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Myelination and the formation of nodes of Ranvier are essential for the rapid conduction of nerve impulses along axons in the peripheral nervous system (PNS). While many animal-based and serum-containing models of peripheral myelination have been developed, these have limited ability when it comes to studying genetic disorders affecting peripheral myelination. We report a fully induced pluripotent stem cell (iPSC)-derived human model of peripheral myelination using Schwann cells (SCs) and motoneurons, cultured in a serum-free medium on patterned and nonpatterned surfaces. Results demonstrated iPSC-derived SC-expressed early growth response protein 2 (Egr2), a key transcription factor for myelination, and after ∼30 days in coculture, hallmark features of myelination, including myelin segment and node of Ranvier formation, were observed. Myelin segments were stained for the myelin basic protein, which surrounded neurofilament-stained motoneuron axons. Clusters of voltage-gated sodium channels flanked by paranodal protein contactin-associated protein 1, indicating node of Ranvier formation, were also observed. High-resolution confocal microscopy allowed for 3D reconstruction and measurement of myelin g-ratios of myelin segments, with an average g-ratio of 0.67, consistent with reported values in the literature, indicating mature myelin segment formation. This iPSC-based model of peripheral myelination provides a platform to investigate numerous PNS diseases, including Charcot-Marie Tooth disorder, Guillian-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, and antimyelin-associated glycoprotein peripheral neuropathy, with the potential for greater translatability to humans for improving the applicability for drug-screening programs.

Malaria remains a global health problem, and the standard membrane feeding assay (SMFA) is a key functional assay for development of new interventions to stop malaria transmission from human to mosquito. For SMFA, media with ~ 10% of human serum has been used for infectious gametocyte cultures, however, there are multiple challenges to obtain a suitable human serum. Here we show a human-serum-free culture medium (HSF), which was a mixture of two stem cell culture media and AlbuMAX, supported infectious gametocyte growth. Moreover, the HSF-induced gametocytes elicited significantly higher numbers of oocysts compared to gametocytes cultured with conventional human serum medium (Conv). While some caution is required when comparing percent transmission reducing activity data generated from HSF-SMFA and Conv-SMFA, the HSF method can facilitate the establishment of gametocyte cultures or SMFA by bypassing the need for human serum. Thus, this study will support future development of P. falciparum transmission-blocking interventions., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

ARPE-19 cells are derived from adult human retinal pigment epithelium (RPE). The response of these cells to the stress of serum deprivation mimics some important processes relevant to age-related macular degeneration (AMD). Here we extend the characterization of this response using RNASeq and EGSEA gene set analysis of ARPE-19 cells over nine days of serum deprivation. This experiment confirmed the up-regulation of cholesterol and lipid-associated pathways that increase cholesterol levels in these cells. The gene expression analysis also identified other pathways relevant to AMD progression. There were significant changes in extracellular matrix gene expression, notably a switch from expression of collagen IV, a key component of Bruch's membrane (part of the blood-retina barrier), to expression of a fibrosis-like collagen type I matrix. Changes in the expression profile of the extracellular matrix led to the discovery that amelotin is induced in AMD and is associated with the development of the calcium deposits seen in late-stage geographic atrophy. The transcriptional profiles of other pathways, including inflammation, complement, and coagulation, were also modified, consistent with immune response patterns seen in AMD. As previously noted, the cells resist apoptosis and autophagy but instead initiate a gene expression pattern characteristic of senescence, consistent with the maintenance of barrier function even as other aspects of RPE function are compromised. Other differentially regulated genes were identified that open new avenues for investigation. Our results suggest that ARPE-19 cells maintain significant stress responses characteristic of native RPE that are informative for AMD. As such, they provide a convenient system for discovery and for testing potential therapeutic interventions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The complement system and neutrophils play crucial roles in innate immunity. Neutrophils release neutrophil extracellular traps (NETs), which are composed of decondensed DNA entangled with granular contents, as part of their innate immune function. Mechanisms governing complement-mediated NET formation remain unclear. In this study, we tested a two-step NETosis mechanism, as follows: classical complement-mediated neutrophil activation in serum and subsequent NET formation in serum-free conditions, using neutrophils from healthy donors, endothelial cells, and various assays (Fluo-4AM, DHR123, and SYTOX), along with flow cytometry and confocal microscopy. Our findings reveal that classical complement activation on neutrophils upregulated the membrane-anchored complement regulators CD46, CD55, and CD59. Additionally, complement activation increased CD11b on neutrophils, signifying activation and promoting their attachment to endothelial cells. Complement activation induced calcium influx and citrullination of histone 3 (CitH3) in neutrophils. However, CitH3 formation alone was insufficient for NET generation. Importantly, NET formation occurred only when neutrophils were in serum-free conditions. In such environments, neutrophils induced NADPH oxidase-dependent reactive oxygen species (ROS) production, leading to NET formation. Hence, we propose that complement-mediated NET formation involves a two-step process, as follows: complement deposition, neutrophil priming, calcium influx, CitH3 formation, and attachment to endothelial cells in serum. This is followed by NADPH-dependent ROS production and NET completion in serum-free conditions. Understanding this process may unveil treatment targets for pathologies involving complement activation and NET formation.

