Storage conditions affect the composition of the lyophilized secretome of multipotent mesenchymal stromal cells.

The widespread use of multipotent mesenchymal stromal cell-derived secretome (MSC-sec) requires optimal preservation methods. Lyophilization offers benefits like concentrating the secretome, reducing the storage volume, and making storage conditions more flexible. This study evaluated the influence of storage duration and temperature on lyophilized MSC-sec. The conditioned medium from Wharton's jelly MSCs was stored at - 80 °C or lyophilized with or without trehalose. Lyophilized formulations were kept at - 80 °C, - 20 °C, 4 °C, or room temperature (RT) for 3 and 30 months. After storage and reconstitution, the levels of growth factors and cytokines were assessed using multiplex assay. The storage of lyophilized MSC-sec at - 80 °C ensured biomolecule preservation for 3 and 30 months. Following 3 month storage at 4 °C and RT, a notable decrease occurred in BDNF, bNGF, and sVCAM-1 levels. Prolonged 30 month storage at the same temperatures significantly reduced BDNF, bNGF, VEGF-A, IL-6, and sVCAM-1, while storage at - 20 °C decreased BDNF, bNGF, and VEGF- A levels. Trehalose supplementation of MSC-sec improved the outcome during storage at 4 °C and RT. Proper storage conditions were crucial for the preservation of lyophilized MSC-sec composition. Short-term storage at various temperatures maintained over 60% of the studied growth factors and cytokines; long-term preservation was only adequate at -80 °C.

Mechanical Regulation of Mitochondrial Dynamics and Function in a 3D-Engineered Liver Tumor Microenvironment.

It has become evident that physical stimuli of the cellular microenvironment transmit mechanical cues regulating key cellular functions, such as proliferation, migration, and malignant transformation. Accumulating evidence suggests that tumor cells face variable mechanical stimuli that may induce metabolic rewiring of tumor cells. However, the knowledge of how tumor cells adapt metabolism to external mechanical cues is still limited. We therefore designed soft 3D collagen scaffolds mimicking a pathological mechanical environment to decipher how liver tumor cells would adapt their metabolic activity to physical stimuli of the cellular microenvironment. Here, we report that the soft 3D microenvironment upregulates the glycolysis of HepG2 and Alexander cells. Both cell lines adapt their mitochondrial activity and function under growth in the soft 3D microenvironment. Cells grown in the soft 3D microenvironment exhibit marked mitochondrial depolarization, downregulation of mitochondrially encoded cytochrome c oxidase I, and slow proliferation rate in comparison with stiff monolayer cultures. Our data reveal the coupling of liver tumor glycolysis to mechanical cues. It is proposed here that soft 3D collagen scaffolds can serve as a useful model for future studies of mechanically regulated cellular functions of various liver (potentially other tissues as well) tumor cells.

Hypothermic Storage of 3D Cultured Multipotent Mesenchymal Stromal Cells for Regenerative Medicine Applications.

The regulatory requirements in cell processing, in the choice of a biomaterial scaffold and in quality control analysis, have to be followed in the clinical application of tissue-engineered grafts. Confirmation of sterility during quality control studies requires prolonged storage of the cell-based construct. After storage, preservation of the functional properties of the cells is an important prerequisite if the cells are to be used for cell-based tissue therapies. The study presented here shows the generation of 3D constructs based on Wharton's jelly multipotent mesenchymal stromal cells (WJ-MSCs) and the clinically-acceptable HyaloFast® scaffold, and the effect of two- and six-day hypothermic storage of 3D cell-based constructs on the functional properties of populated cells. To study the viability, growth, gene expression, and paracrine secretion of WJ-MSCs within the scaffolds before and after storage, xeno-free culture conditions, metabolic, qPCR, and multiplex assays were applied. The WJ-MSCs adhered and proliferated within the 3D HyaloFast®. Our results show different viability of the cells after the 3D constructs have been stored under mild (25 °C) or strong (4 °C) hypothermia. At 4 °C, the significant decrease of metabolic activity of WJ-MSCs was detected after 2 days of storage, with almost complete cell loss after 6 days. In mild hypothermia (25 °C) the decrease in metabolic activity was less remarkable, confirming the suitability of these conditions for cell preservation in 3D environment. The significant changes were detected in gene expression and in the paracrine secretion profile after 2 and 6 days of storage at 25 °C. The results presented in this study are important for the rapid transfer of tissue engineering approaches into clinical applications.

Generation of bone grafts using cryopreserved mesenchymal stromal cells and macroporous collagen-nanohydroxyapatite cryogels.

