Serum-free Medium Research Articles

Cryopreservation of primary neonatal rat oligodendrocytes and recapitulation of in vitro oligodendrocyte characteristics

IntroductionIn vitro, primary rat oligodendrocytes (OLs) are widely used for research on OL development, physiology, and pathophysiology in demyelinating diseases such as multiple sclerosis. Primary culture methods for OLs from rats have been developed and improved over time, but there are still multiple aspects in which efficiency can be boosted.MethodsTo make use of excess oligodendrocyte progenitor cells (OPCs) from primary cultures, a cryopreservation process utilizing a commercially available serum-free cryopreservation medium was established to passage and freeze OPCs at −80°C for later use.ResultsCryopreserved OPCs stored for up to 6 months were viable, and retained their OL lineage purity of ~98%. While OPCs cryopreserved for 3–6 months showed a decrease in cell density after two days of proliferation, ~17% of cryopreserved OPCs maintained the potential for proliferation comparable to control OPCs that had not frozen. After induction of differentiation for four days, ~43% of both control and cryopreserved OPCs differentiated into mature OLs, and when differentiation was induced on aligned nanofibers mimicking axonal structure, myelin sheath-like structures indicative of in vitro myelination was observed in all experimental groups.ConclusionThe validation of cryopreserved primary OLs as a functionally robust in vitro model can help improve the efficiency of primary OL culture, expand its applications, and reduce the inevitable sacrifice of animals.

Expression of PAX6 and Keratocyte-Characteristic Markers in Human Limbal Stromal Cells of Congenital Aniridia and Healthy Subjects, In Vitro

Purpose Our aim was to examine the expression of PAX6 and keratocyte-specific markers in human limbal stromal cells (LSCs) in congenital aniridia (AN) and in healthy corneas, in vitro. Methods Primary human LSCs were extracted from individuals with aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8). The cells were cultured in either normal-glucose serum-containing cell culture medium (NGSC-medium) or low-glucose serum-free cell culture medium (LGSF-medium). Analysis of PAX6 and keratocyte-specific markers was conducted using qPCR and Western blotting. The keratocyte-specific markers included Collagen I (COL1A1), Collagen III (COL3A1), Collagen V (COL5A1), α-smooth muscle actin (ACTA2), Aldehyde Dehydrogenase 3 Family, Member A1 (ALDH3A1), Keratocan (KER), Lumican (LUM), and CD34. Results PAX6 mRNA expression exhibited a significant decrease in AN-LSCs compared to LSCs in both NGSC- and LGSF-medium (p = 0.04; p = 0.014). There was a marked reduction in COL5A1 mRNA expression (p = 0.011), accompanied by notably higher ALDH3A1 and KER mRNA levels (p = 0.007; p = 0.013) in AN-LSCs compared to LSCs when using NGSC-medium. In LGSF-medium, AN-LSCs showed a significant increase in COL1A1 and COL5A1 mRNA expression compared to LSCs (p = 0.048; p = 0.002). Moreover, COL1A1 and α-SMA protein expression were significantly elevated in AN-LSCs compared to LSCs in LGSF-medium (p = 0.048, p = 0.008). Conclusions Our investigation affirms the altered expression of PAX6 and keratocyte-specific markers in AN-LSCs relative to healthy controls. Both NGSC- and LGSF-medium exerted distinct effects on both LSCs and AN-LSCs. The observed variations in PAX6 and keratocyte-specific marker expression in AN-LSCs may play a pivotal role in the development and progression of aniridia-associated keratopathy.

An overview of recent progress in cultured meat: focusing on technology, quality properties, safety, industrialization and public acceptance.

