Hypoxic conditions for expansion of hMSC; selected studies from 2007-2020. Information is given on the cell source, exposure to hypoxic conditions and on stem cell characteristics.

Hypoxic conditions for expansion of hMSC; selected studies from 2007-2020. Information is given on the cell source, exposure to hypoxic conditions and on stem cell characteristics.

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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 amou...

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... a general improvement of cell yield under hypoxic conditions can be concluded (see Table 5) [61,113,[169][170][171][172][173][174][175][176][177][178][179][180][181], some research groups could not confirm the beneficial effects of hypoxic culture conditions [182][183][184][185]. This might also be caused by high variations within the applied parameters. ...

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... Furthermore, approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion are required. Several studies suggest some improvements in 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 MSC phenotypes and functionality during the primary phase of in vitro culture [16]. Collectively, this Special Issue, managed and supervised by Dr. Mehdi Najar, successfully gathers a great collection of research articles and reviews highlighting recent fundamental and applied advances in different types of stem cells. ...
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We are pleased to present this Special Issue of Cells, entitled ‘Feature Papers in Stem Cells’ [...]
... It has been shown that MSCs secrete crucial anti-and proinflammatory factors (PGE2, Indoleamine 2,3-dioxygenase, TGFβ1, TSG-6, HGF, IL-10, IL-6, IFNγ, TNF-α, LIF), which, depending on their ratio, regulate the pro-or anti-inflammatory activity of MSCs. In addition, final immunoregulatory properties may be influenced by cell culture conditions that can prime/enhance MSCs' properties [21,84,[102][103][104][105][106]. ...
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Lung transplantation (LTx) has become the gold standard treatment for end-stage respiratory failure. Recently, extended lung donor criteria have been applied to decrease the mortality rate of patients on the waiting list. Moreover, ex vivo lung perfusion (EVLP) has been used to improve the number/quality of previously unacceptable lungs. Despite the above-mentioned progress, the morbidity/mortality of LTx remains high compared to other solid organ transplants. Lungs are particularly susceptible to ischemia-reperfusion injury, which can lead to graft dysfunction. Therefore, the success of LTx is related to the quality/function of the graft, and EVLP represents an opportunity to protect/regenerate the lungs before transplantation. Increasing evidence supports the use of mesenchymal stromal/stem cells (MSCs) as a therapeutic strategy to improve EVLP. The therapeutic properties of MSC are partially mediated by secreted factors. Hence, the strategy of lung perfusion with MSCs and/or their products pave the way for a new innovative approach that further increases the potential for the use of EVLP. This article provides an overview of experimental, preclinical and clinical studies supporting the application of MSCs to improve EVLP, the ultimate goal being efficient organ reconditioning in order to expand the donor lung pool and to improve transplant outcomes.
... In standard culture conditions, MSCs need the optimal density and cell contacts; thus, the separation by the hydrogel is not their natural state that would increase the proliferation [36]. Three dimensional (3D) cell culture can influence MSC proliferation; however, the positive or negative effect on cell division largely depends on applied cell culture protocols [37,38]. ...
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Cell transplantation has been studied extensively as a therapeutic strategy for neurological disorders. However, to date, its effectiveness remains unsatisfactory due to low precision and efficacy of cell delivery; poor survival of transplanted cells; and inadequate monitoring of their fate in vivo. Fortunately, different bio-scaffolds have been proposed as cell carriers to improve the accuracy of cell delivery, survival, differentiation, and controlled release of embedded stem cells. The goal of our study was to establish hydrogel scaffolds suitable for stem cell delivery that also allow non-invasive magnetic resonance imaging (MRI). We focused on alginate-based hydrogels due to their natural origin, biocompatibility, resemblance to the extracellular matrix, and easy manipulation of gelation processes. We optimized the properties of alginate-based hydrogels, turning them into suitable carriers for transplanted cells. Human adipose-derived stem cells embedded in these hydrogels survived for at least 14 days in vitro. Alginate-based hydrogels were also modified successfully to allow their injectability via a needle. Finally, supplementing alginate hydrogels with Mn ions or Mn nanoparticles allowed for their visualization in vivo using manganese-enhanced MRI. We demonstrated that modified alginate-based hydrogels can support therapeutic cells as MRI-detectable matrices.