Bone tissue engineering strategy involves the 3D scaffolds and appropriate cell types promoting the replacement of the damaged area. In this work, we aimed to develop a fast and reliable clinically relevant protocol for engineering viable bone grafts, using cryopreserved adipose tissue-derived mesenchymal stromal cells (MSCs) and composite 3D collagen-nano-hydroxyapatite (nanoHA) scaffolds. Xeno- and DMSO-free cryopreserved MSCs were perfusion-seeded into the biomimetic collagen/nanoHA scaffolds manufactured by cryotropic gelation and their osteoregenerative potential was assessed in vitro and in vivo. Cryopreserved MSCs retained the ability to homogenously repopulate the whole volume of the scaffolds during 7 days of post-thaw culture. Moreover, the scaffold provided a suitable microenvironment for induced osteogenic differentiation of cells, confirmed by alkaline phosphatase activity and mineralization. Implantation of collagen-nanoHA cryogels with cryopreserved MSCs accelerated woven bone tissue formation, maturation of bone trabeculae, and vascularization of femur defects in immunosuppressed rats compared to cell-free collagen-nanoHA scaffolds. The established combination of xeno-free cell culture and cryopreservation techniques together with an appropriate scaffold design and cell repopulation approach accelerated the generation of viable bone grafts.

Hepatic Tumor Cell Morphology Plasticity under Physical Constraints in 3D Cultures Driven by YAP-mTOR Axis.

Recent studies undoubtedly show that the mammalian target of rapamycin (mTOR) and the Hippo-Yes-associated protein 1 (YAP) pathways are important mediators of mechanical cues. The crosstalk between these pathways as well as de-regulation of their signaling has been implicated in multiple tumor types, including liver tumors. Additionally, physical cues from 3D microenvironments have been identified to alter gene expression and differentiation of different cell lineages. However, it remains incompletely understood how physical constraints originated in 3D cultures affect cell plasticity and what the key mediators are of such process. In this work, we use collagen scaffolds as a model of a soft 3D microenvironment to alter cellular size and study the mechanotransduction that regulates that process. We show that the YAP-mTOR axis is a downstream effector of 3D cellular culture-driven mechanotransduction. Indeed, we found that cell mechanics, dictated by the physical constraints of 3D collagen scaffolds, profoundly affect cellular proliferation in a YAP-mTOR-mediated manner. Functionally, the YAP-mTOR connection is key to mediate cell plasticity in hepatic tumor cell lines. These findings expand the role of YAP-mTOR-driven mechanotransduction to the control hepatic tumor cellular responses under physical constraints in 3D cultures. We suggest a tentative mechanism, which coordinates signaling rewiring with cytoplasmic restructuring during cell growth in 3D microenvironments.

A Comparative Analysis of Multipotent Mesenchymal Stromal Cells derived from Different Sources, with a Focus on Neuroregenerative Potential.

Multipotent mesenchymal stromal cells (MSCs) can be considered an accessible therapeutic tool for regenerative medicine. Here, we compared the growth kinetics, immunophenotypic and immunomodulatory properties, gene expression and secretome profile of MSCs derived from human adult bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and Wharton's jelly (WJ-MSCs) cultured in clinically-relevant conditions, with the focus on the neuroregenerative potential. All the cell types were positive for CD10/CD29/CD44/CD73/CD90/CD105/HLA-ABC and negative for CD14/CD45/CD235a/CD271/HLA-DR/VEGFR2 markers, but they differed in the expression of CD34/CD133/CD146/SSEA-4/MSCA-1/CD271/HLA-DR markers. BM-MSCs displayed the highest immunomodulatory activity compared to AT- and WJ-MSCs. On the other hand, BM-MSCs secreted the lower content and had the lower gene expression of neurotrophic growth factors compared to other cell lines, which may be caused by the higher sensitivity of BM-MSCs to nutrient limitations. Despite the differences in growth factor secretion, the MSC secretome derived from all cell sources had a pronounced neurotrophic potential to stimulate the neurite outgrowth of DRG-neurons and reduce the cell death of neural stem/progenitor cells after H2O2 treatment. Overall, our study provides important information for the transfer of basic MSC research towards clinical-grade manufacturing and therapeutic applications.

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

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.

Clinically Relevant Solution for the Hypothermic Storage and Transportation of Human Multipotent Mesenchymal Stromal Cells.

The wide use of human multipotent mesenchymal stromal cells (MSCs) in clinical trials requires a full-scale safety and identity evaluation of the cellular product and subsequent transportation between research/medical centres. This necessitates the prolonged hypothermic storage of cells prior to application. The development of new, nontoxic, and efficient media, providing high viability and well-preserved therapeutic properties of MSCs during hypothermic storage, is highly relevant for a successful clinical outcome. In this study, a simple and effective trehalose-based solution was developed for the hypothermic storage of human bone marrow MSC suspensions for further clinical applications. Human bone marrow MSCs were stored at 4°C for 24, 48, and 72 hrs in the developed buffered trehalose solution and compared to several research and clinical grade media: Plasma-Lyte® 148, HypoThermosol® FRS, and Ringer's solution. After the storage, the preservation of viability, identity, and therapeutically associated properties of MSCs were assessed. The hypothermic storage of MSCs in the new buffered trehalose solution provided significantly higher MSC recovery rates and ability of cells for attachment and further proliferation, compared to Plasma-Lyte® 148 and Ringer's solution, and was comparable to research-grade HypoThermosol® FRS. There were no differences in the immunophenotype, osteogenic, and adipogenic differentiation and the immunomodulatory properties of MSCs after 72 hrs of cold storage in these solutions. The obtained results together with the confirmed therapeutic properties of trehalose previously described provide sufficient evidence that the developed trehalose medium can be applied as a low-cost and efficient solution for the hypothermic storage of MSC suspensions, with a high potential for translation into clinical practice.

