Xeno-free

Because of the limitations associated with traditional methods for stem cell cultivation, there is a need for novel technologies to facilitate large-scale, cost-effective and reproducible stem cell culture using xeno-free, defined components. Several commercially available, chemically defined culture media for stem cell culture are available [124]. These incorporate ECM components such as laminin, collagen type IV and heparan sulphate to promote adhesion and a variety of growth factors to stimulate cell growth and proliferation [125]. 

Ellerstrom, C., Strehl, R., Moya, K. et al. 2006. Derivation of a xeno-free human embryonic stem cell line. Stem Cells 24(10) 2170-6. 

Lindroos, B., Boucher, S., Chase, L., Kuokkanen, H., Huhtala, H., Haataja, R., Vemuri, M.,Suuronen, R., and Miettinen, S. 2009. Serum-free, xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cells in vitro. Cytotherapy, 11,958-72. 

Figure 6.1 Culture methods for human pluripotent stem cells (hPSCs). hPSCs can be cultured on mouse embryonic fibroblasts (MEFs) or human feeder cells. Feeder-free culturing of hPSCs is possible on xeno-containing Matrigel. Several types of feeder-free and xeno-free cultures of hPSCs can be developed on extracellular matrix (ECM)-coated surfaces and ohgopeptide-immobilLzed surfaces. hPSCs can also be cultured on polysaccharide hydrogels or on synthetic polymer surfaces by selecting a specific GAG or a specific polymer with optimal water content. Reproduced with permission from [2] Copyright 2014 Elsevier Inc.

Medical engineering scientists and molecular biologists have recently examined the effects of culture conditions on stem cell fate. Additionally, since 2010, several novel biomaterials and methods for hPSC culture on these biomaterials have been developed under feeder-free and xeno-free culture conditions. Therefore, this chapter mainly focuses on the current developments in hPSC culture materials and describes the biomaterial-assisted regulation of hPSCs xmder feeder-free and xeno-free culture conditions. 

Several investigators have evaluated that fibronectin-coated substrates maintain hESC pluripotency (Table 6.1) [51-58], whereas other researchers have reported unfavorable results when culturing hESCs on fibronectin-coated substrates [31,59]. Amit et al. cultured several hESC lines (1-6,1-3, and H-9) on bovine and human fibronectin-coated dishes (5 og/cm ) in knockout DMEM (KO-DMEM) supplemented with 15% serum replacement (SR). The fibronectin-specific integrin receptor aSpi was expressed in the undifferentiated hESCs (Figure 6.7) [52]. Under these conditions, the hESCs maintained pluripotency for more than six months, whereas the hESCs cultured on gelatin-coated dishes tended to differentiate [52]. Human fibronectin was found to be relatively more favorable for maintaining the pluripotency of the hESCs compared with bovine fibronectin [52]. The applied SR contained Albumax, which is aHpid-enriched bovine serum. Therefore, this work was not performed under xeno-free culture conditions. 

EB formation. The hESCs were reported to be cultured in a chemically defined medium, mTeSRl, for five days in this study [73]. Dishes with optimal elasticity that are grafted with cyclic RGD peptide might improve the culture of hESCs in xeno-free and chemically defined medium for long passages (10-20 passages). 

The culture of hPSCs on chemically defined substrates synthesized from monomers eliminates the variables associated with feeder cells and natural protein coatings, which can range from batch-to-batch inconsistencies to biosafety issues [23]. In most cases, synthetic polymer matrices support only short-term hPSC propagation or culture in culture media containing xenobiotic products, such as fetal bovine serum (FBS) or MEF-CM [42,84], Recently, several synthetic polymer materials were reported to support hPSC proliferation in xeno-free conditions or in chemically defined media (Table 6.2 and Figure 6.11) [85-89]. In this section, we will discuss (a) several types of synthetic polymers that sustain the long-term culture of hPSCs, (b) the thermore-sponsive synthetic polymers that hold hPSC culture, and (c) synthetic nanofibers that maintain the long-term culture of hPSCs. 

