Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media

Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium. This work was conducted in conjunction with the Walls Medical Research Foundation.     

Comparison of three culture media for the establishment of melanoma cell lines

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols.     

Production of nerve growth-stimulating factor(s) from chick embryo heart cells : Use of Cytodex 3 microcarriers and serum-free media

Medium conditioned by embryonic chick heart cells is known to support extensive neurite outgrowth from autonomic and sensory neurons. In the present report we describe the use of microcarrier cell culture with serum-free media to scale up the production of the nerve growth-stimulating factors. A growth medium composed of DME /F10 supplemented with insulin, transferrin, human serum albumin and fibronectin in combination with a low molecular weight (MW) fraction of fetal calf serum (FCS) or a mixture of FGF, dexamethasone, calmodulin and thrombin supported the heart cell proliferation at a rate similar to that of medium with 10% FCS. Furthermore, the level of successively accumulated nerve growth activity measured in a bioassay with sympathetic ganglia proved to be nearly equivalent to what was obtained when cells were grown in medium containing serum. The results confirm the potential of microcarrier cell culture in serum-free media for the production and subsequent recovery of a specific cell product.     

Human glioma cell culture: two FCS-free media could be recommended for clinical use in immunotherapy

Immunotherapy, particularly active vaccination, may be developed as an effective and safe treatment modality for malignant gliomas, which continue to have a poor prognosis, despite advances in surgical techniques and adjuvant chemotherapy and radiotherapy. Since no glioma-specific tumor-associated antigens (TAAs) have been discovered, autologous tumor cells or well-established glioma cell lines could be used in future vaccination protocols to induce antitumour immunity against unknown TAAs. One obstacle for clinical use of these tumour cell vaccines is related to foetal calf serum (FCS). Efforts are currently being directed toward developing FCS-free media and serum-free alternatives to culture these cell vaccines. In this study, a medium containing human serum and one serum-free medium (UltraCulture), supplemented or not with epidermal growth factor, were tested on morphology, survival, DNA content and TAA expression of human glioma cell lines and glioma biopsy primary cultures. Their effects were compared on FCS-containing medium. Results show that, whatever the medium used, no significant variations in morphology and survival were observed. Furthermore, human serum-containing medium or UltraCulture preserved at early passage cultures the cell population of interest present in the biopsies before culture. In addition, the expression profile of eight TAAs was similar between these media. These data indicate that human serum-containing medium and UltraCulture serum-free medium could be promising candidates to produce tumour-cell vaccines.     

Growth, drug susceptibility, and gene expression profiling of Plasmodium falciparum cultured in medium supplemented with human serum or lipid-rich bovine serum albumin [corrected

In vitro cultivation of Plasmodium falciparum has been extremely useful in understanding the biology of the human malaria parasite as well as research on the discovery of new antimalarial drugs and vaccines. A chemically defined serum-free medium supplemented with lipid-rich bovine serum albumin (AlbuMAX I) offers the following advantages over human serum-supplemented media for the in vitro culture of P. falciparum: 1) improved growth profile, with more than a 2-fold higher yield of the parasites at any stage of the growth cycle; 2) suitability for in vitro antimalarial screening, as the parasites grown in AlbuMAX and human serum-supplemented media show similar sensitivity to standard and novel antimalarials as well as natural product extracts in the in vitro drug susceptibility assays; and 3) DNA microarray analysis comparing the global gene expression profile of sorbitol-synchronized P. falciparum trophozoites grown in the 2 different media, indicating minimal differences.     

Growth of NS0 cells in protein-free, chemically defined medium

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein. CD Hybridoma Medium (a protein-free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX-CYTE VLE. None of the tested supplements supported long-term growth of NS0 cells in CD Hybridoma Medium. Sustained long-term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal- or plant-derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin-lipid complex reached peak cell densities that were more than double those observed in serum-supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin-based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal-derived components if the supplement is made with plant-derived or synthetic lipids.     

Application of percent labeled mitoses (PLM) analysis to the investigation of melanoma cell responsiveness to MSH stimulation throughout the cell cycle

Dissociated embryonic chick dorsal root ganglionic cells were plated on collagen-coated tissue culture dishes in Eagle's basal medium containing 10% fetal calf serum (FCS). After 48 h, which allowed adequate cell attachment, the cultures were washed with serum-free medium and then received fresh medium supplemented with 10% FCS or serum-free defined medium (N1), which was supplemented with insulin, transferrin, progesterone, putrescine and selenium. In addition, both media required the addition of Nerve Growth Factor (NGF). N1 medium selectively maintained the neurons and did not support proliferation or even survival of almost all non-neuronal elements (fibroblasts and Schwann cells). Survival of neurons in N1 was initially as good and eventually better than in serum-containing medium. After 6 days in N1 the cultures consisted almost entirely of neurons (>95%), which had smaller cell bodies but more extensive process formation than in serum-supplemented medium. The omission of any one of the supplements resulted in a reduction of neuron survival. The ability to generate cultures of pure neurons in a serum-free defined medium may be useful for studying (i) the role of specific hormones and growth factors normally supplied by serum in the maintenance of neurons and (ii) biochemical parameters of neurons in the absence of the substantial background due to non-neuronal elements.     

