scholarly journals Novel Cryopreservation Approach Providing Off-the-Shelf Availability of Human Multipotent Mesenchymal Stromal Cells for Clinical Applications

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Olena Rogulska ◽  
Olga Tykhvynska ◽  
Olena Revenko ◽  
Viktor Grischuk ◽  
Svitlana Mazur ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Positive Impact ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Long Term Storage ◽  
Wound Model ◽  
Novel Approach ◽  
Localized Delivery ◽  
Additional Support

Cryopreservation is the only established method to provide long-term storage and fast availability of cellular product for therapeutic applications. The overwhelming majority of cryopreservation media contain toxic concentrations of dimethyl sulfoxide (DMSO) limiting the possibility for the direct administration of cryopreserved cells to the patients. Here, we propose a novel approach for nontoxic xeno-free cryopreservation of human multipotent mesenchymal stromal cells (MSCs) aimed at ensuring high viability, ready-to-use availability, and localized delivery of the cell-based graft into damaged tissues. For MSC cryopreservation, we applied sucrose pretreatment procedure and xeno-free cryoprotective medium containing human platelet-poor blood plasma (PPP), sucrose, and nontoxic concentration of DMSO. Using the combination of PPP, 0.2 M sucrose, and 1% DMSO, the recovery rate of cryopreserved MSCs reached 73% of the values obtained for noncryopreserved cells. Moreover, the presence of PPP in the cryoprotective medium provided the possibility to create a ready-to-use 3D hydrogel for the localized delivery and additional support of MSCs in vivo. In a proof-of-concept study, we assessed the regenerative capacity of cryopreserved MSCs in a full-thickness wound model in mice. The positive impact of MSCs within 3D gel on wound healing rates was confirmed by morphometric and histological examinations. Our results demonstrate the possibility to apply cryopreserved cells immediately after thawing using a cryoprotective medium as the vehicle solution.

scholarly journals Preconditioning in an Inflammatory Milieu Augments the Immunotherapeutic Function of Mesenchymal Stromal Cells

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 462 ◽  
Author(s):  
Luis A. Rodriguez ◽  
Arezoo Mohammadipoor ◽  
Lucero Alvarado ◽  
Robin M. Kamucheka ◽  
Amber M. Asher ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Comprehensive Evaluation ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Human Mscs ◽  
Post Injury ◽  
Anti Inflammatory ◽  
And Function ◽  
Inflammatory Milieu

Multipotent mesenchymal stromal cells (MSCs) have emerged as potent therapeutic agents for multiple indications. However, recent evidence indicates that MSC function is compromised in the physiological post-injury milieu. In this study, bone marrow (BM)- and adipose-derived (AD)-MSCs were preconditioned in hypoxia with or without inflammatory mediators to potentiate their immunotherapeutic function in preparation for in vivo delivery. Human MSCs were cultured for 48 h in either normoxia (21% O2) or hypoxia (2% O2) with or without the addition of Cytomix, thus creating 4 groups: (1) normoxia (21%); (2) Cytomix-normoxia (+21%); (3) hypoxia (2%); and (4) Cytomix-hypoxia (+2%). The 4 MSC groups were subjected to comprehensive evaluation of their characteristics and function. Preconditioning did not alter common MSC surface markers; nonetheless, Cytomix treatment triggered an increase in tissue factor (TF) expression. Moreover, the BM-MSCs and AD-MSCs from the +2% group were not able to differentiate to chondrocytes and osteoblasts, respectively. Following Cytomix preconditioning, the metabolism of MSCs was significantly increased while viability was decreased in AD-MSCs, but not in BM-MSCs. MSCs from both tissues showed a significant upregulation of key anti-inflammatory genes, increased secretion of IL-1 receptor antagonist (RA), and enhanced suppression of T-cell proliferation following the Cytomix treatment. Similarly, following a lipopolysaccharide challenge, the Cytomix-treated MSCs suppressed TNF-α secretion, while promoting the production of IL-10 and IL-1RA. These preconditioning approaches facilitate the production of MSCs with robust anti-inflammatory properties. AD-MSCs preconditioned with Cytomix under normoxia appear to be the most promising therapeutic candidates; however, safety concerns, such as thrombogenic disposition of cells due to TF expression, should be carefully considered prior to clinical translation.

