Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells

The potential of mesenchymal stem cells (MSC) to differentiate into different cell types has opened up the possibility of using these cells clinically to treat a variety of disorders. In this study we describe the use of human umbilical cord blood serum (CBS) as a replacement for fetal bovine serum (FBS) for culturing MSC from different sources. MSC from human and swine bone marrow and human umbilical cord blood were cultured in the presence of DMEM/F12 containing either FBS or CBS. Human MSC cultured in presence of FBS or CBS showed typical fibroblast-like morphology, which is characteristic of MSC. 99% of the cells cultured in FBS had a CD73+/CD105+/CD45- phenotype compared to 96% of cells cultured in CBS. Cells cultured in CBS had a significantly higher cell count as compared to cells cultured in FBS. Swine Bone Marrow MSC cultured in the presence of FBS and CBS were morphologically and phenotypically similar. Human umbilical cord blood serum supports the growth of MSC. While no significant differences were observed in the MSC numbers in swine cells cultured in the presence of FBS or CBS, human cells showed a greater proliferation potential in the presence of CBS as compared to FBS. Therefore, CBS can be used as an effective substitute to FBS for developing clinically useful protocols for culturing MSC.     

Role of serum in survival ofTreponema pallidum in tissue culture

Summary During attempts to cultivateTreponema pallidum, it was determined that length of time for survival of virulent treponemes was highly dependent on the quality of the fetal bovine serum (FBS) used as a protein supplement in the culture medium. Eighteen lots of commercial FBS were tested for their ability to maintain survival ofT. pallidum in cultures of cottontail rabbit epithelial (SflEp) cells. All were capable of supporting growth of these cells. However, in tests on five of the lots, attachment of treponemes to the SflEp cells was either extremely poor or the 50% survival time (ST₅₀) was less than 5 days. With two of these lots, no treponemes survived for 5 days. By contrast, in tests with 11 of the FBS lots, the ST₅₀ of the treponemes was 12 days or greater; however, there was a great variation in the number of treponemes that attached. Selection of lots of FBS for ultimate experimental use was based on their influence both to extend length of time for survival of treponemes and to increase the number of treponemes that attached to the SflEp cells during that period.     

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.     

Characterization of normal human embryo cells grown to over 100 population doublings

Summary Normal human embryonic cells were subcultured for over 100 population doublings without modification of the basic medium. The cells were evaluated for growth rate, confluent density, chromosome stability, growth in soft agar, ability to hydrolyze casein and tumorigenicity. The cells possessed the characteristics of normal cells. The batch of serum used to supplement the medium was found to be of primary importance in the long-term growth of this cell culture. Research sponsored by the National Cancer Institute under Contract No. NO1-CO-25423 with Litton Bionetics, Inc.     

Effect of fetal bovine serum on 3-methylcholanthrene-induced transformation of hamster cells in vitro

Summary Eighteen lots of fetal bovine serum were tested for their ability to support clonal growth and 3-methylcholanthrene-induced morphological transformation of hamster embryo cells in vitro. Most of them supported cloning efficiencies of over 11%. However, cloning efficiency alone was an inadequate criterion for selecting serum for transformation studies, since no transformation was observed with some lots, even though their cloning efficiencies were over 16%. This shows the importance of pretesting serum for its ability to support morphological transformation before it is used in mammalian cell carcinogenesis tests. Research sponsored by the National Cancer Institute under Contract No. N01-CO-75380 with Litton Bionetics, Inc.     

Changes in serum influence the fatty acid composition of established cell lines

Summary The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms: number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fattyacid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines. These studies were supported by Arteriosclerosis Specialized Center of Research Grant HL 14,230 from the National Heart, Lung, and Blood Institute, National Institutes of Health.     

Modelling baculovirus infection of insect cells in culture

Conclusions Infection of insect cells with baculovirus is a potentially attractive means for producing both viral insecticides and recombinant proteins. The continuation of mathematical modelling studies such as those reviewed in this paper are essential in order to realise the full potential of the system. Through mathematical models it is possible to predict complex behaviours such as those observed when infecting cells at low MOI or when propagating virus in a continuous culture system. A purely empirical analysis of the same phenomena is very difficult if not impossible.The present three models are — despite their complexity and the effort that has gone into developing them — all first generation models. They summarise, to a large extent, our present quantitative understanding of the interaction between baculovirus and insect cells, when looked upon as a black box system. The binding and initial infection processes are still quantitatively poorly understood and further work in this area is much needed. On the longer term, a second generation of models is likely to consider interior processes such as viral DNA and RNA accumulation in much more detail using a structured model of the infection cycle.     