Human allogeneic liver-derived progenitor cells (HALPCs) display advanced ability to differentiate into hepatocyte-like cells and exhibit potent immunomodulatory, anti-inflammatory, and anti-fibrotic properties. HALPCs have been successfully manufactured under good manufacturing practice (GMP) and are currently in clinical development. A previous phase 2a trial demonstrated the safety of peripheral intravenous infusions of HALPCs and preliminary evidence of the cells' properties to restore liver function in patients with acute-on-chronic liver failure (ACLF), thus potentially improving their survival. A phase 2b trial is currently ongoing across multiple centers (NCT04229901) to obtain proof-of-concept on efficacy and additional safety. HALPCs are currently manufactured using fetal bovine serum (FBS), which can reveal qualitative and quantitative variations between batches. The use of serum-free medium (SFM) represents an alternative means to overcome this variability while also complying fully with regulations. The aim of this study was to compare current FBS-containing culture conditions with two industry-available GMP-compliant SFMs: StemMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and PRIME-XV (FUJIFILM Irvine Scientific, Santa Ana, California, USA). The proliferation of HALPCs was significantly stimulated by both SFMs, which shortened both their emergence period and population doubling time. This effect was correlated with a significant improvement in their genetic stability as analyzed by conventional karyotyping. The expression profile (identity and purity) and functionality of HALPCs cultured in SFM were maintained, as demonstrated by flow cytometry and enzyme-linked immunoassay (ELISA), respectively. Their potency, evaluated via prostaglandin E2 (PGE2) secretion, showed a similar effect on CD4 + T-cell proliferation in FBS and SFM conditions. Furthermore, a greater proportion of HALPCs cultured in SFM showed enhanced expression of tissue factor (CD142) compared with the FBS condition. Altogether, SFM conditions enabled consistent HALPC quality to be achieved without altering their expression and functional profiles., Competing Interests: Declaration of competing interest PDB, EP, NG, and FL are employees of Cellaïon. ED has nothing to disclose. ES is a founder, patent holder, consultant and board member of Cellaïon and employee of UCLouvain. MN is a patent holder, consultant of Cellaïon and employee of UCLouvain., (Copyright © 2024. Published by Elsevier Inc.)

Background: The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium., Methods: We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions., Results: The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions., Conclusions: These results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies., (© 2024. The Author(s).)

Background: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells., Methods: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method., Results: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells., Conclusions: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gong, Johnsson, Säfholm, Al-Ameri, Sachs, Vali, Nilsson and Rönnberg.)

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., (© 2024. The Author(s).)

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.

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.

The use of fetal bovine serum (FBS) and the price of cell culture media are the key constraints for developing serum-free cost-effective media. This study aims to replace or reduce the typical 10% serum application in fish cell culture media by applying protein hydrolysates from insects and marine invertebrate species for the growth of Zebrafish embryonic stem cells (ESC) as the model organism. Protein hydrolysates were produced from black soldier flies (BSF), crickets, oysters, mussels, and lugworms with a high protein content, suitable functional properties, and adequate amino-acid composition, with the degree of hydrolysis from 18.24 to 33.52%. Protein hydrolysates at low concentrations from 0.001 to 0.1 mg/mL in combination with 1 and 2.5% serums significantly increased cell growth compared to the control groups (5 and 10% serums) ( p < 0.05). All protein hydrolysates with concentrations of 1 and 10 mg/mL were found to be toxic to cells and significantly reduced cell growth and performance ( p < 0.05). However, except for crickets, all the hydrolysates were able to restore or significantly increase cell growth and viability with 50% less serum at concentrations of 0.001, 0.01, and 0.1 mg/mL. Although cell growth was enhanced at lower concentrations of protein hydrolysates, the cell morphology was altered due to the lack of serum. The lactate dehydrogenase (LDH) activity results indicated that BSF and lugworm hydrolysates did not alter the cell membrane. In addition, light and fluorescence imaging revealed that the cell morphological features were comparable to those of the 10% serum control group. Overall, lugworm and BSF hydrolysates reduced the serum by up to 90% while preserving excellent cell health.

Background: Given the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents., Methods: Seeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality., Results: The authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 10 5 cells/cm 2 ) initially had high growth rates, viability and FOXP3 expression, but these parameters decreased with time and were less stable than those observed in cultures of Tregs restimulated at high cell densities (5 × 10 5 cells/cm 2 ), which had slower growth rates. High-density expansion was associated with expression of inhibitory molecules and lower intracellular oxygen and extracellular nutrient concentrations as well as extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposure to low oxygen levels had little impact on expansion, viability or phenotype. Similarly, blockade of inhibitory molecules had little effect. By contrast, replenishing nutrients by increasing the feeding frequency between 2 days and 4 days after restimulation increased FOXP3, viability and expansion in high-density cultures., Conclusion: These data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Cell-based therapies are emerging as a viable modality to treat challenging diseases, resulting in an increasing demand for their large-scale, high-quality production. Production facilities face the issue of batch-to-batch consistency while producing a safe and efficient cell-based product. Controlling culture conditions and particularly media composition is a key factor of success in this challenge. Serum and Xeno-Free Media (SXFM) represent an interesting option to achieve this goal. By reducing batch to batch variability, they increase Good Manufacturing Practices (GMP)-compliance and safety regarding xenogenic transmission, as compared to fetal bovine serum (FBS) supplemented-media or human platelet lysate supplemented medium., Methods: In this study, the isolation, expansion and characteristics including the anti-inflammatory function of human mesenchymal stromal cells (MSC) are compared after culture in MEM α supplemented with human Concentrate Platelet Lysate (hCPL, reference medium) or in MSC-Brew GMP Medium. The latter is a GMP SXFM manufactured in bags under strictly controlled conditions in volumes suitable for expansion to a clinical scale and does not require neither pre-coating of the cell culture units nor the addition of blood derivatives at the isolation step., Results: We showed that MSC derived from human bone-marrow and adipose tissue can be successfully isolated and expanded in this SXFM. Number and size of Colony-Forming Unit fibroblast (CFU-F) is increased compared to cells cultivated in hCPL medium. All cells retained a CD90 + , CD73 + , CD105 + , HLADR - , CD34 - , CD45 - phenotype. Furthermore, the osteogenic and adipocyte potentials as well as the anti-inflammatory activity were comparable between culture conditions. All cells reached the release criteria established in our production facility to treat inflammatory pathologies., Conclusions: The use of MSC-Brew GMP Medium can therefore be considered for clinical bioprocesses as a safe and efficient substitute for hCPL media., Competing Interests: The authors declare there are no competing interests., (©2022 Aussel et al.)

