Three-Dimensional (3D) Methods of PSC Expansion

Recently, 3D methods of stem cell culture have gained traction due to the need for scalable stem cell expansion to obtain therapeutically relevant number of cells. 3D cell culture methods offer the opportunity to significantly scale-up the expansion of hPSCs. The 3D methods of PSC culture are divided into three categories: (i) PSC encapsulation in hydrogels, (ii) microcarrier-based 3D PSC culture, and (iii) PSC suspension culture.

A growing body of literature suggests that 3D cell culture systems recapitulate the in vivo microenvironment of cells that could help to improve stem cells expansion. In a landmark study of PSC encapsulation, a defined and scalable 3D cell encapsulation system in a thermo-responsive hydrogel was developed for hPSC expansion and differentiation; it also enabled efficient retrieval of the cells from the hydrogels following expansion, without using the cell dissociation enzymes (Lei and Schaffer, 2013). The cells expanded ∼80-fold in 3D culture compared to ∼9-fold expansion in 2D over 15 days. Cumulatively, 3D cell culture led to 10⁷²-fold expansion over 60 passages (Lei and Schaffer, 2013). PSCs are mechanosensitive as previously discussed; however, the role of biophysical signals and cell-matrix interactions in the context of 3D PSC expansion was not investigated. Scaffolds used for 3D expansion of PSCs should provide a balance between cell–cell contact and cell–matrix interactions (Li et al., 2012). An alginate-based hydrogel with tethered polypeptides comprising of a cell-binding sequence of E-cadherin for 3D PSC expansion has shown to improve the proliferation rate of PSCs without compromising pluripotency marker expression resulting in up to 23-fold higher expansion in HAV10 peptide conjugated gels (Banerjee et al., 2018). Matrix degradability and remodeling by encapsulated PSCs are other parameters that affect PSC fate. Encapsulated PSCs are known to remodel their environment during proliferation and differentiation (Khetan et al., 2013; Madl et al., 2017). However, the capacity of PSCs to remodel the environment and their effect on the pluripotency related markers are poorly understood. This should be studied in greater detail to design more informed and tailor-made 3D scaffolds for enhanced stem cell expansion.

Microcarrier-based systems are another method for PSC expansion, which combine 2D cell adhesion in microcarriers with a 3D configuration of the bioreactor system to expand the area available for PSC expansion. Microcarriers act as supporting substrates for adherent cell culture with a diameter varying from 10 μm up to 5 mm (Le and Hasegawa, 2019). The major benefit of using microcarriers is their capacity to provide large surface areas for cell growth while being compatible with adherent cell culture systems. Cells can form a confluent layer around the microporous microcarrier; while in macroporous microcarriers, they are entrapped inside the pores of the microcarriers (Badenes et al., 2014). Factors including the type of materials used to fabricate the microcarriers, the shape of the microcarrier, and the type of ECM coating used for cell adhesion influence the yield and pluripotency of the PSC culture in a microcarrier system (Chen A.K.L. et al., 2011). For instance, use of matrigel coating led to up to 18-fold higher PSC expansion compared to uncoated microcarriers (Chen A.K.L. et al., 2011). Recently, dissolvable microcarriers have been developed, which allow the retrieval of cells without using enzymatic dissociation (Badenes et al., 2014; Shekaran et al., 2016; Rodrigues et al., 2019).

By leveraging the self-aggregation property of PSCs, suspension-based cell culture systems are being developed to improve yield. Such systems promote cell-cell interactions while inhibiting the cell-matrix interactions. Usually, such systems consist of single cell culture in the presence of rho kinase (ROCK) inhibitor (Olmer et al., 2010; Abbasalizadeh et al., 2012), which supports long-term PSC survival in an undifferentiated state. PSCs grow in a monoclonal fashion and form suspended spheroids of varying sizes. Microfabrication technology has been used to further improve the homogeneity of PSC colonies (Hsiao and Palecek, 2012; Hookway et al., 2016). Optimization of bioreactor hydrodynamic conditions using combinations of static and stirred culture has enabled size-controlled aggregates of hPSCs (Abbasalizadeh et al., 2012). Traditionally, the yield of hPSCs is lower compared to mPSCs in suspension bioreactor cultures (Lipsitz et al., 2018). Recently, Lipsitz et al. found that the use of naïve hPSCs, as opposed to primed hPSCs, was a critical element for enabling high-yield expansion of PSCs (Over all 25-fold expansion; up to 5.7-fold higher compared to primed hPSCs) in suspension culture (Lipsitz et al., 2018). Despite tremendous progress in suspension-based cell culture, more research is needed in maintaining the homogeneity of cell aggregates in scalable suspension culture. Additionally, cells on the surface of the suspension aggregates experience uncontrolled shear stress, which could also lead to heterogeneous cell populations, as shear stress is known to affect stem cell fate (Toh and Voldman, 2011; Vining and Mooney, 2017).