This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos.

Standard cell culture practices require the addition of animal-derived serum to culture media to achieve adequate cell growth. Typically, 5-10% by volume of fetal bovine serum (FBS) is used, which accounts for a vast majority of the media cost while also imposing environmental and ethical concerns associated with the use of animal serum. Here we tested the efficacy of culturing cells by replacing serum in the media with algae extract and select additives. Using LC-MS, we compared molecular signatures of FBS to Chlorella algae extracts and identified NAD(H)/NADP(H) as common and relatively abundant features in their characteristic profiles. Bovine fibroblasts, cultured in serum-free media supplemented with C. vulgaris extract and just two growth factors plus insulin, showed significant growth with enhanced viability compared to control cells cultured without serum, albeit still lower than that of controls cultured with 10% FBS. Moreover, C. vulgaris extract enhanced cell viability beyond that of cells cultured with the two growth factors and insulin alone. These results suggest that key components in serum which are essential for cell growth may also be present in C. vulgaris extract, demonstrating that it may be used at least as a partial alternative to serum for cell culture applications.

The generation of vasculature is one of the most important challenges in tissue engineering and regeneration. Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types to induce vasculogenesis and angiogenesis as they not only secrete vascular endothelial growth factor (VEGF) but can also differentiate in vitro into both endotheliocytes and pericytes in serum-free culture media. Moreover, hDPSCs can generate complete blood vessels containing both endothelial and mural layers in vivo, upon transplantation into the adult brain. However, many of the serum free media employed for the growth of hDPSCs contain supplements of an undisclosed composition. This generates uncertainty as to which of its precise components are necessary and which are dispensable for the vascular differentiation of hDPSCs, and also hinders the transfer of basic research findings to clinical cell therapy. In this work, we designed and tested new endothelial differentiation media with a fully defined composition using standard basal culture media supplemented with a mixture of B27, heparin and growth factors, including VEGF-A165 at different concentrations. We also optimized an in vitro Matrigel assay to characterize both the ability of hDPSCs to differentiate to vascular cells and their capacity to generate vascular tubules in 3D cultures. The description of a fully defined serum-free culture medium for the induction of vasculogenesis using human adult stem cells highlights its potential as a relevant innovation for tissue engineering applications. In conclusion, we achieved efficient vasculogenesis starting from hDPSCs using serum-free culture media with a fully defined composition, which is applicable for human cell therapy purposes.

The demand for meat is expected to exceed production capacity by livestock in the coming decennia. Therefore, cultured beef might be a viable alternative to traditional livestock-derived beef. One of the problems however is the sustainability of cultured beef through the use of fetal bovine serum. We aimed to identify a serum-free medium or a serum-replacement that is as effective as the current method used for culturing bovine myoblasts. Cells were harvested from a female Blanc Bleu Belge cow and myoblasts were subsequently isolated. Cells were cultured in either Advanced DMEM containing 20% FBS and 10% HS or one of the chemically-defined, serum-free media for 6 days. MTS was used as a measure of cell proliferation at day 1, 4 or 6 and microscopic pictures were taken to assess cell morphology. FBM™, TesR™ and Essential 8™ are commercially available xeno-free media developed for human PSCs and fibroblasts, with the highest potential to sustain bovine myoblast proliferation. Of the supplements tested, XenoFree™ and a custom-prepared growth factor mix failed to stimulate cell proliferation. LipoGro™ stimulated cell proliferation in some cases but also changed the phenotype of myoblasts to an adipocyte-like phenotype. We conclude that serum-free media stimulate exponential cell expansion, albeit not to the extent of the current growth medium containing up to 30% serum. Further research is needed to investigate whether prolonged cell culture or an adaptation period could further increase cell proliferation.

In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for in vitro studies is advocated to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EV-depleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA (snRNA, Y RNA, snoRNA, and piRNA) were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media.

