For Fetal Bovine Serum Research Articles

Enhancement of intracellular extraction from Oscillatoria okine and the potential use of the extract as a supplement to fetal bovine serum in animal cell culture

A robust cell wall and well-organized thylakoid cyanobacteria can be candidates as a promising resource for C-phycocyanin (C-PC) and intracellular applications in various biotechnological areas. However, the development of extraction techniques with minimal chemical contamination to obtain the components remains an ongoing challenge. This study aimed to assess the efficacy of C-PC and intracellular extraction from Ocillatoria okeni TISTR8549 utilizing freezing-thaw (FT), pulsed electric field (PEF), and high-pressure homogenization (HPH) techniques. Additionally, the potential of O. okeni extract as a supplement and substitute for fetal bovine serum (FBS) in HaCaT cell culture was investigated. FT appeared to be the most proper method for C-PC extraction, yielding the highest purity and yield, although requiring 18 cycles for product accomplishment. PEF seemed unsuitable for intracellular component extraction from cyanobacteria with thick cell walls. The need for increased pulses resulted in thermal elevation and prolonged incubation times led to protein degradation. HPH proves to be an effective method for intracellular extraction, yielding high protein content suitable for the potential substitution of FBS in mammalian cell culture. Particularly, increasing pressure during HPH extraction leads to a decrease in protein yield. Resazurin and SRB assays revealed that adding algal extract in culture medium at concentrations of 1 % (w/v) improved HaCaT cell viability without disrupting cell morphology and metabolic processes. However, substituting algal extract in FBS resulted in cell proliferation decrease. Therefore, supplementing O. okeni extract during cell culture improved HaCaT cell proliferation, but it was unsuitable substitute for FBS in the culture medium. However, the feasibility of employing algal extracts as FBS replacements in cell culture is interesting and warrants further detailed investigation into the specific intracellular components that could serve as substitutes for FBS. Such an approach could offer an alternative source, mitigating ethical concerns and reducing costs associated with FBS usage.

Impact of different formulations of platelet lysate on proliferative and immune profile of equine mesenchymal stromal cells.

Platelet lysate (PL) is investigated as a potential replacement for fetal bovine serum (FBS) in cell culture. However, there is limited research on its impact on the immune profile of equine mesenchymal stromal cells (eMSCs). This study aimed to evaluate the effects of different PL formulations on the proliferative capacity, multipotentiality, and immune profile of equine adipose tissue-derived MSCs (eAD-MSCs). In vitro growth kinetics and trilineage differentiation of eAD-MSCs (n = 7) were assessed under three culture conditions: medium-concentration PL (MPL), high-concentration PL (HPL), and FBS as a control. The immune profile was evaluated by studying the expression of immunogenic receptors such as MHC I, MHC II, and immunomodulatory molecules IL-6, IL-10, and TNF-α, determined by gene expression, surface marker expression, and cytokine quantification. Both PL formulations, pooled from 5 donors, exhibited 3.3 and 6.5-fold higher platelet counts than baseline plasma for MPL and HPL, respectively. Higher concentrations of TGF-β and PDGF were found in both PL formulations compared to baseline. Furthermore, MPL and HPL subcultures demonstrated proliferative, clonogenic, and multipotent capacities similar to FBS. The immune profile of PL-cultured cells exhibited gene expression levels related to immunogenicity and immunomodulation similar to the reference condition, and the surface antigen presence of MHC II was also similar. However, HPL media exhibited higher IL-6, IL-10, and TNF-α concentrations in the culture supernatant. In conclusion, both PL media contained higher concentrations of growth factors compared to FBS, supporting the in vitro culture of eAD-MSCs with proliferative, clonogenic, and multipotent capacity similar to the reference medium. Nonetheless, PL usage led to a variation in the immunomodulatory cytokine microenvironment, with higher concentrations of IL-6, IL-10, and TNF-α in HPL media compared to MPL and FBS.