Cultured meat technology represents an innovative food production approach that enables the large-scale cultivation of animal cells to obtain muscle, fat, and other tissues, which are then processed into meat products. Compared to traditional meat production methods, cell-cultured meat may significantly reduce energy consumption by 7% to 45%, greenhouse gas emissions by 78% to 96%, land use by 99%, and water use by 82% to 96%. This technology offers several advantages, including a shorter production cycle and enhanced environmental sustainability, resource efficiency, and overall sustainability. However, numerous technical challenges remain, such as the development of serum-free media, maintenance of seed cell functionality, large-scale cell culture techniques, three-dimensional (3D) culture methods, and innovations in scaffold materials. These hurdles must be addressed to achieve low-cost, high-efficiency industrial production in the cultivated meat sector. Furthermore, as a supplement or substitute for traditional meat, cultured meat products must possess similar sensory characteristics and nutritional value, ensure high food safety standards, and maintain low production costs to enhance market competitiveness. Therefore, achieving the industrialization of cultured meat necessitates careful consideration of several additional challenges related to sensory attributes, nutritional quality, food safety, and consumer acceptance. This review systematically examines these aspects to provide a theoretical and practical foundation for the sustainable biomanufacturing of cultured meat.

Recombinant Porcine FGF1 Promotes Muscle Stem Cell Proliferation and Mitochondrial Function for Cultured Meat Production.

Cultured meat is an emerging technology with the potential to meet future protein demands while addressing the challenges associated with traditional livestock farming. The production of cultured meat requires efficient, animal component-free in vitro systems for muscle stem cell (MuSC) expansion. Fibroblast growth factor 1 (FGF1) is a critical growth factor that regulates the MuSC function. In this study, we established an efficient method for the soluble expression and purification of recombinant porcine FGF1 (rpFGF1) in Escherichia coli, achieving a yield of 48 mg of purified protein per liter of culture. Treatment with rpFGF1 significantly enhanced the proliferation of porcine MuSC under serum-free conditions. Furthermore, rpFGF1 induced mitochondrial fission and mitophagy by activating the ERK-dependent phosphorylation of DRP1 at Ser616, resulting in improved mitochondrial function and proliferation capacity in porcine MuSC. These findings highlight the potential of rpFGF1 in the development of serum-free media for scalable and sustainable cultured meat production.

Alginate-Based Hydrogels with Amniotic Membrane Stem Cells for Wound Dressing Application.

Chronic wounds are a common clinical problem that necessitate the exploration of novel regenerative therapies. We report a method to investigate the in vitro wound healing capacity of an innovative biomaterial, which is based on amniotic membrane-derived stem cells (AMSCs) embedded in an alginate hydrogel matrix. The aim of this study was to prepare an sodium alginate-based hydrogel, cross-linked calcium chloride (CaCl2) with the active ingredient AMSC (AMSC/Alg-H) and to evaluate its in vitro effectiveness for wound closure. This hydrogel preparation involved combining sterile solutions of AMSC, sodium alginate, and CaCl2, followed by rinsing with serum-free media. The cells were cultured in different 6-well plates, namely sodium alginate, calcium chloride, AMSC, Alg-H, and AMSC/Alg-H, in complete medium with 10% FBS. The hydrogel was successfully formulated, as confirmed by characterization techniques including Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, Differential Scanning Calorimetry (DSC), Cytotoxicity Studies, TGF-β1 Level Measurement by ELISA, and Cell Scratch Wound Assay. Cryo-EM characterization of the Alg-H preparation successfully demonstrated the encapsulation of MSCs. FTIR and DSC analyses indicate that crosslinking transpires in Alg-H encapsulating AMSC. The AMSC/Alg-H preparation showed no significant difference in toxicity compared to HaCaT cells (p < 0.05), indicating it was not toxic to HaCaT cells. Furthermore, in the scratch wound assay test at 24hours, the AMSC/Alg-H preparation achieved 100% wound closure, outperforming both AMSC and Alg-H alone. In vitro assessment revealed that AMSC/Alg-H significantly enhanced key wound healing processes, including cell proliferation and migration, compared to Alg-H. Our study demonstrated the promising potential of AMSC/Alg-H as an enhanced regenerative therapy for in vitro wound healing. AMSC/Alg-H was able to maintain the viability of AMSCs and facilitate the formation of tissue-like structures.

