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.