Stem cells are attractive candidates for the regeneration of tissue and organ. Mesenchymal stem cells (MSCs) have been extensively investigated for their potential applications in regenerative medicine and cell therapy. For developing effective stem cell therapy, the mass production of consistent quality cells is required. The cell culture medium is the most critical aspect of the mass production of qualified stem cells. Classically, fetal bovine serum (FBS) has been used as a culture supplement for MSCs. Due to the undefined and heterologous composition of animal origin components in FBS, efforts to replace animal-derived components with non-animal-derived substances led to safe serum free media (SFM). Adipose derived mesenchymal stem cells (ADSCs) cultivated in SFM provided a more stable population doubling time (PDT) to later passage and more cells in a shorter time compared to FBS containing media. ADSCs cultivated in SFM had lower cellular senescence, lower immunogenicity, and higher genetic stability than ADSCs cultivated in FBS containing media. Differential expression analysis of mRNAs and proteins showed that the expression of genes related with apoptosis, immune response, and inflammatory response were significantly up-regulated in ADSCs cultivated in FBS containing media. ADSCs cultivated in SFM showed similar therapeutic efficacy in an acute pancreatitis mouse model to ADSCs cultivated in FBS containing media. Consideration of clinical trials, not only pre-clinical trial, suggests that cultivation of MSCs using SFM might offer more safe cell therapeutics as well as repeated administration due to low immunogenicity.

The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco's Modified Eagle's Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

Successful Drosophila cell culture relies on media containing xenogenic components such as fetal bovine serum to support continuous cell proliferation. Here, we report a serum-free culture condition that supports the growth and proliferation of Drosophila S2R+ and Kc167 cell lines. Importantly, the gradual adaptation of S2R+ and Kc167 cells to a media lacking serum was supported by supplementing the media with adult Drosophila soluble extract, commonly known as fly extract. The utility of these adapted cells lines is largely unchanged. The adapted cells exhibited robust proliferative capacity and a transfection efficiency that was comparable to control cells cultured in serum-containing media. Transcriptomic data indicated that the S2R+ cells cultured with fly extract retain their hemocyte-specific transcriptome profile, and there were no global changes in the transcriptional output of cell signaling pathways. Our metabolome studies indicate that there were very limited metabolic changes. In fact, the cells were likely experiencing less oxidative stress when cultured in the serum-free media supplemented with fly extract. Overall, the Drosophila cell culture conditions reported here consequently provide researchers with an alternative and physiologically relevant resource to address cell biological research questions.

Chemically defined serum-free media are increasingly used as a tool to help standardize experiments by eliminating the potential variability contributed by pooled serum. These media are formulated for the culture and expansion of specific cell types, maintaining cell viability without the need for exogenous animal proteins. Formulated serum-free media could thus help improve viability and reduce variability during sample preparation for flow cytometry, yet a thorough analysis of how such media impact fluorochrome-Ab conjugates has not been performed. In this study, we expose fluorescent Ab-labeled cells or Ab capture beads to white light in the presence of various hematopoietic cell culture media and provide evidence that formulated serum-free media permit rapid light-initiated fluorescent dye degradation in a cell-independent manner. We observed fluorescence signal loss of several dyes, which included fluorescence spillover into adjacent detectors. Finally, photostability of Ab-fluorochrome conjugates in formulated serum-free media is partially restored in the presence of either serum or vitamin C, implicating reactive oxygen species in the observed signal loss. Thus, our data indicate that formulated serum-free media designed to standardize cell culture are not currently optimized for use with fluorochrome-Ab conjugates, and thus, extreme caution should be exercised when using these media in cytometric experiments.

It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 10⁸ ± 1.39 × 10⁸ EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from cultured cells may co-purify RNAs derived from media supplements such as fetal bovine serum (FBS) confounding EV-associated RNA. Defined culture media supplemented with a range of nutrient components provide an alternative to FBS addition and allow EV-collection under full medium conditions avoiding starvation and cell stress during the collection period. However, the potential contribution of serum-free media supplements to EV-RNA contamination has remained elusive and has never been assessed. Here, we report that RNA isolated from EVs harvested from cells under serum-replacement conditions includes miRNA contaminants carried into the sample by defined media components. Subjecting unconditioned, EV-free medium to differential centrifugation followed by reverse transcription quantitative PCR (RT-qPCR) on RNA isolated from the pellet resulted in detection of miRNAs that had been classified as EV-enriched by RNA-seq or RT-qPCR of an isolated EV-fraction. Ribonuclease (RNase-A) and detergent treatment removed most but not all of the contaminating miRNAs. Further analysis of the defined media constituents identified Catalase as a main source of miRNAs co-isolating together with EVs. Hence, miRNA contaminants can be carried into EV-samples even under serum-free harvesting conditions using culture media that are expected to be chemically defined. Formulation of miRNA-free media supplements may provide a solution to collect EVs clean from confounding miRNAs, which however still remains a challenging task. Differential analysis of EVs collected under full medium and supplement-deprived conditions appears to provide a strategy to discriminate confounding and EV-associated RNA. In conclusion, we recommend careful re-evaluation and validation of EV small RNA-seq and RT-qPCR datasets by determining potential medium background.

Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study was to determine if specific MSC culture conditions, MSC expansion in HYPERFlasks® or MSC expansion in a commercially available SFM, would alter MSC proliferation, phenotype or immunomodulatory properties in vitro. MSCs cultured in HYPERFlasks® were similar in phenotype, proliferative capacity and immunomodulatory functions to MSCs grown in standard flasks however MSC yield was markedly increased. HYPERFlasks® therefore provide a viable option to generate greater cell numbers in a streamlined manner. Canine and equine MSCs expanded in SFM displayed similar proliferation, surface phenotype and inhibitory effect on lymphocyte proliferation in vitro. However, MSCs cultured in the absence of FBS secreted significantly less PGE2, and were significantly less able to inhibit IFNγ secretion by activated T-cells. Immunomodulatory functions altered by expansion in SFM were species dependent. Unlike equine MSCs, in canine adipose-derived MSCs, the inhibition of lymphocyte proliferation was not principally modulated by PGE2. The removal of FBS from both canine and equine MSC culture systems resulted in altered immunomodulatory properties in vitro and warrants further investigation prior to moving towards FBS-free culture conditions.

Endothelial cells (ECs), isolated from peripheral blood (PB), bone marrow (BM) and cord blood (CB), are limited in numbers and expansion has had limited success. We used a novel serum-free medium (EndoGo) to evaluate effects on ex vivo expansion of CB-derived ECs.

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T‑cells. In order to overcome a lot‑to‑lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum‑free media [Prime‑XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T‑Cell Expansion Xeno‑Free Medium, Takara Bio™ (TB) and TCM GMP‑Prototype, CellGenix™ (CG)] to the standard CAR T‑cell medium containing FBS (RCF). After 12 days of CD19.CAR T‑cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T‑cells was evaluated using intracellular staining, a chromium release assay and a long‑term co‑culture assay. Expansion and viability did not differ between the CAR T‑cells generated in serum‑free media compared to the standard FBS‑containing medium. The CG CAR T‑cells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNF‑α+ CAR T‑cells than the CAR T‑cells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR T‑cells generated with serum‑free media exhibited a higher cytotoxicity than the CAR T‑cells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR T‑cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte‑activation gene 3 and T‑cell immunoglobulin and mucin‑domain containing‑3. The CAR T‑cells cultured in FF had a higher percentage of central memory CAR T‑cells (40.0 vs. 14.3%, P=0.0470) than the CAR T‑cells cultured with FBS, whereas the CAR T‑cells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR T‑cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum‑free media. Given the advantages of freedom from bovine material and consistent quality, serum‑free media hold promise for the future development of the field of GMP manufacturing of CAR T‑cells.

Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. Here we show that purified Wnt3a protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature, and its solubilization requires a detergent, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), that interferes with stem cell self-renewal. By stabilizing the Wnt3a protein using phospholipids and cholesterol as carriers, we address both problems: Wnt activity remains stable in serum-free media, while non-toxic carriers allow the use of high Wnt concentrations. Stabilized Wnt3a supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased intestine and liver. Moreover, the lipophilicity of Wnt3a protein greatly facilitates its purification. Our findings remove a major obstacle impeding clinical applications of adult stem cells and offer advantages for all cell culture uses of Wnt3a protein.

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

Extracellular vesicles (EVs) play a significant role in cell-cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM) conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun) conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors), some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

Type 2 innate lymphoid cells (ILC2s) were discovered approximately ten years ago and their clinical relevance is gaining greater importance. However, their successful isolation from mammalian tissues and in vitro culture and expansion continues to pose challenges. This is partly due to their scarcity compared to other leukocyte populations, but also because our current knowledge of ILC2 biology is incomplete. This study is focused on ST2+ IL-25Rlo lung resident ILC2s and demonstrate for the first time a methodology allowing mouse type 2 innate lymphoid cells to be cultured, and their numbers expanded in serum-free medium supplemented with Interleukins IL-33, IL-2, IL-7 and TSLP. The procedures described methods to isolate ILC2s and support their growth for up to a week while maintaining their phenotype. During this time, they significantly expand from low to high cell concentrations. Furthermore, for the first time, sub-cultures of primary ILC2 purifications in larger 24- and 6-well plates were undertaken in order to compare their growth in other media. In culture, ILC2s had doubling times of 21 h, a growth rate of 0.032 h-1 and could be sub-cultured in early or late phases of exponential growth. These studies form the basis for expanding ILC2 populations that will facilitate the study and potential applications of these rare cells under defined, serum-free conditions.