The culture of equine adipose tissue-derived mesenchymal cells in serum-free media

Mesenchymal stem/stromal cells (MSCs) isolated from equine adipose tissue (AT) represent a promising material for the creation of bioveterinary products for the prevention and treatment of many diseases. The production of these cells for clinical use requires improved serum-free culture conditions. The microenvironment can influence the properties of MSCs. It is believed that the requirements for culture conditions without animal blood serum are species specific. The purpose of this study was to evaluate the commercially available serum-free media (SFM) MesenCult (STEMCELL Technologies, USA), created for human MSCs, for the cultivation of equine MSC(AT). One part of the cells was propagated for 10 passages in the standard DMEM medium with a low glucose content (1 g/l) and 10% fetal bovine serum (FBS), and the second in SFM. The results show that the propagation of equine MSCs in MesenCult serum free, intended for the cultivation of human MSCs, is possible, since the cells adapt well to it and retain properties characteristic of cells that are cultured in DMEM with FBS: morphology, growth rate, doubling time and mitotic index, clone-forming abilities, diploid set of chromosomes, a large number of cells with the CD90 phenotype (90.8%) and low with the CD31 (0.8%), CD34 (0.9%) phenotype, as well as the potency for induction of differentiation into adipo-, osteo- and chondrogenic directions. Equine MSC(AT) showed stable characteristics after being cultured for 10 passages in SFM, providing a promising basis for their further use. Our results demonstrate that MesenCult media may be an alternative for serum-free culture of equine MSC(AT) for expansion in preclinical studies.

The Topology of Poly(2-methyl-2-oxazine) Shells on Nanoparticles Determines Their Interaction with Serum and Uptake by Immune Cells.

Cyclic poly(2-methyl-2-oxazine) (c-PMOZI) brush shells on Au nanoparticles (NPs) exhibit enhanced stealth properties toward serum and different cell lines compared to their linear PMOZI (l-PMOZI) counterparts. While selectively recruiting immunoglobulins, c-PMOZI shells reduce overall human serum (HS) protein binding and alter the processing of complement factor 3 (C3) compared to chemically identical linear shells. Polymer cyclization significantly decreases NP uptake by nonphagocytic cells and macrophages in both complement-deficient fetal bovine serum (FBS) and complement-expressing HS, indicating ineffective functional opsonization. Even in serum-free media, c-PMOZI-coated NPs show reduced internalization by macrophages compared to l-PMOZI-coated NPs, suggesting lower opsonin-independent cell surface affinity. This study demonstrates that cyclic PMOZI suppresses interactions of NPs with proteins and cells, highlighting how control over chain topology expands the polymer chemistry toolbox for modulating the behavior of core-shell NPs within physiological environments.

Conditioned serum-free culture medium accomplishes adhesion and proliferation of bovine myogenic cells on uncoated dishes

Conditioned serum-free culture medium accomplishes adhesion and proliferation of bovine myogenic cells on uncoated dishes

In vitro model of retinoblastoma derived tumor and stromal cells for tumor microenvironment (TME) studies

Retinoblastoma (RB) is an intraocular tumor arising from retinal cone progenitor cells affecting young children. In the last couple of years, RB treatment evolved towards eye preserving therapies. Therefore, investigating intratumoral differences and the RB tumor microenvironment (TME), regulating tumorigenesis and metastasis, is crucial. How RB cells and their TME are involved in tumor development needs to be elucidated using in vitro models including RB derived stromal cells. In the study presented, we established primary RB derived tumor and stromal cell cultures and compared them by RNAseq analysis to identify their gene expression signatures. RB tumor cells cultivated in serum containing medium were more differentiated compared to RB tumor cells grown in serum-free medium displaying a stem cell like phenotype. In addition, we identified differentially expressed genes for RB tumor and stromal derived cells. Furthermore, we immortalized cells of a RB1 mutated, MYCN amplified and trefoil factor family peptid 1 (TFF1) positive RB tumor and RB derived non-tumor stromal tissue. We characterized both immortalized cell lines using a human oncology proteome array, immunofluorescence staining of different markers and in vitro cell growth analyses. Tumor formation of the immortalized RB tumor cell line was investigated in a chicken chorioallantoic membrane (CAM) model. Our studies revealed that the RB stromal derived cell line comprises tumor associated macrophages (TAMs), glia and cancer associated fibroblasts (CAFs), we were able to successfully separate via magnetic cell separation (MACS). For co-cultivation studies, we established a 3D spheroid model with RB tumor and RB derived stromal cells. In summary, we established an in vitro model system to investigate the interaction of RB tumor cells with their TME. Our findings contribute to a better understanding of the relationship between RB tumor malignancy and its TME and will facilitate the development of effective treatment options for eye preserving therapies.

