A serum-free medium formulation supporting growth of human umbilical cord vein endothelial cells in long-term cultivation

A serum-free medium formulation – TUD-1 – was developed supporting growth of HUVEC in tissue culture. Special features of the basal medium formulation are highly elevated levels of glutamine and serine as well as the inclusion of N-acetylcysteine and phosphoascorbic acid. The cellular mitogenic needs are satisfied by bFGF, VEGF, EGF and liver growth factor. Further hormone supplementation consists of insulin and hydrocortisone. A protocoll for serum-free passage of HUVEC was established for serum-free long-term cultivation of freshly isolated HUVEC for up to 20 cumulative population doublings without significant differences in final cell density compared to controls cultivated with serum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth regulation of serum-free cultures of epithelial cells from normal human buccal mucosa

Summary Human buccal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins 5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings per day on fibronectin/collagen-coated dishes at clonal density. Studies of markers of proliferation and differentiation in buccal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factorβ-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate or an elevated Ca²⁺ concentration (from 0.1 to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area, involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reproducibly derived from explant cultures of human buccal mucosa specimens and the cells transferred under serum-free conditions. Buccal epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably respond to several agents shown to modulate growth of cells that originate from other types of epithelia. This work was supported in part by grants from the Swedish National Board of Laboratory Animals, the Swedish Medical Research Council, the Swedish Natural Science Research Council, the Swedish Fund for Scientific Research Without Animal Experiments, the Swedish Cancer Society, the Swedish Tobacco Company, and Lions Club International, Djurg?rden, Stockholm, Sweden.     

Characterization of proliferation and differentiation of opossum kidney cells in a serum-free defined medium

Summary Proliferation and differentiation of opossum kidney cells in a serum-free defined medium was investigated and compared to that under conditions in which fetal bovine serum FBS (10%) was employed. Monolayers were grown in Dulbecco's modified Eagle's medium-Ham's F12 nutrient mixture containing insulin (10 μg/ml), bovine serum albumin fraction V (1 mg/ml) and fetuin (1 mg/ml). Cells in serum-free medium seeded at 1×10⁴ per cm² grew to confluency within 6 to 8 d and formed hemicysts or domes at a frequency equivalent to those in serum-containing medium. Electron microscopy of cultures grown in serum-free medium revealed polarized monolayers with the presence of microvilli and tight junctions. The differentiated characteristics, including sodium-dependent phosphate transport, the inhibition of this transport by parathyroid hormone (PTH), and the generation of cyclic AMP in response to PTH, were preserved in opossum kidney cells grown in serum-free medium.     

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human -interferon (-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.Abbreviation HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Adaptation of BHK cells producing a recombinant protein to serum-free media and protein-free medium

In this work a recombinant BHK21 clone producing a fusion protein with potential application in tumour target therapy was adapted to five different serum-free media (SFM) and to a protein-free medium (PFM). Only the PFM did not require a gradual adaptation to cell growth in the absence of serum. All tested SFM required a gradual adaptation (up to 35 days). For the majority of the SFM tested, cell specific productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was significantly affected by the serum decrease. Both cell growth and productivity were increased when PFM SMIF6 was used instead of the control medium. Long term measurements (approximately 100 days) of cell specific productivity for PFM and the two best SFM showed that productivity was maintained. This indicates the media capability to be used in long term production processes.     

Characterization and serial propagation of mouse prostate epithelial cells in serum-free medium

Epithelial cell cultures derived from the ventral prostate of normal adult mice have been propagated in serum-free medium. The cultures were initiated and maintained in Ham's F-12 nutrient mixture supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), cholera toxin (10 ng/ml), bovine pituitary extract (25 micrograms protein/ml) and antibiotics. The cells exhibited microvilli on cell surfaces, interdigitations and junctional complexes including desmosomes between cells, and cytokeratins in cytoplasm which are characteristic of epithelial cells. In addition, the cells exhibited the tissue-specific markers, prostatic acid phosphatase and prostate-specific antigen.     

A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells

Summary Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free “attachment medium” was first devised in which cells would attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed. Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation. This research was sponsored by the National Cancer Institute, DHHS, under Contract NO1-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government.     

Clonal growth and culture life span of bovine adrenocortical cells in a serum-free medium

Summary The life span and growth from clonal density of bovine adrenocortical cell cultures were studied in serum-supplemented medium and a serum-free defined medium, which supported sustained cell proliferation and steroid production. The total culture life span was 79 population doublings in serum-supplemented medium with fibroblast growth factor (FGF) and 36 population doublings in the defined medium without serum. Older passage cell cultures grown in the defined medium progressively lost the ability to produce 11β- and 21-hydroxylated steroids, which was observed previously for cultures in serum-supplemented medium, and also had a decline of 17α-hydroxylated steroid production. The cloning efficiency in the defined medium was 12.2% as compared to 24% in serum-supplemented medium with FGF. Five isolated clonal cell lines grown in the defined medium were characterized for steroid function in response to steroidogenic agents. All five clonal cell lines had stimulated steroid production with 8-bromo-cAMP, but only four of the clonal lines were stimulated also by adrenocorticotropin. None of the clonal cell lines produced 11β-, 21- or 17α-hydroxylated steroids in response to treatment with either steroidogenic agent, results that were similar to data obtained from older mass cultures. The apparent deficiency of the defined medium as compared to serum-supplemented medium for maximum support of the culture life span and cloning efficiency may be useful in studies of cellular aging and its relation to differentiated function for this cell culture system. This study was supported by the Iowa Diabetes and Endocrinology Research Center (grant AM25295 from the National Institutes of Health, Bethesda, MD). D.A.F. was supported by a National Research Service Award from the National Institutes of Health (grant HL07485).     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

