Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture.

BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture.

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

Identification of critical process parameters for expansion of clinical grade human Wharton's jelly-derived mesenchymal stromal cells in stirred-tank bioreactors.

Cell therapies based on multipotent mesenchymal stromal cells (MSCs) are traditionally produced using 2D culture systems and platelet lysate- or serum-containing media (SCM). Although cost-effective for single-dose autologous treatments, this approach is not suitable for larger scale manufacturing (e.g., multiple-dose autologous or allogeneic therapies with banked MSCs); automated, scalable and Good Manufacturing Practices (GMP)-compliant platforms are urgently needed. The feasibility of transitioning was evaluated from an established Wharton's jelly MSCs (WJ-MSCs) 2D production strategy to a new one with stirred-tank bioreactors (STRs). Experimental conditions included four GMP-compliant xeno- and serum-free media (XSFM) screened in 2D conditions and two GMP-grade microcarriers assessed in 0.25 L-STRs using SCM. From the screening, a XSFM was selected and compared against SCM using the best-performing microcarrier. It was observed that SCM outperformed the 2D-selected medium in STRs, reinforcing the importance of 2D-to-3D transition studies before translation into clinical production settings. It was also found that attachment efficiency and microcarrier colonization were essential to attain higher fold expansions, and were therefore defined as critical process parameters. Nevertheless, WJ-MSCs were readily expanded in STRs with both media, preserving critical quality attributes in terms of identity, viability and differentiation potency, and yielding up to 1.47 × 109 cells in a real-scale 2.4-L batch.

The Characteristics and Function of Small Extracellular Vesicles Derived from Human Bone Marrow and Umbilical Cord Mesenchymal Stromal Cells Are Influenced by Cell Culture Conditions.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC-EVs) have been proposed as a novel therapeutic tool with numerous clinically related advantages. However, their characteristics and functionality are dependent on the source of MSCs and their cell culture conditions. Fetal bovine serum (FBS) provides a source of nutrients and growth factors to the cultured cells. However, certain pitfalls are associated with its supplementation to the culture media, including introduction of exogenous FBS-derived EVs to the cultured cells. Thus, recent practices recommend utilization of serum-free (SF) media or EV-depleted FBS. On the contrary, evidence suggests that the immunomodulatory ability of MSC-EVs can be improved by exposing MSCs to an inflammatory (IF) environment. The objective of this study was to (1) compare EVs isolated from two tissue sources of MSCs that were exposed to various cell culture conditions and (2) to evaluate their anti-inflammatory effects. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) and umbilical cord-derived mesenchymal stromal cells (UC-MSCs) were exposed to either a SF media environment, an IF environment, or media supplemented with 5% EV-depleted FBS. Following isolation of MSC-EVs, the isolates were quantified and evaluated for particle size, phenotypic changes, and their immunomodulatory potential. A statistically significant difference was not identified on the yield and protein concentration of different isolates of EVs from BM-MSCs and UC-MSCs, and all isolates had a circular appearance as evaluated via electron microscopy. A significant difference was identified on the phenotype of different EVs isolates; however, all isolates expressed classical markers such as CD9, CD63, and CD81. The addition of BM-derived MSC-EVs from FBS environment or UC-derived MSC-EVs from IF environment resulted in statistically significant downregulation of IL-6 messenger RNA (mRNA) in stimulated leukocytes. This study confirms that EVs produced by different MSC sources and cell culture conditions affect their phenotype and their immunomodulatory capacities.

LIPCAR levels in plasma-derived extracellular vesicles is associated with left ventricle remodeling post-myocardial infarction.