Investigation of the physical, chemical, and biological properties of the cockle shell-derived calcium silicate-based pulp capping material: a pilot study.

Hard-setting calcium hydroxide-based materials, e.g., Dycal and Life, have been widely used for direct pulp capping. However, various studies have shown undesirable effects such as high solubility and unpredictable dentine bridge formation. Bioceramic, mainly composed of tricalcium and dicalcium silicates, e.g., mineral trioxide aggregate and Biodentine, have provided more desirable physical and biological properties. This study aims to measure the physical properties, chemical properties, and biological response of human dental pulp cells (HDPCs) on three dental pulp-capping materials, Dycal, Life, and cockle shell-derived tricalcium silicate pulp capping material (C-Cap). C-Cap was prepared from cockle shells and rice husk ash. Its chemical composition was identified using X-ray diffractometry. The setting time, flow, solubility, and radiopacity tests were performed following the International Organization for Standardization 6876:2012. pH and calcium ion release were measured. The materials were subjected to an extraction medium at various concentrations and subsequently measured for cytotoxicity and migration on HDPCs, from three healthy, mature permanent teeth from different donors. Osteogenic differentiation was assessed by examining alkaline phosphatase enzyme activity and alizarin red staining assay. The data were tested for a normal distribution. The differences among groups were statistically analyzed using ANOVA and Tukey's multiple comparison test (p < 0.05). The setting time of each material was approximately 1-2 min. C-Cap showed the lowest solubility (10.27% ± 1.02%) compared to Dycal (12.67% ± 0.94%) and Life (12.74% ± 1.33%), with a significant difference (p < 0.05). All materials exhibited radiopacity ranging from 2.4 to 2.9 mm of aluminum. C-Cap had the highest flow, alkalinity, and calcium ion release. C-Cap was significantly less cytotoxic than Dycal and Life (p < 0.05). The migration of HDPCs cultured in C-Cap extraction medium (27.74% ± 0.12%) was comparable to that in serum-free medium (27.09% ± 0.08%) with a significant difference (p < 0.05). The mineralization by HDPCs maintained in C-Cap extraction medium was significantly higher than those in Dycal and Life extraction mediums with a significant difference (p < 0.05). C-Cap, a tricalcium silicate-based pulp capping material has potential for further development. C-Cap exhibited comparable physical properties and superior biological properties when compared to Dycal and Life.

Immunocompetent murine glioblastoma stem-like cell models exhibiting distinct phenotypes

