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.

The likelihood that potential new drugs will successfully navigate the current translational pipeline is poor, with fewer than 10% of drug candidates making this transition successfully, even after their entry into clinical trials. Prior to this stage, candidate drugs are typically evaluated by using models of increasing complexity, beginning with basic in vitro cell culture studies and progressing through to animal studies, where many of these candidates are lost due to lack of efficacy or toxicology concerns. There are many reasons for this poor translation, but interspecies differences in functional and physiological parameters undoubtedly contribute to the problem. Improving the human-relevance of early preclinical in vitro models may help translatability, especially when targeting more nuanced species-specific cell processes. The aim of the current study was to define a set of guidelines for the effective transition of human primary cells of multiple lineages to more physiologically relevant, translatable, animal-free in vitro culture conditions. Animal-derived biomaterials (ADBs) were systematically replaced with non-animal-derived alternatives in the in vitro cell culture systems, and the impact of the substitutions subsequently assessed by comparing the kinetics and phenotypes of the cultured cells. ADBs were successfully eliminated from primary human dermal fibroblast, uterine fibroblast, pulmonary fibroblast, retinal endothelial cell and peripheral blood mononuclear cell culture systems, and the individual requirements of each cell subtype were defined to ensure the successful transition toward growth under animal-free culture conditions. We demonstrate that it is possible to transition (\'humanise\') a diverse set of human primary cell types by following a set of simple overarching principles that inform the selection, and guide the evaluation of new, improved, human-relevant in vitro culture conditions.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson\'s disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-β), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.

OBJECTIVE: The need for innovative techniques to preserve microbiota for extended periods, while maintaining the species composition and quantitative balance of the bacterial community, is becoming increasingly important. To address this need, we propose an efficient approach to cryopreserve human gut microbiota using a two-component cryoprotective composition comprising fetal bovine serum (FBS) and 5% dimethyl sulfoxide (DMSO). Fetal serum is a commonly utilized component in the freezing media for eukaryotic cells, however, its effects on prokaryotic cells have not been extensively researched.
RESULTS: In our study, we demonstrated the high efficiency of using a two-component cryoprotective medium, FBS + 5% DMSO, for cryopreservation of human gut microbiota using three different methods. According to the obtained results, the intact donor microbiota was preserved at a level of 85 ± 4% of the initial composition based on fluorescent analysis using the LIVE/DEAD test. No differences in survival were observed when comparing with pure DMSO and FBS media. The photometric measurement method for growth of aerobic bacteria (A. johnsoni), facultative anaerobes (E. coli, E. faecalis), microaerophilic (L. plantarum), and obligate anaerobic bacterial cultures (E. barkeri, B. breve) also demonstrated high viability rates of 94-98% in the two-component protective medium, reaching intact control levels. However, for anaerobic microflora representatives, serum proved to be a more suitable cryoprotectant. Also, we demonstrated that using cryoprotective media with 50-75% FBS content is enough to preserve a significant level of bacterial cell viability, from an economic standpoint.

OBJECTIVE: Hepatitis E virus (HEV) is responsible for ∼20 million human infections worldwide every year. The genotypes HEV-3 and HEV-4 are zoonotic and are responsible for most of the autochthonous HEV cases in high-income countries. There are several cell culture systems that allow for propagation of different HEV genotypes in vitro. One of these systems uses human lung carcinoma cells (A549), and was further optimized for propagation of HEV-3 47832c strain. In this study, we investigated the effect of different media supplements as well as microRNA-122 (miR-122) on improving the replication of HEV-3 47832c in A549 cells.
RESULTS: We observed that supplementation of maintenance media with 5% fetal bovine serum was sufficient for efficient replication of HEV-3, and verified the positive effect of media supplementation with Amphotericin B, MgCl2, and dimethyl sulfoxide on replication of HEV-3. We have also demonstrated that adding miR-122 mimics to the culture media does not have any significant effect on the replication of HEV-3 47832c.
CONCLUSIONS: Herein, we detected over a 6-fold increase in HEV-3 replication in A549/D3 cells by adding all three supplements: Amphotericin B, MgCl2, and dimethyl sulfoxide to the culture media, while demonstrating that miR-122 might not play a key role in replication of HEV-3 47832c.

Extracellular vesicles (EVs) are produced by all kinds of cells, including endothelial cells. It has been observed that EVs present in fetal bovine serum (FBS), broadly used in cell culture, can be a confounding factor and lead to misinterpretation of results. To investigate this phenomenon, human brain microvascular endothelial cells (HBMECs) were cultured for 2 or 24 h in the presence of EV-depleted FBS (EVdS). Cell death, gene and protein expression, and the presence of EVs isolated from these cells were evaluated. The uptake of EVs, intercellular adhesion molecule 1 (ICAM-1) expression, and monocyte adhesion to endothelial cells exposed to EVs were also evaluated. Our results revealed higher apoptosis rates in cells cultured with EVdS for 2 and 24 h. There was an increase in interleukin 8 (IL8) expression after 2 h and a decrease in interleukin 6 (IL6) and IL8 expression after 24 h of culture. Among the proteins identified in EVs isolated from cells cultured for 2 h (EV2h), several were related to ribosomes and carbon metabolism. EVs from cells cultured for 24 h (EV24h) presented a protein profile associated with cell adhesion and platelet activation. Additionally, HBMECs exhibited increased uptake of EV2h. Treatment of endothelial cells with EV2h resulted in greater ICAM-1 expression and greater adherence to monocytes than did treatment with EV24h. According to our data, HBMEC cultivated with EVdS produce EVs with different physical characteristics and protein levels that vary over time.

Autologous fat transfers show promise in treating fibrotic skin diseases, reversing scarring and stiffness, and improving quality of life. Adipose-derived stem cells (ADSCs) within these grafts are believed to be crucial for this effect, particularly their secreted factors, though the specific mechanisms remain unclear. This study investigates transcriptomic changes in ADSCs after in vitro fibrotic, inflammatory, and hypoxic conditioning. High-throughput gene expression assays were conducted on ADSCs exposed to IL1-β, TGF-β1, and hypoxia and in media with fetal bovine serum (FBS). Flow cytometry characterized the ADSCs. RNA-Seq analysis revealed distinct gene expression patterns between the conditions. FBS upregulated pathways were related to the cell cycle, replication, wound healing, and ossification. IL1-β induced immunomodulatory pathways, including granulocyte chemotaxis and cytokine production. TGF-β1 treatment upregulated wound healing and muscle tissue development pathways. Hypoxia led to the downregulation of mitochondria and cellular activity.

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.

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R\'s (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R\'s; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

In light of the growing demand for alternative protein sources, laboratory-grown meat has been proposed as a potential solution to the challenges posed by conventional meat production. Cultured meat does not require animal slaughter and uses sustainable production methods, contributing to animal welfare, human health, and environmental sustainability. However, some challenges still need to be addressed in cultured meat production, such as the use of fetal bovine serum for medium supplementation. This ingredient has limited availability, increases production costs, and raises ethical concerns. This review explores the potential of non-animal protein hydrolysates derived from agro-industrial wastes as substitutes for critical components of fetal bovine serum in cultured meat production. Despite the lack of standardization of hydrolysate composition, the potential benefits of this alternative protein source may outweigh its disadvantages. Future research holds promise for increasing the accessibility of cultured meat.