Expansion Of Mesenchymal Stem/stromal Cells Research Articles

Automated Manufacturing Processes and Platforms for Large-scale Production of Clinical-grade Mesenchymal Stem/ Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) hold immense potential for regenerative medicine due to their remarkable regenerative and immunomodulatory properties. However, their therapeutic application requires large-scale production under stringent regulatory standards and Good Manufacturing Practice (GMP) guidelines, presenting significant challenges. This review comprehensively evaluates automated manufacturing processes and platforms for the scalable production of clinical-grade MSCs. Various large-scale culture vessels, including multilayer flasks and bioreactors, are analyzed for their efficacy in MSCs expansion. Furthermore, automated MSCs production platforms, such as Quantum® Cell Expansion System, CliniMACS Prodigy®, NANT001/ XL, CellQualia™, Cocoon® Platform, and Xuri™ Cell Expansion System W25 are reviewed and compared as well. We also underscore the importance of optimizing culture media specifically emphasizing the shift from fetal bovine serum to humanized or serum-free alternatives to meet GMP standards. Moreover, advances in alternative cryopreservation methods and controlled-rate freezing systems, that offer promising improvements in MSCs preservation, are discussed as well. In conclusion, advancing automated manufacturing processes and platforms is essential for realizing the full potential of MSCs-based regenerative medicine and accomplishing the increasing demand for cell-based therapies. Collaborative initiatives involving industry, academia, and regulatory bodies are emphasized to accelerate the translation of MSCs-based therapies into clinical practice.

The Wisdom in Teeth: Neuronal Differentiation of Dental Pulp Cells.

Mesenchymal stem/stromal cells (MSCs) are found in almost all postnatal organs. Under appropriate environmental cues, multipotency enables MSCs to serve as progenitors for several lineage-specific, differentiated cell types. In vitro expansion and differentiation of MSCs give the opportunity to obtain hardly available somatic cells, such as neurons. The neurogenic potential of MSCs makes them a promising, autologous source to restore damaged tissue and as such, they have received much attention in the field of regenerative medicine. Several stem cell pool candidates have been studied thus far, but only a few of them showed neurogenic differentiation potential. Due to their embryonic ontology, stem cells residing in the stroma of the dental pulp chamber are an exciting source for in vitro neural cell differentiation. In this study, we review the key properties of dental pulp stem cells (DPSCs), with a particular focus on their neurogenic potential. Moreover, we summarize the various presently available methods used for neural differentiation of human DPSCs also emphasizing the difficulties in reproducibly high production of such cells. We postulate that because DPSCs are stem cells with very close ontology to neurogenic lineages, they may serve as excellent targets for neuronal differentiation in vitro and even for direct reprogramming.

Universal or Personalized Mesenchymal Stem Cell Therapies: Impact of Age, Sex, and Biological Source.

Mesenchymal stem/stromal cells (MSCs) hold great promise for the treatment of autoimmune conditions given their immunomodulatory properties. Based on the low immunogenicity of MSCs, it is tempting to consider the expansion of MSCs from a “universal donor” in culture prior to their allogeneic applications for immediate care. This raises the critical question of the criteria we should use to select the best “universal donor”. It is also imperative we compare the “universal” approach with a “personalized” one for clinical value. In addition to the call for MHC-matching, recent studies suggest that factors including age, sex, and biological sources of MSCs can have significant impact on therapy outcome. Here, we will review findings from these studies, which shed light on the variables that can guide the important choice of “universal” or “personalized” MSC therapy for autoimmune diseases.

Stress Relaxation and Composition of Hydrazone-Crosslinked Hybrid Biopolymer-Synthetic Hydrogels Determine Spreading and Secretory Properties of MSCs.

The extracellular matrix plays a critical role in mechanosensing and thereby influences the secretory properties of bone-marrow-derived mesenchymal stem/stromal cells (MSCs). As a result, interest has grown in the development of biomaterials with tunable properties for the expansion and delivery of MSCs that are used in cell-based therapies. Herein, stress-relaxing hydrogels are synthesized as hybrid networks containing both biopolymer and synthetic macromer components. Hyaluronic acid is functionalized with either aldehyde or hydrazide groups to form covalent adaptable hydrazone networks, which are stabilized by poly(ethylene glycol) functionalized with bicyclononyne and heterobifunctional small molecule crosslinkers containing azide and benzaldehyde moieties. Tuning the composition of these gels allows for controlled variation in the characteristic timescale for stress relaxation and the amount of stress relaxed. Over this compositional space, MSCs are observed to spread in formulations with higher degrees of adaptability, with aspect ratios of 1.60± 0.18, and YAP nuclear:cytoplasm ratios of 6.5± 1.3. Finally, a maximum MSC pericellular protein thickness of 1.45± 0.38µm occurred in highly stress-relaxing gels, compared to 1.05± 0.25µm in non-adaptable controls. Collectively, this study contributes a new understanding of the role of compositionally defined stress relaxation on MSCs mechanosensing and secretion.

Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for use in Humans

Adipose tissue (AT) represents a commonly used source of mesenchymal stem/stromal cells (MSCs) whose proregenerative potential has been widely investigated in multiple clinical trials worldwide. However, the standardization of the manufacturing process of MSC-based cell therapy medicinal products in compliance with the requirements of the local authorities is obligatory and will allow us to obtain the necessary permits for product administration according to its intended use. Within the research phase (RD), we optimized the protocols used for the processing and ex vivo expansion of AT-derived MSCs (AT-MSCs) for the development of an Advanced Therapy Medicinal Product (ATMP) for use in humans. Critical process parameters (including, e.g., the concentration of enzyme used for AT digestion, cell culture conditions) were identified and examined to ensure the high quality of the final product containing AT-MSCs. We confirmed the identity of isolated AT-MSCs as MSCs and their trilineage differentiation potential according to the International Society for Cellular Therapy (ISCT) recommendations. Based on the conducted experiments, in-process quality control (QC) parameters and acceptance criteria were defined for the manufacturing of hospital exemption ATMP (HE-ATMP). Finally, we conducted a validation of the manufacturing process in a GMP facility. In the current study, we presented a process approach leading to the optimization of processing and the ex vivo expansion of AT-MSCs for the development of ATMP for use in humans.

Development of bioreactor protocols for stem cell‐based therapies

AbstractStem cell‐based therapies offer a new treatment alternative for individuals suffering from devastating disorders such as Parkinson's disease (PD) and Huntington's disease (HD). Derivation and transplantation of stem cells or their derivatives have shown significant therapeutic outcomes in animal model studies and clinical trials. However, commercialization of stem cell‐based therapies and widespread access to these treatments are largely hampered by access to large quantities of high‐quality products generated under standardized, robust, and controlled conditions. Over the last three decades, the Pharmaceutical Production Research Facility (PPRF) of the University of Calgary, founded by the late Professor Leo A. Behie, has carried out groundbreaking research focusing on the fundamentals of bioprocessing for various stem cells including human neural precursor cells (NPCs) and mesenchymal stem/stromal cells (MSCs). These efforts led to the development of serum‐free growth media and expansion protocols for large‐scale production of therapeutically‐relevant NPCs for the treatment of neurodegenerative disorders. Similarly, high‐performance, defined growth media and reliable, large‐scale computer‐controlled bioreactor protocols were developed for the expansion of MSCs derived from human bone marrow and other sources and the generation of a therapeutically‐relevant secretome. Important bioprocessing considerations such as the hydrodynamics of the bioreactor, mass transfer, cellular microenvironment and synergistic impact of media components were evaluated in these studies. Undoubtedly, the diverse topics of research carried out at PPRF significantly contributed to the translation of the laboratory‐based stem cell culture studies into production of clinical‐grade materials and eventually commercialization of stem cell‐based therapies through applying principles of bioengineering and bioprocessing.

Influences of Xeno-Free Media on Mesenchymal Stem Cell Expansion for Clinical Application.

Mesenchymal stem/stromal cells (MSCs) are multipotent somatic stem/progenitor cells that can be isolated from various tissues and have attracted increasing attention from the scientific community. This is due to MSCs showing great potential for incurable disease treatment, and most applications of MSCs involve tissue degeneration and treatment of immune- and inflammation-mediated diseases. Conventional MSC cultures contain fetal bovine serum (FBS), which is a common supplement for cell development but is also a risk factor for exposure to animal-derived pathogens. To avoid the risks resulting from the xenogeneic origin and animal-derived pathogens of FBS, xeno-free media have been developed and commercialized to satisfy MSC expansion demands for human clinical applications. This review summarized and provided an overview of xeno-free media that are currently used for MSC expansion. Additionally, we discussed the influences of different xeno-free media on MSC biology with particular regard to cell morphology, surface marker expression, proliferation, differentiation and immunomodulation. The xeno-free media can be serum-free and xeno-free media or media supplemented with some human-originating substances, such as human serum, human platelet lysates, human umbilical cord serum/plasma, or human plasma-derived supplements for cell culture medium. These media have capacity to maintain a spindle-shaped morphology, the expression of typical surface markers, and the capacity of multipotent differentiation and immunomodulation of MSCs. Xeno-free media showed potential for safe use for human clinical treatment. However, the influences of these xeno-free media on MSCs are various and any xeno-free medium should be examined prior to being used for MSC cultures.