Cultivation of HeLa cells with fetal bovine serum or ultroser G: Effects on the plasma membrane constitution

Summary Plasma membranes isolated from HeLa cells cultivated in suspension cultures supplemented with 3.5% fetal bovine serum or 2% of the commercially available serum substitute Ultroser G contained the same amounts of protein, cholesterol, and phosphate on a cellular basis. Minor differences in the plasma membrane fatty acid composition were seen, with the most pronounced alteration observed for palmitic acid, which amounted to 27 and 20% in fetal bovine serum- and Ultroser G-supplemented cells, respectively. Plasma membranes from cells growth with Ultroser G contained almost twice as much phosphatidylethanolamine and displayed two thirds of the phosphatidylcholine content, compared to plasma membranes obtained from fetal bovine serum supplemented cells. The former membranes also showed a 3 times higher specific [³H]acetate labeling of cholesterol, indicating a higherde novo synthesis of cholesterol. Both quantitative and qualitative alterations were revealed among the plasma membrane polypeptides when these were subjected to immuno- and lectin blottings. Fluorescence anisotropy measurements at different temperatures produced similar results irrespective of the growth medium supplement when the plasma membrane specific probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene was used on intact cells. However, the average cellular rigidity was higher for Ultroser G supplemented cells, determined with 1,6-diphenyl-1,3,5-hexatriene as a probe. This investigation was supported by grants from the Swedish Natural Science Research Council, Anders Otto Sw?rds Stiftelse, Stockholm, Crafoordska Stiftelsen, Lund and Kungl. Fysiografiska S?llskapet, Lund.     

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.     

Reversible change in the fibroblast lysosomal enzyme dipeptidyl aminopeptidase-1 (cathepsin c) related to the commercial source of fetal bovine serum in the culture medium

Summary The commercial source of fetal bovine serum used to supplement the growth medium of human skin fibroblasts alters the activity of the lysosomal enzyme dipeptidyl aminopeptidase-1 (DAP-1). Cells grown with one serum were found to have a threefold higher level of DAP-1 than those grown with serum from another source (P<0.001). The effect on DAP-1 activity was specific inasmuch as no differences were found in the activities of a variety of other lysosomal and nonlysosomal hydrolases: DAP-II, DAP-III, DAP-IV, β-glucosidase, β-glucuronidase, andN-acetyl-β-galactosaminidase. The effect is reversible and is observed over a wide range of cell population doublings. Cell growth kinetics were not significantly different with the different sera. This work was supported in part by grants from the National Institutes of Health, Bethesda, MD (NS 16287).     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum

Summary Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a K_M for putrescine of 2.58×10⁻⁶ m and a V_max of 0.53 nmol per hr per 50 μl serum. Aminoguanidine competitively inhibited the enzyme with a K_I of 1.8×10⁻⁸ m. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The V_max for the ABS putrescine oxidase was 2.10 nmol per hr per 50 μl serum, and the K_M for putrescine, 50.3×10⁻⁶ m. The K₁ of the ABS putrescine oxidase for aminoguanidine was 41×10⁻⁶ m. On the basis of both the K_M and K_I values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56°C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed. This work was supported in part by a grant from the Cystic Fibrosis Foundation.     

Variable responsiveness of rat tracheal epithelial cells to bovine serum albumin in serum-free culture

Summary The colony-forming efficiency of rat tracheal epithelial (RTE) cells was determined in serum-free media containing different types of commercially available bovine serum albumin (BSA): crude fraction V, essentially globulin-free, essentially fatty-acid-free, and essentially globulin- and fatty-acid-free BSA. RTE cells exhibited a concentration-dependent increase in colony-forming efficiency in response to crude fraction V BSA. Similar results were obtained using essentially globulin-free BSA. However, deletion of cholera toxin from the medium resulted in a decrease in the colony-forming efficiency for cells plated in high concentrations (>2 mg/ml) of globulin-free, but not one type of fraction V, BSA. Essentially fatty-acid-free or essentially fatty-acid- and globulin-free BSA stimulated RTE cell colony formation at low concentrations (less than 2.5 to 5 mg BSA/ml) but resulted in concentration-dependent decreases in colony-forming efficiency at higher concentrations. The response of cells to these BSAs was not dependent on cholera toxin. Finally, commerically available fraction V BSA prepared by heat shock, dialysis, charcoal treatment, and deionization was stimulatory at low concentrations but inhibitory at high concentrations. These data suggest that impure preparations of BSA can, under different conditions, stimulate or inhibit cell proliferation and that the expression, of these activities is affected by the method of BSA preparation, the concentration of BSA used, and, in some cases, by the presence or absence of cholera toxin. Research conducted with support from the Office of Health and Environmental Research, U.S. Department of Energy, Washington, DC, under contract no. DE-AC04-76EV01013 in facilities fully acredited by the American Association for Laboratory Animal Care.     