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

The modulation of transcobalamin II (TC-II) production by cyclic adenosine 3',5'-monophosphate in the murine macrophage cell line J774: relationship to growth behavior

We undertook a study to define the role of cyclic AMP [cAMP] in modulating the secretion of transcobalamin II (TC-II) in the mouse macrophage like cell line J774. J774 was observed to secrete large amounts of TC-II, particularly in the presence of 8-bromo cAMP or cholera toxin or when grown in medium supplemented with low concentrations of horse serum (1% or 5%) or in serum-free medium. Variant cell lines derived from J774 and deficient either in adenylate cyclase (ac -) or cAMP-dependent protein kinase (pk -) activity showed very low and intermediate levels of basal secretory activity of TC-II, respectively, compared to J774. Maximum secretory activity of TC-II was observed in J774 under conditions in which growth was poorest (in the presence of 8-bromo-cAMP or 1% or 5% horse serum-supplemented medium or in serum-free medium). Cells grown in serum-free medium were found to have elevated basal adenylate cyclase activity and cAMP levels compared to those grown in medium supplemented with 20% horse serum. The data from this study demonstrate a negative correlation between growth activity and TC-II secretion in the J774 cell line. The stimulatory effect of exogenous cAMP on TC-II secretion by J774, the reduced secretory activity of the variant lines ac- and pk- and the observed increase in cell cAMP levels under conditions of serum starvation in which TC-II secretion is considerably enhanced, suggest that cell cAMP is an important modulator of TC-II secretion and growth behavior in the J774 cell line.     

Cellular selenoproteins and the effects of selenite on cell proliferation

The incorporation of radioactive selenium into cellular proteins and the effect of selenite on proliferation were examined in human (HeLa, HT-29, and IMR-90) and mouse (3T3 and CMT-93) cell lines. Proteins incorporating selenium were detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major polypeptide subunits at 60, 23, 21, 19, and 16 kD were detected in the two tumorigenic and one normal human cell lines. The 23 kD polypeptide migrated to the same position on the gel as the major subunit of human erythrocyte glutathione peroxidase. In the mouse cells, the 60 kD polypeptide was almost entirely absent; four other major selenoproteins were detected, with molecular weights similar to those in the human cells. In both mouse and human cells, the same pattern of selenoproteins was observed irrespective of whether the cells were grown in medium containing 10% fetal bovine serum or in defined medium supplemented with 0.1 or 1 microM selenite, or with 1% serum. The effect of selenite on proliferation of HeLa, HT-29, and CMT-93 cells in medium supplemented with 10% fetal bovine serum and in serum-free medium was examined. At concentrations up to about 1 microM, selenite stimulated proliferation of the human cells slightly in serum-free medium but not in serum-supplemented medium. At concentrations of about 5 microM and higher selenite significantly inhibited proliferation of all cells in both types of media. In CMT-93 cells, this inhibition was greater in serum-free medium, but there were no significant differences in this regard in the human cells. These results demonstrate that selenium is stably incorporated into several polypeptides in human and mouse cells, that there are no significant differences in this regard among several cell lines, and slight differences between human and mouse cells. They further confirm that selenium can have a slight stimulatory effect on cell growth, and a much larger inhibitory effect, depending on its concentration.     

The effects of epidermal growth factor on cell proliferation and prolactin production by GH3 rat pituitary cells

The effects of epidermal growth factor (EGF) were studied in rat pituitary tumor cells, GH₃, grown in serum-supplemented and serum-free chemically defined media. EGF (1 nM) increased the cell number to 132% of the control cultured in the defined medium during a 6-day incubation period, while it decreased the cell number to 60% of the control in the serum-supplemented medium. EGF altered the morphology of the cells grown in the defined medium more markedly to an elongated conformation than that of cells grown in the serum-supplemented medium. EGF also stimulated prolactin (PRL) production by culture in the presence or absence of serum. The effects of the cell density of GH₃ on the action of EGF were shown to appear in two ways. The mitogenic influence of EGF was more effective on, and more responsive to, high-density cells, whereas the stimulatory action on PRL production was less effective on high-density cells. However, the inhibitory effects on cellular growth appeared independently of cell densities. The results obtained with ¹²⁵I-EGF binding experiments indicated that the number of binding sites, affinity, and internalization of EGF receptors were similar in either serum-supplemented or serum-free culture. At low cell density, the number of available ¹²⁵I-EGF binding sites per cell was larger than at high cell density. These results suggested that there was no apparent correlation between EGF binding and its differing effects on the growth of GH₃ cultured in the serum-supplemented and the defined medium.     

