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.     

Production of human immunodeficiency virus from T-lymphoblastoid cells using serum-free medium

Summary A serum-free medium was constructed for the suspension culture of mammalian cells. Effects of the serum-free medium on the cultivation of T-lymphoblastoid cells secreting human immunodeficiency virus (HIV) were observed. The cell growth and HIV production in the serum-free medium were comparable to those in the medium supplemented with 10% fetal bovine serum (FBS), which showed a possibility of economical industrial application.     

Serum-free medium for murine and human lymphoid and hybridoma cell lines

We established a serum-free medium of low protein content(125g/ml) TYI 100, consisting of three hormones and five growth factors for the growth of lymphoid and hybridoma cell lines. In TYI 100 medium, mouse and human hybridomas grew equally well as in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS) without adaptation to the serum-free medium. TYI 100 medium allowed several passages of mouse hybridoma lines and the total cell number was more than in 10% FBS. TYI 100 medium also supported growth of myelomas and anchorage dependent cell lines, Bowes and CHO, well. TYI 100 medium is composed of inexpensive supplements and is therefor applicable to large scale culture.Abbreviations FBS Fetal Bovine Serum - IMDM Iscove's Modification of Dulbecco's Medium - PBS Phosphate-Buffered Saline - TPA Tissue Plasminogen Activator     

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.     

Growth of human-human hybridomas in serum-free media enhances antibody secretion

Summary Human-human hybridomas derived from fusing lymph node lymphocytes with UC 729-6 were adapted to grow in commercially available serum-free medium and were compared with serum-supplemented [10% fetal bovine serum (FBS)] cultures. Over a 6-d period, no significant changes occurred in the growth of the cells in 10% FBS or serum-free medium. In cultures supplemented with 10% FBS more total proteins were secreted than in serum-free cultures. However, there was an enhanced secretion of three- to four-fold of both immunoreactive human IgG and IgM under serum-free conditions compared to serum-supplemented conditions. Serum-free conditions may provide the appropriate milieu for the increased level of Ig secretion from human hybridomas derived from UC 729-6 in that there are no inhibitors that may be present in serum. The work described in this report was partially supported by grants from the National Institutes of Health (CA 32047, CA 37497), Bethesda, MD, and the University of California Cancer Research Coordinating Committee. R.E.P. was a postdoctoral fellow supported by the UCSD Cancer Center and M.C.G. was the recipient of an NIH New Investigator Research Award. A.M. was a visiting scientist from the Medical Academy of Bulgaria, Sofia.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.     

Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media

To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H₂O₂ exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The precoating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H₂O₂ to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 M) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required.     

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.     

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.     

Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system

Embryonic stem (ES) cells have the ability to differentiate in vitro into a wide variety of cell types with potential applications for tissue regeneration. However, a large number of cells are required, thus strengthening the need to develop large-scale systems using chemically defined media for ES cell production and/or controlled differentiation. In the present studies, a stirred culture system (i.e. spinner flask) was used to scale-up mouse ES (mES) cell expansion in serum-containing (DMEM/FBS) or serum-free medium, both supplemented with leukemia inhibitory factor (LIF), using either Cytodex 3 or Cultispher S microcarriers. After 8 days, maximal cell densities achieved were (1.9+/-0.1), (2.6+/-0.7) and 3.5x10(6)cells/mL for Cytodex 3 in DMEM/FBS, Cultispher S in DMEM/FBS and Cultispher S in serum-free cultures, respectively, with fold increases of 38+/-2, 50+/-15 and 70. Both microcarriers were suitable to sustain mES cell expansion, though the macroporous Cultispher S seemed to be advantageous in providing a more protective environment against shear stress forces, which harmful effects are exacerbated in serum-free conditions. Importantly, mES cells expanded under stirred conditions using serum-free medium retained their pluripotency and the ability to commit to the neural lineage.     

Evaluation of methods for cultivating limbal mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSC) with similar properties to bone marrow-derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We have evaluated methods for culturing human limbal MSC (L-MSC).MethodsFour basic strategies were compared: serum-supplemented medium (10% fetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor and fibroblast growth factor 2, or one of two commercial serum-free media, defined keratinocyte serum-free medium (Invitrogen) and MesenCult-XF® (Stem Cell Technologies). The resulting cultures were examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141 and CD271), immunocytochemistry (alpha-smooth muscle actin; α-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis) and co-culture experiments with human limbal epithelial (HLE) cells.ResultsWhile all techniques supported the establishment of cultures to varying degrees, sustained growth and serial propagation were only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70–80% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, approximately 25% α-sma⁺ and displayed multipotency. Cultures established in MesenCult-XF were > 95% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, 40% CD141⁺, rarely expressed α-sma, and displayed multipotency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ?Np63, along with the corneal differentiation marker cytokeratin 3.ConclusionsMesenCult-XF is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.     

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.     

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.     