... Regardless of promising results from preclinical and clinical studies of the simple administration of naïve MSCs, conflicting outcomes regarding their clinical benefits have been reported in some clinical trials (Herreros et al., 2012;Weiss et al., 2013) necessitating the need for a more developed understanding of molecular and cellular mechanisms that affect clinical efficacy of these stem cells. As therapeutic effectiveness of MSCs can be influenced by a number of factors such as the donor, harvest site, and how they are expanded in vitro (Choudhery et al., 2014;Heo et al., 2016;Nikolits et al., 2021), there have been efforts to strengthen or fine-tune their therapeutic effects for certain diseases through physical, chemical, or genetic modulation. A recent study reported that ectopic expression of Nanog could restore myogenic differentiation potential of MSCs (Han et al., 2012). ...
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Mesenchymal stem cells (MSCs) are recognized as potential treatments for multiple degenerative and inflammatory disorders as a number of animal and human studies have indicated their therapeutic effects. There are also several clinically approved medicinal products that are manufactured using these cells. For such large-scale manufacturing requirements, the in vitro expansion of harvested MSCs is essential. Multiple subculturing of MSCs, however, provokes cellular senescence processes which is known to deteriorate the therapeutic efficacy of the cells. Strategies to rejuvenate or selectively remove senescent MSCs are therefore highly desirable for fostering future clinical applications of these cells. In this present study, we investigated gene expression changes related to cellular senescence of MSCs derived from umbilical cord blood and found that CD26, also known as DPP4, is significantly upregulated upon cellular aging. We further observed that the inhibition of CD26 by genetic or pharmacologic means delayed the cellular aging of MSCs with their multiple passaging in culture. Moreover, the sorting and exclusion of CD26-positive MSCs from heterogenous cell population enhanced in vitro cell attachment and reduced senescence-associated cytokine secretion. CD26-negative MSCs also showed superior therapeutic efficacy in mouse lung emphysema model. Our present results collectively suggest CD26 is a potential novel target for the rejuvenation of senescent MSCs for their use in manufacturing MSC-based applications.
... Moreover, with ubiquitous culture conditions such as oxygen and glucose availability, which are vital for general cellular behavior and viability [49], demonstrating a vast difference, the impact might be even more pronounced for specific parameters for development, differentiation, inflammation, or wound healing. Besides specific tissue models and applications, the monitoring platform can be utilized to improve general culture conditions of 3D cell constructs investigating the availability of oxygen, pH, glucose, lactate, but also of amino acids, lipids, growth factors, trace elements and serum, or platelet lysates [50]. ...
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The use of 3D cell cultures has gained increasing importance in medical and pharmaceutical research. However, the analysis of the culture medium is hardly representative for the culture conditions within a 3D model which hinders the standardization of 3D cultures and translation of results. Therefore, we developed a modular monitoring platform combining a perfusion bioreactor with an integrated minimally invasive sampling system and implemented sensors that enables the online monitoring of culture parameters and medium compounds within 3D cultures. As a proof-of-concept, primary cells as well as cell lines were cultured on a collagen or gelatin methacryloyl (GelMA) hydrogel matrix, while monitoring relevant culture parameters and analytes. Comparing the interstitial fluid of the 3D models versus the corresponding culture medium, we found considerable differences in the concentrations of several analytes. These results clearly demonstrate that analyses of the culture medium only are not relevant for the development of standardized 3D culture processes. The presented bioreactor with an integrated sampling and sensor platform opens new horizons for the development, optimization, and standardization of 3D cultures. Furthermore, this technology holds the potential to reduce animal studies and improve the transferability of pharmaceutical in vitro studies by gaining more relevant results, bridging the gap towards clinical translation.
... Of interest, it has been demonstrated that BMSCs expanded in these 3D culture systems produce a significantly larger amount of EVs [35,36,47], although the effects of bioprocess forces such as shear stress generated during 3D culture on the EV quality have not been fully elucidated [30,37]. In addition to culture systems, evolution has been made to culture media to allow rapid expansion of BMSCs without xenogeneic supplements optimized for clinical use [48]. These HPMs support the rapid expansion of BMSCs both in 2D and 3D culture systems. ...
