FBS-supplemented Medium Research Articles

Calcium chloride declotted human platelet lysate promotes the expansion of mesenchymal stromal cells and allows manufacturing of immunomodulatory active extracellular vesicle products

BackgroundMesenchymal stromal cells (MSCs) exert immunomodulatory effects, primarily through released extracellular vesicles (EVs). For the clinical-grade manufacturing of MSC-EV products culture conditions need to support MSC expansion and allow the manufacturing of potent MSC-EV products. Traditionally, MSCs are expanded in fetal bovine serum-supplemented media. However, according to good manufacturing practice (GMP) guidelines the use of animal sera should be avoided. To this end, human platelet lysate (hPL) has been qualified as an animal serum replacement. Although hPL outcompetes animal sera in promoting MSC expansion, hPL typically contains components of the coagulation system that need to be inhibited or removed to avoid coagulation reactions in the cell culture. Commonly, heparin is utilized as an anticoagulant; however, higher concentrations of heparin can negatively impact MSC viability, and conventional concentrations alone do not sufficiently prevent clot formation in prepared media. MethodsTo circumvent unwanted coagulation processes, this study compared various clotting prevention strategies, including different anticoagulants and calcium chloride (CaCl2)-mediated declotting methods, which in combination with heparin addition was found effective. We evaluated the influence of the differently treated hPLs on the proliferation and phenotype of primary bone marrow-derived MSCs and identified the CaCl2-mediated declotting method as the most effective option. To determine whether CaCl2 declotted hPL allows the manufacturing of immunomodulatory MSC-EV products, EVs were prepared from conditioned media of MSCs expanded with either conventional or CaCl2 declotted hPL. In addition to metric analyses, the immunomodulatory potential of resulting MSC-EV products was assessed in a recently established multi-donor mixed lymphocyte reaction assay. Results and ConclusionsOur findings conclusively show that CaCl2-declotted hPLs support the production of immunomodulatory-active MSC-EV products.

Characteristics of Buccal Fat Adipose-Derived Stem Cells Cultured in Autologous Human Serum- and Fetal Bovine Serum-Supplemented Media: A Comparative Study

Characteristics of Buccal Fat Adipose-Derived Stem Cells Cultured in Autologous Human Serum- and Fetal Bovine Serum-Supplemented Media: A Comparative Study

Longitudinal characterization of TK6 cells sequentially adapted to animal product-free, chemically defined culture medium: considerations for genotoxicity studies.

Introduction: In vitro approaches are an essential tool in screening for toxicity of new chemicals, products and therapeutics. To increase the reproducibility and human relevance of these in vitro assessments, it is advocated to remove animal-derived products such as foetal bovine serum (FBS) from the cell culture system. Currently, FBS is routinely used as a supplement in cell culture medium, but batch-to-batch variability may introduce inconsistency in inter- and intra-lab assessments. Several chemically defined serum replacements (CDSR) have been developed to provide an alternative to FBS, but not every cell line adapts easily and successfully to CDSR-supplemented medium, and the long-term effect on cell characteristics remains uncertain. Aim: The aim of this study was to adapt the TK6 cell line to animal-product free CDSR-supplemented medium and evaluate the long-term effects on cell health, growth, morphology, phenotype, and function. This included a provisional assessment to determine the suitability of the transitioned cell line for standardised genotoxicity testing using the "in vitro mammalian cell micronucleus test" (OECD TG 487). Materials and methods: Gradual adaptation and direct adaptation methodologies were compared by assessing the cell proliferation, size and viability every passage until the cells were fully adapted to animal-free CDSR. The metabolic activity and membrane integrity was assessed every 4-8 passages by PrestoBlue and CytoTox-ONE™ Homogeneous Membrane Integrity Assay respectively. A detailed morphology study by high content imaging was performed and the expression of cell surface markers (CD19 and CD20) was conducted via flow cytometry to assess the potential for phenotypic drift during longer term culture of TK6 in animal-free conditions. Finally, functionality of cells in the OECD TG 487 assay was evaluated. Results: The baseline characteristics of TK6 cells cultured in FBS-supplemented medium were established and variability among passages was used to set up acceptance criteria for CDSR adapted cells. TK6 were adapted to CDSR supplemented medium either via direct or gradual transition reducing from 10% v/v FBS to 0% v/v FBS. The cell growth rate was compromised in the direct adaptation and therefore the gradual adaptation was preferred to investigate the long-term effects of animal-free CDSR on TK6 cells. The new animal cells showed comparable (p > 0.05) viability and cell size as the parent FBS-supplemented cells, with the exception of growth rate. The new animal free cells showed a lag phase double the length of the original cells. Cell morphology (cellular and nuclear area, sphericity) and phenotype (CD19 and CD20 surface markers) were in line (p > 0.05) with the original cells. The new cells cultured in CDSR-supplemented medium performed satisfactory in a pilot OECD TG 487 assay with compounds not requiring metabolic activation. Conclusion: TK6 cells were successfully transitioned to FBS- and animal product-free medium. The new cell cultures were viable and mimicked the characteristics of FBS-cultured cells. The gradual transition methodology utilised in this study can also be applied to other cell lines of interest. Maintaining cells in CDSR-supplemented medium eliminates variability from FBS, which in turn is likely to increase the reproducibility of in vitro experiments. Furthermore, removal of animal derived products from cell culture techniques is likely to increase the human relevance of in vitro methodologies.