Platelet-Rich Plasma Promotes the Expansion of Human Myoblasts and Favors the In Vitro Generation of Human Muscle Reserve Cells in a Deeper State of Quiescence

Stem cell therapy holds significant potential for skeletal muscle repair, with in vitro-generated human muscle reserve cells (MuRCs) emerging as a source of quiescent myogenic stem cells that can be injected to enhance muscle regeneration. However, the clinical translation of such therapies is hampered by the need for fetal bovine serum (FBS) during the in vitro generation of human MuRCs. This study aimed to determine whether fresh allogeneic human platelet-rich plasma (PRP) combined or not with hyaluronic acid (PRP-HA) could effectively replace xenogeneic FBS for the ex vivo expansion and differentiation of human primary myoblasts. Cells were cultured in media supplemented with either PRP or PRP-HA and their proliferation rate, cytotoxicity and myogenic differentiation potential were compared with those cultured in media supplemented with FBS. The results showed similar proliferation rates among human myoblasts cultured in PRP, PRP-HA or FBS supplemented media, with no cytotoxic effects. Human myoblasts cultured in PRP or PRP-HA showed reduced fusion ability upon differentiation. Nevertheless, we also observed that human MuRCs generated from PRP or PRP-HA myogenic cultures, exhibited increased Pax7 expression and delayed re-entry into the cell cycle upon reactivation, indicating a deeper quiescent state of human MuRCs. These results suggest that allogeneic human PRP effectively replaces FBS for the ex vivo expansion and differentiation of human myoblasts and favors the in vitro generation of Pax7High human MuRCs, with important implications for the advancement of stem cell-based muscle repair strategies.Graphical

Human venous blood derivatives as fetal bovine serum substitute for fibroblast culture cells in a fibrin construct

Aim: Venous blood derivatives (VBDs) have been suggested as substitutes for Fetal Bovine Serum (FBS) to improve the clinical transition of cell-based therapies. The literature is not clear about which is the best VBDs substitute. The present study aimed to evaluate the influence of VBDs on cell viability and describe a new method to seed these cells in a 3D Platelet-Rich Fibrin (PRF). Methods: Blood was processed to obtain Platelet-Poor Plasma from PRF (P-PRF), Human Serum (HS), Platelet-Poor Plasma from PRP (P-PRP), activated-PRP (a-PRP), and Platelet lysate (PL). Cells were supplemented with each VBD at 10% and FBS at 10% was the control. Cell viability (fibroblast 3T3/NIH) test was evaluated with MTT assay in two ways: i) cell-seeded and expanded with VBD; ii) cell-seed with FBS and expanded with VBD. To seed the Fibrin construct, cells were suspended in PBS and dropped into the blood sample before performing Choukroun’s protocol for PRF. Constructs were cultured for 7 days in VBD supplements and FBS. Histological and Immunohistochemical analysis with vimentin was performed. Cell viability was analyzed by one-way ANOVA. Results: VBD’s production time was very heterogeneous. Cells expanded in HS and a-PRP has grown faster. VBD-supplemented culture media provided cell culture highly sensible to trypsin/EDTA 0.25%. Cells seeded and expanded with VBD presented viability comparable to FBS in HS, a-PRP, and P-PRP (p>0.05) and lower in P-PRF and PL groups (p<0.05). The viability of cell seed with FBS and expanded with VBD was similar between P-PRF, a-PRP, PL, and FBS (p>0.05) and lower in HS and P-PRP (p<0.005). PRF-seeded cells showed a positive expression of vimentin and were able to maintain all cells supplemented with VBD. Conclusion: VBD supplements were able to maintain fibroblast cells in 2D and 3D cultures. The new method of the fibrin-cell construct was efficient to insert the cells into the fibrin network.

Use of platelet-rich plasma on in vitro maturation during bovine embryo production.

One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.

Evaluation of fermented soybean meal and edible insect hydrolysates as potential serum replacement in pig muscle stem cell culture