The therapeutic potential of three-dimensional multipotent mesenchymal stromal cell spheroids.

The efficiency of clinical trials involving transplantation of multipotent mesenchymal stromal cells (MSCs) is often insufficient due to harsh conditions present within the target tissue including hypoxia, low nutrient supply as well as inflammatory reactions. This indicates the necessity for optimization of cell-based therapy approaches which might include either modification of the cell manufacturing process or specific cell pretreatment procedures prior to transplantation. Recent reports confirm evidence that the aggregation of MSCs into three-dimensional (3D) multicellular spheroids results in enhancement of the overall therapeutic potential of cells, by improving the anti-inflammatory and angiogenic properties, stemness and survival of MSCs after transplantation. Such an MSCs spheroid generation approach may open new opportunities for the enlargement of MSCs applications in clinical research and therapy. However, the unification and optimization of 3D spheroid generation techniques, including the selection of appropriate clinical-grade culture conditions and methods for their large-scale production, are still of great importance. The current review addresses questions regarding therapeutic-associated properties of 3D multicellular MSCs spheroids in vitro and during preclinical animal studies, with special attention to the possibilities of translating these research achievements toward further clinical manufacturing and applications.

Towards ready-to-use 3-D scaffolds for regenerative medicine: adhesion-based cryopreservation of human mesenchymal stem cells attached and spread within alginate-gelatin cryogel scaffolds.

Cultivation and proliferation of stem cells in three-dimensional (3-D) scaffolds is a promising strategy for regenerative medicine. Mesenchymal stem cells with their potential to differentiate in various cell types, cryopreserved adhesion-based in fabricated scaffolds of biocompatible materials can serve as ready-to-use transplantation units for tissue repair, where pores allow a direct contact of graft cells and recipient tissue without further preparation. A successful cryopreservation of adherent cells depends on attachment and spreading processes that start directly after cell seeding. Here, we analyzed different cultivation times (0.5, 2, 24 h) prior to adhesion-based cryopreservation of human mesenchymal stem cells within alginate-gelatin cryogel scaffolds and its influence on cell viability, recovery and functionality at recovery times (0, 24, 48 h) in comparison to non-frozen control. Analysis with confocal laser scanning microscopy and scanning electron microscopy indicated that 2 h cultivation time enhanced cryopreservation success: cell number, visual cell contacts, membrane integrity, motility, as well as spreading were comparable to control. In contrast, cell number by short cultivation time (0.5 h) reduced dramatically after thawing and expanded cultivation time (24 h) decreased cell viability. Our results provide necessary information to enhance the production and to store ready-to-use transplantation units for application in bone, cartilage or skin regenerative therapy.

Use of osteogenic protein-1 in patients at high risk for spinal pseudarthrosis: a prospective cohort study assessing safety, health-related quality of life, and radiographic fusion. Invited submission from the Joint Section on Disorders of the Spine and Peripheral Nerves, March 2007.

OBJECT: The capability of osteogenic protein (OP)-1 to induce bone formation has led to an increasing interest in its use in fusion surgery. This prospective study examines the safety and efficacy of OP-1 use in patients considered to be at a high risk for developing pseudarthrosis following reconstructive spinal surgery. METHODS: Outcome measures included documentation of adverse events, radiographic evaluation of fusion by an independent musculoskeletal radiologist blinded to treatment, the Oswestry Disability Index (ODI), and the 36-Item Short Form Health Survey (SF-36). The health-related quality of life (HRQOL) assessments (ODI and SF-36) were given at baseline and at 3, 6, 12, 18, and 24 months after the surgical OP-1 implant. RESULTS: The study consisted of 17 male and 13 female patients, with a mean age of 53 years (range 20-77 years). Fourteen patients underwent operations for cervical disease, and 16 for lumbar disease, with a median postoperative follow-up of 24 months (range 13-46 months). There were significant improvements in the physical health (from 28.7 +/- 1.5 to 34.2 +/- 3; p = 0.025) and mental health (from 43.7 +/- 2 to 47.5 +/- 3.1; p = 0.015) summary scores on the SF-36. The mean postoperative ODI score at 6, 9, 12, and 18 months was significantly lower than the baseline ODI score, after taking into consideration a 10-point measurement error (p = 0.0003, p = 0.003, p = 0.004, and p = 0.032, respectively). At 24 months, however, the differences in ODI scores were no longer significant. Of the 30 patients, 24 (80%) were deemed to have a solid fusion. There were no allergic reactions to OP-1 and no symptomatic postoperative hematomas. CONCLUSIONS: Our results suggest that the use of OP-1 is safe and may contribute to high fusion rates, as demonstrated by radiographs, reduced levels of disability, and improved HRQOL in patients considered to be at a high risk for developing a nonunion after spinal reconstructive surgery.