Intra-stromal injection of honey-treated keratocytes as a cell-based therapy for experimental corneal laceration.

This study aimed to investigate the potential of honey-supplemented medium (HSM) for expanding corneal keratocytes and its transplantation in a model of corneal laceration. Keratocytes were cultured in 1 % HSM- or 10 % fetal bovine serum (FBS)-supplemented medium for 24 h. The effect of HSM on keratocyte proliferation was evaluated using the MTT assay. The relative expression of Lum, Kera, and ALDH3A1, known markers of native keratocytes, was quantified by real-time PCR. The safety and efficacy of HSM-treated keratocyte intrastromal injection in a rabbit model of corneal laceration were also evaluated. The MTT assay showed that HSM treatment didnot significantly affect cell viability compared to FBS-supplemented medium (84.71±2.38 vs. 100.08±10.92, respectively; p=0.076). Moreover, HSM-treated keratocytes had significantly increased expression of Lum, Kera,andALDH3A1 compared to cells treated with FBS, while theexpression of the proliferation biomarker Thy-1 did notsignificantly differ between the two treatments. Intrastromal injection of HSM-treated keratocytes in the laceration animal model was safe and uneventful, resulting in less stromal inflammation and neovascularization, and consequently, better final architecture with less residual haze compared to the group injected with FBS-treated keratocytes. These findings suggest that honey is a suitable supplement for keratocyte treatment and corneal cell therapy. The use of HSM may have potential applications in the treatment of corneal injuries and diseases.

Differential Development of Umbilical Cord-Derived Mesenchymal Stem Cells During Long-Term Maintenance in Fetal Bovine Serum-Supplemented Medium and Xeno- and Serum-Free Culture Medium

Umbilical cord-derived mesenchymal stem/stromal cells (UC-MSCs) are believed to have potential for the treatment of various diseases; thus, many scientists have investigated the molecular mechanisms underlying the function of UC-MSCs and, for example, the appropriate media for large-scale UC-MSC expansion to prepare cells for real-world application. In this study, we investigated the cellular morphology, proliferation capacity, surface markers, cellular senescence signals, clonogenic potential, trilineage differentiation capacity, and secreted factors of human primary UC-MSCs in long-term culture from passage 2 (P2) to passage 10 (P10) with either conventional fetal bovine serum (FBS)-supplemented medium or commercial xeno- and serum-free medium (StemMACS™). We found that the cells cultured in both media had similar morphology and marker expression. However, the proliferation kinetics as measured by the cell population doubling time differed in a passage (P2-P10)-dependent manner between the cells cultured in the two media; sustainable growth was observed in cells maintained in xeno- and serum-free medium. Moreover, significant differences in cellular senescence signals were observed, with more aging cells in the cell population cultured in FBS-containing medium. Colony numbers and the day that the first colony appeared were similar; however, UC-MSC colony sizes were smaller when cultured in FBS-containing medium. In addition, the multidifferentiation potential of UC-MSCs cultured in xeno- and serum-free StemMACS medium was maintained during long-term culture, but this potential was lost for adipogenic differentiation at P9. Moreover, secreted epidermal growth factor and vascular endothelial growth factor (VEGF)-A were detected in the conditioned media from UC-MSCs, whereas platelet-derived growth factor was not. Similar expression of these factors was observed in conditioned media of UC-MSCs cultured in StemMACS, but the VEGF level was higher in young UC-MSCs (P6) than in aged UC-MSCs cultured in FBS-supplemented Dulbecco's modified Eagle's medium/F12. Thus, StemMACS is better for UC-MSC expansion than conventional FBS-supplemented culture medium, especially when culturing UC-MSCs for real-world applications.

