Cryopreservation of Human Bone Marrow Derived Mesenchymal Stem Cells at High Concentration Is Feasible.

Introduction: For stem cell therapies to be adopted in mainstream health care, robust, reliable, and cost-effective storage and transport processes must be developed. Cryopreservation remains the best current platform for this purpose, and freezing cells at high concentration may have many benefits, including savings on cost and storage space, facilitating transport logistics, and reducing cryoprotectant volume. Cells, such as mesenchymal stem cells (MSCs), are typically frozen at 1 million cells per milliliter (mL), but the aim of this study is to examine the post-thaw attributes of human bone marrow derived MSCs (hBM-MSCs) frozen at 1, 5, and 10 million cells per mL. Methods: Thawed cells were assessed for their morphology, phenotypic marker expression, viability, apoptosis level, metabolic activity, proliferation, and osteogenic and adipogenic differentiation. Results: In this study, for the first time, it is shown that all assessed cells expressed the typical MSC markers (CD90, CD105, and CD73) and lacked the expression of CD14, CD20, CD34, CD45, and HLA-DR. In addition, all cells showed elongated fibroblastic morphology. Post-thaw viability was retained with no difference among the three concentrations. Moreover, no significant statistical difference was observed in the post-thaw apoptosis level, metabolic activity, proliferation, and osteogenic potential, indicating that these cells are amenable to cryopreservation at higher concentrations. Conclusion: The results of this study are of paramount importance to the development of manufacturing processes around a useful freezing concentration when cells are targeted to be stored for at least 6 months.

Evaluating patient perspectives on participating in scientific research and clinical trials for the treatment of spinal cord injury.

A questionnaire was developed to evaluate patients' perspective on research aimed at improving functions and overcoming complications associated with spinal cord injury (SCI). The first three sections were based on published and validated assessment tools. The final section was developed to assess participant perspectives on research for SCI. One thousand patients were approached, of which 159 participated. Fifty-eight percent of participants were satisfied with their 'life as a whole'. Two factors could be generated that reflected the variance in the data regarding participants' life with a SCI: "Psychosocial and physical wellbeing" and "Independent living". The majority of participants stated they would be involved in research (86%) or clinical trials (77%). However, the likelihood of participation dropped when potential risks of the research/trials were explained. Which participants would be willing to participate in research could not be predicted based on the severity of their injury, their psychosocial and physical wellbeing or their independent living. Despite participant establishment of a life with SCI, our data indicates that individuals strive for improvements in function. Participant willingness to be included in research studies is noteworthy and scientists and clinicians are encouraged to involve more patients in all aspects of their research.

Quantitative assessment of the impact of cryopreservation on human bone marrow-derived mesenchymal stem cells: up to 24 h post-thaw and beyond.

BACKGROUND: The effects of cryopreservation on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are still ill-defined. In this study, a quantitative approach was adopted to measure several post-thaw cell attributes in order to provide an accurate reflection of the freezing and thawing impact. METHODS: Fresh and cryopreserved passage-matched cells from three different donors were discretely analysed and compared for their viability, apoptosis level, phenotypic marker expression, metabolic activity, adhesion potential, proliferation rate, colony-forming unit ability (CFUF) and differentiation potentials. RESULTS: The results of this study show that cryopreservation reduces cell viability, increases apoptosis level and impairs hBM-MSC metabolic activity and adhesion potential in the first 4 h after thawing. At 24 h post-thaw, cell viability recovered, and apoptosis level dropped but metabolic activity and adhesion potential remained lower than fresh cells. This suggests that a 24-h period is not enough for a full recovery. Beyond 24 h post-thaw, the observed effects are variable for the three cell lines. While no difference is observed in the pre- and post-cryopreservation proliferation rate, cryopreservation reduced the CFUF ability of two of the cell lines and variably affected the adipogenic and osteogenic differentiation potentials of the three cell lines. CONCLUSION: The data collected in this study clearly show that fresh and cryopreserved hBM-MSCs are different, and these differences will inevitably introduce variabilities to the product and process development and subsequently imply financial losses. In order to avoid product divergence pre- and post-cryopreservation, effective strategies to mitigate freezing effects must be developed and implemented.

