Fetal Bovine Serum In Culture Research Articles

LHPL as Substitute for FBS in Stem Cell Culture by FC

With the advent of regenerative medicine, new methods for treating many incurable diseases might be used to replace damaged, dead, or diseased cells using cell-based methodologies. Mesenchymal stem cells possess immunomodulatory, tissue-protective, and pro-angiogenic properties that make them highly promising for cell-based therapies. Umbilical cord tissue (UCT) is the most common source of mesenchymal stem cells (MSCs) used for explant culture because of its potential for ex-vivo proliferation. Fetal bovine serum (FBS) is the conventional and promising present medium used to culture the MSCs. It is derived from the blood of cow fetus on cardiac puncture without anaesthesia that causes a lot of pain and discomfort to the fetus. This makes its availability limited for research work. Also using a xenogeneic supplement like a foetal bovine serum (FBS) to grow MSCs outside the body increases the risk of zoonotic infection transmission and unfavourable immunological reactions. The self-life of MHC is also very limited. A lot of research is going on to find an alternate to FBS of the above reasons like, human serum, adult bovine serum, lyophilized human platelet lysate (LHPL and chemical formulations. Our research here experimented the potency of lyophilized human platelet lysate (LHPL) as an alternate for FBS in culturing MSCs by using flow cytometry and proved, LHPL is a non-xenogeneic growth supplement with a longer self-life that will sustain the development of MSCs effective and can be stored and reconstituted on demand.

Impact of Three Different Serum Sources on Functional Properties of Equine Mesenchymal Stromal Cells.

Culture and expansion of equine mesenchymal stromal cells (MSCs) are routinely performed using fetal bovine serum (FBS) as a source of growth factors, nutrients, and extracellular matrix proteins. However, the desire to minimize introduction of xenogeneic bovine proteins or pathogens and to standardize cellular products intended for clinical application has driven evaluation of alternatives to FBS. Replacement of FBS in culture for several days before administration has been proposed to reduce antigenicity and potentially prolong survival after injection. However, the functional consequences of MSC culture in different serum types have not been fully evaluated. The objective of this study was to compare the immunomodulatory and antibacterial properties of MSCs cultured in three serum sources: FBS or autologous or allogeneic equine serum. We hypothesized that continuous culture in FBS would generate MSCs with improved functionality compared to equine serum and that there would not be important differences between MSCs cultured in autologous vs. allogeneic equine serum. To address these questions, MSCs from three healthy donor horses were expanded in medium with FBS and then switched to culture in FBS or autologous or allogeneic equine serum for 72 h. The impact of this 72-h culture period in different sera on cell viability, cell doubling time, cell morphology, bactericidal capability, chondrogenic differentiation, and production of cytokines and antimicrobial peptides was assessed. Altering serum source did not affect cell viability or morphology. However, cells cultured in FBS had shorter cell doubling times and secreted more interleukin 4 (IL-4), IL-5, IL-17, RANTES, granulocyte–macrophage colony-stimulating factor, fibroblast growth factor 2, eotaxin, and antimicrobial peptide cathelicidin/LL-37 than cells cultured in either source of equine serum. Cells cultured in FBS also exhibited greater spontaneous bactericidal activity. Notably, significant differences in any of these parameters were not observed when autologous vs. allogeneic equine serum was used for cell culture. Chondrogenic differentiation was not different between different serum sources. These results indicate that MSC culture in FBS will generate more functional cells based on a number of parameters and that the theoretical risks of FBS use in MSC culture should be weighed against the loss of MSC function likely to be incurred from culture in equine serum.

Influence of l-carnitine on lipid metabolism of buffalo cumulus-oocyte complexes matured in either fetal bovine serum or fatty acid-free bovine serum albumin

Consequences of oocyte supplementation with l-carnitine may vary depending on species-specific cellular lipid profile, level of mitochondrial activity, or even on ipid availability in culture medium. This study aimed to evaluate l-carnitine supplementation on competence and gene expression of enzymes related to lipid metabolism in oocytes and cumulus cells from buffalo COCs matured in the presence or absence of fetal bovine serum (FBS). COCs were matured in vitro in FBS (10%) or bovine serum albumin fatty acid-free (BSA-FAF) (0.4%) and with or without supplementation with l-carnitine (3.03 mM). COCs matured in the presence of FBS or BSA-FAF were fertilized and cultured, then supplemented with l-carnitine during in vitro maturation or in vitro embryo culture. Finally, in vivo mature and immature COCs were included for gene expression analysis. COCs matured in culture medium with FBS in the presence of l-carnitine produced a lower blastocyst rate (p ≤ 0.05) compared to controls. In turn, the blastocyst rate from COCs matured with BSA-FAF in the presence of l-carnitine was similar to controls (p > 0.05), and higher than FBS + L-carnitine treated COCs (p ≤ 0.05). Addition of l-carnitine during embryo culture showed no differences in blastocyst production between experimental groups and controls (p > 0.05). In cumulus cells, gene expression of ACACA, SCD and FASN was upregulated in COCs matured in the presence of BSA-FAF + L-carnitine, while all genes in oocytes were significantly expressed upregulated by COCs matured in vivo, and only BSA-FAF + L-carnitine group showed similar expression of the FASN gene. In conclusion, the consequences of l-carnitine supplementation during in vitro maturation of buffalo COCs on oocyte competence vary depending on presence or absence of FBS in culture. With FBS, l-carnitine impairs oocyte competence, while in its absence, gene expression suggests adequate lipid metabolism and increased oocyte competence.

Human platelet lysate as an alternative to fetal bovine serum for culture and endothelial differentiation of human amniotic fluid mesenchymal stem cells.