A critical appraisal of humanized alternatives to fetal bovine serum for clinical applications of umbilical cord derived mesenchymal stromal cells.

The study is aimed to verify the possibility of using humanized alternatives to fetal bovine serum (FBS) such as umbilical cord blood plasma (CBP) and AB+ plasma to support the long-term growth of mesenchymal stromal cells (MSCs) derived from the umbilical cord. We hypothesized that umbilical CBP would be a potential substitute to FBS, especially for small scale autologous clinical transplantations. The MSCs were cultured for six consecutive passages to evaluate xeno-free media's ability to support long-term growth. Cell proliferation rates, colony-forming-unit (CFU) efficiency and population doublings of expanded MSCs, were investigated. Ex vivo expanded MSCs were further characterized using flow cytometry and quantitative PCR. The impact of cryopreservation and composition of cryomedium on phenotype, viability of MSC was also assessed. Our results on cell proliferation, colony-forming unit efficiency suggested that the expansion of the cells was successfully carried out in media supplemented with humanized alternatives. MSCs showed lower CFU counts in FBS (~ 25) than humanized alternatives (~ 35). The gene expression analysis revealed that transcripts showed significant differential expression by two to three folds in the FBS group compared with MSCs grown in medium with humanized alternatives (p < 0.05). In addition, MSCs grown in a medium with FBS had more osteogenic activity, a signatureof unwanted differentiation. The majority of ex vivo expanded MSCs at early and late passages expressed CD44+, CD73+, CD105+, CD90+, and CD166+ in all the experimental groups tested (~ 90%). In contrast to the other MSC surface markers, expression levels of STRO-1+ (~ 21-10%) and TNAP+ (~ 29-11%) decreased with the increase in passage number for MSCs cultured in a FBS-supplemented medium (p < 0.05). Our results established that CBP supported culture of umbilical cord tissue-derived MSCs and is a safer Xeno free replacement to FBS. The use of CBP also enables the storage of umbilical cord tissue derived MSCs in patient-specific conditions to minimize adverse events if cells are delivered directly to the patient.

An assessment of serum-dependent impacts on intracellular accumulation and genomic response of per- and polyfluoroalkyl substances in a placental trophoblast model.

Per- and polyfluoroalkyl substances (PFAS), a class of environmental contaminants, have been detected in human placenta and cord blood. The mechanisms driving PFAS-induced effects on the placenta and adverse pregnancy outcomes are not well understood. This study investigated the impact of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and a replacement PFAS known as hexafluoropropylene oxide dimer acid (HFPO-DA, tradename GenX) on placental trophoblasts in vitro. Several key factors were addressed. First, PFAS levels in cell culture reagents at baseline were quantified. Second, the role of supplemental media serum in intracellular accumulation of PFAS in a human trophoblast (JEG3) cell line was established. Finally, the impact of PFAS on the expression of 96 genes involved in proper placental function in JEG3 cells was evaluated. The results revealed that serum-free media (SFM) contained no detectable PFAS. In contrast, fetal bovine serum-supplemented media (SSM) contained PFNA, PFUdA, PFTrDA, and 6:2 FTS, but these PFAS were not detected internally in cells. Intracellular accumulation following 24 hr treatments was significantly higher when cultured in SFM compared to SSM for PFOS and PFOA, but not HFPO-DA. Treatment with PFAS was associated with gene expression changes (n = 32) in pathways vital to placental function, including viability, syncytialization, inflammation, transport, and invasion/mesenchymal transition. Among the most robust PFAS-associated changes were those observed in the known apoptosis-related genes, BAD and BAX. These results suggest a complex relationship between PFAS, in vitro culture conditions, and altered expression of key genes necessary for proper placentation.