Abstract Background Glioblastoma (GBM) treatment is hindered by a dearth of representative mouse GBM preclinical models in immunocompetent mice. Here, we characterized five murine GBM stem-like cell (mGSC) models derived from lentivirus-induced tumors in transgenic mice that are driven by the activation of the Nf1-Ras signaling pathway and inactivation of Tp53. Methods MGSC lines (005, RIG, NF53, C1, and C3) were cultured as spheres in serum-free stem cell media. Whole exome sequencing (WES) was employed to quantify single nucleotide polymorphisms (SNPs). Stem cell properties were characterized by stemness in vitro and tumorigenicity after intracerebral implantation in C57BL/6 mice. Tumor phenotypes and the immune microenvironment were characterized by immunohistochemistry, flow cytometry, and RNA sequencing. Results WES revealed a large variation in coding sequence SNPs across mGSC lines (~20-fold), likely influenced by the mixed backgrounds of the parental mice. MGSCs exhibited variable clonogenic sphere formation and CD133 expression levels. In vivo, they consistently initiated lethal malignant gliomas, with median survival ranging from 29 to 82 days, and showed strong CD44 expression and variable invasiveness. The tumor microenvironment featured an abundance of CD68+ macrophages and uniform high PD-L1+ myeloid cells, while T cell infiltration varied among the models, with low mutation burden C1 and C3 exhibiting fewer tumor-infiltrating T cells. Conclusions Upon orthotopic implantation in immunocompetent mice, mGSCs generate tumors characteristic of human GBM. Despite similar strategies to generate these mGSCs, they exhibited a range of phenotypes and immune profiles in mGSC-derived orthotopic tumors. These mGSCs provide new preclinical GBM models for developing GBM immunotherapies.

Development of peptone-based serum-free media to support Vero CCL-81 cell proliferation and optimize SARS-CoV2 viral production.

Developing an optimal media for Vero cell lines is crucial as it directly influences cell survival, proliferation, and virus production. The use of serum in cell culture raises safety concerns in biological production. The United States Food and Drug Administration (FDA) and the European Medicines Agency have implemented stricter regulations on the use of animal-derived components in commercial protein manufacturing to ensure patient safety. This study aims to develop new chemically defined media using peptone hydrolysate as a serum-free alternative. A rational experimental design was employed to screen and optimize potential serum substitutes. Six types of peptones were tested at varying concentrations to determine the top three candidates. These candidates were subsequently combined into five mixtures to identify the two most effective serum-free media for Vero CCL-81cells. The evaluation criteria included morphology observation and proliferation assay. The two most promising serum-free formulations were further evaluated for their ability to support virus production and using TCID50 and absolute quantification by RT-qPCR. The results of this study indicate that Mixture 4, consisting of 0.5g/L Phytone™ Peptone, 0.5g/L Difco™ Soytone, and 2g/L Bacto™ Malt, significantly enhanced Vero CCL-81cell proliferation and produced high viral titers. The developed medium demonstrated comparable performance to commercially available serum-free media. Peptone-based serum-free media provide a robust alternative for supporting Vero CCL-81cell growth and are suitable for virus production.

The culture and identification of human placenta-derived mesenchymal stem cells

Objective: This study aimed to establish a reliable protocol for the cultivation and characterization of human placenta-derived mesenchymal stem cells (hPMSCs) to evaluate their growth dynamics and immunophenotype. Methods: hPMSCs were thawed and cultured under controlled conditions using specialized serum-free medium. Cell viability and morphology were assessed using an inverted microscope, and medium changes were performed bi-daily. For cell identification, immunofluorescence staining was conducted with antibodies CD44, CD90, and CD45, and cells were characterized based on surface marker expression. Results: Cultured hPMSCs exhibited a fibroblast-like morphology with rapid proliferation, particularly after reaching a seeding density of 50%. Growth curves indicated peak proliferation between days 3 and 4. Immunofluorescence analysis confirmed that hPMSCs were positive for CD90 and CD44, but negative for CD45, aligning with typical mesenchymal stem cell profiles. Conclusion: The established protocol successfully cultivated and characterized hPMSCs, demonstrating their viability and specific surface markers. These findings support their potential application in regenerative medicine and therapeutic research.