Chicken egg yolk-supplemented medium and the serum-free growth of normal mammalian cells

Summary Supplementation of tissue culture medium with chicken egg yolk can support the proliferation of low density bovine vascular and corneal endothelial cells and vascular smooth muscle cells maintained on basement lamina-coated dishes. The optimal growth-promoting effect was observed at concentrations of 7.5 to 10% egg yolk (vol/vol). The average doubling time of bovinn vascular endothelial cells during their logarithmic growth phase when exposed to egg yolk-supplemented medium was longer than that of their counterparts grown in serum-supplemented medium (21 versus 15 h, respectively). Cultures grown in egg yolk-supplemented medium on basement lamina-coated dishes could be serially passaged, but their in vitro life span (15 generations) was less than that of serum-grown cultures (50 generations). The egg white was devoid of any grwoth-promoting activity. This work was supported by Grants HL 20197 and HL 23678 from the National Institutes of Health, Bethesda, MD.     

Microcarrier culture of bowes melanoma cells in serum-free medium with Human plasma fraction IV-4+ V

Bowes melanoma cells were cultivated successfully in a serum-free medium which was constructed by the concept of maximum retention of proteins from fractionated human plasma having growth stimulatory activities. The cells could be cultivated in the serum-free medium without any adaptation period. The major serum-free component of the medium was the fraction IV-4 + V of the Cohn fractionation process of human plasma. Approximately six times increase of tissue-type plasminogen activator (t-PA) activity as compared with that in serum-free medium even though the cell growth was much slower. In addition, the growth stimulatory activities of thrombin and fibronectin were investigated during the cultivation of Bowes melanoma cells in this serum-free medium. These proteins contributed significantly to the enhanced growth of cells by reducing doubling time to 25 and 35 h as compared with 55 h in the serum-free medium without them. Especially, fibronectin supported cells to propagate near to the maximum cell density achieved in the medium with 10% FBS.     

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

Growth of human malignant lymphoid cell lines in serum-free medium

Summary Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products. This research was supported by a grant from the National Institutes of Health, CA 12800, and the Concern Foundation of Los Angeles, and CA 09120 (C. U.)     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Summary Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described. This work was supported by the “Centre National de la Recherche Scientifique” (Grant 1208-Biochimie du Développement and Grant 4162-Endocrinologie), by the “Ministère de la Recherce et de la Technologie” (Grant 81-L-1322), by the “Fondation pour la Recherche Médicale,” by NATO (Grant 1704), and by the “Institut National de la Santé et de la Recherche Médicale” (Grant 827006).     

Egg yolk lipoprotein,a new supplement for the growth of mammalian cells in serum-free medium

Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity.     

A serum-free medium for the culture of insect cells and production of recombinant proteins

Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.     

Development of a new serum-free medium,USC-HC1, for growth and normal phenotype in postembryonic chicken growth plate chondrocytes

Summary A serum-free medium for postembryonic chicken epiphyseal growth plate chondrocytes has been developed from 104 MCDB medium. To enable these fastidious cells to survive, grow, and express normal phenotype, a substantial increase over MCDB 104 in the level of many of the amino acids was required, as well as a change in the buffer system and the addition of SerXtend, a defined, serum-free product containing various growth factors, including fibroblast growth factor. Also required was the provision of cell attachment factors, either by coating culture surfaces with type II collagen, or better, by allowing the freshly released cells to recover for several hours in a medium supplemented with 10% fetal bovine serum before plating. Ths new serum-free medium, which we call USC-HC1, supports growth and replication, the retention of normal polygonal morphology, the expression of significant levels of cellular alkaline phosphatase activity, the production of sulfated proteoglycans, type II collagen, and the formation of alkaline phosphatase-rich matrix vesicles by the chondrocytes. The major advantage of USC-HC1, however, is that it will provide for the first time an opportunity to examine the effects of various defined growth and hormonal factors on the phenotypic expression and differentiation of growth plate chondrocytes, in the absence of the variable (stimulatory and inhibitory) factors present in fetal bovine serum. This work was supported by grant AM18983 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, Bethesda, MD.     

The growth of Vero cells in suspension as cell-aggregates in serum-free media

Vero cell lines, usually considered anchorage-dependent, could be grown as cell-aggregates in suspension culture with serum-free media. Several different combinations of base media gave growth results above 10⁶ cells/ml (NCTC 135:SFRE 199-1; NCTC 135:Waymouth MB 752/1; NCTC 135:RPMI 1640). Insulin was not essential for growth and Bovine Serum Albumin could be diluted out of the media if linoleic acid was present. The size and density of the aggregates formed varied depending on the media used.