The impact of cryopreservation on bone marrow-derived mesenchymal stem cells: a systematic review.

Mesenchymal stem cells (MSCs) represent an invaluable asset for the field of cell therapy. Human Bone marrow-derived MSCs (hBM-MSCs) are one of the most commonly used cell types in clinical trials. They are currently being studied and tested for the treatment of a wide range of diseases and conditions. The future availability of MSCs therapies to the public will require a robust and reliable delivery process. Cryopreservation represents the gold standard in cell storage and transportation, but its effect on BM-MSCs is still not well established. A systematic review was conducted to evaluate the impact of cryopreservation on BM-MSCs and to attempt to uncover the reasons behind some of the controversial results reported in the literature. Forty-one in vitro studies were analysed, and their results organised according to the cell attributes they assess. It was concluded that cryopreservation does not affect BM-MSCs morphology, surface marker expression, differentiation or proliferation potential. However, mixed results exist regarding the effect on colony forming ability and the effects on viability, attachment and migration, genomic stability and paracrine function are undefined mainly due to the huge variabilities governing the cryopreservation process as a whole and to the lack of standardised assays.

Determining Conditions for Successful Culture of Multi-Cellular 3D Tumour Spheroids to Investigate the Effect of Mesenchymal Stem Cells on Breast Cancer Cell Invasiveness.

Mesenchymal stem cells have been widely implicated in tumour development and metastases. Moving from the use of two-dimensional (2D) models to three-dimensional (3D) to investigate this relationship is critical to facilitate more applicable and relevant research on the tumour microenvironment. We investigated the effects of altering glucose concentration and the source of foetal bovine serum (FBS) on the growth of two breast cancer cell lines (T47D and MDA-MB-231) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) to determine successful conditions to enable their co-culture in 3D tumour spheroid models. Subsequently, these 3D multi-cellular tumour spheroids were used to investigate the effect of hBM-MSCs on breast cancer cell invasiveness. Findings presented herein show that serum source had a statistically significant effect on two thirds of the growth parameters measured across all three cell lines, whereas glucose only had a statistically significant effect on 6%. It was determined that the optimum growth media composition for the co-culture of 3D hBM-MSCs and breast cancer cell line spheroids was 1 g/L glucose DMEM supplemented with 10% FBS from source A. Subsequent results demonstrated that co-culture of hBM-MSCs and MDA-MB-231 cells dramatically reduced invasiveness of both cell lines (F(1,4) = 71.465, p = 0.001) when embedded into a matrix comprising of growth-factor reduced base membrane extract (BME) and collagen.

The Role of Dissolved Oxygen Levels on Human Mesenchymal Stem Cell Culture Success, Regulatory Compliance, and Therapeutic Potential.

Most cells in the human body, including human mesenchymal stem cells (hMSCs), have evolved to survive and function in a low physiological oxygen (O2) environment. Investigators have become increasingly aware of the effects of O2 levels on hMSC biology and culture and are mimicking the natural niche of these cells in vitro to improve cell culture yields. This presents many challenges in relation to hMSC identity and function and in the maintenance of a controlled O2 environment for cell culture. The aim of this review was to discuss an "hMSC checklist" as a guide to establishing which identity and potency assays to implement when studying hMSCs. The checklist includes markers, differentiation potential, proliferation and growth, attachment and migration, genomic stability, and paracrine activity. Evidence drawn from the current literature demonstrates that low O2 environments could improve most "hMSC checklist" attributes. However, there are substantial inconsistencies around both the terminology and the equipment used in low O2 studies. Therefore, "hypoxia" as a term and as a culture condition is discussed. The biology of short-term (acute) versus long-term (chronic) hypoxia is considered, and a nascent hypothesis to explain the behavior of hMSCs in long-term hypoxia is presented. It is hoped that by establishing an ongoing discourse and driving toward a regulatory recognizable "hMSC checklist," we may be better able to provide the patient population with safe and efficacious regenerative treatments.