Abstract 4147630: Towards transplantation of hearts from donation after circulatory death - stem cell derived therapy for ameliorating donor organ damage

The use of heart transplantation (HTx), a key life-saving therapy for end-stage heart failure, is limited by the shortage of acceptable donor hearts. Although donation after circulatory death (DCD) offers a potentially promising resource for clinical transplantation, its adoption for HTx is concerned by warm ischemic damage during the death process. Mesenchymal stem cell (MSC)-derived paracrine action has been proved to mediate cardioprotection. Based on our previous study showing that MSC conditioned media (MSC CM) and MSC-exosomes (MSC Exo) confer improved donor heart preservation during cold ischemic storage, we aimed to test the effect of MSC CM and MSC Exo on ex vivo recovery of ischemia-damaged myocardium and on promoting the implanted DCD heart function using an in vivo murine heterotopic heart transplantation model. Methods: Donor hearts from adult male C57BL/6 mice with 0, 30’ or 50’ in vivo warm ischemia (WI) were divided into (n=5-7/group): 1) no WI; 2) 30’-WI; 3) 50’-WI; 4) no WI+4h cold storage at 0-4°C (no WI+CS); 5) 30’-WI+CS; 6) 50’-WI+CS; 7) 50’-WI+CS+vehicle; 8) 50’-WI+CS+MSC CM; and 9) 50’-WI+CS+MSC Exo. The CM and Exo were obtained from cultivation of human bone marrow-MSCs in serum-free media for 72h and was added into preservation solution. Donor hearts were implanted into C57BL/6 male recipients using cervical heterotopic heart transplantation. At 24h post-surgery, left ventricular (LV) function was detected in transplanted hearts. Abdominal artery pressure was measured in recipients to validate technical reproducibility of the HTx model. Mitochondrial structure was detected by electron microscopy on the fixed human LV (hDCD&amp;hDBD). Results: WI significantly impaired LV function in transplanted DCD hearts vs. no-WI (Fig. A, B). Intriguingly, markedly improved graft function was observed in MSC CM- or Exo-treated DCD hearts vs. vehicle (Fig. C). Short and disrupted cristae in mitochondria were noticed in hDCD vs. hDBD (Fig. D). Conclusions: Our results represent the initial evidence that MSC secretome (CM&amp;Exo) mediates ex vivo cardiac repair in DCD hearts and validate an approach on MSC derived therapy to rescue marginal DCD donor hearts and improve post-transplant graft function.

BIOM-04. SINGLE-CELL RNA SEQUENCING PROTOCOL OF THE HUMAN METASTATIC PROSTATE SPINE TUMOR MICROENVIRONMENT

Abstract INTRODUCTION 90% of spine tumor patients can develop metastases with debilitating complications, such as sensorimotor dysfunction, paralysis, and spinal instability. As spine tumor patients have limited eligibility to participate in clinical trials and have heterogenous tumor pathology, there is a distinct lack of therapeutic targets and prognostication markers of disease. Thus, there is a distinct need for molecular profiling of spine tumors. OBJECTIVE We describe an OR-to-bench-top processing of spine tumors for single-cell omic studies, with samples from a metastatic prostate cancer patient to demonstrate the ability to compare molecular markers and microenvironments of difficult-to-process spine tumors and adjacent normal tissue. METHODS Under IRB protocol #Pro00101198, patients undergoing spine surgery with the Department of Neurosurgery at Duke University were consented for tissue collection. Vertebral osseous metastases and adjacent normal tissue were confirmed histologically (H&amp;E, immunohistochemistry) and radiologically (MRI, PET, CT, and x-ray). Tissues were manually and enzymatically homogenized in serum-free media with collagenase A and DNAse I to obtain a single cell suspension. Cells were then filter sterilized, erythrolyzed, counted to determine live-death ratio, resuspended to 10-20 million cells per mL, then cryopreserved for downstream standard library preparation for single-cell sequencing using the 10X genomics platform. Downstream analysis was performed using Seurat pipeline, InferCNV analysis, and SingleR. RESULTS P404S and PSA expression confirmed tumor was prostate in origin. Radiological imaging and H&amp;E staining confirmed the presence of T7 osseous metastasis. Uniform manifold approximation and project (UMAP) plots identified 12 unique clusters of immune cell subpopulations between normal and tumorous vertebrae, with their own subsets of differentially expressed genes. CONCLUSION We have developed a protocol to generate granular single cell molecular profiling for spine tumors and metastases. Applications of this protocol can aid in identifying therapeutic targets, molecular predictors of disease, and potentiate increased eligibility of patients for clinical trials.