Regulatory-compliant conditions during cell product manufacturing enhance in vitro immunomodulatory properties of infrapatellar fat pad-derived mesenchymal stem/stromal cells

Background aimsMesenchymal stem/stromal cell (MSC)-based therapies have gained attention as potential alternatives for multiple musculoskeletal indications based on their trophic and immunomodulatory properties. The infrapatellar fat pad (IFP) serves as a reservoir of MSCs, which play crucial roles modulating inflammatory and fibrotic events at the IFP and its neighboring tissue, the synovium. In an effort to comply with the existing regulatory framework regarding cell-based product manufacturing, we interrogated the in vitro immunomodulatory capacity of human-derived IFP-MSCs processed under different conditions, including a regulatory-compliant protocol, in addition to their response to the inflammatory and fibrotic environments often present in joint disease. MethodsImmunophenotype, telomere length, transcriptional and secretory immunomodulatory profiles and functional immunopotency assay were assessed in IFP-MSCs expanded in regular fetal bovine serum (FBS)-supplemented medium and side-by-side compared with same-donor cells processed with two media alternatives (i.e., regulatory-compliant pooled human platelet lysate [hPL] and a chemically reinforced/serum-reduced [Ch-R] formulation). Finally, to assess the effects of such formulations on the ability of the cells to respond to pro-inflammatory and pro-fibrotic conditions, all three groups were stimulated ex vivo (i.e., cell priming) with a cocktail containing TNFα, IFNγ and connective tissue growth factor (tumor-initiating cells) and compared with non-induced cohorts assessing the same outcomes. ResultsNon-induced and primed IFP-MSCs expanded in either hPL or Ch-R showed distinct morphology in vitro, similar telomere dynamics and distinct phenotypical and molecular profiles when compared with cohorts grown in FBS. Gene expression of IL-8, CD10 and granulocyte colony-stimulating factor was highly enriched in similarly processed IFP-MSCs. Cell surface markers related to the immunomodulatory capacity, including CD146 and CD10, were highly expressed, and secretion of immunomodulatory and pro-angiogenic factors was significantly enhanced with both hPL and Ch-R formulations. Upon priming, the immunomodulatory phenotype was enhanced, resulting in further increase in CD146 and CD10, significant CXCR4 presence and reduction in TLR3. Similarly, transcriptional and secretory profiles were enriched and more pronounced in IFP-MSCs expanded in either hPL or Ch-R, suggesting a synergistic effect between these formulations and inflammatory/fibrotic priming conditions. Collectively, increased indoleamine-2,3-dioxygenase activity and prostaglandin E2 secretion for hPL- and Ch-R-expanded IFP-MSCs were functionally reflected by their robust T-cell proliferation suppression capacity in vitro compared with IFP-MSCs expanded in FBS, even after priming. ConclusionsCompared with processing using an FBS-supplemented medium, processing IFP-MSCs with either hPL or Ch-R similarly enhances their immunomodulatory properties, which are further increased after exposure to an inflammatory/fibrotic priming environment. This evidence supports the adoption of regulatory-compliant practices during the manufacturing of a cell-based product based on IFP-MSCs and anticipates a further enhanced response once the cells face the pathological environment after intra-articular administration. Mechanistically, the resulting functionally enhanced cell-based product has potential utilization as a novel, minimally invasive cell therapy for joint disease through modulation of local immune and inflammatory events.