scholarly journals Allogeneic Adipose-Derived Mesenchymal Stromal Cells Ameliorate Experimental Autoimmune Encephalomyelitis by Regulating Self-Reactive T Cell Responses and Dendritic Cell Function

2017 ◽  
Vol 2017 ◽  
pp. 1-15 ◽  
Author(s):  
Per Anderson ◽  
Elena Gonzalez-Rey ◽  
Francisco O’Valle ◽  
Francisco Martin ◽  
F. Javier Oliver ◽  
...  
Keyword(s):  
T Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cell Function ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Brain Inflammation ◽  
Inos Inhibitor ◽  
Cox 1

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising therapy for autoimmune diseases, including multiple sclerosis (MS). Administration of MSCs to MS patients has proven safe with signs of immunomodulation but their therapeutic efficacy remains low. The aim of the current study has been to further characterize the immunomodulatory mechanisms of adipose tissue-derived MSCs (ASCs) in vitro and in vivo using the EAE model of chronic brain inflammation in mice. We found that murine ASCs (mASCs) suppress T cell proliferation in vitro via inducible nitric oxide synthase (iNOS) and cyclooxygenase- (COX-) 1/2 activities. mASCs also prevented the lipopolysaccharide- (LPS-) induced maturation of dendritic cells (DCs) in vitro. The addition of the COX-1/2 inhibitor indomethacin, but not the iNOS inhibitor L-NAME, reversed the block in DC maturation implicating prostaglandin (PG) E2 in this process. In vivo, early administration of murine and human ASCs (hASCs) ameliorated myelin oligodendrocyte protein- (MOG35-55-) induced EAE in C57Bl/6 mice. Mechanistic studies showed that mASCs suppressed the function of autoantigen-specific T cells and also decreased the frequency of activated (CD11c+CD40high and CD11c+TNF-α+) DCs in draining lymph nodes (DLNs). In summary, these data suggest that mASCs reduce EAE severity, in part, through the impairment of DC and T cell function.

Decellularized extracellular matrix microparticles seeded with bone marrow mesenchymal stromal cells for the treatment of full-thickness cutaneous wounds

2019 ◽  
Vol 33 (8) ◽  
pp. 1070-1079 ◽  
Author(s):  
Amanda M Westman ◽  
Rachel L Goldstein ◽  
Gino Bradica ◽  
Scott M Goldman ◽  
Mark A Randolph ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Full Thickness ◽  
Decellularized Extracellular Matrix ◽  
Wound Model ◽  
Cellular Attachment ◽  
Healing Environments

Extracellular matrix materials mechanically dissociated into submillimeter particles have a larger surface area than sheet materials and enhanced cellular attachment. Decellularized porcine mesothelial extracellular matrix microparticles were seeded with bone marrow-derived mesenchymal stromal cells and cultured in a rotating bioreactor. The mesenchymal stromal cells attached and grew to confluency on the microparticles. The cell-seeded microparticles were then encapsulated in varying concentrations of fibrin glue, and the cells migrated rapidly off the microparticles. The combination of microparticles and mesenchymal stromal cells was then applied to a splinted full-thickness cutaneous in vivo wound model. There was evidence of increased cell infiltration and collagen deposition in mesenchymal stromal cells-treated wounds. Cell-seeded microparticles have potential as a cell delivery and paracrine therapy in impaired healing environments.

scholarly journals Potential of Extracellular Vesicle-Associated TSG-6 from Adipose Mesenchymal Stromal Cells in Traumatic Brain Injury

2020 ◽  
Vol 21 (18) ◽  
pp. 6761
Author(s):  
Santiago Roura ◽  
Marta Monguió-Tortajada ◽  
Micaela Munizaga-Larroudé ◽  
Marta Clos-Sansalvador ◽  
Marcella Franquesa ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Current Knowledge ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Cell Contact

