Large-scale compatible methods for the preservation of human embryonic stem cells: current perspectives

Human embryonic stem cells (hESCs) and hESC-derived cells are of great interest, not only because of their therapeutic potential but also their prospective uses in in vitro drug and toxicity screening. The ability to preserve these cells is critical, allowing for the generation of quality-controlled stocks of cells, transport of cells between sites, and avoiding the need for expensive and time-consuming continuous culture. Current methodologies, namely conventional slow freezing and vitrification, can successfully preserve hESCs and their differentiated progeny, retaining the key characteristics of the cells. However, there is a significant gap between the number of cells potentially needed to either treat patients or run a high-throughput drug screen and how many cells can be preserved using these techniques. Therefore, this review focuses on the scalability of slow freezing and vitrification, identifying key barriers to success and whether they can be overcome. Given the precedent with other mammalian cells in using slow freezing to successfully preserve large quantities of cells and its compatibility with current and emerging culture methods for hESCs, it is likely to become the method of choice for cryopreserving these cells at scale. However, issues other than scale still exist; therefore, alternatives to cryopreservation should also be explored. Here, the potential to lyophilize hESCs for long-term storage is considered as one such alternative

Alternatives to cryopreservation for the short and long-term storage of mammalian cells

The ability to preserve mammalian cells has long been a critical part of cell-based research for several reasons. It allows for the transport of cells between laboratories or sites, ensures the availability of consistent starting material for the research through the establishment of cell banks and can uncouple cell-based assays from the culture process. Although cryopreservation, whether conventional slow freezing or vitrification, is widely used as a method for preserving cells long-term, it can result in low cell recovery post-thaw and the cryoprotective agents used in the freezing medium can be cytotoxic. With the emergence of a cell-based therapies industry, where clinical grade cells will need to be stored and transported, there is a growing need to develop scalable GMPcompatible preservation methods that retain not just high cell viability but also clinical efficacy. With these issues in mind, this review will explore two alternatives to cryopreservation: cell desiccation for long-term storage of cells and the short-term storage of cells under hypothermic (>0°C) conditions. © 2013 by Nova Science Publishers, Inc. All rights reserved

From production to patient: challenges and approaches for delivering cell therapies.

The cell therapy industry is rapidly growing with several new products approved for clinical use over the past few years and many more currently in clinical trials. Nonetheless, a number of challenges remain in getting cell therapies to the market and into routine use. For instance, cell therapy bioprocesses still need to be optimised, but if these therapies are to be widely adopted there must also be clear routes for their delivery from the site of manufacture to the clinic and the patients. This review explores the currently available routes, the factors that need to be taken into consideration when choosing a route and the challenges that remain with respect to cell therapy logistics

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

Mesenchymal stem cells (MSCs) represent an invaluable asset for the feld of cell therapy. Human Bone marrowderived 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 efect 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 afect BMMSCs morphology, surface marker expression, diferentiation or proliferation potential. However, mixed results exist regarding the efect on colony forming ability and the efects on viability, attachment and migration, genomic stability and paracrine function are undefned mainly due to the huge variabilities governing the cryopreservation process as a whole and to the lack of standardised assay

Conductive PANI fibres and determining factors for the electrospinning window

Polyaniline doped with CSA / PEO conductive nanofibres were produced by electrospinning. The electrospinning window was determined by using a three level, full factorial experimental design. The combined effects of the humidity, voltage and flow rate on the fibre morphology and diameter were examined demonstrating that the ambient humidity is the critical factor affecting the electrospinning process and determining the electrospinning window for a conductive polymer. Low humidity favors the formation of defect free fibres while high humidity either hinders fibre formation or causes the formation of defects on the fibres either due to jet discharge or due to water absorption and phase separation. High level of doping with CSA led to the formation of crystalline structures. Data fitting was used to explore the behavior of conductive polymers in electrospinning and very good agreement between experimental and theoretical predictions was obtained for only a limited range of experimental conditions, whereas deviation was observed for all other sets of conditions

A quantitative approach for understanding small-scale human mesenchymal stem cell culture implications for large-scale bioprocess development

Human mesenchymal stem cell (hMSC) therapies have the potential to revolutionise the healthcare industry and replicate the success of the therapeutic protein industry; however, for this to be achieved there is a need to apply key bioprocessing engineering principles and adopt a quantitative approach for large-scale reproducible hMSC bioprocess development. Here we provide a quantitative analysis of the changes in concentration of glucose, lactate and ammonium with time during hMSC monolayer culture over 4 passages, under 100% and 20% dissolved oxgen (dO2), where either a 100%, 50% or 0% growth medium exchange was performed after 72h in culture. Yield coefficients, specific growth rates (h-1) and doubling times (h) were calculated for all cases. The 100% dO2 flasks outperformed the 20% dO2 flasks with respect to cumulative cell number, with the latter consuming more glucose and producing more lactate and ammonium. Furthermore, the 100% and 50% medium exchange conditions resulted in similar cumulative cell numbers, whilst the 0% conditions were significantly lower. Cell immunophenotype and multipotency were not affected by the experimental culture conditions. This study demonstrates the importance of determining optimal culture conditions for hMSC expansion and highlights a potential cost savings from only making a 50% medium exchange, which may prove significant for large-scale bioprocessing

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 is to discuss a “hMSC checklist” as a guide to establishing which identity and potency assays to implement when studying hMSCs. The checklist includes markers, differentiation potential, proliferation & growth, attachment & 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 are discussed. The biology of short (acute) vs long-term (chronic) hypoxia is considered and a nascent hypothesis to explain the behaviour of hMSCs in long-term hypoxia is presented. It is hoped that by establishing an ongoing discourse, and driving towards a regulatory recognisable “hMSC checklist”, we may be better able to provide the patient population with safe and efficacious regenerative treatments

Multiparameter flow cytometry for the characterisation of extracellular markers on human mesenchymal stem cells

Extracellular surface proteins are used to identify fully-functional human mesenchymal stem cells (hMSCs) in a mixed population. Here, a multiparameter flow cytometry assay was developed to examine the expression of several bone marrow-derived hMSC markers simultaneously at the single cell level. The multiparameter approach demonstrates a depth of analysis that goes far beyond the conventional single or dual staining methods. CD73, CD90 and CD105 were chosen as positive markers as they are expressed on multipotent hMSCs, whilst CD34 and HLA-DR were chosen as negative indicators. Single colour analysis suggested a population purity of 100 %; in contrast, when analysed via the multiparameter method, the CD73/CD105/CD90/HLA-DR/CD34 phenotypes represented 94.5 ± 1.3 % of the total cell population. Also, although CD271 has been posited as a definite early stage hMSC marker, here we show it is not present on pre-passage cells, highlighting the need for careful marker selection. © 2013 Springer Science+Business Media Dordrecht

Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor

For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O2 concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 105 cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O2 uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity

The use of bioreactors as in vitro models in pharmaceutical research

Bringing a new drug to market is costly in terms of capital and time investments, and any development issues encountered during late-stage clinical trials can often be the result of in vitro-in vivo extrapolations (IVIVE) not accurately reflecting clinical outcome. In the discipline of drug metabolism and pharmacokinetics (DMPK), current in vitro cellular methods do not provide the 3D structure and function of organs found in vivo; therefore, new dynamic methods need to be established to aid improvement of IVIVE. In this review, we highlight the importance of model progression into dynamic systems for use within drug development, focusing on devices developed currently in the areas of the liver and blood-brain barrier (BBB), and the potential to develop models for other organ systems, such as the kidney. We discuss the development of dynamic 3D bioreactor-based systems as in vitro models for use in DMPK studies