Changes in the sulfation extent of membrane-associated proteoglycans produced by sertoli cells in culture

Sertoli cells in culture synthesize two different membrane-associated proteoglycans (MA-PG): a proteoglycan containing heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains and a CS-PG containing only CS-GAG chains. The structure of these molecules is regulated by the presence of fetal calf serum (FCS) in the culture medium. Changes in the concentration of FCS resulted in changes in the total ³⁵SO₄ incorporation into MA-PG and a shift in the elution profile of each component subjected to ion-exchange chromatography. Thus, without FCS, the incorporation was low, while in 1% and 10% FCS, the uptake of the precursor was 1.7 and 4.5 times higher, respectively. MA-PG synthesized by Sertoli cells cultured in 10% FCS eluted from DEAE-Sephacel columns at higher salt concentration than the MA-PG synthesized by cells cultured in 0% or 1% FCS. Double-labeled experiments showed that the ³⁵SO_4/3H-glucosamine ratio incorporated into MA-PG produced by Sertoli cells, increased from 17.6 to 23.6 and 50.9 in cells cultured at 0, 1, and 10% FCS, respectively. However, the presence of FCS affected neither the hydrodynamic size nor the chemical nature of GAG chains of MA-PG. These results show that changes in the FCS concentration promote changes in the sulfation extent of MA-PG molecules produced by Sertoli cells.     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.     

Calcium oxalate crystals in fetal bovine serum: implications for cell culture, phagocytosis and biomineralization studies in vitro

Cell culture methods and models are key investigative tools for cell and molecular biology studies. Fetal bovine serum (FBS) is commonly used as an additive during cell culture since its constituents promote cell survival, proliferation and differentiation. Here we report that commercially available FBS from different major suppliers consistently contain precipitated, calcium oxalate crystals-either in the monohydrate (COM) or dihydrate (COD) form. Mineral structure and phase identification of the crystals were determined by X-ray diffraction, chemical composition by energy-dispersive X-ray microanalysis, and imaging and measurement of crystal growth steps by atomic force microscopy-all identified and confirmed crystallographic parameters for COM and COD. Proteins binding to the crystals were identified by immunoblotting, revealing the presence of osteopontin and fetuin-A (alpha(2)HS-glycoprotein)--known regulators of crystal growth found in serum. Macrophage cell cultures exposed to calcium oxalate crystals showed internalization of the crystals by phagocytosis in a process that induced disruption of cell-cell adhesion, release of reactive oxygen species and membrane damage, events that may be linked to the release of inflammatory cytokines by these cells into the culture media. In conclusion, calcium oxalate crystals found in commercially available FBS are toxic to cells, and their presence may confound results from in vitro studies where, amongst others, phagocytosis, biomineralization, renal cell and molecular biology, and drug and biomaterial testing are being examined.     

Shear sensitivity of insect cells

Conclusions While insect cells can be easily damaged in bioreactors as a result of hydrodynamic forces, it is also relatively easy to prevent this damage. Of several possible damage mechanisms, the best understood and preventable is the attachment of cells to gas-liquid interfaces and the subjection of these attached cells to the hydro-dynamic forces and/or physical forces associated with these interfaces. For example, cells attached to gas bubbles in a bioreactor can be transported into the foam layer where they are physically removed from the cell suspension, or they can be killed when the gas bubble they are attached to ruptures at the medium-air interface at the top of the bioreactor. The easiest method to prevent this damage is through the use of specific surface active compounds, such as Pluronic F-68 or Methocel E-50 which prevent the cells from attaching to the gas-medium interface.     

Human marrow-derived mesenchymal progenitor cells

A number of adult mesenchymal tissues contain subpopulations of undifferentiated cells, which retain the capacity to differentiate along multiple lineages. These mesenchymal progenitor cells may be cultured in an undifferentiated state and, when given the appropriate signals, differentiate into an expanding list of several mesenchymal and a few ectodermal derived tissues. The maintenance and propagation of the multipotential nature of these progenitor cell populations are crucially dependent on the isolation protocol, the culture expansion conditions, particularly the properties of the fetal bovine serum supplement in the culture medium. This article describes a method for selection of the appropriate serum lot, and introduces a simplified isolation technique to optimize the yield of progenitor cells that maintain the capability of undergoing multilineage differentiation in response to appropriate cues. Cell populations isolated and culture expanded in this manner, by virtue of their multiple differentiation potential, should serve as ideal candidate cells for tissue engineering applications for the repair and regeneration of tissue damaged by disease and or trauma.