Purpose: of the research: To achieve male fertility preservation and restoration, experimental strategies for in vitro germ cell differentiation are required. The effects of two different culture conditions on in vitro maintenance and differentiation of non-human primate germ cells was studied. Three testes from three 6-month-old marmosets were cultured using a gas-liquid interphase system for 12 days. Testicular maturation in pre-culture control and samples cultured in gonadotropin and serum supplemented and non-supplemented culture samples was evaluated using Periodic Acid-Schiff (PAS) and immunohistochemical stainings., Principle Results: Gonadotropins and serum-supplemented tissues demonstrate up to meiotic differentiation (BOULE + Pachytene spermatocyte) and advanced localization of germ cells (MAGEA4+). Moreover, complex (with gonadotropin and marmoset monkey serum) conditions induced progression in somatic cell maturation with advanced seminiferous epithelial organization, maintenance of encapsulation of cultured fragments with peritubular-myoid cells, preservation of tubular structural integrity and architecture., Major Conclusions: We report stimulation-dependent in vitro meiotic transition in non-human primate testes. This model represents a novel ex vivo approach to obtain crucial developmental progression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-β1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-β1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-β are described, including prostaglandin E 2 receptor downmodulation, TGF-β insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy., Competing Interests: J.M. is chief scientific officer, scientific founder, and shareholder of Leucid Bio; is a member of the scientific advisory board of Suda Pharmaceutics; and has undertaken consultancy work for Bristol-Meyers-Squibb, Juno, Celgene, Ellipses Pharma, and Biotest. A.C.P.-P. is currently an employee of GlaxoSmithKline. L.H. and B.D. undertook Ph.D. studentships funded by Leucid Bio. C.H. and D.M.D are currently employees of Leucid Bio. L.M.W. and D.M.D. were formerly consultants to Leucid Bio. L.M.W. is currently an employee of Oxford Biomedica. T.Z. is currently an employee of Orchard Therapeutics. J.X., F.A., and A.S. were formerly employees of Bayer AG. A.S. is a shareholder of Bayer AG. J.M., A.C.P.-P., and R.E.B. are co-inventors on patent filings in relation to the manufacture of γδ T cells in the presence of TGF-β. C.-Y.R.J. is currently an employee of TCR2 Therapeutics. H.G.R. and H.H.-S. are employees of Bayer AG. B.D. and D.A. are shareholders of Autolus Therapeutics. D.A. is a former employee of Autolus Therapeutics. F.M. and R.T.M.d.R. are co-inventors on patent filings in relation to the manufacture of 89Zr-oxine for cell labeling. A.N.J.T. is/has been a consultant for AstraZeneca, Merck KGaA, Artios, Pfizer, Vertex, GE Healthcare, Inbiomotion, and MD Anderson Cancer Center; has received grant/research support from AstraZeneca, Myriad, Medivation, and Merck KGaA; is a stockholder in Inbiomotion; and stands to gain from the use of PARP inhibitors as part of the ICR’s “rewards to inventors” scheme. The other authors declare no competing interests., (© 2021 The Author(s).)

It is increasingly recognized that brain microvascular endothelial cells (BMECs), the principal component of the blood-brain barrier (BBB), are highly sensitive to soluble cues from both the bloodstream and the brain. This concept extends in vitro, where the extracellular milieu can also influence BBB properties in cultured cells. However, the extent to which baseline culture conditions can affect BBB properties in vitro remains unclear, which has implications for model variability and reproducibility, as well as downstream assessments of molecular transport and disease phenotypes. Here, we explore this concept by examining BBB properties within human-induced pluripotent stem cell (iPSC)-derived BMEC-like cells cultured under serum-free conditions in DMEM/F12 and Neurobasal media, which have fully defined compositions. We demonstrate notable differences in both passive and active BBB properties as a function of basal media composition. Further, RNA sequencing and phosphoproteome analyses revealed alterations to various signaling pathways in response to basal media differences. Overall, our results demonstrate that baseline culture conditions can have a profound influence on the performance of in vitro BBB models, and these effects should be considered when designing experiments that utilize such models for basic research and preclinical assays., (© 2021 International Society for Neurochemistry.)

Background: We have observed an increased expression of negative markers in some clinical-grade, xeno- and serum-free cultured adipose-derived mesenchymal stem/stromal cell (ADMSC) samples. It gave rise to concern that xeno- and serum-free conditions might have unexpected effects on human ADMSCs. This study aims to test this hypothesis for two xeno- and serum-free media, PowerStem MSC1 media (PS) and StemMACS MSC Expansion Media (SM), that support the in vitro expansion of ADMSCs., Methods: We investigated the expression of negative markers in 42 clinical-grade ADMSC samples expanded in PS. Next, we cultured ADMSCs from seven donors in PS and SM and examined their growth and colony-forming ability, surface marker expression, differentiation, cell cycle and senescence, as well as genetic stability of two passages representing an early and late passage for therapeutic MSCs., Results: 15 of 42 clinical-grade PS-expanded ADMSC samples showed an increased expression of negative markers ranging from 2.73% to 34.24%, which positively correlated with the age of donors. This rise of negative markers was related to an upregulation of Human Leukocyte Antigen - DR (HLA-DR). In addition, the PS-cultured cells presented decreased growth ability, lower frequencies of cells in S/G2/M phases, and increased ß-galactosidase activity in passage 7 suggesting their senescent feature compared to those grown in SM. Although MSCs of both PS and SM cultures were capable of multilineage differentiation, the PS-cultured cells demonstrated chromosomal abnormalities in passage 7 compared to the normal karyotype of their SM counterparts., Conclusions: These findings suggest that the SM media is more suitable for the expansion of therapeutic ADMSCs than PS. The study also hints a change of ADMSC features at more advanced passages and with increased donor's age. Thus, it emphasizes the necessity to cover these aspects in the quality control of therapeutic MSC products., (© 2021. The Author(s).)

