Cryopreservation of human umbilical cord-derived mesenchymal stem cells in complex sugar based cryoprotective solutions

Mesenchymal stem cells (MSCs) are able to differentiate in vivo and in vitro giving rise to different cell types including osteoblasts, adipocytes, chondrocytes and neuronal cells, providing a valuable source for treatment of degenerative and age-associated diseases. Im-provement of protocols and procedures for human MSCs cryopreservation will contribute significantly to the development of cell replacement therapies. We developed an alternative cryopreservation solutions for stem cell cryopreservation. Most cryoprotectants need to be removed from the cells by washing after thawing, a procedure that can lead to a loss of precious stem cells. Additionally, the procedure is time and cost-consuming. In our study we used a combination of transfusable and non-toxic substances such as hydroxyethylstarch, sorbitol and dextran replacing DMSO and FCS. We found that a cryosolution containing 5% HES, 0.3M sorbitol and 5% dextran provide successful protection for human umbilical cord derived mesenchymal stem cells. These MSC retain a high viability and show multilineage differentiation

Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch–based cryoprotectants

Background: Preservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers. Results: We cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution. Conclusion: We conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols

Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch–based cryoprotectants

Background: Preservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers. Results: We cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution. Conclusion: We conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols

Protective effects of alpha phenyl-tert-butyl nitrone and ascorbic acid in human adipose derived mesenchymal stem cells from differently aged donors.

Adipose-derived mesenchymal stem cells (ADSCs) are multipotent stem cells that promote therapeutic effects and are frequently used in autologous applications. Little is known about how ADSCs respond to genotoxic stress and whether or not donor age affects DNA damage and repair. In this study, we used the comet assay to assess DNA damage and repair in human ADSCs derived from young (20-40 years), middle-aged (41-60 years), and older (61+ years) donors following treatment with H2O2 or UV light. Tail lengths in H2O2-treated ADSCs were substantially higher than the tail lengths in UV-treated ADSCs. After 30 minutes of treatment with H2O2, ADSCs preconditioned with alpha phenyl-tert-butyl nitrone (PBN) or ascorbic acid (AA) showed a significant reduction in % tail DNA. The majority of ADSCs treated with PBN or AA displayed low olive tail movements at various timepoints. In general and indicative of DNA repair, % tail length and % tail DNA peaked at 30 minutes and then decreased to near-control levels at the 2 hour and 4 hour timepoints. Differently aged ADSCs displayed comparable levels of DNA damage in the majority of these experiments, suggesting that the age of the donor does not affect the DNA damage response in cultured ADSCs

Distribution pattern following systemic mesenchymal stem cell injection depends on the age of the recipient and neuronal health

BACKGROUND: Mesenchymal stem cells (MSCs) show therapeutic efficacy in many different age-related degenerative diseases, including Alzheimer's disease. Very little is currently known about whether or not aging impacts the transplantation efficiency of MSCs. METHODS: In this study, we investigated the distribution of intravenously transplanted syngeneic MSCs derived from young and aged mice into young, aged, and transgenic APP/PS1 Alzheimer's disease mice. MSCs from male donors were transplanted into female mice and their distribution pattern was monitored by PCR using Y-chromosome specific probes. Biodistribution of transplanted MSCs in the brains of APP/PS1 mice was additionally confirmed by immunofluorescence and confocal microscopy. RESULTS: Four weeks after transplantation into young mice, young MSCs were found in the lung, axillary lymph nodes, blood, kidney, bone marrow, spleen, liver, heart, and brain cortex. In contrast, young MSCs that were transplanted into aged mice were only found in the brain cortex. In both young and aged mouse recipients, transplantation of aged MSCs showed biodistribution only in the blood and spleen. Although young transplanted MSCs only showed neuronal distribution in the brain cortex in young mice, they exhibited a wide neuronal distribution pattern in the brains of APP/PS1 mice and were found in the cortex, cerebellum, hippocampus, olfactory bulb, and brainstem. The immunofluorescent signal of both transplanted MSCs and resident microglia was robust in the brains of APP/PS1 mice. Monocyte chemoattractant-1 levels were lowest in the brain cortex of young mice and were significantly increased in APP/PS1 mice. Within the hippocampus, monocyte chemoattractant-1 levels were significantly higher in aged mice compared with younger and APP/PS1 mice. CONCLUSIONS: We demonstrate in vivo that MSC biodistribution post transplantation is detrimentally affected by aging and neuronal health. Aging of both the recipient and the donor MSCs used attenuates transplantation efficiency. Clinically, our data would suggest that aged MSCs should not be used for transplantation and that transplantation of MSCs into aged patients will be less efficacious