Development of standard protocol for the cGMP production of human mesenchymal stem/stromal cells from different source tissues

Background & Aim Mesenchymal stem/stromal cells (MSC) hold great potential for tissue engineering and regenerative medicine because of their ability for self-renewal and differentiation into tissue-specific cells such as osteoblasts, chondrocytes, and adipocytes. MSCs are important pieces in the process of tissue development, maintenance and repair, producing secretory factors that support engraftment and trophic functions. For that reason, they are currently being used in hundreds of clinical trials as a promising treatment option for a variety of clinical conditions, with an increasing evidence of therapeutic efficacy. MSC constitute a heterogeneous subset of stromal regenerative cells which can be harvested from several adult tissues. Initially MSC were identified in bone marrow but currently they can be harvested from several autologous and allogeneic sources, including adipose tissue, peripheral blood, lungs, marrow spaces of long bone, synovial fluids, periodontal ligament, dental pulp and muscle. In addition, MSCs are also obtained from placenta, umbilical cord and cord blood. Notably, MSCs obtained from various sources differ in their biological characteristics, CFU-F efficiency, specific markers, multi-lineage differentiation as well as paracrine functions which may determine their different clinical applications. The use of MSC from different sources together with the fact that most laboratories use their own standardized protocols for cell expansion results in a clear variability in cell preparations between labs which can lead to variability in the outcome of experiments and clinical trials across different laboratories and institutions. Investigations have attempted to address this issue of unpredictable outcomes by seeking to establish standard practices for the isolation, characterization, and maintenance of cells in culture. The development of uniform protocols for the expansion and preparation of MSCs is crucial to improve comparison of results, for the exploitation of MSC clinical potential and for a successful translation of approaches to the clinic. Methods, Results & Conclusion Here we will present the development of a standardized protocol for a consistent and efficient cGMP production of clinically relevant numbers of MSCs obtained from different source tissues, using MSC NutriStem® XF Basal Medium supplemented with PLTGold®, a fully xenogeneic free human platelet lysate.

Rapid expansion of mesenchymal stem/stromal cells using optimized media supplemented with human platelet lysate PLTMax® or PLTGold®, suitable for cGMP expansion at large scale.

Background & Aim Mesenchymal Stem/Stromal Cells (MSCs) are a promising candidate for tissue engineering and regenerative medicine applications due to their capacity of self-renewal and multilineage differentiation, ease of isolation and ability to grow in vitro. They are currently being used in hundreds of clinical trials and have been safely administered to thousands of patients with an expanding body of evidence of therapeutic efficacy. Mill Creek Life Sciences’ PLTMax®, derived from normal human donor platelets, arose as an alternative to FBS to be used as a supplement for in vitro expansion of human cells used in clinical applications. It is the only media supplement to date demonstrating clinical cultures of MSCs with long term genetic fidelity, rapid expansion and potent clinical activity. PLTMax® is being used worldwide in over 30 clinical trials including Phase I, II and III. Our latest product PLTGold®, is a second-generation human platelet lysate that does not require the addition of heparin to remain clot free, thus providing a fully xenogeneic free alternative supplement. Methods, Results & Conclusion In traditional monolayer expansion systems, we have demonstrated growth of adipose derived and bone marrow derived MSCs using an optimized, fully cGMP compliant culture system. We have found that MSC NutriStem® XF Basal Medium (Biological Industries USA, Inc, Cromwell, CT) supplemented with PLTMax® or PLTGold® exceeds the performance of other conventional media products, obtaining up to 5 × 108 cells in only 5 passages (5 times more cells than with other commercially available products). Furthermore, when we use MSC NutriStem® XF Complete Medium supplemented with PLTMax® or PLTGold®, we obtained up to 2 × 1010 cells in only 5 passages (200 times more cells than with other commercially available products). Cells grown in these media maintained MSC phenotype and capacity to undergo differentiation. The effective transfer into the clinic of allogeneic cell therapies using MSCs will depend predominantly on the development of large scale and cost effective manufacturing platforms that allow production of functional cells at the scale required to meet clinical demand. We also present the extension of these studies using this optimized method to evaluate 9 different microcarriers. Together, we will present an optimized protocol for the establishment of large scale expansion of MSCs in bioreactors.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Abstract PR12: Targeting FSTL1 augments therapeutic activities of immune checkpoint inhibitors