BACKGROUND: Long Intergenic noncoding RNA predicting CARdiac remodeling (LIPCAR) is a long noncoding RNA identified in plasma of patients after myocardial infarction (MI) to be associated with left ventricle remodeling (LVR). LIPCAR was also shown to be a predictor of early death in heart failure (HF) patients. However, no information regarding the expression of LIPCAR and its function in heart as well as the mechanisms involved in its transport to the circulation is known. The aims of this study are (1) to characterize the transporter of LIPCAR from heart to circulation; (2) to determine whether LIPCAR levels in plasma isolated-extracellular vesicles (EVs) reflect the alteration of its expression in total plasma and could be used as biomarkers of LVR post-MI. METHODS: Since expression of LIPCAR is restricted to human species and the limitation of availability of cardiac biopsy samples, serum-free conditioned culture media from HeLa cells were first used to characterize the extracellular transporter of LIPCAR before validation in EVs isolated from human cardiac biopsies (non-failing and ischemic HF patients) and plasma samples (patients who develop or not LVR post-MI). Differential centrifugation at 20,000g and 100,000g were performed to isolate the large (lEVs) and small EVs (sEVs), respectively. Western blot and nanoparticle tracking (NTA) analysis were used to characterize the isolated EVs. qRT-PCR analysis was used to quantify LIPCAR in all samples. RESULTS: We showed that LIPCAR is present in both lEVs and sEVs isolated from all samples. The levels of LIPCAR are higher in lEVs compared to sEVs isolated from HeLa conditioned culture media and cardiac biopsies. No difference of LIPCAR expression was observed in tissue or EVs isolated from cardiac biopsies obtained from ischemic HF patients compared to non-failing patients. Interestingly, LIPCAR levels were increased in lEVs and sEVs isolated from MI patients who develop LVR compared to patients who did not develop LVR. CONCLUSION: Our data showed that large EVs are the main extracellular vesicle transporter of LIPCAR from heart into the circulation independently of the status, non-failing or HF, in patients. The levels of LIPCAR in EVs isolated from plasma could be used as biomarkers of LVR in post-MI patients.

Housekeeping Gene Stability in Adipose Mesenchymal Stromal Cells Cultivated in Serum/Xeno-Free Media for Osteoarthritis.

Among the available therapeutics for the conservative treatment of osteoarthritis (OA), mesenchymal stromal cells (MSCs)-based products appear to be the most promising. Alongside minimally manipulated cell-based orthobiologics, where MSCs are the engine of the bioactive properties, cell expansion under good manufacturing practice (GMP) settings is actively studied to obtain clinical-grade pure populations able to concentrate the biological activity. One of the main characteristics of GMP protocols is the use of clinical-grade reagents, including the recently released serum-free/xeno-free (SFM/XFM) synthetic media, which differ significantly from the traditional reagents like those based on fetal bovine serum (FBS). As SFM/XFM are still poorly characterized, a main lack is the notion of reliable housekeeping genes (HKGs) for molecular studies, either standalone or in combination with standard conditions. Indeed, the aim of this work was to test the stability of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) in adipose-derived MSCs (ASCs) cultivated in two commercially available SFM/XFM and to compare outcomes with those obtained in FBS. Four different applets widely recognized by the scientific community (NormFinder, geNorm, comparative ΔCt method, and BestKeeper) were used and data were merged to obtain a final stability order. The analysis showed that cells cultured in both synthetic media had a similar ranking for HKGs stability (GAPDH being best), albeit divergent from FBS expanded products (EF1A at top). Moreover, it was possible to identify specific HKGs for side by side studies, with EF1A/TBP being the most reliable normalizers for single SFM/XFM vs. FBS cultured cells and TBP the best one for a comprehensive analysis of all samples. In addition, stability of HKGs was donor-dependent. The normalization effect on selected genes coding for factors known to be involved in OA pathology, and whose amount should be carefully considered for the selection of the most appropriate MSC-based treatment, showed how HKGs choice might affect the perceived amount for the different media or donor. Overall, this work confirms the impact of SFM/XFM conditions on HKGs stability performance, which resulted similarly for both synthetic media analyzed in the study.

Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12 mmol cell/h) in SF and for glutamine (30.8 × 10-12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Satellite cells sourced from bull calves and dairy cows differs in proliferative and myogenic capacity - Implications for cultivated meat.