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease, with no present cure. The progressive loss of MNs is the hallmark of ALS. We have previously shown the therapeutic effects of the phosphatase and tensin homolog (PTEN) inhibitor, potassium bisperoxo (picolinato) vanadium (bpV[pic]), in models of neurological injury and demonstrated significant neuroprotective effects on MN survival. However, accumulating evidence suggests PTEN is detrimental for MN survival in ALS. Therefore, we hypothesized that treating the mutant superoxide dismutase 1 G93A (mSOD1 G93A ) mouse model of ALS during motor neuron degeneration and an in vitro model of mSOD1 G93A motor neuron injury with bpV(pic) would prevent motor neuron loss. To test our hypothesis, we treated mSOD1 G93A mice intraperitoneally daily with 400 μg/kg bpV(pic) from 70 to 90 days of age. Immunolabeled MNs and microglial reactivity were analyzed in lumbar spinal cord tissue, and bpV(pic) treatment significantly ameliorated ventral horn motor neuron loss in mSOD1 G93A mice (p = 0.003) while not significantly altering microglial reactivity (p = 0.701). Treatment with bpV(pic) also significantly increased neuromuscular innervation (p = 0.018) but did not affect muscle atrophy. We also cultured motor neuron-like NSC-34 cells transfected with a plasmid to overexpress mutant SOD1 G93A and starved them in serum-free medium for 24 h with and without bpV(pic) and downstream inhibitor of Akt signaling, LY294002. In vitro, bpV(pic) improved neuronal viability, and Akt inhibition reversed this protective effect (p < 0.05). In conclusion, our study indicates systemic bpV(pic) treatment could be a valuable neuroprotective therapy for ALS., (© 2021. The Author(s).)

Background: Flagellated protozoan of the genus Leishmania is the causative agent of vector-borne parasitic diseases of leishmaniasis. Since the production of recombinant pharmaceutical proteins requires the cultivation of host cells in a serum-free medium, the elimination of FBS can improve the possibility of large-scale culture of Leishmania parasite. In the current study, we aimed at evaluating a new serum-free medium in Leishmania parasite culture for future live Leishmania vaccine purposes., Methods: Recombinant L. tarentolae secreting PpSP15-EGFP and wild type L. major were cultured in serum-free (complete serum-free medium [CSFM]) and serum-supplemented medium. The growth rate, protein expression, and infectivity of cultured parasites in both conditions was then evaluated and compared., Results: Diff-Quick staining and epi-fluores¬cence microscopy examination displayed the typical morphology of L. major and L. tarentolae-PpSP15-EGFP promastigote grown in CSFM medium. The amount of EGFP expression was similar in CSMF medium compared to M199 supplemented with 5% FBS in flow cytometry analysis of L. tarentolae-PpSP15-EGFP parasite. Also, a similar profile of PpSP15-EGFP proteins was recognized in Western blot analysis of L. tarentolae-PpSP15-EGFP cultured in CSMF and the serum-supplemented medium. Footpad swelling and parasite load measurements showed the ability of CSFM medium to support the L. major infectivity in BALB/C mice., Conclusion: This study demonstrated that CSFM can be a promising substitute for FBS supplemented medium in parasite culture for live vaccination purposes.

Human induced pluripotent stem cells (iPSCs) and macrophages derived from them are increasingly popular tools for research into both infectious and degenerative diseases. However, as the field strives for greater modeling accuracy, it is becoming ever more challenging to justify the use of undefined and proprietary media for the culture of these cells. Here, we describe a defined, serum-free, open-source medium for the differentiation of iPSC-derived macrophages. This medium is equally capable of maintaining these cells compared with commercial alternatives. The macrophages differentiated in this medium display improved terminally differentiated cell characteristics, reduced basal expression of induced antiviral response genes, and improved polarization capacity. We conclude that cells cultured in this medium are an appropriate and malleable model for tissue-resident macrophages, on which future differentiation techniques can be built., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The biopharmaceutical industry prefers to culture the mammalian cells in suspension with a serum-free media (SFM) due to improved productivity and process consistency. However, mammalian cells preferentially grow as adherent cells in a complete medium (CM) containing serum. Therefore, cells require adaptation from adherence in CM to suspension culture in SFM. This work proposes an adaptation method that includes media supplementation during the adaption of Chinese hamster ovary cells. As a result, the adaptation was accelerated compared to the traditional repetitive subculturing. Ca 2+ /Mg 2+ supplementation significantly reduced the doubling time compared to the adaptation without supplementation during the adaptation of adherent cells from 100% CM to 75% CM (p < 0.05). Furthermore, a definitive screening design (DSD) was applied to select essential nutrients during the adaptation from 10% CM to 0% CM. The main effects of Ca 2+ and Dulbecco's modified essential medium (DMEM) were found significant to both viable cell density and viability at harvest. Additionally, the interaction term between Ca 2+ and DMEM was found significant, which highlights the ability of DSD to capture interaction terms. Eventually, the media supplementation method resulted in adaptation SFM in 27 days, compared to the previously reported 66 days. Additionally, the membrane surface integrin expression was found significantly decreased when adherent cells were adapted to suspension. Moreover, the Ca 2+ /Mg 2+ supplementation correlated with faster integrin recovery after trypsinization. However, faster integrin recovery did not contribute to the accelerated cell growth when subculturing from 100% CM to 75% CM., (© 2021 American Institute of Chemical Engineers.)

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

Numerous studies have shown that hedgehog inhibitors (iHHs) only partially block the growth of tumor cells, especially in vivo. Leukemia often expands in a nutrient-depleted environment (bone marrow and thymus). In order to identify putative signaling pathways implicated in the adaptive response to metabolically adverse conditions, we executed quantitative phospho-proteomics in T-cell acute lymphoblastic leukemia (T-ALL) cells subjected to nutrient-depleted conditions (serum starvation). We found important modulations of peptides phosphorylated by critical signaling pathways including casein kinase, mammalian target of rapamycin, and 5'AMP-activated kinase (AMPK). Surprisingly, in T-ALL cells, AMPK signaling was the most consistently downregulated pathway under serum-depleted conditions, and this coincided with increased GLI1 expression and sensitivity to iHHs, especially the GLI1/2 inhibitor GANT-61. Increased sensitivity to GANT-61 was also found following genetic inactivation of the catalytic subunit of AMPK (AMPKα1) or pharmacological inhibition of AMPK by Compound C. Additionally, patient-derived xenografts showing high GLI1 expression lacked activated AMPK, suggesting an important role for this signaling pathway in regulating GLI1 protein levels. Further, joint targeting of HH and AMPK signaling pathways in T-ALL cells by GANT-61 and Compound C significantly increased the therapeutic response. Our results suggest that metabolic adaptation that occurs under nutrient starvation in T-ALL cells increases responsiveness to HH pathway inhibitors through an AMPK-dependent mechanism and that joint therapeutic targeting of AMPK signaling and HH signaling could represent a valid therapeutic strategy in rapidly expanding tumors where nutrient availability becomes limiting.