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Extracellular vesicles (EVs) released by bone marrow stromal cells (BMSCs) have been shown to act as a transporter of bioactive molecules such as RNAs and proteins in the therapeutic actions of BMSCs in various diseases. Although EV therapy holds great promise to be a safer cell-free therapy overcoming issues related to cell therapy, manufacturing processes that offer scalable and reproducible EV production have not been established. Robust and scalable BMSC manufacturing methods have been shown to enhance EV production; however, the effects on EV quality remain less studied. Here, using human BMSCs isolated from nine healthy donors, we examined the effects of high-performance culture media that can rapidly expand BMSCs on EV production and quality in comparison with the conventional culture medium. We found significantly increased EV production from BMSCs cultured in the high-performance media without altering their multipotency and immunophenotypes. RNA sequencing revealed that RNA contents in EVs from high-performance media were significantly reduced with altered profiles of microRNA enriched in those related to cellular growth and proliferation in the pathway analysis. Given that pre-clinical studies at the laboratory scale often use the conventional medium, these findings could account for the discrepancy in outcomes between pre-clinical and clinical studies. Therefore, this study highlights the importance of selecting proper culture conditions for scalable and reproducible EV manufacturing.
... Our group was one of the firsts to describe that culturing MSC in hypoxia (with 5% O2) improved MSC proliferation and expansion [12]. Several studies have also reported that hypoxia preconditioning enhances the therapeutic potential of MSC [34][35][36][37][38]. However, maintaining in vitro the hypoxic conditions that MSC have physiologically in the human bone marrow niche is difficult to preserve only by regulating culture conditions, since the exposure to a normoxic environment rapidly reverses the gained features induced by hypoxia. ...
... However, maintaining in vitro the hypoxic conditions that MSC have physiologically in the human bone marrow niche is difficult to preserve only by regulating culture conditions, since the exposure to a normoxic environment rapidly reverses the gained features induced by hypoxia. There is extensive information about the key role of HIF-1α regulating the hypoxic metabolism [35,37,39,40]. That is why in the current work, we have opted by overexpressing HIF-1α in MSC by lentiviral transduction in order to mimic some of the effects induced by hypoxia and maintain those effects over time. ...
... Delving further into the mechanisms involved in the changes induced in MSC by low oxygen concentrations, it has been shown that the most remarkable is the increase in HIF-1α expression, promoting their regenerative and angiogenic capacity, activating Notch downstream genes, increasing cell proliferation and survival and the retention of stem cell properties as self-renewal and senescence inhibition [35,[37][38][39]41]. In previous works, we have observed that most of these changes are also induced when transducing MSC with HIF-1α lentivirus vectors [17,[20][21][22]. ...
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Background Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34⁺ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo. Methods Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34⁺ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34⁺ cells. In addition, CD34⁺ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34⁺ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice. Results We did not observe significant differences in viability, cell cycle and ROS expression between CD34⁺ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34⁺ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34⁺ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34⁺ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34⁺ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively). Conclusions Co-transplantation of human CD34⁺ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.
... To increase the relevance of data from basic research and develop robust and safe large-scale manufacturing processes, it is essential to establish physiologic culture conditions throughout the entire phase of ex vivo culture, including isolation, expansion and eventually differentiation of MSCs. Although the standard protocols for 2D isolation and expansion can be further optimized towards more physiologic culture conditions [15], true physiological conditions can only be achieved by incorporating 3D culture, dynamic culture systems, and physiologic oxygen conditions ( Figure 1). TA B L E 1 Impact of 3D isolation on the functionality of MSCs Bone marrow aspirate • Lower yield compared to 2D explant isolation • Higher clonogenicity and proliferation after bioreactor culture • Upregulation of stemness genes [33] The aim of this review is to present novel concepts for the physiologic isolation and expansion of MSCs to maintain or increase therapeutically relevant functionalities. ...
Article
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The utilization of mesenchymal stem/stromal cells raises new hopes in treatment of diseases and pathological conditions, while at the same time bringing immense challenges for researchers, manufacturers and physicians. It is essential to consider all steps along the in vitro fabrication of cell‐based products in order to reach efficient and reproducible treatment outcomes. Here, the optimal protocols for isolation, cultivation and differentiation of mesenchymal stem cells are required. In this review we discuss these aspects and their influence on the final cell‐based product quality. We demonstrate that physiological in vitro cell cultivation conditions play a crucial role in therapeutic functionalities of cultivated cells. We show that three‐dimensional cell culture, dynamic culture conditions and physiologically relevant in vitro oxygen concentrations during isolation and expansion make a decisive contribution towards the improvement of cell‐based products in regenerative medicine.