Cultivated meat produced with primary muscle satellite cells (SCs) will need a continuous supply of isolated cell material from relevant animal donors. Factors such as age, sex, and breed, along with the sustainability and availability of donor animals, could determine the most appropriate donor type for an efficient production. In this study, we focus on the proliferation and differentiation of bovine SCs isolated from bull calf and dairy cow muscle samples. The proliferative performance of bull calf SCs was significantly better than SCs from dairy cows, however a dynamic differentiation assay revealed that the degree of fusion and formation of myotubes were similar between donor types. Furthermore, the proliferation of SCs from both donor types was enhanced using an in-house developed serum-free media compared to 10% FBS, which also delayed myogenic differentiation and increased final cell population density. Using gene chip transcriptomics, we identified several differentially expressed genes between the two donor types, which could help explain the observed cellular differences. This data also revealed a high biological variance between the three replicate animals within donor type, which seemed to be decreased when using our in-house serum-free media. With the use of the powerful imaging modalities of Cytation 5, we developed a novel high contrast brightfield-enabled label-free myotube quantification method along with a more efficient end-point fusion analysis using Phalloidin-staining. The results give new insights into the bovine SC biology and potential use of bull calves and dairy cows as relevant donor animals for cultivated beef cell sourcing. The newly developed differentiation assays will further enhance future research within the field of cultivated meat and SC biology.

Generation of Human Regulatory Dendritic Cells from Cryopreserved Healthy Donor Cells and Hematopoietic Stem Cell Transplant Recipients.

Acute graft versus host disease (GVHD) remains a significant complication following hematopoietic stem cell transplant (HSCT), despite improved human leukocyte antigen (HLA) matching and advances in prophylactic treatment regimens. Previous studies have shown promising results for future regulatory dendritic cell (DCreg) therapies in the amelioration of GVHD. This study evaluates the effects of cryopreservation on the generation of DCreg, the generation of young and older DCreg in serum-free media, and the feasibility of generating DCreg from young and older HSCT patient monocytes. DCregs were generated in X-vivo 15 serum-free media from donor or patient monocytes. This study includes the use of monocytes from young and older healthy, donor, and HSCT patients with varying hematological diseases. Phenotypic differences in cell populations were assessed via flow cytometry while pro-inflammatory and anti-inflammatory cytokine production was evaluated in culture medium. The number of DCreg generated from cryopreserved monocytes of healthy donors was not significantly different from freshly isolated monocytes. DCreg generated from cryopreserved monocytes had comparable levels of co-stimulatory molecule expression, inhibitory molecule expression, and cytokine production as freshly isolated monocytes. Young and older healthy donor monocytes generated similar numbers of DCreg with similar cytokine production and phenotype. Although monocytes from older HSCT patients generated significantly fewer DCreg, DCreg from young and older HSCT patients had comparable phenotypes and cytokine production. Monocytes from young and older myelodysplastic syndrome (MDS) patients generated reduced numbers of DCreg compared to non-MDS-derived DCreg. We demonstrate that the cryopreservation of monocytes from HSCT patients of varying hematological diseases allows for the cost-effective generation of DCreg on an as-needed basis. Although the generation of DCreg from MDS patients requires further assessment, these data support the possibility of in vitro-generated DCreg as a therapy to reduce GVHD-associated morbidity and mortality in young and older HSCT recipients.

A simple and robust serum-free media for the proliferation of muscle cells.