Figure 6.11 Chemical structures of the synthetic polymers used as substrates, hydrogels, and scaffolds for the proliferation of pluripotent hPSCs in defined media under feeder-free and xeno-free conditions.

In most cases, hPSC cultmes on microcarriers require a Matrigel coating and/or MEF-CM. These conditions are not xeno-free culture conditions and make it difficult to use hPSCs in clinical applications. 

Although Heng s work [113] achieved feeder layer-free and xeno-free cultures of hESCs in chemically defined medium, the researchers only verified a few ES cell lines (HES-3 and H7). It is not clear whether the microcarriers and cell culture protocol used by Heng et al. could support all hESC and hiPSC lines. It is preferable to prepare or synthesize microcarriers composed of other types of biomaterials and to develop and design optimal microcarriers for hPSC culture in feeder layer-free and xeno-free culture conditions by referencing Heng s work [113]. 

T. Lei, S. Jacob, I. Ajil-Zaraa, J.B. Dubuisson, O. Irion, M. Jaconi, and A. Feki, Xeno-free derivation and culture of human embryonic stem cells current status, problems and challenges, Cell Res., 17 (8) 682-688, Aug. 2007. 

A. Swistowski, J. Peng, Y. Han, A.M. Swistowska, M.S. Rao, andX. Zeng, Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them, PLoS One, 4 (7) e6233, Jul. 2009. 

R. Bergstrom, S. Strom, F. Holm, A. Feki, and O. Hovatta, Xeno-free culture of human pluripotent stem cells, Methods Mol. Biol, 767,125-136,2011. 

G.L. Meng, S.Y. Liu, and D.E. Rancourt, Synergistic effect of medium, matrix, and exogenous factors on the adhesion and growth of human pluripotent stem cells under defined, xeno-free conditions. Stem Cells Develop., 21 (11) 2036-2048, Jul. 2012. 

D. Ilic, E. Stephenson, V. Wood, L. Jacquet, D. Stevenson, A. Petrova, N. Kadeva, S. Codognotto, H. Patel, M. Semple, G. Cornwell, C. Ogilvie, and P. Braude, Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions,... 

G. Meng, S. Liu, X. Li, R. Krawetz, and D.E. Rancourt, Extracellular matrix isolated from foreskin fibroblasts supports long-term xeno-free human embryonic stem cell culture. Stem Cells Develop., 19 (4) 547-556, Apr. 2010. 

K. Rajala, H. Hakala, S. Panula, S. Aivio, H. Pihlajamaki, R. Suuronen, O. Hovatta, and H. Skottman, Testing of nine different xeno-free culture media for human embryonic stem cell cultures. Hum. Reprod., 22 (5) 1231-1238, May. 2007. 

PA. Marinho, D.T. Vareschini, I.C. Gomes, S. Paulsen Bda, D.R. Furtado, R. Castilho Ldos, and S.K. Rehen, Xeno-free production of human embryonic stem cells in stirred microcarrier systems using a novel animal/human-component-free medium. Tissue Eng. Part C, Methods, 19 (2) 146-155, Feb. 2013. 

Substantial progress has been made in developing defined, xeno-free media for human pluripotent stem cell culture. However, implementation of defined extracellular matrices for iPSC and hESC culture lags media development. While lab-scale culture is now relatively straightforward, there are substantial challenges in larger-scale culture for generating industrial- or clinical-quality pluripotent stem cells... 

Rodriguez-Piza, L, Richaud-Patin, Y, Vassena, R. et al. 2010. Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions. Stem Cells 28 36-44. 

Ross, P. J., Suhr, S., Rodriguez, R. M. et al. 2010. Human induced pluripotent stem ceUs produced under xeno-free conditions. Stem Cells Dev 19 1221-9. 

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