Multipotent mesenchymal stromal cells (MSC) represent a promising strategy for a variety of medical applications. Although only a limited number of MSC engraft and survive after in vivo cellular infusion, MSC have shown beneficial effects on immunomodulation and tissue repair. This indicates that the contribution of MSC exists in paracrine signaling, rather than a cell-contact effect of MSC. In this review, we focus on current knowledge about tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) and mechanisms based on extracellular vesicles (EV) that govern long-lasting immunosuppressive and regenerative activity of MSC. In this context, in particular, we discuss the very robust set of findings by Jha and colleagues, and the opportunity to potentially extend their research focus on EV isolated in concentrated conditioned media (CCM) from adipose tissue derived MSC (ASC). Particularly, the authors showed that ASC-CCM mitigated visual deficits after mild traumatic brain injury in mice. TSG-6 knockdown ASC were, then, used to generate TSG-6-depleted CCM that were not able to replicate the alleviation of abnormalities in injured animals. In light of the presented results, we envision that the infusion of much distilled ASC-CCM could enhance the alleviation of visual abnormalities. In terms of EV research, the advantages of using size-exclusion chromatography are also highlighted because of the enrichment of purer and well-defined EV preparations. Taken together, this could further delineate and boost the benefit of using MSC-based regenerative therapies in the context of forthcoming clinical research testing in diseases that disrupt immune system homeostasis.

scholarly journals Biologic Characteristics of Bone Substituting Tissue Engineering Construction Based on Calcium Phosphate Ceramics, Autologous Mesenchymal Stromal Cells and Fibrin Hydrogel

2015 ◽  
pp. 52-59
Author(s):  
V. E. Mamonov ◽  
A. G. Chemis ◽  
V. S. Komlev ◽  
A. L. Berkovskiy ◽  
E. M. Golubev ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Calcium Phosphate ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Orthotopic Implantation ◽  
Calcium Phosphate Ceramic ◽  
Engineering Construction

Biological characteristics of bone substituting tissue engineering construction (TEC) that contained porous calcium phosphate ceramic granulate (CPC) of phase structure ((tricalcium phosphate (TCP)), fibrin hydrogel and autologous multipotent mesenchymal stromal cells (auto-MMSC) induced and non-induced to osteogenic differentiation were studied in vivo. The following characteristics of TEC were determined: ability to transfer within its structure the viable auto-MMSC with preservation of their regeneration potential; ability to osteogenesis only under conditions of orthotopic implantation; ability of induced to osteogenic differentiation auto-MMSC to participate in the reparative processes for not more than within 6 weeks after implantation; negative affect of fibrin hydrogel on the osteoinductive properties of CPC within TCP structure. It was shown that to provide osteogenesis in the implanted TEC not only the viable auto-MMSC but simultaneous presence of osteoinductive and osteoconductive factors was required. No bone formation in a critical bone defect and in ectopic implantation takes place without observance of these conditions.

Human Multipotent Mesenchymal Stromal Cells Inhibit Proliferation of PBMC in the Absence of Functional IFNγR1 and IDO Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2567-2567
Author(s):  
Friederike Gieseke ◽  
Susanne Viebahn ◽  
Sandra Kordowich ◽  
Ewa Koscielniak ◽  
Wilhelm Friedrich ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Molecular Mechanism ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Interferon Γ ◽  
Hematopoietic Stem ◽  
Indoleamine 2,3 Dioxygenase