Isolation of thecal cells: an assessment of purity and steroidogenic potential

In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E(2)) and progesterone (P(4)) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results confirm that no E(2) could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The production of P(4) in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E(2) was detected from the same culture media. In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P(4) (P<0.05) in the serum-free cell culture media. The stimulation of P(4) production was greater at higher LH concentration (50 ng/ml) (P<0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhibited the production of P(4) by LH-stimulated thecal cell cultures (P<0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC(50)<1 ng/ml at LH-20 ng/ml vs. EC(50)>1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E(2) production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.     

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID₅₀) titers of the two cell lines reached 4.51×10⁶ cells/mL, 2.94Log₁₀(HAU/50 μL) and 8.49Log₁₀(virions/mL), and 5.97×10⁶ cells/mL, 3.88Log₁₀(HAU/50 μL), and 10.34Log₁₀(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.     

Cell growth stimulating effect of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> spores and their potential application for Chinese hamster ovary K1 cell cultivation

In this work, water-soluble extracts of Ganoderma lucidum spores (Gls), a Chinese medicinal herb that possesses cell growth stimulating function, were found to be an effective growth factor for Chinese hamster ovary (CHO) cell cultivation. The Gls extract was prepared and supplemented to CHO K1 cell culture media with various serum levels. Our results obtained from both the static culture and the spinner-flask suspension culture showed that use of small-amount Gls extract effectively promoted cell growth and suppressed cell apoptosis induced by serum deprivation with normal cell cycle maintained in a low-serum medium. The low-serum medium containing 1 % (v/v) fetal bovine serum (FBS) and 0.01 % (w/v) Gls extract showed a comparable performance on both cell growth and fusion protein productivity with the conventional CHO culture medium containing 10 % (v/v) FBS and a commercial serum-free medium. This is the first study of the potential of Gls extracts for use as an alternative cell growth factor and nutrient for CHO cells. The findings have presented a new approach to economic cultivation of CHO cells for therapeutic protein production.     

The growth requirements of BHK-21 cells in serum-free culture

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm², this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.     

Rabbit chondrocytes maintained in serum-free medium : I. Synthesis and secretion of hydrodynamically-small proteoglycans

The biosynthesis of sulfated proteoglycan in vitro by rabbit articular chondrocytes in first passage monolayer culture maintained in fetal bovine serum (FBS) or in serum-free conditions was compared. Neosynthesized proteoglycan in the culture medium in the most dense fraction of an associative CsCl density gradient (fraction dAl) declined with increasing time under serum-free conditions, but not when cells were maintained in the presence of serum. After one day, the major peak of incorporated 35SO4 in medium fraction dAl eluted as a retarded peak (Kav 0.28) on Sepharose CL-2B, whether cells were maintained under serum-free or serum-containing conditions. The hydrodynamic size of proteoglycan monomer fraction dAlDl obtained after one day of exposure to serum-free culture media was smaller than dAlDl from serum-containing cultures. The hydrodynamic size of dAlDl obtained from serum-free culture media became even progressively smaller after 2 and 3 days' exposure to these conditions. Hydrodynamically small sulfated proteoglycans were identified in the cell-associated dAlDl fraction as early as one day after switching chondrocytes from serum-containing to serum-free medium. Culture medium fraction dAlDl from serum-free culture medium aggregated poorly when incubated with human hyaluronic acid (HA) in the presence of bovine link protein or when dialysed against bovine nasal cartilage proteoglycan aggregate. Proteoglycan monomer from serum-containing medium reaggregated more efficiently under both conditions. No change in the size of glycosaminoglycan chains was seen in the smaller proteoglycan subpopulations, nor was there any indication of marked changes in the glycosaminoglycan types.     

Expansion of human bone marrow-derived mesenchymal stromal cells: serum-reduced medium is better than conventional medium

Background aimsHuman mesenchymal stromal cells (hMSC) are of enormous interest for various clinical applications. For the expansion of isolated hMSC to relevant numbers for clinical applications, 10% fetal bovine serum (FBS)-supplemented medium is commonly used. The main critical disadvantage of FBS is the possibility of transmission of infectious agents as well as the possibility of immune rejection of the transplanted cells in response to the bovine serum. Therefore, we tested a commercially available medium, Panserin 401, that was specifically developed for serum-free cell cultivation.MethodshMSC were isolated from bone marrow (BM) and expanded in either Dulbecco's modified Eagle medium (DMEM) or Panserin 401 alone, or combined with FBS (2% or 10%), with or without supplementary growth factors. Cell proliferation and cytotoxicity were monitored twice a week for 3 weeks.Results and ConclusionsNo proliferation was observed in any of the serum-free media. However, DMEM/10% FBS (the conventional culture medium for hMSC) and DMEM/2% FBS with growth factors revealed moderate proliferation. Interestingly, the best proliferation was obtained using Panserin 401 supplemented with 2% FBS and growth factors (as well as with 10% FBS). Analysis of cell growth in Panserin 401 supplemented with 2% FBS only or with growth factors only revealed no proliferation, demonstrating the necessity of the combination of 2% FBS and growth factors. Efficient isolation and expansion of hMSC from cancellous bone could also be performed using Panserin 401 with 2% FBS and growth factors. Furthermore, these isolated cultures maintained multipotency, as demonstrated by adipogenic and osteogenic differentiation.