Human serum enhances the proliferative capacity and immunomodulatory property of MSCs derived from human placenta and umbilical cord

BackgroundMesenchymal stromal cells (MSCs) are considered potential candidates that hold great promise in the treatment of immune-related diseases. For therapeutic applications, it is necessary to isolate and expand MSCs with procedures complying with good manufacturing practice (GMP). Recent studies reported the use of human serum (HS) instead of fetal bovine serum (FBS) for the expansion of bone marrow-derived MSCs. Nevertheless, there are only limited data on HS as an alternative to FBS for the isolation and expansion of umbilical (UC-MSCs) and placenta-derived MSCs (PL-MSCs). In this study, we evaluate the effect of HS compared to FBS on the proliferative and immunosuppressive capacities of these MSCs.MethodsPL-MSCs and UC-MSCs were isolated and cultured in HS- or FBS-supplemented media. The MSC characteristics, including morphology, immunophenotype, and differentiation ability, were verified. The proliferative and immunosuppressive capacities were also examined. In addition, the proliferative-enhancing factors in both sera were explored using proteomic analysis.ResultsPL-MSCs and UC-MSCs proliferated faster in HS-supplemented medium than in equivalent levels of FBS-supplemented medium. Adipogenic and osteogenic differentiations occurred at nearly identical levels in HS- and FBS-supplemented media. Interestingly, MSCs cultured in HS-supplemented medium had a similar immunosuppressive effect as MSCs cultured in FBS-supplemented medium. Proteomic analysis revealed that Con-A binding glycoproteins with a molecular weight > 100 kDa in FBS could significantly enhance MSC proliferation. In contrast, the proliferative enhancing factors in HS were found in the Con-A non-binding fraction and WGA binding fraction with a molecular weight > 100 kDa.ConclusionsTaken together, our results suggest applications for the use of HS instead of FBS for the isolation and expansion of PL-MSCs and UC-MSCs for cell therapy in the future. Furthermore, this study identifies factors in HS that are responsible for its proliferative and immunosuppressive effects and might thus lead to the establishment of GMPs for the therapeutic use of MSCs.

Vitrification of immature oocytes of goats in Bangladesh

Cryopreservation of oocytes and embryos by vitrification can have advantages in assisted reproductive technologies (ARTs) in mammals. The aim of this study was to establish an effective vitrification procedure and cryodevice for goat’s oocytes in Bangladesh. Cumulus oocyte complexes (COCs) were collected from ovaries from slaughterhouse. COCs with more than 3 layers of cumulus cells were selected. COCs were vitrified by two-step procedure using 7.5% and 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA), loaded on Cryotop or French mini-straw, then directly plunged into liquid nitrogen (LN2). Then the COCs containing Cryotop or French mini-straws were warmed in 0.25 M sucrose and 20% FBS-supplemented tissue culture medium (TCM) 199 followed by in vitro culture in 50 μl droplets of bicarbonate-buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 h at 39°C with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. The in vitro maturation rate of goat’s oocytes after vitrification and warming was 39.3 ± 6.8%, 31.3 ± 9.4%, 61.6 ± 14.2% when using Cryotop (cryodevice), French mini-straws and without vitrification (control), respectively. Maturation rate was significantly higher (P&lt;0.05) without vitrification. It is suggested that both Cryotop and French mini-straw are efficient cryodevices for vitrification of goat’s oocytes and further investigation is required to optimize the protocol for vitrification and warming procedure for the satisfactory survival of goat’s oocytes.&#x0D; The Bangladesh Veterinarian (2018) 35(1&amp;2): 7-12