In this study, we investigated the potential of edible hydrolysates as a replacement for fetal bovine serum (FBS) in pig muscle stem cell culture. Hydrolysates of two fermented soybean meals (fermented with a combination of Aspergillus oryzae and Bacillus subtilis and fermented with Bacillus subtilis) and two edible insects (mealworm and cricket) were prepared (FAB-H, FB-H, TM-H, and GB-H, respectively). All hydrolysates prepared showed antioxidant activity and provided a suitable cell culture environment as the pH of media containing each hydrolysate (at concentrations ranging from 0.01 to 1%) was within the acceptable range. The supplementation with hydrolysate (0.01–5% FAB-H and FB-H, 0.01–1% TM-H, or 0.01–0.1% GB-H) promoted cell proliferation in media containing 10% FBS. Furthermore, 0.01 and 0.1% of FAB-H, FB-H, and TM-H were able to replace up to 50% of FBS while maintaining proliferation and differentiation capacity. Notably, 0.1% of FB-H and TM-H in 50% FBS-reduced media further enhanced differentiation compared to 10% FBS media. However, further investigations are required to improve the effects of these hydrolysates on the long-term culture of pig muscle stem cells. Nevertheless, the partial FBS replacement with edible and cost-effective natural materials (FAB-H, FB-H, and TM-H) could significantly help reduce the cost of cultured meat significantly.

Employing active learning in the optimization of culture medium for mammalian cells

Medium optimization is a crucial step during cell culture for biopharmaceutics and regenerative medicine; however, this step remains challenging, as both media and cells are highly complex systems. Here, we addressed this issue by employing active learning. Specifically, we introduced machine learning to cell culture experiments to optimize culture medium. The cell line HeLa-S3 and the gradient-boosting decision tree algorithm were used to find optimized media as pilot studies. To acquire the training data, cell culture was performed in a large variety of medium combinations. The cellular NAD(P)H abundance, represented as A450, was used to indicate the goodness of culture media. In active learning, regular and time-saving modes were developed using culture data at 168 h and 96 h, respectively. Both modes successfully fine-tuned 29 components to generate a medium for improved cell culture. Intriguingly, the two modes provided different predictions for the concentrations of vitamins and amino acids, and a significant decrease was commonly predicted for fetal bovine serum (FBS) compared to the commercial medium. In addition, active learning-assisted medium optimization significantly increased the cellular concentration of NAD(P)H, an active chemical with a constant abundance in living cells. Our study demonstrated the efficiency and practicality of active learning for medium optimization and provided valuable information for employing machine learning technology in cell biology experiments.

National Blood Foundation 2021 Research and Development summit: Discovery, innovation, and challenges in advancing blood and biotherapies.

National Blood Foundation 2021 Research and Development summit: Discovery, innovation, and challenges in advancing blood and biotherapies.

An Evaluation of Different Types of Peptone as Partial Substitutes for Animal-derived Serum in Vero Cell Culture

Vero cells are one of the most frequently used cell types in virology. They can be used not only as a vehicle for the replication of viruses, but also as a model for investigating viral infectivity, cytopathology and vaccine production. There is increasing awareness of the need to limit the use of animal-derived components in cell culture media for a number of reasons, which include reducing the risk of contamination and decreasing costs related to the downstream processing of commercial products obtained via cell culture. The current study evaluates the use of protein hydrolysates (PHLs), also known as peptones, as partial substitutes for fetal bovine serum (FBS) in Vero cell culture. Eleven plant-based, two yeast-based, and three casein-based peptones were assessed, with different batches evaluated in the study. We tested the effects of three concentration ratios of FBS and peptone on Vero cell proliferation, four days after the initial cell seeding. Some of the tested peptones, when in combination with a minimal 1% level of FBS, supported cell proliferation rates equivalent to those achieved with 10% FBS. Collectively, our findings showed that plant-based peptones could represent promising options for the successful formulation of serum-reduced cell culture media for vaccine production. This is especially relevant in the context of the current COVID-19 pandemic, in view of the urgent need for SARS-CoV-2 virus production for certain types of vaccine. The current study contributes to the Three Rs principle of reduction, as well as addressing animal ethics concerns associated with FBS, by repurposing PHLs for use in cell culture.

Addressing Animal Welfare Issues in Fetal Blood Collection for Fetal Bovine Serum Production

There is ethical debate over whether fetal calves suffer when their dam is slaughtered and fetal blood extracted by cardiac puncture for fetal bovine serum (FBS) production. Yet, the serum industry does not follow best practice, as recommended by the European Food Safety Authority (EFSA), to avoid fetal distress. We discuss the key elements of this debate, and recommend how the serum industry can alter its practices to improve animal welfare.

