Stem cells expansion

Keywords stem cell expansion stem cell culture in vitro diffusion chambers... 

We currently established cultural system (amphycultural diffusion capsules) that allowed for conditions favorable for stem cell expansion in vitro. Many cell types and culture protocols and their combination with cytokines, growth factors, feeder layers can be implemented with ADC. Capsules are characterized by high perfusion rates that ensure that allow dilution of inhibitory autocrine factors and support long-term cell expansion. We have shown that ADC in vitro provides optimal cellular microenvironment that supports long term hematopoiesis (Bilko et al. 2005). 

SCEPF stem cell expansion promoting factor... 

Three different approaches for the cultivation of isolated hematopoietic cells have been described, the static, the stirred and the immobilized culture. Static cultivation takes place in very simple culture systems like well plates, tissue-culture flasks or gas-permeable culture bags [62, 63]. As the first two systems do not allow cell cultivation on a clinical scale, the latter is actually the most often used technique for stem cell expansion. All these systems have the advantage of being easy to handle, single-use devices, which enable an uncomplicated cell harvest. But all of them do not offer possibilities for process control or continuous feeding. This causes variations in culture conditions during fermentation (e.g., oxygen tension, pH, substrate, metabolite and cytokine concentrations). 

The most sophisticated technique for stem cell expansion is the Aastrom-Rephcell system (Aastrom Biosciences Inc., Ann Arbor, Ml, USA), which is an automated clinical system for the onsite expansion of stem cells in cancer therapy. It consists of a grooved perfusion chamber for the retention of the hematopoietic cells, with the medium flow perpendicular to the channel grooves resulting in a continuous supply of fresh nutrients while metabolites are simultaneously removed [47,71,72]. This technique has already been used in a number of clinical studies [73,74]. No incompatibihty of the expanded cells was found,but the expansion of the early progenitor cells was rather low [75]. 

The main attraction of stromal culture of hematopoietic cells is their superior ability for stem cell expansion especially in direct co-culture. However, despite this, a clinical use has not been realized until now. The major drawback is the origin of the stromal support. While autologous stroma would be feasible, in many cases this is not reaHzable. Furthermore, the use of cell Hnes (although irradiated to prevent further proliferation) is problematic as all stromal cells have to be removed completely prior to transplantation, a demand which is difficult to fulfill. In the case of murine cell Hnes a transfer of residue cells into a patient would be a xenotransplantation, which is faced with extensive regulatory hurdles. 

Doucet, C, Ernou, I., Zhang, Y. Z., et al. (2005), Platelet lysates promote mesenchymal stem cell expansion A safety substitute for animal serum in cell-based therapy applications. J. Cell. Physiol, 205(2), 228-236. 

Most stem cells can divide via either asymmetric or symmetric modes (22). Because each asymmetric division generates one daughter with a stem-cell fate (self-renewal) and one daughter that differentiates, it was postulated that asymmetric division could be a barrier to stem cell expansion. Therefore, conversion of asymmetric division to symmetric division would promote self-renewal and long-term culture of stem cells. Indeed, this idea was confirmed by using one small molecule, the purine nucleoside xanthosine (Xs) (23). This small molecule promotes guanine ribonucleotide biosynthesis that reversibly converts cells from asymmetric division kinetics to symmetric division kinetics. It was found that Xs derived from stem cell lines exhibit Xs-dependent symmetric kinetics, and this derived stable line shows enhanced self-renewal. This study underscores the importance of balance between the two modes of division, both to stem cell expansion and to the regenerative capacity of adult stem cells. 

Y. Lei, D. V. Schaffer, A fuUy defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. Proc Natl Acad Sci USA 110, E5039-E5048 (2013). 

Chen S, Oh SKW. (2010) Human embryonic stem cell expansion and differentiation for clinical applications. Available online on www.aiche.org/SBE/Publications/Articles.aspx http //www.lw20.eom/201205191054769.html cf Cortes-Caminero M. (2010) The engineering of stem cells. SBE supplement Stem cell Eng. Chem. Eng. Prog., Nov. 34. 

Small Molecules to Promote Stem Cell Expansion... 

Table 4.1 Selected small molecules to promote stem cell expansion/proliferation. 

Liao, T., Moussallem, M. D., Kim, J., Schlenoff, J. B., Ma, T. (2010). V-isopropylacrylamide-based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion. Biotechrwlogy Progress, 26, 1705—1713. 

Fernandes, A. M., Fernandes, T. G., Diogo, M. M. et al. 2007. Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system. J Biotechnol 132(2) 227-36. 

Fok, E. Y. L. and Zandstra, P. W. 2005. Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation. Stem Cells 23(9) 1333-42. 

DeUatore, S. M., A. S. Garcia et al. 2008. Mimicking stem cell niches to increase stem cell expansion. Curr Opin Biotechnol 19 534-40. 

Efficient stem cell expansion is a key bottleneck for clinical application and commercialization of stem cell therapy. Membrane bioreactors may make a significant contribution due to its important features such as possibility for uniform chemical and biochemical conditions within the bioreactor, low or even zero hydrodynamic shears, large surface-to-volume ratios, and physical separation between two cell types but allowing biochemical signaling between them. For example, it may be possible to culture the feeder cells on one side of the membrane, while culturing human embryonic stem cells on the other. In this way human embryonic stem cells are not mixed with the feeder cells, which eliminates the need for later difficult separation, but get the biochemical signals from the feeder cells that are necessary to proliferate embryonic stem cells (e.g., Choo et al., 2006 Klimanskaya et al., 2005). 

Cells Deriving Induced Pluripotent Stem Cells Characterizing Human Pluripotent Stem Cells Expansion of Human Pluripotent Stem Cells Large-Scale Expansion of Human Pluripotent Stem Cells Summary References... 

Bioreactors for Stem Cell Expansion and Differentiation Carlos A.V. 

Traditional 2D static methods can culture only a hmited number of cells and is generally considered time-consuming and labor-intensive. Unhke traditional static 2D culture methods, bioreactor systems have the ability to achieve scale-up, which makes bioreactors critical for potential clinical apphcations. Additionally, the dynamic flow of bioreactors creates a more homogenous environment and increases nutrient availabihty when compared to traditional static culture (Nielsen 1999). Stem cell expansion and differentiation has been typically performed in static cultures, but recently the expansion of adult HPCs and efficiency of ES cell differentiation into various lineages has been studied in several different types of bioreactors, including stirred flasks, rotary wall, perfusion cultures, and packed bed bioreactors. Bioreactors and their apphcation in the culture of hematopoietic cells are summarized in Table 35.2 and discussed in detail in the following sections. 

Stirred culture vessels, including stirred-tank bioreactors and spinner flasks, are widely used in the expansion of mammalian cells for protein production. Not surprisingly, stirred culture systems have been adapted to pluripotent stem cell expansion and differentiation. The expansion of undifferentiated... 

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