Abstract Purpose: Tumor heterogeneity is composed of genetic and phenotypic changes in tumor itself, and also of the consequence of reciprocal evolution together with the tumor microenvironment containing a variety of cells such as stromal cells, vascular cells and immune cells in the host. Imbalanced immunity is one of the major problems supporting tumor progression and then amplification of the heterogeneity. Many inhibitors specific for immune checkpoint molecules interfering induction of anti-tumor immunity have been recently developed in clinical settings, and the clinical responses have convinced us of its efficaciousness. However, good results are limited to a part of the treated patients, and additional but another strategy is needed for treating patients more efficiently and effectively. For the purpose, we have been investigating the complex network in the tumor milieu and the systemic responses in the whole body comprehensively and collectively, and previously identified a BMP family FSTL1, which is significantly secreted from Snail+ cancer stem-like cells, as a key effector molecule in the cancer-associated immune suppression and dysfunction. FSTL1 expands pluripotent mesenchymal stem/stromal cells (MSCs), which generate immunoregulatory cells such as Tregs and tolerogenic DCs, and functionally impaired CD8-low T cells. FSTL1 and its induced MSCs also confer higher invasive and metastatic properties on tumor cells leading to further promotion of tumor growth and metastasis. To prevent such FSTL1-caused vicious circulation, we established anti-FSTL1 blocking mAbs, and evaluated the anti-tumor efficacy in combination with immune checkpoint inhibitors (ICIs) using mouse FSTL1+ tumor models. Experimental Procedures: We generated monoclonal antibodies (mAbs) specific for both mouse and human FSTL1 utilizing the chicken immunization system followed by constructing cDNA phage library. After in vitro screening, the established anti-FSTL1 mAbs were tested for the in vivo anti-tumor activity using two syngeneic tumor models: a bone metastatic tumor model that C57BL/6 was implanted with snail-transduced melanoma B16-F10 cells, and a lung metastatic tumor model that BALB/c was implanted with colon cancer intrinsic FSTL1+ CT26 cells. The mAbs (10mg/kg) were intraperitoneally injected into these mice twice on day 4 and day 7 after tumor implantation. On day 14, immunological analysis was conducted using tumor-infiltrating cells, spleen cells and bone marrow cells obtained from the mice. Results: We established chicken/mouse chimeric anti-FSTL1 mAb capable of inhibiting the FSTL1-elicited events including MSC expansion, immunoregulatory cell induction, and tumor invasion in vitro. In both bone and lung metastatic tumor models, treatment with the anti-FSTL1 mAb induced tumor-specific CD8+ T cells with higher CTL activities by suppressing increase of MSCs, but not Tregs and MDSCs, and significantly suppressed subcutaneous tumor growth and metastasis in the treated mice as compared to those of the control mice. The ICI mono-therapy was insufficient to suppress increase of MSCs, although significantly reduced immunoregulatory cells, and the anti-tumor efficacy was almost similar. Combination with the anti-FSTL1 mAb synergistically and significantly enhanced the ICI mono-therapeutic efficacy, particularly anti-PDL1 mAb, by inducing potent tumor-specific CTLs, resulting in tumor-free in some of the treated mice. Conclusions: Blocking FSTL1 reprograms and properly induces anti-tumor immunity by switching the tumor-supportive immune directivity to the active mode against cancer. Anti-FSTL1 mAb having totally different molecular mechanisms from the conventional ICIs may be a promising partner for radically correcting cancer-manipulated immunity in clinical settings. Citation Format: Chie Kudo-Saito, Yamato Ogiwara, Marina Hennmi, Kazunori Aoki. Targeting FSTL1 augments therapeutic activities of immune checkpoint inhibitors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR12.

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGROTM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11±2days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGROTM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGROTM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. 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 (R(2) ≥ 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 R(2) 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.

Clinical-Grade Manufacturing of Therapeutic Human Mesenchymal Stem/Stromal Cells in Microcarrier-Based Culture Systems.