Human amniotic fluid (hAF) mesenchymal stem cells (MSCs) are commonly cultured in medium containing FBS. However, there are concerns about using animal serum in therapeutic applications due to the potential for immunogenic reactions and the risk of transmission of pathogens. For safety reasons, human platelet lysate (hPL) has been suggested as a replacement for FBS because it appears to be a natural source of growth factors. In this present study, it was investigated whether FBS could be substituted with hPL in hAF-MSCs culture without affecting their properties. Pooled hPL was generated by the freeze-thaw method. The concentration of hPL was selected after evaluation by MTT assay. The hAF-MSCs were cultured in FBS- or hPL-supplemented conditions and shared a fibroblast-like morphology. Cell proliferation assays showed that the growth characteristic of hAF-MSCs cultured in 10% hPL-supplemented media was similar to those cultured in 10% FBS-supplemented media. The expression of MSC markers did not differ between the cells cultured in the different conditions. The endothelial differentiation potential was also investigated. Reverse transcription-quantitative (RT-q)PCR revealed that induced cells supplemented with hPL showed an increase level of endothelial specific gene expression compared to the FBS-supplemented cells. Immunofluorescence analysis showed specific protein localization in both induced cell groups. Additionally, induced cells supplemented with hPL had the potential to form networks on Matrigel. This present study indicated that hPL could be used to culture and enhance the endothelial differentiation potential of hAF-MSCs.

Potential use of adult bovine serum obtained during the slaughtering process as a biological reagent

Starting from 2013, the Korean ‘Marine Environment Management Act,’ in response to international regulation on marine pollution, will prohibit the marine disposal of land wastes such as animal husbandry by-products and waste produced during the slaughtering process. Currently, most bovine blood is wasted during the slaughtering process and causes environmental problems in Korea. Therefore, long-term measures are required to process blood waste produced during the slaughtering process. The aim of this research is to investigate the potential use of adult bovine serum obtained during the slaughtering process for use in cell cultures. Primary mouse astrocytes, primary human fibroblasts, and L6 cells were cultured with adult male, adult female, and castrated male bovine sera for comparison with fetal bovine serum (FBS). The performance of adult bovine serum was comparable to that of FBS in culture of astrocytes and fibroblasts. Compared to FBS, adult bovine serum supported enhanced proliferation and differentiation of L6 cells. These results suggest that adult bovine serum obtained from the slaughtering process has a potential use as a biological reagent. Moreover, recycling adult bovine serum will contribute to solving the environmental issues arising from the disposal of waste produced during the slaughtering process.

Reprogramming of Sheep Fibroblasts into Pluripotency under a Drug-Inducible Expression of Mouse-Derived Defined Factors

Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes.

Optimum Combination of Insulin-Transferrin-Selenium and Fetal Bovine Serum for Culture of Rabbit Articular Chondrocytes in Three-Dimensional Alginate Scaffolds

Fetal bovine serum (FBS) has been reported to affect chondrocyte biosynthesis in monolayer culture. Insulin-Transferrin-Selenium (ITS) was investigated as a partial replacement for FBS during in vitro culture of rabbit articular chondrocytes in three-dimensional alginate scaffold. Chondrocyte-seeded alginate hydrogels were cultured in Dulbecco's modified Eagle's medium plus 10% FBS, 1% ITS plus 2% FBS, 1% ITS plus 4% FBS, or 1% ITS plus 8% FBS. At designed time point, the Chondrocyte-seeded alginate hydrogels were harvested and evaluated with histological staining, immunohistochemistry, and quantitative gene expression analysis. Viable cell density and cell division were also evaluated. Chondrocytes biosynthesis and cell division in 1% ITS with 2% FBS medium were similar to that in medium added with 10% FBS. For a total culture of 3 weeks, phenotypic gene expression in chondrocyte-seeded hydrogels was maintained at high levels in medium with 1% ITS plus 2% FBS, while it was decreased to varying degrees in the other groups. In conclusion, with 1% ITS, medium with 2% FBS could promote chondrocyte biosynthesis and cell division, and prevented cell dedifferentiation in three-dimensional alginate scaffolds.

In VitroProliferation and Differentiation of Human Mesenchymal Stem Cells Cultured in Autologous Plasma Derived from Bone Marrow

Expansion of human mesenchymal stem cells (hMSCs) in medium supplemented with fetal bovine serum (FBS) has a potential risk of transmitting viral and prion diseases and causing immunological rejection. The aim of our present study was to find a substitute for the traditional FBS in culture of hMSCs to facilitate the clinical application of hMSCs. We used autologous plasma derived from bone marrow (APM) as a substitute for FBS and found that, when cultured with APM, the cell surface markers and the proportion of hMSCs in the G(0)/G(1) phase and the S+G(2)/M phase resembled those cultured with FBS. However, there were fewer early apoptotic cells in APM-supplemented medium than in FBS (p < 0.01). APM resulted in much greater thymidine incorporation than FBS (p < 0.001). There were significantly more alkaline phosphatase (ALP)-positive fibroblast colony-forming units (CFU-Fs) covering larger areas in APM than in FBS (p < 0.01). Also, APM induced greater ALP activity, more mineralized nodules, and greater expression of osteogenic genes than did FBS. In addition, when cultured in adipogenic medium, there were fewer oil-red O-positive cells and lower expression of adipogenic gene with APM than with FBS. In conclusion, expansion of hMSCs in APM-supplemented medium instead of traditional FBS is more advantageous. It could promote cell proliferation, enhance osteogenic differentiation, and suppress adipogenic differentiation of hMSCs and is therefore a safer and more effective substitute for FBS in clinical cytotherapy processes.