A comparative study of pathogen inactivation technologies in human platelet lysate and its optimal efficiency in human placenta-derived stem cells culture

BackgroundPathogen inactivation (PI) is necessary for the pooled components derived from a biological source. Recently, the use of human platelet lysate (hPL) has increased in the cell manufacturing process as a xeno-free substitute for Fetal Bovine Serum (FBS). Therefore, an effective PI process to produce a pathogen-free hPL with the optimal efficiency in the manufacturing of cell therapy products is a vital requirement. Study design and methodsTo evaluate the efficacy of gamma irradiation and riboflavin/ultraviolet light (RB/UV) as PI methods for hPL, the reduction factor (RF) of titer of model viruses and bacteria were examined. Furthermore, the effect of different PI methods on the hPL performance was evaluated by the in vitro expansion of human placenta-derived mesenchymal stem cells (PLMSCs). To compare different study groups, the growth kinetic, immunophenotype, colony formation, and differentiation capacity (osteogenic and adipogenic) of PLMSCs were examined. In addition, the concentration of growth factors was assayed in each study group. ResultsAchievement to the RF more than 5 log10 for all pathogens, showed the effectiveness of two PI methods. In comparison with the other study groups, the dose of 45 kGy gamma irradiation considerably decreased the growth factor level of the hPL. It also showed a significant adverse effect on PLMSCs growth kinetics. The dose of 30 KGy gamma irradiation and RB/UV demonstrated a favorable effect on different assays of the in vitro expanded PLMSCs. ConclusionThe 30 KGy gamma irradiation and RB/UV were effective in the RF of the viral and bacterial models of the contaminated hPL. The efficacy of these PI-hPLs for PLMSCs expansion was preserved. To increase the safety of cell therapy products, PI methods should be considered for the hPL preparations.

Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same.

Simple SummaryRegenerative medicine using platelet-based blood products or adult stem cells offers the prospect of better clinical outcomes with many diseases. In veterinary medicine, most progress has been made with the development and therapeutic use of these regenerative therapeutics in horses, but the clinical need is given in dogs as well. Our aim was to transfer previous advances in the development of horse regenerative therapeutics, specifically the use of platelet lysate for feeding stem cell cultures, to the dog. Here, we describe the scalable production of canine platelet lysate, which could be used in regenerative biological therapies. We also evaluated the canine platelet lysate for its suitability in feeding canine stem cell cultures in comparison to equine platelet lysate used for equine stem cell cultures. Platelet lysate production from canine blood was successful, but the platelet lysate did not support stem cell culture in dogs in the same beneficial way observed with the equine platelet lysate and stem cells. In conclusion, canine platelet lysate can be produced in large scales as described here, but further research is needed to improve the cultivation of canine stem cells.Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.

Marine cyanobacterium Spirulina maxima as an alternate to the animal cell culture medium supplement

Serum is a stable medium supplement for in vitro cell culture. Live cells are used in stem cell research, drug toxicity and safety testing, disease diagnosis and prevention, and development of antibiotics, drugs, and vaccines. However, use of serum in culture involves concerns such as an ethical debate regarding the collection process, lack of standardized ingredients, and high cost. Herein, therefore, we evaluated the possibility of using edible cyanobacterium (Spirulina maxima), which is a nutrient-rich, sustainable, and ethically acceptable source, as a novel substitute for fetal bovine serum (FBS). H460 cells were cultured to the 10th generation by adding a mixture of spirulina animal cell culture solution (SACCS) and FBS to the culture medium. Cell morphology and viability, cell cycle, apoptosis, proteomes, and transcriptomes were assessed. We observed that SACCS had better growth-promoting capabilities than FBS. Cell proliferation was promoted even when FBS was replaced by 50–70% SACCS; there was no significant difference in cell shape or viability. There were only slight differences in the cell cycle, apoptosis, proteomes, and transcriptomes of the cells grown in presence of SACCS. Therefore, SACCS has the potential to be an effective, low-cost, and eco-friendly alternative to FBS in in vitro culture.