The therapeutic potential of mesenchymal stem/stromal cells (MSC) has triggered the need for high cell doses in a vast number of clinical applications. This demand requires the development of good manufacturing practices (GMP)-compliant ex vivo expansion protocols that should be effective to deliver a robust and reproducible supply of clinical-grade cells in a safe and cost-effective manner. Controlled stirred-tank bioreactor systems under xenogeneic (xeno)-free culture conditions offer ideal settings to develop and optimize cell manufacturing to meet the standards and needs of human MSC for cellular therapies. Herein we describe two microcarrier-based stirred culture systems using spinner flasks and controlled stirred-tank bioreactors under xeno-free conditions for the efficient ex vivo expansion of human bone marrow and adipose tissue-derived MSC.

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells.

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.

A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue.

Human mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and the development of microcarrier-based cultures in scalable bioreactors with well-defined xenogeneic-free components represent important milestones towards the clinical-scale production of these cells. In this work, we optimized our previously developed xeno-free microcarrier-based system for the scalable expansion of human MSC isolated from bone marrow (BM MSC) and adipose-derived stem/stromal cells (ASC). By adapting the agitation/feeding protocol at the initial cell seeding/cultivation stage in spinner flasks, we were able to maximize cell expansion rate and final cell yield. Maximal cell densities of 3.6 × 10(5) and 1.9 × 10(5) cells/mL were obtained for BM MSC (0.60 ± 0.04 day(-1) ) and ASC (0.9 ± 0.1 day(-1) ) cultures, upon seven and eight days of cultivation, respectively. Ready-to-use microcarriers Synthemax® II and Enhanced Attachment® supported identical expansion performance of BM MSC, turning those effective alternatives to the pre-coated plastic microcarriers used in our xeno-free scalable culture system. Importantly, expanded MSC maintained their immunophenotype and multilineage differentiation potential. Moreover, secretome analysis suggested a priming effect of stirred culture conditions on cytokine production by MSC. This culture system yielded considerable final cell densities that can be scaled-up to controlled large-scale bioreactors allowing a more efficient, safe and cost-effective MSC production for clinical settings.

A human platelet lysate-based culture supplement for the successful isolation and scalable expansion of umbilical cord matrix-derived mesenchymal stem/stromal cells

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSC) are considered promising therapeutic agents. However, low cell yields per UC unit and high cell doses required in clinical settings emphasize the need for ex vivo expansion of UCM MSC. We recently demonstrated the ability to isolate and ex-vivo expand UCM MSC under serum-/xeno-free culture conditions using static systems.1 Also, we have previously established a xeno-free microcarrier-based system for the scalable expansion of human MSC from bone marrow and adipose tissue, using plastic microcarriers and a commercially available xeno-free medium formulation.2-3 However, the scalable expansion of UCM MSC under dynamic conditions, using plastic microcarriers combined with different commercially available serum-/xeno-free media, remains challenging. Here, we demonstrate the ability of using a human platelet lysate (HPL)-based culture supplement (UltraGROTM, HELIOS BioScience Inc.) for the successful isolation and scalable expansion of UCM MSC. MSC-like cells were isolated from UCM explant cultures after 11±2 days and upon 5 passages cells retained their immunophenotype and multilineage differentiation potential. In addition, UCM MSC expanded using UltraGROTM-supplemented medium expanded faster compared to cells expanded using the previously established protocol (1.0 vs. 0.54 day-1, respectively)1. Importantly, UCM MSC were successfully expanded under dynamic conditions on plastic microcarriers using UltraGROTM-supplemented medium in spinner flasks. Upon an initial 42-60% cell adhesion to the beads, UCM MSC were able to expand by >10 fold after 4-5 days. Upon dynamic culture, cells maintained their immunophenotype and multilineage differentiation ability. To our knowledge, this is the first study reporting the successful UCM MSC expansion in a fully scalable xeno-free microcarrier-based system, representing an important advance to obtain safer and clinically meaningful MSC numbers for Cell Therapy.

A xenogeneic‐free bioreactor system for the clinical‐scale expansion of human mesenchymal stem/stromal cells

The large cell doses (>1 × 10(6) cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC production system.

Scalable ex vivo expansion of human mesenchymal stem/stromal cells in microcarrier-based stirred culture systems.

The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different microcarriers and xeno-free medium formulations.