Cultivated meat production requires an efficient, robust and highly optimized serum-free cell culture media for the needed upscaling of muscle cell expansion. Existing formulations of serum-free media are complex, expensive and have not been optimized for muscle cells. Thus, we undertook this work to develop a simple and robust serum-free media for the proliferation of bovine satellite cells (SCs) through Design of Experiment (DOE) and Response Surface Methodology (RSM) using precise and high-throughput image-based cytometry. Proliferative attributes were investigated with transcriptomics and long-term performance was validated using multiple live assays. Here we formulated a media based on three highly optimized components; FGF2 (2 ng/mL), fetuin (600 µg/mL) and BSA (75 µg/mL) which together with an insulin-transferrin-selenium (1x) supplement, sustained the proliferation of bovine SCs, porcine SCs and murine C2C12 muscle cells. Remarkably, cells cultured in our media named Tri-basal 2.0+ performed better than cell cultured in 10% FBS, with respect to proliferation. Hence, the optimized Tri-basal 2.0+ enhanced serum-free cell attachment and long-term proliferation, providing an alternative solution to the use of FBS in the production of cultivated meat.

Aligned skeletal muscle assembly on a biofunctionalized plant leaf scaffold.

Decellularized plant scaffolds have drawn attention as alternative tissue culture platforms due to their wide accessibility, biocompatibility, and diversity of innate microstructures. Particularly, in this work, monocot leaves with innate uniaxial micropatterned topography were utilized to promote cell alignment and elongation. The leaf scaffold was biofunctionalized with poly(PEGMEMA-r-VDM-r-GMA) copolymer that prevented non-specific protein adsorption and was modified with cell adhesive RGD peptide to enable cell adhesion and growth in serum-free media. The biofunctionalized leaf supported the adhesion, growth, and alignment of various human cells including embryonic stem cells (hESC) derived muscle cells. The hESC-derived myogenic progenitor cells cultured on the biofunctionalized leaf scaffold adopted a parallel orientation and were elongated along the leaf topography. These cells showed significant early myogenic differentiation and muscle-like bundled myotube formation. The aligned cells formed compact myotube assemblies and showed uniaxial muscle contraction under chemical stimulation, a critical requirement for developing functional skeletal muscle tissue. Polymer-functionalized plant leaf scaffolds offer a novel human cell culture platform and have potential in human tissue engineering applications that require parallel alignment of cells. STATEMENT OF SIGNIFICANCE: Plant scaffolds are plentiful sources in nature and present a prefabricated construct to present topographical cues to cells. Their feature width is ideal for human cell alignment and elongation, especially for muscle cells. However, plant scaffolds lack proteins that support mammalian cell culture. We have developed a polymer coated leaf scaffold that enables cell adhesion and growth in serum-free media. Human muscle cells cultured on the biofunctionalized leaf, aligned along the natural parallel micro-patterned leaf topography, and formed muscle-like bundled myotube assemblies. These assemblies showed uniaxial muscular contraction, a critical requirement for developing functional skeletal muscle tissue. The biodiversity of the plant materials offers a novel human cell culture platform with potential in human tissue engineering.

Production of Tissue-Engineered Skin Substitutes for Clinical Applications: Elimination of Serum.

Tissue-engineered skin substitutes (TESs) are used as a treatment for severe burn injuries. Their production requires culturing both keratinocytes and fibroblasts. The methods to grow these cells have evolved over the years, but bovine serum is still commonly used in the culture medium. Because of the drawbacks associated with the use of serum, it would be advantageous to use serum-free media for the production of TESs. In a previous study, we developed a serum-free medium (Surge SFM) for the culture of keratinocytes. Herein, we tested the use of this medium, together with a commercially available serum-free medium for fibroblasts (Prime XV), to produce serum-free TESs. Our results show that serum-free TESs are macroscopically and histologically similar to skin substitutes produced with conventional serum-containing media. TESs produced with either culture media expressed keratin 14, Ki-67, transglutaminase 1, filaggrin, type I and IV collagen, and fibronectin comparably. Mechanical properties, such as contraction and tensile strength, were comparable between TESs cultured with and without serum. Serum-free TESs were also successfully grafted onto athymic mice for a six-month period. In conclusion, Surge SFM and Prime XV serum-free media could be used to produce high quality clinical-grade skin substitutes.

P04-A115†Serum containing organ culture of the human cornea - still state of the art?