Osteoarthritis (OA) is the most common type of arthritis, afflicting millions of people in the world. Elevation of inflammatory mediators and enzymatic matrix destruction is often associated with OA. Therefore, the objective of this study was to investigate the effects of conditioned medium from periodontal ligament-derived stem cells (PDLSCs) on inflammatory and catabolic gene expressions of chondrocytes, synoviocytes, and meniscus cells under in vitro inflammatory condition. Stem cells were isolated from human periodontal ligaments. Conditioned medium was collected and concentrated 20 × . Chondrocytes, synoviocytes, and meniscus cells were isolated from pig knees and divided into four experimental groups: serum-free media, serum-free media+interleukin-1β (IL-1β) (10 ng/mL), conditioned media (CM), and CM+IL-1β. Protein content and extracellular vesicle (EV) miRNAs of CM were analyzed by liquid chromatography-tandem mass spectrometry and RNA sequencing, respectively. It was found that the IL-1β treatment upregulated the expression of IL-1β, tumor necrosis factor-α (TNF-α), MMP-13, and ADAMTS-4 genes in the three cell types, whereas PDLSC-conditioned medium prevented the upregulation of gene expression by IL-1β in all three cell types. This study also found that there was consistency in anti-inflammatory effects of PDLSC CM across donors and cell subcultures, while PDLSCs released several anti-inflammatory factors and EV miRNAs at high levels. OA has been suggested as an inflammatory disease in which all intrasynovial tissues are involved. PDLSC-conditioned medium is a cocktail of trophic factors and EV miRNAs that could mediate different inflammatory processes in various tissues in the joint. Introducing PDLSC-conditioned medium to osteoarthritic joints could be a potential treatment to prevent OA progression by inhibiting inflammation.

Acid bone lysates (ABLs) represent the growth factors and other molecules released during autologous graft resorption. However, the impact of these bone-derived growth factors on the healing of bone defects has not yet been investigated. The aim of the present study was, therefore, to examine the impact of ABLs adsorbed to collagen membranes on bone regeneration. To this end, in 16 female Sprague Dawley rats, a standardized 5-mm-diameter critical size defect on the calvarial bone was created. The defects were covered with collagen membranes that had been soaked either in serum-free media or ABLs followed by lyophilization. After a healing period of 4 weeks, micro-computed tomography (μCT) and histological analyses by means of undecalcified thin ground sections were performed. μCT analysis of the inner 4 mm of the calvaria defect showed a greater bone defect coverage in the control group when compared to ABL group, 29.8% (confidence interval [CI]: 17.7-50.3) versus 5.6% (CI: 1.0-29.8, p = .03), respectively. Moreover, we found significantly more absolute bone volume (BV) in the control group when compared to ABL group, 0.59 mm 3 (CI: 0.27-1.25) versus 0.07 mm 3 (CI: 0.06-0.59, p = .04), respectively. Histomorphometry confirmed these findings with a relative BV in the central compartment of 14.1% (CI: 8.4-20.6) versus 5.6% (CI: 3.4-7.9, p = .004), respectively. These findings indicate that bone-derived growth factors contained in ABLs are able to attenuate bone regeneration within collagen membranes., (© 2020 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Mesenchymal stem cells (MSCs) are of great interest for their use in cell-based therapies due to their multipotent differentiation and immunomodulatory capacities. In consequence of limited numbers following their isolation from the donor tissue, MSCs require extensive expansion performed in traditional 2D cell culture setups to reach adequate amounts for therapeutic use. However, prolonged culture of MSCs in vitro has been shown to decrease their differentiation potential and alter their immunomodulatory properties. For that reason, preservation of these physiological characteristics of MSCs throughout their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications. With this objective in mind, many studies already investigated certain parameters for enhancing current standard MSC culture protocols with regard to the effects of specific culture media components or culture conditions. Although there is a lot of diversity in the final therapeutic uses of the cells, the primary stage of standard isolation and expansion is imperative. Therefore, we want to review on approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion. The reviewed studies investigate and suggest improvements focused on culture media components (amino acids, ascorbic acid, glucose level, growth factors, lipids, platelet lysate, trace elements, serum, and xenogeneic components) as well as culture conditions and processes (hypoxia, cell seeding, and dissociation during passaging), in order to preserve the MSC phenotype and functionality during the primary phase of in vitro culture.

Parkinson's disease (PD) is a chronic neurodegenerative condition characterized by motor symptoms such as bradykinesia, resting tremor, and rigidity. PD diagnosis is based on medical history, review of signs, symptoms, neurological and physical examinations. Unfortunately, by the time the disease is diagnosed, dopamine (DA) neuronal loss is often extended, thereby resulting in ineffective therapies. Recent evidence suggests that neuroinflammation may be pivotal during PD onset and progression. However, suitable cellular models and biomarkers to detect early signs of neuroinflammation are still missing. In this study, we developed a well-differentiated DAergic neuronal cell line where we triggered a neuroinflammatory response to assess the temporal expression of the tissue- and urokinase plasminogen activators (tPA and uPA) and their endogenous inhibitor (PAI-1) along with that of pro-inflammatory mediators and the neuronal marker nNOS. Human neuroblastoma cells SH-SY5Y were differentiated into DAergic neuronal-like cells using a combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum depletion. Terminally-differentiated neurons were then exposed to lipopolysaccharide (LPS) for short (up to 24 h) or long term (up to 10 days) to mimic acute or chronic inflammation. Results demonstrated that uPA protein expression was stably upregulated during chronic inflammation, whereas the expression of nNOS protein better reflected the cellular response to acute inflammation. Additional studies revealed that the temporal induction of uPA was associated with increased AKT phosphorylation, but did not seem to involve cAMP-responsive element-binding protein (CREB) activation, nor the mitogen-activated protein kinase (MAPK) pathway. In conclusion, our in vitro data suggests that nNOS and uPA may serve as viable candidate biomarkers of acute and chronic neuroinflammation.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

Cell adhesion has tremendous impact on the function of culture platforms and implants. Cell-adhesive proteins and peptides have been extensively used for decades to promote cell adhesion, however, their application suffers from their easy enzymatic degradation, difficulty in large-scale preparation and expensiveness. To develop the next-generation cell-adhesive materials, we mimic the cell adhesion functions and mechanisms of RGD and KRSR peptides and design cell-adhesive cationic-hydrophobic amphiphilic β-amino acid polymers that are stable upon proteolysis and easily prepared in large scale at low cost. The optimal polymer strongly promotes cell adhesion, using preosteoblast cell as a model, by following dual mechanisms that are independent of sequence and chirality of the statistic copolymer. Our strategy opens avenues in designing the next-generation cell-adhesive materials and may guide future studies and applications.

