Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system

The need for efficient and reliable technologies for clinical‐scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood‐derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10⁷ cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10⁸ cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra‐Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier‐based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical‐scale production system. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 568–572, 2013     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Monitoring the ex‐vivo expansion of human mesenchymal stem/stromal cells in xeno‐free microcarrier‐based reactor systems by MIR spectroscopy

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell‐based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex‐vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi‐analytical methods, some of them time‐consuming. The present work evaluates the use of mid‐infrared (MIR) spectroscopy, through rapid and economic high‐throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno‐free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R² ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R² of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:447–455, 2016     

Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.     

Periodic harvesting of embryonic stem cells from a hollow‐fiber membrane based four‐compartment bioreactor

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale‐up of stem cell culture is necessary. Bioreactors for dynamic three‐dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow‐fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10⁶ mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10⁶ mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four‐compartment bioreactor including further cultivation of remaining cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:141–151, 2016     

Expansion,harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot‐sizes required for commercial production. The use of animal‐derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot‐to‐lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large‐scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum‐free hMSC manufacturing process. Human bone‐marrow derived hMSCs were expanded on fibronectin‐coated, non‐porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 10⁵ cells.mL⁻¹ in serum‐free medium. The hMSCs were successfully harvested by our recently‐developed technique using animal‐free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post‐harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony‐forming potential. The hMSCs were held in suspension post‐harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum‐free vehicle solution using a controlled‐rate freezing process. Post‐thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component‐free hMSC production process from expansion through to cryopreservation. Biotechnol. Bioeng. 2015;112: 1696–1707. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

Neurotrophic and neuroprotective potential of human limbus-derived mesenchymal stromal cells

Background aimsThe purpose of this study was to examine neurotrophic and neuroprotective effects of limbus stroma-derived mesenchymal stromal cells (L-MSCs) on cortical neurons in vitro and in vivo.MethodsCultured L-MSCs were characterized by flow cytometry and immunofluorescence through the use of specific MSC marker antibodies. Conditioned media were collected from normoxia- and hypoxia-treated L-MSCs to assess neurotrophic effects. Neuroprotective potentials were evaluated through the use of in vitro hypoxic cortical neuron culture and in vivo rat focal cerebral ischemia models. Neuronal morphology was confirmed by immunofluorescence with the use of anti-MAP2 antibody. Post-ischemic infarct volume and motor behavior were assayed by means of triphenyltetrazolium chloride staining and open-field testing, respectively. Human growth antibody arrays and enzyme-linked immunoassays were used to analyze trophic/growth factors contained in conditioned media.ResultsIsolated human L-MSCs highly expressed CD29, CD90 and CD105 but not CD34 and CD45. Mesenchymal lineage cell surface expression pattern and differentiation capacity were identical to MSCs derived form human bone marrow and adipose tissue. The L-MSC normoxic and hypoxic conditioned media both promoted neurite outgrowth in cultured cortical neurons. Hypoxic conditioned medium showed superior neurotrophic function and neuroprotective potential with reduced ischemic brain injury and improved functional recovery in rat focal cerebral ischemia models. Human growth factor arrays and enzyme-linked immunoassays measurements showed neuroprotective and growth-associated cytokines (vascular endothelial growth factor [VEGF], VEGFR3, brain-derived neurotrophic factor, insulin-like growth factor -2 and hepatocyte growth factor) contained in conditioned media. Hypoxic exposure caused VEGF and brain-derived neurotrophic factor upregulation, possibly contributing to neurotrophic and neuroprotective effects.ConclusionsL-MSCs can secrete various neurotrophic factors stimulating neurite outgrowth and protecting neurons against brain ischemic injury through paracrine mechanism.     

Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System

BackgroundThe use of bone marrow–derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohn's disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times.MethodsWe evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials.ResultsAfter only two passages, we were able to expand a mean of 6.6 × 10⁸ MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10⁸ cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active.ConclusionsThe Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures.     

Multiomics characterization of mesenchymal stromal cells cultured in monolayer and as aggregates

Mesenchymal stromal cells (MSCs) have failed to consistently demonstrate their therapeutic efficacy in clinical trials, due in part to variability in culture conditions used for their production. Of various culture conditions used for MSC production, aggregate culture has been shown to improve secretory capacity (a putative mechanism of action in vivo) compared with standard monolayer culture. The purpose of this study was to perform multiomics characterization of MSCs cultured in monolayer and as aggregates to identify aspects of cell physiology that differ between these culture conditions to begin to understand cellular-level changes that might be related to secretory capacity. Targeted secretome characterization was performed on multiple batches of MSC-conditioned media, while nontargeted proteome and metabolome characterization was performed and integrated to identify cellular processes differentially regulated between culture conditions. Secretome characterization revealed a reduction in MSC batch variability when cultured as aggregates. Proteome and metabolome characterization showed upregulation of multiple protein and lipid metabolic pathways, downregulation of several cytoskeletal processes, and differential regulation of extracellular matrix synthesis. Integration of proteome and metabolome characterization revealed individual lipid metabolites and vesicle-trafficking proteins as key features for discriminating between culture conditions. Overall, this study identifies several aspects of MSC physiology that are altered by aggregate culture. Further exploration of these processes and pathways is needed to determine their potential role in regulating cell secretory capacity.     