Derivation of transplantable human thyroid follicular epithelial cells from induced pluripotent stem cells

The production of mature functioning thyroid follicular cells (TFCs) from human induced pluripotent stem cells (iPSCs) is critical for potential novel therapeutic approaches to post-surgical and congenital hypothyroidism. To accomplish this, we developed a novel human iPSC line that expresses fluorophores targeted to the NKX2-1 and PAX8 loci, allowing for the identification and purification of cells destined to become TFCs. Optimizing a sequence of defined, serum-free media to promote stepwise developmental directed differentiation, we found that bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2) stimulated lineage specification into TFCs from multiple iPSC lines. Single-cell RNA sequencing demonstrated that BMP4 withdrawal after lineage specification promoted TFC maturation, with mature TFCs representing the majority of cells present within 1month. After xenotransplantation into athyreotic immunodeficient mice, engrafted cells exhibited thyroid follicular organization with thyroglobulin protein detected in the lumens of NKX2-1-positive follicles. While our iPSC-derived TFCs presented durable expression of thyroid-specific proteins, they were unable to rescue hypothyroidism invivo.

Production of Oncolytic Measles Virus in Vero Cells: Impact of Culture Medium and Multiplicity of Infection.

Oncolytic measles virus (MeV) is a promising anti-cancer treatment. However, the production of high titers of infectious MeV (typically 107-109 TCID50 per dose) is challenging because the virus is unstable under typical production conditions. The objective of this study was to investigate how the multiplicity of infection (MOI) and different media-a serum-containing medium (SCM), a serum-free medium (SFM) and two chemically defined media (CDM)-affect MeV production. We infected Vero cells at MOIs of 0.02, 0.2 or 2 TCID50 cell-1 and the lowest MOI resulted in the largest number of infected cells towards the end of the production period. However, this did not equate to higher maximum MeV titers, which were similar for all the MOIs. The medium had a moderate effect, generating maximum titers of 0.89-2.17 × 106, 1.08-1.25 × 106 and 4.58-9.90 × 105 TCID50 mL-1 for the SCM, SFM and CDM, respectively. Infection at a low MOI often required longer process times to reach maximum yields. On the other hand, a high MOI requires a large amount of MeV stock. We would therefore recommend a mid-range MOI of 0.2 TCID50 cell-1 for MeV production. Our findings show that SCM, SFM and CDM are equally suitable for MeV production in terms of yield and process time. This will allow MeV production in serum-free conditions, addressing the safety risks and ethical concerns associated with the use of serum.

Use of serum-free media for peripheral blood mononuclear cell culture and the impact on T and B cell readouts.

As part of a wider programme of work developing next-generation risk assessment approaches (NGRA) using non-animal methods (NAMs) for safety assessment of materials, Unilever SEAC is exploring the use of a peripheral blood mononuclear cell (PBMC) system to investigate how cells from different arms of the human immune system are impacted by different treatments. To maximise human relevance, the cell cultures are supported by human serum, but this came with some challenges, including an inability to measure induced levels of immunoglobulins due to high background levels. Therefore, a study comparing use of human sera containing media with three different chemically defined serum-free media was undertaken. PBMC were isolated from healthy donors and cultured in the absence (media alone) or presence of stimulation reagents (CpG-ODN plus IL-15, Pokeweed Mitogen (PWM) or Cytostim (CS)), in RPMI plus human serum, AIM-V, CTS OpTmizer T cell expansion SFM or X-VIVO 15 media. T cell (CD4+ and CD8+) and B cell proliferation and viability were measured after 6days, along with levels of total IgG in the cell culture supernatants. Each of the serum-free media tested supported good levels of viable and proliferating T cells and B cells over the 6 days of culture, with only a few, small differences across the media, when there was no stimulation. They also enabled detection of a stimulation-evoked increase in IgG levels. There were however some differences in the viability and proliferation responses of T and B cells, to different stimuli, across the different media. The serum-free media formulations tested in this study offer defined systems for. measuring B cell IgG responses, in vitro, in either a 'T cell-independent' (CpG + IL-15) or "T cell-dependent" (PWM or CS) manner and for assessing B cell proliferation, particularly in response to a "T cell-independent" stimulus. However, there are some characteristics and features endowed by human serum that appear to be missing. Therefore, further work is required to optimise animal-free, chemically defined culture conditions for PBMC based assays for inclusion in tiered safety assessments.