Human in vitro hepatic models generate faster drug toxicity data with higher human predictability compared to animal models. However, for long-term studies, current models require the use of serum and 3D architecture, limiting their utility. Maintaining a functional long-term human in vitro hepatic culture that avoids complex structures and serum would improve the value of such systems for preclinical studies. This would also enable a more straightforward integration with current multi-organ devices to study human systemic toxicity to generate an alternative model to chronic animal evaluations. A human primary hepatocyte culture system was characterized for 28 days in 2D and serum-free defined conditions. Under the studied conditions, human primary hepatocytes maintained their characteristic morphology, hepatic markers and functions for 28 days. The acute and chronic administration of known drugs validated the sensitivity of the system for drug testing. This human 2D model represents a realistic system to evaluate hepatic function for long-term drug studies, without the need of animal serum, confounding variable in most models, and with less complexity and resultant cost compared to most 3D models. The defined culture conditions can easily be integrated into complex multi-organ in vitro models for studying systemic effects driven by the liver function for long-term evaluations., (© 2020 American Institute of Chemical Engineers.)

Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.

Background Aims: Mesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency., Methods: The authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics., Results: MSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications., Conclusions: The authors' data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products., (Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Aurora kinases despite their similarity have distinct roles in the cell cycle, which is regulated by cell-matrix adhesion and growth factors. This study reveals loss of adhesion and re-adhesion to differentially regulate Aurora kinases. AURKB activation that drops on the loss of adhesion recovers on re-adhesion in serumdeprived conditions but not in the presence of serum growth factors. A rapid 30 min serum treatment of serumdeprived cells blocks the adhesion-dependent recovery of AURKB, which negatively correlates with Erk activation. AZD mediated inhibition of AURKB in serum-deprived re-adherent cells promotes Erk activation and membrane ruffling, comparable to presence of serum. These studies thus define a novel adhesion-growth factor-dependent regulation of AURKB that controls adhesion-dependent Erk activation in re-adherent fibroblasts.

Human induced Pluripotent Stem Cell (hiPSC) models are a valuable new tool for research into neurodegenerative diseases. Neuroinflammation is now recognized as a key process in neurodegenerative disease and aging, and microglia are central players in this. A plethora of hiPSC-derived microglial models have been published recently to explore neuroinflammation, ranging from monoculture through to xenotransplantation. However, combining physiological relevance, reproducibility, and scalability into one model is still a challenge. We examine key features of the in vitro microglial environment, especially media composition, extracellular matrix, and co-culture, to identify areas for improvement in current hiPSC-microglia models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Hedegaard, Stodolak, James and Cowley.)

GOLPH3, an oncoprotein, plays crucial roles in tumor etiology. Compelling evidences have demonstrated that GOLPH3 contributes to regulate tumor cell growth, migration and invasion under normal nutrient condition. However, the oncogenic activity of GOLPH3 under serum starvation remains largely unknown. In this study, we reported that GOLPH3 depletion led to marked reduction in adhesion of glioma U251 cells, particularly under serum deprivation. We found that silencing of GOLPH3 expression reduced the protein amount of ITGB1 only in serum-free medium. Further insights into the mechanism between GOLPH3 and ITGB1, we applied proteasome or lysosome inhibitor to block the degradation of ITGB1, and identified GOLPH3 silencing can prompt ITGB1 lysosomal degradation under serum starvation. Finally, we found the reductions in glioma cell adhesion and ITGB1 protein amount could be rescued by ITGB1 overexpression. Taken together, these results show that GOLPH3 contributes to the adhesion of glioma cells by regulating the lysosomal degradation of ITGB1 under serum starvation., Competing Interests: Declaration of competing interest We have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Perinatal stem cells have been regarded as an attractive and available cell source for medical research and clinical trials in recent years. Multiple stem cell types have been identified in the human placenta. Recent advances in knowledge on placental stem cells have revealed that human amniotic epithelial stem cells (hAESCs) have obvious advantages and can be used as a novel potential cell source for cellular therapy and clinical application. hAESCs are known to possess stem-cell-like plasticity, immune-privilege, and paracrine properties. In addition, non-tumorigenicity and a lack of ethical concerns are two major advantages compared with embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). All of the characteristics mentioned above and other additional advantages, including easy accessibility and a non-invasive application procedure, make hAESCs a potential ideal cell type for use in both research and regenerative medicine in the near future. This review article summarizes current knowledge on the characteristics, therapeutic potential, clinical advances and future challenges of hAESCs in detail.

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application., (Copyright © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Human mesenchymal stromal cells (hMSCs) have enormous potential for the treatment of various inflammatory and degenerative diseases. Their manufacturing for cell-based therapies requires extensive ex vivo expansion and optimal growth conditions. To support cell adhesion, spreading, and growth in serum-free culture conditions, the applied plasticware needs to be functionalized with essential biochemical cues. By employing a recently developed screening tool, a chemically defined functional matrix composed of dextran sulfate and a bone-related extracellular matrix peptide is identified, which supports long-term culture of bone marrow-derived hMSCs in serum-free culture conditions. Cells grown under these conditions display rapid proliferation and high viability while maintaining their differentiation and immunomodulatory capacity, characteristic cell morphology, expression of hMSC-specific surface antigens as well as important markers of stemness and differentiation potential. The chemically defined, serum-free culture environment enables reliable and reproducible expansion of hMSCs important for cell based-therapies, drug screening, and disease modeling., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Stem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.