Comparative Analysis Of Bone Marrow and Wharton's Jelly Mesenchymal Stem/Stromal Cells

Bone narrow (BM)- derived mesenchymal stem/stromal cells (MSCs) represent the most extensively studied population of adult MSCs and are considered as the gold-standard for MSC-based clinical applications. Yet, it is now becoming increasingly clear that BM may not represent the most suitable source for MSC collection. Indeed, Umbilical cord (UC) has emerged as a more abundant and easily attainable source of MSCs and several reports have shown that MSCs can be efficiently isolated from the connective tissue that surrounds UC vessels, namely the Wharton's jelly (WJ). According to the existing literature, WJ-MSCs display typical MSC characteristics, however a head-to-head comparison with BM-MSCs is still lacking.Provided that ex vivo MSC expansion is a prerequisite for clinical MSC-applications, in the present study we seek to comparatively investigate the characteristics of WJ- and BM-MSCs, cultured under identical conditions.MSCs were isolated and expanded from consenting healthy donors' BM aspirates (n=5) and from the WJ of full-term neonates (n=10) after written informed consent of the family. MSCs were in vitro expanded and re-seeded for a total of 10 passages (P) and phenotypically characterized by flow cytometry (FC). MSCs were induced to differentiate in vitro to adipocytes and osteoblasts. Differentiation was assessed by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes. Relative gene expression was calculated by the ΔCt method. MSC growth characteristics were assessed by evaluating the population doubling time (DT) and by a methyl-triazolyl-tetrazolium (MTT)-assay throughout passages. Cell-cycle analysis was performed using propidium iodide (PI) staining. MSC survival was evaluated by FC with 7-Aminoactinomycin D (7-AAD) and senescence was estimated by the percentage of SA-b-gal+ cells in cultures. Moreover, MSC karyotypic stability was assessed with classic G-banding. Finally the expression of genes related to Wnt-mediated signal transduction was also investigated, using a PCR array. Total RNA was thus isolated from 6 representative BM- and 6 WJ-MSC cultures at P2. The fold change (FC) for each gene between the group of WJ- and the group of BM-MSCs was calculated with the ΔΔCt method (FC=2-ΔΔCt).WJ-MSCs displayed a spindle-shape morphology, similar to BM-MSCs. Furthermore, WJ- and BM-MSCs displayed identical immunophenotype, as evidenced by the expression of CD90,CD105,CD44,CD29,CD73 and the lack of expression of CD45,CD14,CD34,CD31. WJ-MSCs displayed superior proliferative potential compared to BM-MSCs throughout passages (p<0.05). Moreover, the proportion of proliferating (S/G2/M) WJ-MSCs was higher compared to BM-MSCs at P4 (p<0.001), while there was no significant difference between two MSC populations in the proportion of 7-AADbright/dim –cells at P4. Regarding senescence, significantly fewer SA-b-gal+ cells were observed in WJ-MSC cultures, as compared to BM-MSCs at P10 (p<0.05). Compared to their bone marrow counterparts, WJ-MSCs displayed inferior capacity to differentiate into adipocytes and osteoblasts as evidenced by Oil Red O and Alizarin Red staining, respectively, and also by the weaker expression of adipocyte- (PPAR-g, p<0.0002; CEBP-a, p<0.0001) and osteocyte-specific markers (RUNX2, p<0.0006; DLX5, p<0.0001; ALP, p<0.0042). No chromosomal abnormalities were observed in either WJ- or BM-MSCs during in vitro expansion. Regarding the Wnt-pathway signaling molecules, the Wnt antagonist sFRP4, which induces adipogenesis, as well the Wnt/b-catenin target gene Wisp-1, a regulator of osteogenesis were significantly down-regulated in WJ-MSCs (FC=22.3825, p<0,05; FC=20.18, p<0.0001, respectively). On the other hand, the expression of Wnt/b-catenin target gene Cyclin D1, which induces MSC proliferation and represses adipogenesis, was up-regulated in WJ-MSCs (FC=2.8, p<0.05).Taken together WJ-MSCs display decreased cellular senescence after extended in vitro culture, increased proliferative capacity and reduced potential to differentiate in vitro to adipocytes and osteocytes, as compared to BM-MSCs. The last two observations can be explained, at least partly, by the aberrant expression of Wnt-signaling molecules in WJ-MSCs. The emerging role of Wnt-signaling pathway in WJ-MSC biology is currently under investigation. Disclosures:No relevant conflicts of interest to declare.