... Furthermore, the preservation of the physiological characteristics of MSCs during their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications, as recently reviewed [39]. In view of this document, there is considerable research focus on the optimization of specific culture media, culture conditions, and protocols for MSCs. ...
Article
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Foreskin, considered a biological waste material, has been shown to be a reservoir of therapeutic cells. The immunomodulatory properties of mesenchymal stromal/stem cells (MSCs) from the foreskin (FSK-MSCs) are being evaluated in cell-based therapy for degenerative, inflammatory and autoimmune disorders. Within the injured/inflamed tissue, proinflammatory lymphocytes such as IL-17-producing T helper cells (Th17) may interact with the stromal microenvironment, including MSCs. In this context, MSCs may encounter different levels of T cells as well as specific inflammatory signals. Uncovering the cellular and molecular changes during this interplay is central for developing an efficient and safe immunotherapeutic tool. To this end, an in vitro human model of cocultures of FSK-MSCs and T cells was established. These cocultures were performed at different cell ratios in the presence of an inflammatory setting. After confirming that FSK-MSCs respond to ISCT criteria by showing a typical phenotype and multilineage potential, we evaluated by flow cytometry the expression of Th17 cell markers IL-17A, IL23 receptor and RORγt within the lymphocyte population. We also measured 15 human Th17 pathway-related cytokines. Regardless of the T cell/MSC ratio, we observed a significant increase in IL-17A expression associated with an increase in IL-23 receptor expression. Furthermore, we observed substantial modulation of IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40, and TNF-α secretion. These findings suggest that FSK-MSCs are receptive to their environment and modulate the T cell response accordingly. The changes within the secretome of the stromal and immune environment are likely relevant for the therapeutic effect of MSCs. FSK-MSCs represent a valuable cellular product for immunotherapeutic purposes that needs to be further clarified and developed.
... To date, 10% FCS continues to be the most common media supplement employed in clinical trials for kidney disease, although some consideration has been given to the use of xenogeneic-free media such as hPL (5 or 10%), human serum albumin (HSA) and a CDM ( Table 2). Currently, some commercial and non-commercial CDM formulations have been investigated (359)(360)(361)(362)(363), however, there is still limited availability of some of these media for large-scale manufacturing at GMP quality level. ...
Article
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Advanced therapy medicinal products (ATMPs) offer new prospects to improve the treatment of conditions with unmet medical needs. Kidney diseases are a current major health concern with an increasing global prevalence. Chronic renal failure appears after many years of impairment, which opens a temporary window to apply novel therapeutic approaches to delay or halt disease progression. The immunomodulatory, anti-inflammatory, and pro-regenerative properties of mesenchymal stromal cells (MSCs) have sparked interest for their use in cell-based regenerative therapies. Currently, several early-phase clinical trials have been completed and many are ongoing to explore MSC safety and efficacy in a wide range of nephropathies. However, one of the current roadblocks to the clinical translation of MSC therapies relates to the lack of standardization and harmonization of MSC manufacturing protocols, which currently hinders inter-study comparability. Studies have shown that cell culture processing variables can have significant effects on MSC phenotype and functionality, and these are highly variable across laboratories. In addition, heterogeneity within MSC populations is another obstacle. Furthermore, MSCs may be isolated from several sources which adds another variable to the comparative assessment of outcomes. There is now a growing body of literature highlighting unique and distinctive properties of MSCs according to the tissue origin, and that characteristics such as donor, age, sex and underlying medical conditions may alter the therapeutic effect of MSCs. These variables must be taken into consideration when developing a cell therapy product. Having an optimal scale-up strategy for MSC manufacturing is critical for ensuring product quality while minimizing costs and time of production, as well as avoiding potential risks. Ideally, optimal scale-up strategies must be carefully considered and identified during the early stages of development, as making changes later in the bioprocess workflow will require re-optimization and validation, which may have a significant long-term impact on the cost of the therapy. This article provides a summary of important cell culture processing variables to consider in the scale-up of MSC manufacturing as well as giving a comprehensive review of tissue of origin-specific biological characteristics of MSCs and their use in current clinical trials in a range of renal pathologies.