Background: Optimal expansion of therapeutic natural killer (NK) cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-cell expansion, but few SFM systems have been developed for NK cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum-containing medium for their ability to support NK cell expansion and function., Methods: Human peripheral blood NK cells were expanded in selected media by recursive weekly stimulation with K562-based feeder cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts., Results: TexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production., Discussion: The AIM V + ICSR SFM system supports excellent ex vivo expansion of clinical-grade NK cells with the phenotype and function needed for adoptive immunotherapy., (Copyright © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

While reliable transfection methods are essential for Chinese hamster ovary (CHO) cell line engineering, reduced transfection efficiencies have been observed in several commercially prepared media. In this study, we aimed to assess common media additives that impede efficiency mediated by three chemical transfection agents: liposomal-based (Lipofectamine 2000), polymer-based (TransIT-X2), and lipopolyplex-based (TransIT-PRO). An in-house GFP-expressing vector and serum-free medium (BCR-F12: developed for the purposes of this study) were used to analyze transient transfection efficiencies of three CHO cell lines (CHO-K1, DG44, DP12). Compared to a selection of commercially available media, BCR-F12 displayed challenges associated with transfection in vendor-prepared formulations, with no detection when liposomal-based methods were used, reduced (<3%) efficiency observed when polymer-based methods were used and only limited efficiency (25%) with lipopolyplexes. Following a stepwise removal protocol, ferric ammonium citrate (FAC) was identified as the critical factor impeding transfection, with transfection enabled with the liposomal- and polymer-based methods and a 1.3- to 7-fold increased lipopolyplex efficiency observed in all cell lines in FAC-depleted media (-FAC), although lower viabilities were observed. Subsequent early addition of FAC (0.5-5 hr post-transfection) revealed 0.5 hr post-transfection as the optimal time to supplement in order to achieve transfection efficiencies similar to -FAC medium while retaining optimal cellular viabilities. In conclusion, FAC was observed to interfere with DNA transfection acting at early stages in all transfection agents and all cell lines studied, and a practical strategy to circumvent this problem is suggested., (© 2019 American Institute of Chemical Engineers.)

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC., (© 2019 American Institute of Chemical Engineers.)

Natural killer (NK) cells are innate lymphocytes with functions that include target cell killing, inflammation and regulation. NK cells integrate incoming activating and inhibitory signals through an array of germline-encoded receptors to gauge the health of neighbouring cells. The reactive potential of NK cells is influenced by microRNA (miRNA), small non-coding sequences that interfere with mRNA expression. miRNAs are highly conserved between species, and a single miRNA can have hundreds to thousands of targets and influence entire cellular programs. Two miRNA species, miR-155-5p and miR-146a-5p are known to be important in controlling NK cell function, but research to best understand the impacts of miRNA species within NK cells has been bottlenecked by a lack of techniques for altering miRNA concentrations efficiently and without off-target effects. Here, we describe a non-viral and straightforward approach for increasing or decreasing expression of miRNA in primary human NK cells. We achieve >90% transfection efficiency without off-target impacts on NK cell viability, education, phenotype or function. This opens the opportunity to study and manipulate NK cell miRNA profiles and their impacts on NK cellular programs which may influence outcomes of cancer, inflammation and autoimmunity., Competing Interests: The authors declare that no competing interests exist.

Previously, we have shown platelet lysate (PL) can be used as a non-xenogeneic serum supplement for generation of monocyte-derived dendritic cells (DCs). Since DC-based activation protocols are extremely sensitive to microenvironmental changes such as replacement of culture medium, we wanted to examine the behavior of DCs cultured in the presence of PL under various type-1 activation conditions and assess their type 1 polarization capacity. We compared the quality of DCs cultured in 10% PL-supplemented RPMI medium (plDCs) with clinical-grade DCs obtained using commercially available serum-free medium (sfDCs), frequently used in established DC vaccine protocols. The DC maturation protocols consisted of either monophosphoryl lipid A/IFN-γ, poly I:C/TNF-α/IFN-α or poly I:C/R848. In general, plDCs were inferior to sfDCs in most aspects of their functional type 1 polarization characteristics. After maturation, the expression of co-stimulatory, HLA class II and lymph node-homing molecules was strongly up-regulated, with some noticeable differences. The expression of CD80 and CD86 was more extensive on plDCs, which was particularly evident in case of CCR7. However, after observing their functional capacity, plDCs had significantly lower allo-stimulatory capacity both in terms of CD4 + and CD8 + T cell stimulation. The high expression of CCR7 corresponded to higher CCL-19 directed DC migration of plDCs compared to sfDCs. Finally, their capacity to induce granzyme B and IFN-γ production in CD8 + T cells was significantly reduced in comparison to sfDCs. Based on these findings, the use of PL as an alternative serum supplement for generation of monocyte-derived DC anti-tumor vaccines is questionable. Abbreviations : Ag: antigen; CCL: chemokine ligand; CCR: chemokine receptor; DC: dendritic cells; DC-SIGN: dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; FBS: fetal bovine serum; GMP: good manufacturing practice; IFN: interferon; IL: interleukin; MPLA: monophosphoryl lipid A; PGE: prostaglandin E; pI:C: polyinosinic:polycytidylic acid; pl: platelet lysate; sf: serum free; TLR: toll-like receptor; TNF: tumor necrosis factor.

Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification-OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes ( NANOG and OCT-4 ) and increased levels of expression in osteogenic genes ( ALP, BSP, OCN and RUNX2 ). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The most significant cost driver for efficient bio-production of edible animal proteins is the cell culture media, where growth factors account for up to 96% of the total cost. The culture media must be serum-free, affordable, contain only food-grade ingredients, be efficient to promote cell growth and available in massive quantities. The commercially available serum substitutes are expensive and not necessarily food-grade. Identifying inexpensive food-safe alternatives to serum is crucial. By-products from food production are available in massive quantities, contain potential factors that can promote growth and are promising ingredients for serum replacement. The main goal of this study was to explore if food-grade by-product materials can be used as growth promoting agents in skeletal muscle cell culture to develop a tailor-made serum free media. Different by-products, including chicken carcass, cod backbone, eggshell membrane, egg white powder and pork plasma were enzymatically or chemically hydrolyzed. The hydrolysates in addition to lyophilized pork plasma and yeast extract were further characterized by size-exclusion chromatography, elemental combustion analysis and degree of hydrolysis. The materials were used as supplement to or replacement of commercial serum and further evaluated for their effect on metabolic activity, cell proliferation and cell cytotoxicity in muscle cells cultured in vitro. Our results indicate that none of the materials were cytotoxic to the skeletal muscle cells. Hydrolysates rich in peptides with approximately 2-15 amino acids in length were shown to improve cell growth and metabolic activity. Of all the materials tested pork plasma hydrolysates and yeast extract were the most promising. Pork plasma hydrolysates increased metabolic activity by 110% and cell proliferation with 48% when cultured in serum-free conditions for 3 days compared with control cells cultured with full serum conditions. Most interestingly, this response was dependent on both material and choice of enzyme used. We suggest that these materials have the potential to replace serum during cultivation and as such be included in a tailor-made serum-free media.

Introduction: The olfactory neuro-epithelium has an intrinsic capability of renewal during lifetime provided by the existence of globose and horizontal olfactory precursor cells. Additionally, mesenchymal stromal olfactory cells also support the homeostasis of the olfactory mucosa cell population. Under in vitro culture conditions with Dulbecco modified eagle/F12 medium supplemented with 10% fetal bovine serum, tissue biopsies from upper turbinate have generated an adherent population of cells expressing mainly mesenchymal stromal phenotypic markers. A closer examination of these cells has also found co-expression of olfactory precursors and ensheathing cell phenotypic markers. These results were suggestive of a unique property of olfactory mesenchymal stromal cells as potentially olfactory progenitor cells., Objective: To study whether the expression of these proteins in mesenchymal stromal cells is modulated upon neuronal differentiation., Materials and Methods: We observed the phenotype of olfactory stromal cells under DMEM/F12 plus 10% fetal bovine serum in comparison to cells from spheres induced by serum-free medium plus growth factors inducers of neural progenitors., Results: The expression of mesenchymal stromal (CD29+, CD73+, CD90+, CD45-), horizontal basal (ICAM-1/CD54+, p63+, p75NGFr+), and ensheathing progenitor cell (nestin+, GFAP+) proteins was determined in the cultured population by flow cytometry. The determination of Oct 3/4, Sox-2, and Mash-1 transcription factors, as well as the neurotrophins BDNF, NT3, and NT4 by RT-PCR in cells, was indicative of functional heterogeneity of the olfactory mucosa tissue sample., Conclusions: Mesenchymal and olfactory precursor proteins were downregulated by serum-free medium and promoted differentiation of mesenchymal stromal cells into neurons and astroglial cells.

Using stem and progenitor cells to treat retinal disorders holds great promise. Using defined culture conditions to maintain the desires phenotype is of utmost clinical importance. We cultured human retinal progenitor cells (hRPCs) in different conditions: such as normoxia (20% oxygen), and hypoxia (5% oxygen) with and without knock-out serum replacement (KOSR) to evaluate its effect on these cells. KOSR is known nutrient supplement often used to replace bovine serum for culturing embryonic or pluripotent stem cells, especially those destined for clinical applications. The purpose of this study was to identify the impact of different environmental and chemical cues to determine if this alters the fate of these cells. Our results indicate that cells cultured with or without KOSR do not show significant differences in viability, but that the oxygen tension can significantly change their viability (higher in hypoxia than normoxia). However, cells with KOSR in hypoxia condition expressed significantly higher stemness markers such as C-myc and Oct4 (31.20% and 13.44% respectively) in comparison to hRPCs cultured in KOSR at normoxia (12.07% and 4.05%). Furthermore, levels of markers for retinal commitment such as rhodopsin were significantly lower in the KOSR supplemented cells in hypoxia culture compared to normoxia. KOSR is known to improve proliferation and maintain stemness of embryonic cells and our experiments suggest that hRPCs maintain their proliferation and stemness characteristics in hypoxia with KOSR supplement. Normoxia, however, results in mature cell marker expression, suggesting a profound effect of oxygen tension on these cells.

Donor cornea contamination is one of the major risks for corneal transplants. The use of antibiotics in storage media remains as one of the most important security measurements to minimize the contamination risk in corneal preservation. Since antibiotic resistance among microorganisms have been rising gradually, it is important to gain knowledge about the antimicrobial susceptibility pattern for choosing the most suitable antimicrobial agents. Thus, we evaluated the in vitro susceptibility of microorganisms isolated in donor corneas processed at the Center for Blood Transfusion, Tissues and Cells (Córdoba, Spain) during 4 years in order to evaluate the efficiency, and to promote changes for further antibiotics use. Our results show the high rate of resistance to gentamicin, an antibiotic used in corneal preservation media such as Optisol GS and Eusol-C. Conversely, all the analyzed microorganisms were sensitive to vancomycin. This suggests the possibility of replacing gentamicin with another more effective antibacterial agent such as vancomycin.

Brown adipocytes coordinate systemic energy metabolism associated with the pathogenesis of obesity and related metabolic diseases including type 2 diabetes. We have previously reported chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts without using transgenes. In this study, to reveal a precise molecular mechanism underlying the direct conversion and human adipocyte browning, we developed serum-free brown adipogenic medium (SFBAM) with an optimized chemical cocktail consisting of Rosiglitazone, Forskolin, and BMP7. During the direct conversion, treatment with BMP7 enhanced Ucp1 expression rather than the conversion efficiency in the absence of BMP signalling inhibitors. Moreover, treatment with a TGF-β signalling pathway inhibitor was no longer required in the serum-free medium, likely because the TGF-β pathway was already suppressed. SFBAM and the chemical cocktail efficiently converted human dermal fibroblasts into ciBAs within four weeks. The ciBAs exhibited increased mitochondrial levels, elevated oxygen consumption rate, and a response to β-adrenergic receptor agonists. Thus the ciBAs converted by the serum-free medium and the chemical cocktail provide a novel model of human brown (beige) adipocytes applicable for basic research, drug screening, and clinical applications.