A Compact, Automated Cell Culture System for Clinical Scale Cell Expansion from Primary Tissues
ABSTRACT Despite the growing number of clinically practical automated cell culture systems, demand is also increasing for more compact platforms with greater capabilities to prepare primary cells directly from patient tissue. Here we report the development of an automated cell culture system that is also compact. The machinery consisted of a supply unit, an incubation unit, and a collection unit, which fit within a 70 cm x 60 cm x 86 cm space. The compact size was enabled by our concept of using a single culture vessel from the primary culture steps to final cell harvest instead of scaling up with multiple culture vessels. Human fibroblasts and bone marrow stromal cells (BMSCs) were successfully cultured with this system over 19 days without contamination. From three pieces of gingival tissue (2 mm x 2 mm) or from 10 mL of bone marrow aspirate, the system could produce more than 2.0x10(7) cells and up to 3.0x10(7) cells for fibroblasts and BMSCs, respectively. The BMSCs produced by this system were capable of ectopic bone formation after transplantation into the subcutaneous space of nude mice. Our prototype system will provide a foundation for minimizing automatic culture machinery with clinically relevant cell yields while also expanding the automation capabilities to include primary tissue culture.
- SourceAvailable from: Toshiyuki Owaki
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- "Several technical concepts of automated cell culture system have been reported       and based on some of the concepts equipment has been designed and made commercially available. "
ABSTRACT: Substantial progress made in the areas of stem cell research and regenerative medicine has provided a number of innovative methods to repair or regenerate defective tissues and organs. Although previous studies regarding regenerative medicine, especially those involving induced pluripotent stem cells, have been actively promoted in the past decade, there remain some challenges that need to be addressed in order to enable clinical applications. Designed for use in clinical applications, cell sheet engineering has been developed as a unique, scaffold-free method of cell processing utilizing temperature-responsive cell culture vessels. Clinical studies using cell sheets have shown positive outcomes and will be translated into clinical practice in the near future. However, several challenges stand in the way of the industrialization of cell sheet products and the widespread acceptance of regenerative medicine based on cell sheet engineering. This review describes current strategies geared towards the realization of the regenerative medicine approach.Biotechnology Journal 07/2014; 9(7). DOI:10.1002/biot.201300432 · 3.49 Impact Factor
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- "A typical outcome of these inspections is an estimate of confluency, a measure of the fraction of the growth area covered by cells. As a metric, confluency is particularly useful when detachment is not possible, for example to determine when to passage cells (Kato et al., 2010) or when to induce a perturbation (Stewart and Rotwein, 1996; Van den Eijnde et al., 2001). Confluency also informs on the spatial crowding of the cells, a property relatable to in vivo cellular tissues, which was shown to have an impact on gene expression (Ruutu et al., 2004), formation of cell–cell junctions (Lampugnani et al., 1997) and the development potential of embryonic stem cells into viable embryos (Gao et al., 2003). "
ABSTRACT: The quantitative determination of key adherent cell culture characteristics such as confluency, morphology and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post-hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (< 1s per 1208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.Biotechnology and Bioengineering 03/2014; 111(3). DOI:10.1002/bit.25115 · 4.13 Impact Factor
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ABSTRACT: The field of tissue engineering has made considerable strides since it was first described in the late 1980s. The advent and subsequent boom in stem cell biology, emergence of novel technologies for biomaterial development and further understanding of developmental biology have contributed to this accelerated progress. However, continued efforts to translate tissue-engineering strategies into clinical therapies have been hampered by the problems associated with scaling up laboratory methods to produce large, complex tissues. The significant challenges faced by tissue engineers include the production of an intact vasculature within a tissue-engineered construct and recapitulation of the size and complexity of a whole organ. Here we review the basic components necessary for bioengineering organs-biomaterials, cells and bioactive molecules-and discuss various approaches for augmenting these principles to achieve organ level tissue engineering. Ultimately, the successful translation of tissue-engineered constructs into everyday clinical practice will depend upon the ability of the tissue engineer to "scale up" every aspect of the research and development process.Organogenesis 07/2010; 6(3):151-7. DOI:10.4161/org.6.3.12139 · 2.80 Impact Factor