Ex-vivo models of post-surgical residual disease in human glioblastoma

Background Glioblastoma is a highly infiltrative, currently incurable brain cancer. To date, translation of novel therapies for glioblastoma from the laboratory into clinical trials has relied heavily on in vitro cell culture and murine (subcutaneous and orthotopic) xenograft models using cells derived from the main bulk of patient tumours. However, it is the residual cells left-behind after surgery that are responsible for disease progression and death in the clinic. A lack of substantial improvements in patient survival for decades suggests commonly used murine xenograft models, a key step before clinical trials, do not reflect the biology of residual disease in patients. Methods To address this, we have developed the ‘Sheffield Protocol’ to generate ex vivo models that reflect both resected, and post-surgical residual disease from the same patient. The protocol leverages parallel derivation of inherently treatment-resistant glioblastoma stem cells (GSCs) from ‘core’ and distant ‘edge’ regions through careful macrodissection of a large en bloc specimen, such as from a partial lobectomy for tumour, followed by tissue dissociation and propagation in serum-free media. Opportunistic en bloc specimen use can liberate the most distant infiltrative cells feasibly accessible from living patients. Results We provide an example illustrating that resected and residual disease models represent spatially divergent tumour subpopulations harbouring distinct transcriptomic and cancer stem cell marker expression profiles. We also introduce the ‘Sheffield Living Biobank’ of glioma models (SLB) that incorporates over 150 GSC lines from 60+ patients, including 44+ resected and residual models, which are available for academic use via MTA. Conclusions These models provide a novel tool to reduce animal xenograft usage by improving candidate drug triage in early preclinical studies and directly replacing animal studies for some therapies that are post-Phase 1+ clinical trial for other cancers/conditions to, ultimately, deliver more effective treatments for post-surgical residual disease in glioblastoma.

Matrikine stimulation of equine synovial fibroblasts and chondrocytes results in an in vitro osteoarthritis phenotype.

Osteoarthritis (OA) is a debilitating disease that impacts millions of individuals and has limited therapeutic options. A significant hindrance to therapeutic discovery is the lack of in vitro OA models that translate reliably to in vivo preclinical animal models. An alternative to traditional inflammatory cytokine models is the matrikine stimulation model, in which fragments of matrix proteins naturally found in OA tissues and synovial fluid, are used to stimulate cells of the joint. The objective of this study was to determine if matrikine stimulation of equine synovial fibroblasts and chondrocytes with fibronectin fragments (FN7-10) would result in an OA phenotype. We hypothesized that FN7-10 stimulation of equine articular cells would result in an OA phenotype with gene and protein expression changes similar to those previously described for human chondrocytes stimulated with FN7-10. Synovial fibroblasts and chondrocytes isolated from four horses were stimulated in monolayer culture for 6 or 18 h with 1 µM purified recombinant 42 kD FN7-10 in serum-free media. At the conclusion of stimulation, RNA was collected for targeted gene expression analysis and media for targeted protein production analysis. Consistent with our hypothesis, FN7-10 stimulation resulted in significant alterations to many important genes that are involved in OA pathogenesis including increased expression of IL-1β, IL-4, IL-6, CCL2/MCP-1, CCL5/RANTES, CXCL6/GCP-2, MMP-1, MMP-3, and MMP13. The results of this study suggest that the equine matrikine stimulation model of OA may prove useful for in vitro experiments leading up to preclinical trials.