162 Developmental competence of bovine oocytes matured in defined medium

Routinely, bovine oocytes are matured in medium supplemented with 10% fetal bovine serum (FBS). However, due to the undefined nature of FBS and biosafety concerns, a standardized protein supplementation alternative is needed. We hypothesised that oocytes matured in defined medium supplemented with 5% serum substitute (DM) would have higher developmental competence than those matured in FBS-supplemented medium (Control). In 6 replicates, in vitro-produced embryos were produced during the summer season by aspirating cumulus-oocyte complexes from 2- to 8-mm follicles of abattoir ovaries (LAB-COC), or aspirated from FSH-stimulated Holstein and Jersey donors (OPU-COC, n=20; 53 sessions). The LAB-COC (n=1307) and OPU-COC (n=679) were matured for 23h in control medium or DM supplemented with pyruvate and gonadotropins at 6% CO2 in air, fertilized with semen from 1 of 3 or 15 bulls, respectively, and cultured in SCF2 at 38.5°C, 6% CO2, 5% O2 and 89% N2. On Day 7.5 post-fertilization, blastocyst rates were evaluated, and embryos (n=4/replicate) from each group of LAB-COC were stained with 1µg mL−1 Nile Red for lipid quantification, 5 µM Cell Rox® Green (Thermo Fisher Scientific, Waltham, MA, USA) for reactive oxygen species (ROS) assessment and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA) adjusted per cell number. Data were analysed by one-way ANOVA using SAS software (SAS Institute Inc., Cary, NC, USA). Results (Table 1) indicate similar cleavage and blastocyst rates and mitochondrial polarity for embryos from LAB-COC in Control v. DM (P&amp;gt;0.05). However, ROS and lipid content were higher in Control than in DM (P&amp;lt;0.05). For OPU-COC, DM resulted in similar cleavage and blastocyst rates and embryos produced per OPU session compared with Control (P&amp;gt;0.05). Results suggest that maturing oocytes (LAB and OPU) in DM maintain developmental competence, and the resulting embryos had lower lipid content and ROS than those matured in Control medium. Cryopreservation studies and evaluation of pregnancy rates are ongoing. Table 1.Developmental competence of oocytes matured in control or defined medium with 5% serum substitute (DM)

Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions

Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches

BackgroundCanine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL).MethodshMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3′-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL.ResultsHS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives.ConclusionsOur findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.

Xeno-free pre-vascularized spheroids for therapeutic applications

Spheroid culture has gained increasing popularity, arising as a promising tool for regenerative medicine applications. Importantly, spheroids may present advantages over single-cell suspensions in cell-based therapies (CT). Unfortunately, most growth media used for spheroid culture contain animal origin-components, such as fetal bovine serum (FBS). The presence of FBS compromises the safety of CT and presents economic and ethical constraints. SCC (supplement for cell culture) is a novel xeno-free (XF) industrial cell culture supplement, derived from well-controlled pooled human plasma and processed under good manufacturing practice rules. Here, we developed a XF SCC-based formulation for 2D-culture of outgrowth endothelial cells (OEC), and then used it for generating co-culture spheroids of OEC and mesenchymal stem cells (MSC). XF MSC-OEC spheroids were characterized in detail and compared to spheroids cultured in FBS-supplemented medium. XF spheroids presented comparable integrity, size and morphology as the reference culture. The use of both media resulted in spheroids with similar structure, abundant extracellular matrix deposition and specific patterns of OEC distribution and organization. Notably, XF spheroids presented significantly enhanced angiogenic potential, both in vitro (fibrin sprouting assay) and in vivo (CAM assay). These findings are particularly promising in the context of potential therapeutic applications.

Ex vivo Caprine Model to Study Virulence Factors in Keratitis.