Human CD34+ hematopoietic stem cells culture in humanized culture medium for cell therapy

IntroductionHematopoietic stem cell transplantation (HSCT) is a proper treatment for many cancers, including malignant and non-malignant hematologic and immune disorders. However, due to the small quantity of HSCTs, their number should be increased ex vivo. Cell proliferation increase by animal serum can transmit certain diseases and immune problems to the recipient; therefore, replacement of culture media with media of human origin seems to be crucial. Acellular Wharton's jelly (AWJ) can be considered as an alternative medium in this regard. This study aimed to investigate the effect of AWJ substitution for fetal bovine serum (FBS) on the expression of myeloid and lymphoid specific markers and CD34+ cells proliferation pattern following co-culture with mesenchymal stem cells. Materials and methodsIn this study, CD34+ umbilical cord blood HSCs were co-cultured with MSCs in the presence of 10% FBS or AWJ, stem cell factor (SCF), thrombopoietin (TPO), and FLT3-L and the proliferation pattern and expression of myeloid as well as lymphoid lineage markers were then evaluated. ResultsThe expression of myeloid and lymphoid specific markers was increased and decreased in AWJ and FBS containing culture medium, respectively. ConclusionThis study provided evidence for the differentiation of UCB-derived CD34+ cells toward myeloid lineage and lack of their differentiation toward lymphoid lineage in AWJ containing culture medium.

Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate

BackgroundInnovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology.MethodsStromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed.ResultsBiochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na+ were elevated in pHPL preparations, K+ and Fe3+ levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions.ConclusionAll pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.

Characterization of the viral genomes present in commercial batches of horse serum obtained by high-throughput sequencing

Horses are often used as blood donors for commercial horse serum (HS) production and to manufacture biologicals. HS is an alternative for fetal bovine serum (FBS) used as a supplement for cell culture and vaccine production. Furthermore, HS is also frequently obtained in order to produce antisera toxins and pathogens. The advent of high-throughput sequencing (HTS) has promoted changes in virus detection, since previous knowledge of targets is not required. Thus, the present study aimed to describe the virome of five different batches of commercial HS from New Zealand (three batches) and Brazil and the United States (one batch each) using HTS. Each HS pool were processed and sequenced using an Illumina MiSeq platform. Sequences-related to viruses belonging to the Flaviviridae, Herpesviridae, and Parvoviridae families were detected. Particularly, equine hepacivirus (EqHV), equine pegivirus (EPgV), and Theiler's disease-associated virus (TDAV) were more frequent found in the batches analyzed. The presence of viral genomes in cell culture sera illustrates that these commercial sera can contain a mixture of different viruses and, therefore, can be regarded as potentially infectious for susceptible hosts. Moreover, the innocuity of commercial HS is important for the efficiency and security of diagnostics and the production of biological products.

Human Platelet Lysate Improves Bone Forming Potential of Human Progenitor Cells Expanded in Microcarrier-Based Dynamic Culture.

Xenogeneic‐free media are required for translating advanced therapeutic medicinal products to the clinics. In addition, process efficiency is crucial for ensuring cost efficiency, especially when considering large‐scale production of mesenchymal stem cells (MSCs). Human platelet lysate (HPL) has been increasingly adopted as an alternative for fetal bovine serum (FBS) for MSCs. However, its therapeutic and regenerative potential in vivo is largely unexplored. Herein, we compare the effects of FBS and HPL supplementation for a scalable, microcarrier‐based dynamic expansion of human periosteum‐derived cells (hPDCs) while assessing their bone forming capacity by subcutaneous implantation in small animal model. We observed that HPL resulted in faster cell proliferation with a total fold increase of 5.2 ± 0.61 in comparison to 2.7 ± 02.22‐fold in FBS. Cell viability and trilineage differentiation capability were maintained by HPL, although a suppression of adipogenic differentiation potential was observed. Differences in mRNA expression profiles were also observed between the two on several markers. When implanted, we observed a significant difference between the bone forming capacity of cells expanded in FBS and HPL, with HPL supplementation resulting in almost three times more mineralized tissue within calcium phosphate scaffolds. FBS‐expanded cells resulted in a fibrous tissue structure, whereas HPL resulted in mineralized tissue formation, which can be classified as newly formed bone, verified by μCT and histological analysis. We also observed the presence of blood vessels in our explants. In conclusion, we suggest that replacing FBS with HPL in bioreactor‐based expansion of hPDCs is an optimal solution that increases expansion efficiency along with promoting bone forming capacity of these cells. stem cells translational medicine 2019;8:810&821

