Complexity of Stem Cell Bioprocess Design: The Need to Consider Cellular Mechanics
Stem cells have a remarkable ability to undergo self-renewal in an undifferentiated state and differentiation into one or more cell types (Pittenger et al., 1999a; Tewary et al., 2018). These cell types include embryonic stem cells (ESCs) that are derived from the inner cell mass of blastocysts; mesenchymal stem cells (MSCs) that originate from adult tissues; and induced pluripotent stem cells (iPSCs) that can be reprogrammed into their pluripotent state by treatment with defined factors from a somatic cell (Thomson el al., 1998; Yamanaka, 2020; Zhu et al., 2021). Recent stem cell-based technologies have substantially accelerated the applicability of these cells in disease modeling and cell-replacement therapy (Harrison et al., 2018; Madl et al., 2018; McKee and Chaudhry, 2017; Pittenger et al., 1999b). The capacity to develop potential stem cell applications will rest on the ability of users to process large numbers of cells. In vitro stem cell maintenance and propagation constitutes a major technical challenge due to fluctuations in cell properties and complex relationship between culture conditions and process outcomes (Kami et al., 2013; Kim and Kino-oka, 2020b; Mount et al., 2015; Panchalingam et al., 2015; Thomas et al., 2008). Especially, the importance of mechanical force has been extensively studied in single cells, but the behavior of multiple cells is less well understood, largely because of the limited generation of mechanical forces in maintenance of cellular homeostasis. Stem cells monitor their physical and mechanical environment via macromolecular complexes, known as mechanosensors, and initiate an adaptive response in an unfavorable mechanical environment (Figure 1 ). It has recently been suggested that the cell nucleus is exposed to the mechanical forces transmitted through the actin cytoskeleton from outside the cell, and the changes in nuclear morphology possibly affect the regulation of cellular homeostasis to maintain self-renewal and pluripotency (Discher et al., 2009; Dupont et al., 2011; Ingber, 1997; Iskratsch et al., 2014). In particular, cells remember their behavioral changes in past environments. For instance, it has been shown that mechanotransduction through cytoskeletal contractility, Rho family GTPase signaling, and subsequent changes in epigenetic marks, such as DNA methylation, histone modifications, and chromatin state, play major roles in cellular homeostasis (Matthews et al, 2006; Rosowski et al., 2015; Thanuthanakhun et al., 2021). The ability of cells to maintain a constant level of cytoskeletal tension in response to external and internal disturbances is defined by tensional homeostasis (Ambriz et al., 2018; Lenormand et al., 2007; Mizuno et al., 2007; Walker et al., 2020a; Webster et al., 2014). In response to the fluctuating nature of a bioprocess, cells can generate internal forces either by extending membranes or by rearranging their actin cytoskeletons, thereby producing contractile forces. In these processes, the breakdown in tensional homeostasis can be caused by alterations in the biological processes involved in force sensing and conversion, cell adhesion molecules such as integrin and cadherin, cell cytoskeleton, and various cytoplasmic and nuclear signaling molecules. The breakdown in tensional homeostasis can lead to changes in epigenetic state/memory, thereby leading to functional variability in stem cells (Alisafaei et al., 2019).
The field of stem cell mechanobiology is rapidly developing with the growing realization of the importance of biophysical and biomechanical factors and is being adopted in the development of new bioengineering technologies (Argentati et al., 2019; Ingber, 2018; Mammoto et al., 2013; Sun et al., 2012; Roca-Cusachs et al., 2017). Many studies have focused on the development of culture strategies that can recapitulate the physico-chemical properties of the complex native cellular microenvironment (Jo et al., 2020; Li et al., 2021). A large effort has been made over the last decades to propose various strategies to develop affordable high-quality cell-based products in a reproduceable and robust manner (Kim and Kino-oka, 2018; Kim and Kino-oka, 2020a). However, a knowledge gap that still exists in the mechanobiological sciences for the stem cell bioprocess development, optimization and characterization is not currently being filled by the bioprocess research. In addition, no straightforward means exist for the development of new techniques, such as bioprocess equipment operations; process design, optimization, and scale-up; and monitoring, analysis, and prediction. The creation of a conceptual framework as a guide for the strategies involved in bioprocess development and optimization would be a necessary step towards full-fledged bioprocess integration that would serve to eliminate the adverse pressures currently faced by many bioengineers.