Purpose:To develop an infectious keratitis model using caprine (goat) corneas and to investigate the expression of virulence factors during infection.Methods:Goat eyes were surface-sterilized and dissected, and the corneas were placed on an agarose-gelatin solid support (0.5% in phosphate-buffered saline) in a 12-well culture plate containing 10% fetal bovine serum-supplemented culture medium for 3 weeks. Cell viability tests (trypan blue and MTT) were performed on the cultured corneas. Corneas were infected with Pseudomonas aeruginosa and Fusarium solani separately. Infection progression was observed via histological analysis and hematoxylin and eosin (H-E) staining. For Pseudomonas-infected corneas, expression of eight virulence genes (exoS, exoT, exoY, alpR, prpL, lasA, lasB, and algD) was determined via quantitative real-time PCR (qRT-PCR) at 48-h and 72-h time-points. For Fusarium-infected corneas, expression of five proteases (C7Z0E6, C7ZFW9, C7Z7U2, C7ZNV5, and C7YY94) was quantified via qRT-PCR at 2, 4, and 8 days after infection. Protease from infected corneas was detected via gelatin zymography.Results:Goat corneas with a viable epithelium could be maintained for 15 days. Pseudomonas infection progressed rapidly, and complete corneal degradation was observed on day 4 after infection. Fusarium infection progressed more slowly. Histological analysis and H-E staining of Fusarium-infected cornea revealed mycelia penetrating all layers of the cornea. qRT-PCR revealed expression of all eight virulence factors, and statistically significant difference in expression of prpL and alpR in Pseudomonas-infected corneas. Expression of C7ZNV5 was highest in Fusarium-infected corneas.Conclusion:Goat corneas can be used to evaluate the expression of virulence factors involved in Pseudomonas and Fusarium infection.

7-ketocholesterol and 27-hydroxycholesterol decreased doxorubicin sensitivity in breast cancer cells: estrogenic activity and mTOR pathway.

Hypercholesterolemia is one of the risk factors for poor outcome in breast cancer therapy. To elucidate the influence of the main circulating oxysterols, cholesterol oxidation products, on the cell-killing effect of doxorubicin, cells were exposed to oxysterols at a subtoxic concentration. When cells were exposed to oxysterols in fetal bovine serum-supplemented medium, 7-ketocholesterol (7-KC), but not 27-hydroxycholesterol (27-HC), decreased the cytotoxicity of doxorubicin in MCF-7 (high estrogen receptor (ER)α/ERβ ratio) cells and the decreased cytotoxicity was restored by the P-glycoprotein inhibitor verapamil. 7-KC stimulated the efflux function of P-glycoprotein and reduced intracellular doxorubicin accumulation in MCF-7 but not in ERα(-) MDA-MB-231 and the resistant MCF-7/ADR cells. In MCF-7 cells, 7-KC increased the mRNA and protein levels of P-glycoprotein. The 7-KC-suppressed doxorubicin accumulation was restored by the fluvestrant and ERα knockdown. In a yeast reporter assay, the ERα activation by 7-KC was more potent than 27-HC. 7-KC, but not 27-HC, stimulated the expression of an ER target, Trefoil factor 1 in MCF-7 cells. When charcoal-stripped fetal bovine serum was used, both 7-KC and 27-HC induced Trefoil factor 1 expression and reduced doxorubicin accumulation in MCF-7 cells. 7-KC-reduced doxorubicin accumulation could be reversed by inhibitors of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR). These findings demonstrate that 7-KC decreases the cytotoxicity of doxorubicin through the up-regulation of P-glycoprotein in an ERα- and mTOR-dependent pathway. The 7-KC- and 27-HC-elicited estrogenic effects are crucial in the P-glycoprotein induction in breast cancer cells.

Dual contribution of surface charge and protein-binding affinity to the cytotoxicity of polystyrene nanoparticles in nonphagocytic A549 cells and phagocytic THP-1 cells

ABSTRACTKnowledge that links the physicochemical properties of nanoparticles (NP) to their toxicity is key to evaluating and understanding mechanisms underlying toxicity and developing appropriate testing methods for NP; however, this is currently limited since only a small set of NP have been used, with typically poor control of their physical properties. In this study, eight types of polystyrene NP (PLNP) were synthesized with different functional groups, but all based on an identical core. In vitro cell-based assays were performed to determine the influence of changes in physicochemical properties, such as charge, hydrodynamic size, and protein binding potential, in relation to NP-mediated toxicity. The PLNP were incubated with nonphagocytic A549 cells or phagocytic differentiated THP-1 cells for 4 h with/without fetal bovine serum (FBS), followed by incubation for 20 h in FBS-supplemented medium with/without a washing step, to assess cell-type specificity and impact of protein corona formation. The effect of surface charge on cytotoxicity differed between A549 cells and THP-1 cells. In nonphagocytic A549 cells, the zeta potential of PLNP exhibited a negative correlation with cytotoxicity, partly due to the level of coronated protein that might affect cellular uptake. In phagocytic THP-1 cells, the zeta potential of PLNP showed a positive correlation with cytotoxicity but coronated protein levels displayed no marked association with cytotoxicity, owing to the professional uptake efficacy of phagocytic cells. The consistency of our data with THP-1 cells with the surface charge paradigm in nanotoxicology suggests that phagocytic cells are the predominant targets for lung inflammatory reactions induced by PLNP.