Gamma irradiation preserves immunosuppressive potential and inhibits clonogenic capacity of human bone marrow‐derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are promising candidates for the treatment of graft‐versus‐host and autoimmune diseases. Here, by virtue of their immunosuppressive effects, they are discussed to exhibit inhibitory actions on various immune effector cells, including T lymphocytes that promote the underlying pathology. While it becomes apparent that MSCs exhibit their therapeutic effect in a transient manner, they are usually transplanted from third party donors into heavily immunocompromised patients. However, little is known about potential late complications of persisting third party MSCs in these patients. We therefore analysed the effect of gamma irradiation on the potency and proliferation of MSCs to elucidate an irradiation dose, which would allow inhibition of MSC proliferation while at the same time preserving their immunosuppressive function. Bone marrow‐derived MSCs (BM‐MSCs) were gamma‐irradiated at increasing doses of 5, 10 and 30 Gy and subsequently assessed by colony formation unit (CFU)‐assay, Annexin V‐staining and in a mixed lymphocyte reaction, to assess colony growth, apoptosis and the immunosuppressive capacity, respectively. Complete loss of proliferative capacity measured by colony formation was observed after irradiation with a dose equal to or greater than 10 Gy. No significant decrease of viable cells was detected, as compared to non‐irradiated BM‐MSCs. Notably, irradiated BM‐MSCs remained highly immunosuppressive in vitro for at least 5 days after irradiation. Gamma irradiation does not impair the immunosuppressive capacity of BM‐MSCs in vitro and thus might increase the safety of MSC‐based cell products in clinical applications.     

Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells

Abstract Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2⁺, SH3⁺, CD29⁺, CD44⁺, CD166⁺, CD13⁺) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.     

Epigenetic changes in umbilical cord mesenchymal stromal cells upon stimulation and culture expansion

Background

Mesenchymal stromal cells (MSCs) are studied for their immunotherapeutic potential. Prior to therapeutic use, MSCs are culture expanded to obtain the required cell numbers and, to improve their efficacy, MSCs may be primed in vitro. Culture expansion and priming induce phenotypical and functional changes in MSCs and thus standardisation and quality control measurements come in need. We investigated the impact of priming and culturing on MSC DNA methylation and examined the use of epigenetic profiling as a quality control tool.

Evaluation of a low cost cryopreservation system on the biology of human amniotic fluid-derived mesenchymal stromal cells

Background

Human amniotic-derived mesenchymal stromal cells (hAMSC) are a novel population of multipotent stem cells that have been shown to have great potential for use in regenerative medicine. However, procedures to store and preserve hAMSC for future clinical applications have not been explored extensively.

Methods

In this study, we analyzed the influence of cryopreservation, using a protocol based on freezing rate of 1 °C/min, 10% dimethyl sulfoxide as cryoprotectant and a thawing rate >100 °C/min, on hAMSC morphology, proliferation rates, viability, cell cycle, karyotype, immune phenotype and multilineage differentiation potential.

Results

This study found that this cryopreservation protocol does not affect the biological properties of hAMSC.

Discussion

This shows that this protocol is a viable system for banking hAMSC, with the associated advantages that has a low cost in terms of expense, time and personnel involved and is easy to implement.     

N‐isopropylacrylamide‐based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion

Human mesenchymal stem cells (hMSCs) are colony‐forming unit fibroblasts (CFU‐F) derived from adult bone marrow and have significant potential for many cell‐based tissue‐engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N‐isopropylacrylamide (NIPAM)‐based thermoresponsive polyelectrolyte multilayer (N‐PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N‐PEMU films were made via layer‐by‐layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU‐F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer‐by‐layer approach is an efficient way to form PNIPAM‐based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU‐forming ability and their multi‐lineage differentiation potential. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Anti-inflammatory roles of mesenchymal stromal cells during acute Streptococcus pneumoniae pulmonary infection in mice

Background

Pneumonia is the fourth leading cause of death worldwide, and Streptococcus pneumoniae is the most commonly associated pathogen. Increasing evidence suggests that mesenchymal stromal cells (MSCs) have anti-inflammatory roles during innate immune responses such as sepsis. However, little is known about the effect of MSCs on pneumococcal pneumonia.

Expansion strategies for human mesenchymal stromal cells culture under xeno‐free conditions

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost‐effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno‐free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix‐derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno‐free bioprocess. UCM MSC were cultured in a scalable planar (compact 10‐layer flasks and roller bottles) and 3‐D microcarrier‐based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10⁴ and 1.4 ± 0.3 × 10⁴ cells/cm². UCM MSC‐based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5–23.0 × 10⁴ cells/cm²) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno‐free expansion processes represents an important step toward a GMP compliant large‐scale production platform for MSC‐based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358–1367, 2017     

Immunogenic potential of human bone marrow mesenchymal stromal cells is enhanced by hyperthermia

Background aims

Bone marrow–derived mesenchymal stromal cells (MSCs) have been reported to suppress T-cell proliferation and used to alleviate the symptoms of graft-versus-host disease (GVHD). MSCs are a mixed cell population and at this time there are no tools to isolate the cells responsible for the T-cell suppression. We wanted to find a way to enhance the immune-modulatory actions of MSCs and tried varying the temperature at which they were cultured.

DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells

Within 2–3 months of in vitro culture-expansion, mesenchymal stromal cells (MSC) undergo replicative senescence characterized by cell enlargement, loss of differentiation potential and ultimate growth arrest. In this study, we have analyzed DNA methylation changes upon long-term culture of MSC by using the HumanMethylation27 BeadChip microarray assessing 27 578 unique CpG sites. Furthermore, we have compared MSC from young and elderly donors. Overall, methylation patterns were maintained throughout both long-term culture and aging but highly significant differences were observed at specific CpG sites. Many of these differences were observed in homeobox genes and genes involved in cell differentiation. Methylation changes were verified by pyrosequencing after bisulfite conversion and compared to gene expression data. Notably, methylation changes in MSC were overlapping in long-term culture and aging in vivo . This supports the notion that replicative senescence and aging represent developmental processes that are regulated by specific epigenetic modifications.