Abstract Human multipotent mesenchymal stromal cells (MSC) can easily be isolated and propagated from bone marrow. MSC have been shown to inhibit the proliferation and effector functions of immune cells such as T cells, B cells, NK cells and dendritic cells. Over the recent years, they have gained clinical interest in transplantation and regenerative medicine. In allogeneic stem cell transplantation, MSC have been clinically applied not only because of their stromal support for hematopoietic stem cells, but also because of their immunomodulatory effects in graft-versus-host disease (GvHD). However, the molecular mechanism is not fully understood yet. In several studies the enzyme indoleamine 2,3-dioxygenase (IDO) has been described as a candidate be involved in this molecular mechanism. IDO has been shown to suppress T cell responses when expressed in dendritic cells by degradation of the essential amino acid tryptophan. In vivo, IDO is important to induce and maintain tolerance for the fetus during pregnancy. Although IDO is not expressed in MSC constitutively, it can strongly be induced by interferon-γ (IFNγ). Contradictory results have been published regarding the role of IFNγ-induced IDO expression in immunomodulation by MSC and it is still a matter of debate whether IDO is required in this setting. To clarify this contentious issue we carried out proliferation assays with PBMC stimulated by IL-2 and OKT-3 either in the presence or absence of HLA-mismatched MSC. We added exogenous IFNγ in order to induce IDO, which should have enhanced the inhibition of PBMC proliferation mediated by MSC. However, the effect of MSC was similar in cultures with or without exogenous IFNγ. Neither did pre-incubation of MSC with IFNγ before adding PBMC and IL-2 and OKT-3 have any effect on the inhibition. Moreover, we analyzed the effect of the IDO inhibitor 1-methyl-tryptophan. By adding 1-methyl-tryptophan the proliferation of PBMC in the presence of MSC could not be restored. These results suggested that expression of IDO in MSC was not responsible for the inhibition of PBMC. To further rule out that IFNγ produced by PBMC was sufficient to induce IDO and inhibition by 1-methyl-tryptophan was not complete, we isolated MSC from bone marrow of a child with a mutation in the subunit I of the INFγ receptor (IFNγR1) leading to a non-functional IFNg receptor. MSCIFNγR1−/− behaved normal in terms of plasticity and were readily differentiated into osteoblasts and adipocytes by standard protocols. Interestingly, MSCIFNγR1−/− were able to suppress the proliferation of PBMC to the same extent as MSC with functional IFNγ receptor. This showed that MSC inhibit PBMC independent of functional IFNγR1. Subsequently, we analyzed IDO expression in MSCIFNγR1−/− by RT-PCR. Expectedly, we could show that in MSCIFNγR1−/− IDO was neither expressed constitutively nor was it induced by incubation with IFNγ. Taken together, these findings conclusively demonstrate that neither IFNγR1-mediated effects of IFNγ nor indoleamine 2,3-dioxygenase are involved in the molecular mechanism used by MSC to inhibit PBMC proliferation.

scholarly journals Biologic Characteristics of Bone Substituting Tissue Engineering Construction Based on Calcium Phosphate Ceramics, Autologous Mesenchymal Stromal Cells and Fibrin Hydrogel

2015 ◽  
Vol 22 (4) ◽  
pp. 52-59
Author(s):  
V. E Mamonov ◽  
A. G Chemis ◽  
V. S Komlev ◽  
A. L Berkovskiy ◽  
E. M Golubev ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Calcium Phosphate ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Orthotopic Implantation ◽  
Calcium Phosphate Ceramic ◽  
Engineering Construction

Biological characteristics of bone substituting tissue engineering construction (TEC) that contained porous calcium phosphate ceramic granulate (CPC) of phase structure ((tricalcium phosphate (TCP)), fibrin hydrogel and autologous multipotent mesenchymal stromal cells (auto-MMSC) induced and non-induced to osteogenic differentiation were studied in vivo. The following characteristics of TEC were determined: ability to transfer within its structure the viable auto-MMSC with preservation of their regeneration potential; ability to osteogenesis only under conditions of orthotopic implantation; ability of induced to osteogenic differentiation auto-MMSC to participate in the reparative processes for not more than within 6 weeks after implantation; negative affect of fibrin hydrogel on the osteoinductive properties of CPC within TCP structure. It was shown that to provide osteogenesis in the implanted TEC not only the viable auto-MMSC but simultaneous presence of osteoinductive and osteoconductive factors was required. No bone formation in a critical bone defect and in ectopic implantation takes place without observance of these conditions.

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