Notochordal cell conditioned medium (NCCM) regenerates end-stage human osteoarthritic articular chondrocytes and promotes a healthy phenotype.

BackgroundNotochordal cell conditioned medium (NCCM) derived from non-chondrodystrophic dogs has pro-anabolic and anti-catabolic effects upon nucleus pulposus (NP) cells. Here, for the first time, we assessed the ability of NCCM to influence the production of extracellular matrix and inflammatory proteins by healthy and osteoarthritic human chondrocytes within engineered cartilage tissues. We hypothesized that, similar to its action on NP cells, NCCM exerts metabolic and anti-catabolic effects on human articular chondrocytes and has the potential to significantly counteract inflammatory mediators.MethodsChondrocytes from nine non-osteoarthritic patients and from six osteoarthritic (OA) donors at the time of total knee arthroplasty were chondro-differentiated in pellets for 2 weeks. Non-OA pellets were exposed for 72 hours to IL-1β/TNF-α and then cultured up to 14 days in 2 % FBS-supplemented NCCM or 2 % FBS-supplemented medium (control (ctr)). OA pellets were cultured in NCCM or ctr medium without pro-inflammatory treatment. Tissues after each culture phase were analyzed biochemically (GAG/DNA), (immuno-) histologically (collagen I, II and GAG) and by Western blotting. Supernatants were analyzed by ELISA.ResultsResponse to NCCM was age and disease dependent with healthy chondrocyte pellets (from donors >55 years of age) recovering their glycosaminoglycan (GAG) contents to baseline levels only with NCCM. OA pellets treated with NCCM significantly increased GAG content (1.8-fold) and levels of hyaluronic acid link protein (HAPLN), fibromodulin and SOX-9. The catabolic proteins (matrix metalloproteinase (MMP)-3 and MMP-13) and pro-inflammatory enzyme levels (cyclooxygenase-2 (COX-2)) were markedly reduced and there was significantly reduced secretion of pro-inflammatory chemokines (IL-6 and IL-8).ConclusionsNCCM restores cartilage matrix production of end-stage human OA chondrocytes towards a healthy phenotype and suppresses the production of inflammatory mediators. Harnessing the necessary and sufficient factors within NCCM that confers chondroprotection and regenerative effects could lead to a minimally invasive agent for treatment of degenerative and inflammatory joint diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1026-x) contains supplementary material, which is available to authorized users.

Use of human serum for human corneal endothelial cell culture

Background/aimsTo study human corneal endothelial cells (HCECs) cultured in vitro with human serum (HS) supplemented media (HS-SM) compared with HCEC cultured in fetal bovine serum (FBS) supplemented media (FBS-SM).MethodsOne cornea...

Establishment of a xeno-free culture system that preserves the characteristics of placenta mesenchymal stem cells.

Although stem cells are promising candidates for cell replacement therapies, the vast majority are derived using animal sera, which has risk of being contaminated by animal viruses or toxins. To overcome these potential problems, we initially established multiple lines of stem cells from first-trimester human placenta (fPMSC), which were cultivated using human follicular fluid (hFF) instead of fetal bovine serum (FBS). FF provides a very important microenvironment for the development of oocytes. No differences were found in the general morphology, growth rate, karyotype, gene and surface expressions between placental MSCs cultured in 5% hFF-supplemented medium (fPMSC-X) or 10% FBS-supplemented medium (fPMSC). Differentiation experiments confirmed similar levels of potency in cells grown in either condition. Since hFF preserved the unique features of the stem cells and is free from potential pathogens, it should be considered as the main culture medium supplement for the propagation of human stem cells for clinical applications.