PURPOSE: For decades, human corneas are prepared and stored in specialized tissue banks prior to transplantation. Especially in Europe, storage takes place in 'organ culture', the storage in cell culture medium at approximately physiological temperature. Traditionally, a serum-containing medium is used for this purpose. However, the use of fetal calf serum has considerable disadvantages: there is a risk of disease transmission, availability may not always be guaranteed in the necessary quality, there are considerable differences from batch to batch, which is associated with batch testing required in each case, and last but not least, the extraction of serum from unborn calves is an ethical issue. METHODS: In recent years, several studies have focused on the improvement of organ culture conditions for donor corneas, including different serum-free media and alternative deswelling substances. Meanwhile, media are on the market which seem to be equivalent to serum-supplemented MEM. Nevertheless, serum-free medium has not yet found its way into routine organ culture of corneas. RESULTS: Our own preliminary studies have shown that despite the promising approaches, no satisfactory overall result could be achieved. Since only maintenance metabolism is required for storage of corneas until transplantation, in principle cultivation in the conventionally used medium seems possible without addition of serum at all. Corneas stored in this way had comparably endothelial cell density (ECD) to their counterpart stored in serum-supplemented medium. However, during the final evaluation after deswelling, the ECD dropped drastically.Engelmann et al. started research on the use of serum-free culture medium (SFM) for a long time and comparable or even superior ECD and viability could be demonstrated. So far, however, it has not been possible to define a deswelling medium adapted to these conditions.Also, a serum-free storage medium developed by Eurobio (CorneaSyn) could not completely convince, because although ECD of the examined corneas remained constant, the morphology of the cells changed. CONCLUSION: Since it is essential to intensify efforts towards a serum-free system it is planned to test serum substitutes and, if possible, also to replace the de-swelling additive dextran with a less harmful alternative to guarantee the quality of cornea grafts in the future.

Significant improvement of bone marrow-derived MSC expansion from MDS patients by defined xeno-free medium.

BACKGROUND: Robust and reliable in vitro and in vivo models of primary cells are necessary to study the pathomechanisms of Myelodysplastic Neoplasms (MDS) and identify novel therapeutic strategies. MDS-derived hematopoietic stem and progenitor cells (HSPCs) are reliant on the support of bone marrow (BM) derived mesenchymal stroma cells (MSCs). Therefore, isolation and expansion of MCSs are essential for successfully modeling this disease. For the clinical use of healthy MSCs isolated from human BM, umbilical cord blood or adipose tissue, several studies showed that xeno-free (XF) culture conditions resulted in superior growth kinetics compared to MSCs cultured in the presence of fetal bovine serum (FBS). In this present study, we investigate, whether the replacement of a commercially available MSC expansion medium containing FBS with a XF medium is beneficial for the expansion of MSCs derived from BM of MDS patients which are often difficult to cultivate. METHODS: MSCs isolated from BM of MDS patients were cultured and expanded in MSC expansion medium with FBS or XF supplement. Subsequently, the impact of culture media on growth kinetics, morphology, immunophenotype, clonogenic potential, differentiation capacity, gene expression profiles and ability to engraft in immunodeficient mouse models was evaluated. RESULTS: Significant higher cell numbers with an increase in clonogenic potential were observed during culture of MDS MSCs with XF medium compared to medium containing FBS. Differential gene expression showed an increase in transcripts associated with MSC stemness after expansion with XF. Furthermore, immunophenotypes of the MSCs and their ability to differentiate into osteoblasts, adipocytes or chondroblasts remained stable. MSCs expanded with XF media were similarly supportive for creating MDS xenografts in vivo as MSCs expanded with FBS. CONCLUSION: Our data indicate that with XF media, higher cell numbers of MDS MSCs can be obtained with overall improved characteristics in in vitro and in vivo experimental models.

Impact of serum­free media on the expansion and functionality of CD19.CAR T­cells.