Regulating aging in adult stem cells with microRNA

Aging can be defined as the result of accumulated cellular damage and deregulation of the epigenome. These changes cause impaired cell maintenance systems, reduced tissue regeneration, weakening of the immune system and increased risk of malignancy. The higher mortality rate in older individuals is a result of these pathologies. The study of age-related changes in adult stem cells and their regenerative potential is crucial to our understanding of the physical deterioration of organs and tissues. The growing interest and knowledge in the field of microRNAs adds a further dimension to this field of research. MicroRNAs are important posttranscriptional regulators of gene expression. They co-regulate stem cell properties such as potency, differentiation, self-renewal and senescence. Various cell systems, e.g. defense mechanisms against reactive oxygen radicals (ROS), DNA repair and apoptosis are regulated by microRNAs. These properties and the assumption that microRNAs act as some kind of general switch make them highly relevant in aging research

Regulating aging in adult stem cells with microRNA

Aging can be defined as the result of accumulated cellular damage and deregulation of the epigenome. These changes cause impaired cell maintenance systems, reduced tissue regeneration, weakening of the immune system and increased risk of malignancy. The higher mortality rate in older individuals is a result of these pathologies. The study of age-related changes in adult stem cells and their regenerative potential is crucial to our understanding of the physical deterioration of organs and tissues. The growing interest and knowledge in the field of microRNAs adds a further dimension to this field of research. MicroRNAs are important posttranscriptional regulators of gene expression. They co-regulate stem cell properties such as potency, differentiation, self-renewal and senescence. Various cell systems, e.g. defense mechanisms against reactive oxygen radicals (ROS), DNA repair and apoptosis are regulated by microRNAs. These properties and the assumption that microRNAs act as some kind of general switch make them highly relevant in aging research

Alzheimer: nasal transplantation of microglia

Alzheimer’s disease (AD) is an age-related neurodegenerative disease associated with the formation of amyloid plaques, [tau] aggregation and oxidative/inflammatory damage. Microglia play an important role in the early phase of the disease and are known to be involved in AD progression. Senescent microglia accumulate in AD causing inflammation, neuronal damage and increasing the A[beta] load. Microglia could be an attractive target for mesenchymal cell therapies (MSC)

Alzheimer: nasal transplantation of microglia

Alzheimer’s disease (AD) is an age-related neurodegenerative disease associated with the formation of amyloid plaques, [tau] aggregation and oxidative/inflammatory damage. Microglia play an important role in the early phase of the disease and are known to be involved in AD progression. Senescent microglia accumulate in AD causing inflammation, neuronal damage and increasing the A[beta] load. Microglia could be an attractive target for mesenchymal cell therapies (MSC)

Hydroxyethylstarch in cryopreservation - mechanisms, benefits and problems

As the progress of regenerative medicine places ever greater attention on cryopreservation of (stem) cells, tried and tested cryopreservation solutions deserve a second look. This article discusses the use of hydroxyethyl starch (HES) as a cryoprotectant. Charting carefully the recorded uses of HES as a cryoprotectant, in parallel to its further clinical use, indicates that some HES subtypes are a useful supplement to dimethysulfoxide (DMSO) in cryopreservation. However, we suggest that the most common admixture ratio of HES and DMSO in cryoprotectant solutions has been established by historical happenstance and requires further investigation and optimization. © 2012 Elsevier Ltd