A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Use of synthetic serum-free medium for culture of human dermal fibroblasts to establish an experimental system similar to living dermis

In this study, we sought to establish a defined experimental system for fibroblast growth similar to that of the living dermis. To this end, we evaluated the growth and biochemical characteristics of fibroblasts cultured with serum-free HFDM-1, a finely tuned synthetic medium for human fibroblast culture. Three culture conditions were used to grow fibroblasts obtained from primary culture: (1) culture with Dulbecco’s modified Eagle medium (DMEM) plus 10 % fetal bovine serum (serum-supplemented DMEM), (2) culture with DMEM (serum-free DMEM), and (3) culture with HFDM-1 (HFDM-1), and fibroblast morphology, growth, collagen type I production, and lipid composition were analyzed. Fibroblasts grown in HFDM-1 maintained cell numbers at nearly 100 % from days 14 to 21 and produced more collagen type I than cells grown in serum-supplemented and serum-free DMEM. Arachidonic acid (20:4) and total polyunsaturated fatty acids were lower in cells grown in serum-free DMEM and HFDM-1 than in serum-supplemented DMEM. These results suggested that HFDM-1 recapitulated growth conditions in the dermis better than traditional, serum-supplemented DMEM. In addition, the controlled chemical composition of HFDM-1 eliminated a potential source of variability in cell culture conditions.     

Effects of various factors on the growth and function of a human hepatoblastoma cell line, HuH-6--an approach for serum-free cell culture

The effects on a human hepatoblastoma cell line (HuH-6) of serum and Ca2+ were investigated. A higher concentration of serum reduced the production of alpha-fetoprotein, whereas Ca2+ enhanced that of both albumin and alpha-fetoprotein. In view of these facts, a serum-free medium using RPMI-1640 supplemented with lactalbumin hydrolysate, epidermal growth factor, hydrocortisone, insulin, chrelatoxine, glucagone and selenium was then developed. Collagen type IV was superior to collagen type I, laminin, fibronectin and plastic in supporting the growth of HuH-6 in the serum-free medium. Higher amounts of both albumin and alpha-fetoprotein were secreted in HuH-6 cultured in serum-free medium than in serum-supplemented medium.     

Replacement of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60

The human promyelocytic leukemia cell line HL-60 can be grown in serum-free synthetic medium supplemented with insulin and transferrin alone. Growth of HL-60 in this defined medium is at a rate approx. 80% of that in medium containing serum. Moreover, the distinct morphological and histochemical myeloid characteristics of HL-60 are maintained in such serum-free medium. The HL-60 promyelocytes are induced by DMSO to differentiate to mature granulocytes equally well in both serum-supplemented and serum-free medium. However, this defined medium does not support colony growth of HL-60 in semi-solid medium such as methylcellulose.     

Retinoic acid stability in stem cell cultures

It has been reported that retinoids, such as retinoic acid (RA) and retinol (ROL), dissolved in aqueous solutions are susceptible to oxidative damage when exposed to light, air, and relatively high temperatures, conditions that are normal for culturing stem cells. Thus, questions arise regarding the interpretation of results obtained from studies of mouse embryonic stem cells exposed to retinoids because their isomerization state, their stability in culture conditions, and their interactions with other potential differentiation factors in growth media could influence developmental processes under study. Media samples were supplemented with retinoids and exposed to cell culture conditions with and without mouse embryonic stem cells (mESC), and retinoids were extracted and analyzed using HPLC. To determine whether retinoids are stable in media supplemented with fetal bovine serum (FBS) or in chemically-defined, serum-free media, mESC adapted to each type of growth media were investigated. Studies reported here indicate there was little loss or isomerization of at-RA, 9-cis-RA, 13-cis-RA, or ROL in cell cultures grown in serum-supplemented media when cell cultures were maintained in the dark and manipulated and observed under yellow light. In contrast, the stability of both at-RA and ROL were determined to be greatly reduced in serum-free media as compared with serum-supplemented media. Addition of 6 mg/ml bovine serum albumin was found to stabilize retinoids in serum-free media. It was also determined that ROL is less stable than RA in cell culture conditions.     

Effects of thyroliberin (TRH) on cell proliferation and prolactin secretion by GH3/B6 rat pituitary cells: a comparison between serum-free and serum-supplemented media

Numerous studies have shown that prolactin (PRL) production by GH3 cells grown in serum supplemented media is regulated by several hormones including thyroliberin (TRH). The recent availability of hormonally defined, serum-free media for the growth of GH3 cells has made it possible to determine the effect of TRH in absence of other prolactin regulating hormones. Here we demonstrate that transfer of GH3/B6 cells from serum-supplemented medium to serum-free media results in several important changes: (1) altered growth response to TRH, (2) altered cell attachment and morphology, (3) greatly reduced prolactin production, and (4) greater stimulation of prolactin production by TRH. After 4 days in serum-free medium, TRH stimulates prolactin production by as much as 5-fold instead of approximately 2-fold in serum-supplemented medium. Furthermore, this increased responsiveness to TRH in serum-free medium is accompanied by a 10-fold decrease in the ED50 for TRH (concentration needed for half-maximal response) and paradoxically by a 2-fold reduction in the number of high-affinity TRH binding sites without significant change of their association constant.     

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Recombinant Protein Production by the Baculovirus-Insect Cell System in Basal Media Without Serum Supplementation

The production of β-galactosidase by Sf9 cells infected with recombinant Autographa californica nucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the β-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted β-galactosidase production, their removal by medium replacement on post-infection day 1 gave a β-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing β-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.