polyvinylalcohol Essential Lipids), and its use in spin EB differentiation of HESCs. We also describe a more economical variant, BPEL (Bovine Serum Albumin (BSA) Polyvinylalchohol Essential Lipids), in which BSA replaces the recombinant human albumin. The integration of a medium that includes only defined and recombinant components with a defined number of cells to initiate EB formation results in a generally applicable, robust platform for growth factor-directed HESC differentiation.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Effects of synthetic lysophosphatidylcholines on suspension cultures of the Chinese hamster ovary DG44 cells in protein-free media

This study described the effects of synthetic lysophosphatidylcholines on the growth of recombinant CHO-DG44 cells in suspension. Overall, cell growth characteristics were improved when cultivated in suspension in a protein-free medium supplemented with natural soybean lysophosphatidylcholines. To substitute synthetic lysophosphatidylcholines for the naturally occurring lysophosphatidylcholines, we implemented a systematic approach in which twelve synthetic lysophosphatidylcholines were grouped into three lipid mixtures according to the length of their acyl chains. We found that synthetic lysophosphatidylcholines with medium acyl chain lengths (C14-C18), including oleoyl lysophosphatidylcholine (C18:1) could increase cell growth in the protein-free media. The fortified protein-free medium with medium acyl chain length lysophosphatidylcholines (C14-C18) maintained growth of CHO-DG44 cells over five consecutive passages, whereas the cell growth in a CHO protein-free medium was decreased gradually after four passages. We also observed that the restorative effect of oleoyl lysophosphatidylcholine was comparable to that of natural lysophosphatidylcholine in batch and long-term cultivation. These results show that synthetic lysophosphatidylcholines can be used as lipid supplements in either protein-free media or chemically defined media for CHO cell suspension cultures.