Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation.

Easy, cost-effective and reliable cryopreservation protocols are crucial for the successful and effective application of tissue engineering. Several different protocols are in use, but no comprehensive comparisons across different machine-based and manual methods have been made. Here, we compare the effects of different cooling rates on the post-thaw survival and proliferative capacity of two basic cell lines for skin tissue engineering fibroblasts and keratinocytes, cultured and frozen in suspension or as a monolayer. We demonstrate that effectiveness of cryopreservation cannot be reliably determined immediately after thawing: the results at this stage were not indicative of cell growth in culture 3 days post-thaw. Cryopreservation of fibroblasts in an adherent state greatly diminishes their subsequent growth potential. This was not observed when freezing in suspension. In keratinocytes, however, adherent freezing is as effective as freezing in suspension, which could lead to significant cost and labour savings in a tissue-engineering environment. The 'optimal' cryopreservation protocol depends on cell type and intended use. Where time, ease and cost are dominant factors, the direct freezing into a nitrogen tank (straight freeze) approach remains a viable method. The most effective solution across the board, as measured by viability 3 days post-thaw, was the commonly used, freezing container method. Where machine-controlled cryopreservation is deemed important for tissue-engineering Good Manufacturing Practice, we present results using a portfolio of different cooling rates, identifying the 'optimal' protocol depending on cell type and culture method. Copyright © 2013 John Wiley & Sons, Ltd.

Comparing the burden: what can we learn by comparing regulatory frameworks in abortion and fertility services?

In the UK, regulation of clinical services is being restructured. We consider two clinical procedures, abortion and IVF treatment, which have similar ethical and political sensitivities. We consider factors including the law, licensing, inspection, amount of paperwork and reporting requirements, the reception by practitioners and costs, to establish which field has the greater 'regulatory burden'. We test them based on scientific, ethical, social, political factors that might explain differences. We find that regulatory burden borne by IVF services is greater than in abortion, but none of the explanatory theses can provide a justification of this phenomenon. We offer an alternative explanation based on regulatory 'overspill' from research regulation and policy making, conceptualisation of risk regulation and a high public profile that locks a regulator into self-preservation.

The procurement of cells for the derivation of human embryonic stem cell lines for therapeutic use: recommendations for good practice.

The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.

Scientists' perception of ethical issues in nanomedicine: a case study.

AIM: Research and development in nanomedicine has been accompanied by the consideration of ethical issues; however, little is known about how researchers working in this area perceive such issues. This case-study explores scientists' attitude towards and knowledge of ethical issues. METHOD: Data were collected by semi-structured interviews with 22 nanomedicine practitioners and subject to content analysis. RESULTS: We found that scientists reflect with ambiguity on the reputed novelty of nanomedicine and what the ethical issues and risks are in their work. Respondents see no necessity for a paradigm shift in ethical considerations, but view ethical issues in nanomedicine as overlapping with those of other areas of biomedical research. Most respondents discuss ethical issues they faced in scientific work with their colleagues, but expect benefit from additional information and training on ethics. CONCLUSION: Our findings that scientists are motivated to reflect on ethical issues in their work, can contribute to the design of new strategies, including training programs, to engage scientists in ethical discussion and stimulate their responsibility as nanomedicine practitioners.

The implications of "advanced therapies" regulation.

Recent policy developments in the United States and Europe have created new regulatory regimes for advanced and complex treatments such as cell therapies, tissue engineering, and gene therapies that are of particular relevance for regenerative medicine. Where these new legal rules operate as a mere extension of existing regimes, they risk stifling innovation in these areas of life extension technology.

Fusion and regenerative therapies: is immortality really recessive?

Harnessing cellular fusion as a potential tool for regenerative therapy has been under tentative investigation for decades. A look back the history of fusion experiments in gerontology reveals that whereas some studies indicate that aging-related changes are conserved in fused cells, others have demonstrated that fusion can be used as a tool to revoke cellular senescence and induce tissue regeneration. Recent findings about the role of fusion processes in tissue homeostasis, replenishment, and repair link insights from fusion studies of previous decades with modern developments in stem cell biology and regenerative medicine. We suggest that age-associated loss of regenerative capacity is associated with a decline of effectiveness in stem cell fusion. We project how studies into the fusion of stem cells with tissue cells, or the fusion between activator stem cells and patient cells might help in the development of applications that "rejuvenate" certain target cells, thereby strategically reinstating a regeneration cascade. The outlook is concluded with a discussion of the next research milestones and the potential hazards of fusion therapies.