The effect of activated pure platelete rich plasma (P-PRP) on the proliferation of adipose -derived mesenchymal stem cells (AD-MSCs) as a substitute for fetal bovine serum (FBS)

The field of stem cells has attracted the attention of many researchers as a hope to treat many incurable diseases because of their ability of self-renewal and differentiation into a specific type of cells that compensates damaged cells. Many studies have been published to confirm their effectiveness but there are some obstacles that limit their clinical applications. One of these obstacles is xenogenic fetal bovine serum (FBS); the main proliferative source for stem cell culture with subsequent risk of infection transmission or immunogenic problems. So, in our study, we aimed to replace the xenogenic FBS with xeno-free blood derivative. We tested the effect of different concentrations of activated pure platelet rich plasma (P-PRP); one of the blood derivatives on proliferation of adipose derived mesenchymal stem cells (AD-MSCs) in comparison to FBS and found that 20% activated P-PRP followed by 10% activated P-PRP increased the proliferation rate of AD-MSCs more than 10% FBS. KEYWORDS AD-MSCs, P-PRP, FBS.

The effect of activated pure platelete rich plasma (P-PRP) on the proliferation of adipose -derived mesenchymal stem cells (AD-MSCs) as a substitute for fetal bovine serum (FBS)

The field of stem cells has attracted the attention of many researchers as a hope to treat many incurable diseases because of their ability of self-renewal and differentiation into a specific type of cells that compensates damaged cells. Many studies have been published to confirm their effectiveness but there are some obstacles that limit their clinical applications. One of these obstacles is xenogenic fetal bovine serum (FBS); the main proliferative source for stem cell culture with subsequent risk of infection transmission or immunogenic problems. So, in our study, we aimed to replace the xenogenic FBS with xeno-free blood derivative. We tested the effect of different concentrations of activated pure platelet rich plasma (P-PRP); one of the blood derivatives on proliferation of adipose derived mesenchymal stem cells (AD-MSCs) in comparison to FBS and found that 20% activated P-PRP followed by 10% activated P-PRP increased the proliferation rate of AD-MSCs more than 10% FBS.

Platelet Rich in Growth Factors (PRGF): A Suitable Replacement for Fetal Bovine Serum (FBS) in Mesenchymal Stem Cell Culture

ntroduction: Due to high differentiation potential and self-renewality, Mesenchymal stem cells are now widely considered by researchers in several diseases. FBS is used as a supplement in culture media for proliferation, differentiation, and other culture processes of MSCs, which is associated with transmission risk of a variety of infections as well as immune responses. PRGF derived from platelets contains growth factors causing the growth and proliferation of MSCs. This study was conducted to compare the effect of PRGF in comparison to FBS on the expression of h-TERT gene, in umbilical cord-derived MSCs. Materials and Methods: This study is an experimental research. Four expired platelet concentrate bags were obtained from Kerman blood transfusion center, and PRGF was prepared by multiple centrifugation rounds of the platelet bag. Calcium chloride was added as an anticoagulant to PRGF in order to prevent gelatinization of the culture medium. On the other hand, mesenchymal stem cells were isolated from the umbilical cord as a primary culture. The phenotype of the cells was confirmed by flow cytometry, and the cells were randomly cultured as control (using FBS) and experimental (using PRGF) groups. The expression of the gene involved in increasing cell longevity (h-TERT) was investigated by real-time PCR technique after six days. Results: Mesenchymal stem cells were successfully isolated from the umbilical cord. Morphologically, the mesenchymal cells cultured in the experimental group (using PRGF) were similar to the cells in the control medium. The cells exhibited a high expression level of CD73, CD90, and CD105, while the surface markers of hematopoietic cells such as CD45 and CD34 were slightly expressed. Therefore, there was no significant difference in the expression of cell surface markers between control and experimental groups. In this study, using the real-time PCR technique, it was shown that PRGF derived from the platelet could increase the expression of h- TERT gene in the umbilical cord mesenchymal stem cells compared with the control. (P = 0.034)