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T­cells. In order to overcome a lot­to­lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum­free media [Prime­XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T­Cell Expansion Xeno­Free Medium, Takara Bio™ (TB) and TCM GMP­Prototype, CellGenix™ (CG)] to the standard CAR T­cell medium containing FBS (RCF). After 12 days of CD19.CAR T­cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T­cells was evaluated using intracellular staining, a chromium release assay and a long­term co­culture assay. Expansion and viability did not differ between the CAR T­cells generated in serum­free media compared to the standard FBS­containing medium. The CG CAR T­cells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNF­α+ CAR T­cells than the CAR T­cells 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 T­cells generated with serum­free media exhibited a higher cytotoxicity than the CAR T­cells 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 T­cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte­activation gene 3 and T­cell immunoglobulin and mucin­domain containing­3. The CAR T­cells cultured in FF had a higher percentage of central memory CAR T­cells (40.0 vs. 14.3%, P=0.0470) than the CAR T­cells cultured with FBS, whereas the CAR T­cells 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 T­cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum­free media. Given the advantages of freedom from bovine material and consistent quality, serum­free media hold promise for the future development of the field of GMP manufacturing of CAR T­cells.

Protein expression and purification of bioactive growth factors for use in cell culture and cellular agriculture.

Mitogenic growth factors are major cost drivers in serum-free media, contributing up to 95% of the total cost. Here, we present a streamlined workflow detailing cloning, expression testing, protein purification, and bioactivity screening that allows for low-cost production of bioactive growth factors including basic fibroblast growth factor and transforming growth factor ß1. This generalized procedure can be used for multiple families of growth factors with minor modification, and the outputs are bioactive and suitable for cell culture applications. For complete details on the use and execution of this protocol, please refer to Venkatesan, et al.1.

Comparison of rabbit corneal changes during different preservation techniques using optisol-GS and airlift.

A previous study suggested that the airlift condition is superior to the Optisol-GS condition for preserving the limbal tissue of the human cornea. The purpose of this research is to investigate a new preservation device that preserves the cornea while separating epithelial and endothelial areas. The differences after preserving the corneal epithelium under different conditions were compared. A total of 24 corneas of New Zealand rabbits were divided into four groups in which the corneal epithelia were submersed in Optisol-GS or under airlift conditions for 1 and 2 weeks at 4 [Formula: see text]C. Transparency, optical coherence tomography (OCT), hematoxylin and eosin (H &E) staining, and epithelial migration tests were used to assess corneal status. The epithelial migration examination showed significantly greater migration ability after the airlift condition. Corneas in the 1-week Optisol-GS group were the most transparent, followed by the 1-week airlift group. OCT showed a progressive increase in corneal thickness to the end of the study. H &E staining showed that the epithelial cells retained intact cellular structure and morphology of the cells for both 1-week-preserved groups. However, there was disruption of the corneal epithelial cell structure for both 2-week-preserved groups. Corneal epithelium preserved under the hypothermic airlift condition was comparable to that under the Optisol-GS condition.

A Beefy-R culture medium: Replacing albumin with rapeseed protein isolates.

The development of cost-effective serum-free media is essential for the economic viability of cultured meat. A key challenge facing this goal is the high-cost of recombinant albumin which is necessary in many serum-free media formulations, including a recently developed serum-free medium for bovine satellite cell (BSC) culture termed Beefy-9. Here we alter Beefy-9 by replacing recombinant albumin with rapeseed protein isolate (RPI), a bulk-protein solution obtained from agricultural waste through alkali extraction (pH 12.5), isoelectric protein precipitation (pH 4.5), dissolution of physiologically soluble proteins (pH 7.2), and concentration of proteins through 3 kDa ultrafiltration. This new medium, termed Beefy-R, was then used to culture BSCs over four passages, during which cells grew with an average doubling time of 26.6 h, showing improved growth compared with Beefy-9. In Beefy-R, BSCs maintained cell phenotype and myogenicity. Together, these results offer an effective, low-cost, and sustainable alternative to albumin for serum-free culture of muscle stem cells, thereby addressing a key hurdle facing cultured meat production.

A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.