Cellular therapy using microglial cells.

Recent insights into the function and dysfunction of microglia may inform future therapies to combat neurodegeneration. We hypothesise how different aspects of microglial activity including migration, activation, oxidative response, phagocytosis, proteolysis, and replenishment could be targeted by novel therapeutic approaches. A combined approach is suggested, encompassing opsonization and anti-inflammatory strategies in conjunction with an engineering of microglial precursors. Xenoproteases for bioremediation could be used to enhance intracellular and extracellular proteolytic capacity. The capacity of microglial precursors to cross the blood-brain barrier and to home in on sites of neural damage and inflammation might prove to be particularly useful for future therapeutic strategies.

Stressed stem cells: Temperature response in aged mesenchymal stem cells.

Mesenchymal stem cells (MSCs) derived from young (6 week) and aged (56 week) Wistar rats were cultured at standard (37 degrees C) and reduced (32 degrees C) temperature and compared for age markers and stress levels. (ROS, NO, TBARS, carbonyls, lipofuscin, SOD, GPx, apoptosis, proteasome activity) and heat shock proteins (HSP27, -60, -70, -90). Aged MSCs display many of the stress markers associated with aging in other cell types, but results vary across marker categories and are temperature dependant. In young MSCs, culturing at reduced temperature had a generally beneficial effect: the anti-apoptotic heat shock proteins HSP 27, HSP70, and HSP90 were up-regulated; pro-apoptotic HSP60 was downregulated; SOD, GPx increased; and levels in ROS, NO, TBARS, carbonyl, and lipofuscin were diminished. Apoptosis was reduced, but also proteasome activity. In contrast, in aged MSCs, culturing at reduced temperature generally produced no 'beneficial' changes in these parameters, and can even have detrimental effects. Implications for tissue engineering and for stem cell gerontology are discussed. The results suggest that a 'hormesis' theory of stress response can be extended to MSCs, but that cooling cultivation temperature stress produces positive effects in young cells only.

Aging of mesenchymal stem cells.

The role of adult mesenchymal stem cells (MSC) in tissue maintenance and regeneration has received significant attention of late. Questions arise to what extent these cells are either subject to, or causes of aging; whether age-related changes in these cells are due to intrinsic factors or induced by the somatic environment. This review collates and examines recent data in support of these different theories. By means of introduction, a brief overview is given of current MSC definitions and their basic role in tissue regeneration followed by a comparative analysis of gerontological studies involving MSC. Evidence for extrinsic aging and various aging markers relating to morphology, proliferation, signalling, senescence markers, telomeres and telomerase, and other indicators is discussed. We observe that while the literature might often appear to conflict, many apparent discrepancies are attributable to inconsistent methods of extracting and isolating MSC which in fact contains various subsets of adult stem cells, varying not only in their differentiation potential but also in their vulnerability to senescence--ranging from quasi-somatic lifespan to perennial vigour. Thus, mesenchymal stem cells emerge as both subject to and key mediators of organismal aging.

Chronically active: activation of microglial proteolysis in ageing and neurodegeneration.

One of the microglial cell functions is the removal of modified extracellular proteins in the brain. The connection between protein oxidation, proteolysis, and microglial activation is the topic of this review. The effect of various activation agents on microglial cells with regard to changes in substrate uptake, proteolytic capacity and degradation efficiency of different types of oxidized protein materials is reviewed. It is shown that different activation stimuli initiate substrate-specific modulation for uptake and proteolysis, influencing an array of factors including receptor expression, lysosomal pH, and proteasome subunit composition. Age-related alterations in activation and proteolytic capacity in microglial cells are also discussed. In ageing, proteolytic effectiveness is diminished, while microglial cells are chronically activated and lose the oxidative burst ability, possibly supporting a 'vicious circle' of macrophage-induced neurodegeneration.