Supplementary materials section:
SM1:
Scale-up: Since cell damage due to elevated energy dissipation rate (turbulences, shear stress)
is a basic issue in stirred tank reactors, scale-up of such reactor systems is generally
performed by keeping constant either the energy input per unit volume (P = ρN3D5, where ρ =
medium density, N = agitation rate, D = diameter of agitator) or the Reynold’s number across
the reactor scale, in view of keeping constant the energy dissipation rate as well as the mean
Kolmogorov length at the different reactor scales. That this is also valid for the scale-up of
cell aggregate cultures of neural precursors could be shown by Gilbertson et al. 2006).
In principle, stirring is rather simple at a spinner scale however, at a large scale the choice of
the agitation system is highly critical. In view of large scale applications, Ibrahim & Nienow
(2004) presented the hydrofoil HE3 impeller as optimal in order to minimise the mean
specific energy dissipation rate for the suspension of microcarriers presenting thus a potential
agitator for microcarrier cultures at a large (industrial) scale. However, presently, no
information on the type and implementation of agitators for industrial scale microcarrier
cultures is available in the open literature. Nevertheless, the largest reported process scale is a
6000L stirred tank reactor based microcarrier process for the production of influenza virus
vaccine (Barret et al. 2009).
In addition to classical engineering simulations, the implementation of computational fluid
dynamics (CFD) simulations provides a means for the verification of energy dissipation rate
scalability at different scales of stirred tank reactors. Comparing different bioreactor
configurations Johnson et al. (2014) could show that hydrodynamic scalability is achieved as
long as design features, including baffles and impellers, remain consistent across the scales. In
addition, it could be confirmed for single suspension cultures that the mean Kolmogorov
length scale is considerably larger than the average cell size, signifying that substantial cell
damage due to agitation is highly improbable.
SM2:
WAVE reactor (Fig. S1): A culture system characterized by the generation of less shear
stress is the WAVE reactor (Singh 1999) which can be used for classical suspension and also
for microcarrier dependent culture, but with a scale limit of 500L (working volume) due to
oxygen transfer limitations beyond this scale. Using computational fluid dynamics, Öncül et
al. (2009) have investigated flow conditions in 2L and 20L WAVE reactors. Under standard
conditions using the non-dimensional Womersley number (Wo) and a parameter β according
to Kurzweg et al. (1989), they could establish that the liquid flow in both bags was in a
laminar state. Moreover, the maximum shear stress in the cellbags was around 0.01 N/m2
which is very low in comparison to stirred tank reactors where values exceeding 1 N/m2 can
be observed (Joshi et al. 1996). For instance, Croughan & Wang (1991) reported a shear stress
threshold of at least 0.7 N/m2 associated with damage of anchorage-dependent cells. Since
turbulences and shear stress are much lower in this culture system it is well placed for
medium scale cell propagation, in particular, of shear sensitive cells. In this context, Genzel et
al. (2006) could show the beneficial effects of low turbulences and shear on cultivation of
MDCK cells on microcarriers. In comparison to normal stirred tank bioreactors, higher cell
densities on microcarriers were obtained. The WAVE reactor has also been shown to be of
high interest for the expansion of human placental MSCs using microcarriers (Timmins et al.
2012).
Moreover, microcarriers can be replaced by Fibra-cel chips in which adherent cells as well as
those with a tendency to detach, like HEK293 cells, can be propagated but under considerably
reduced shear fields in comparison to a microcarrier approach (Greene et al. 2012). Such an
approach allows also the easy washing and medium exchange of immobilized cells which is
more difficult for cells attached to traditional microcarriers.
In addition to medium scale productions, WAVE type reactors are often used for the
generation of biomass for the inoculation of large scale bioreactors.
Packed bed/fixed bed reactor: A practical alternative for using microcarriers or
macroporous carriers for the propagation of anchorage dependent cells but also of suspension
cells is the use of packed bed reactors. However, from a large scale point of view this type of
reactor system represents a medium scale production system because of scale limitations due
the formation of nutritional and oxygen gradients across the fixed bed.
In this type of bioreactors initially pioneered by Spier & Whiteside (1976) for the production
of Foot-and-Mouth Disease Virus using BHK cells, the cells are attached to, immobilized on
or entrapped in carriers: solid (Bliem et al. 1990) or macroporous carriers (Looby & Griffiths
1990), glass spheres, stainless steel gauze and coupons (Aboud et al. 1994), or porous spongelike materials, such as Fibra-cel (Kadouri & Zipori 1989). There are two types of fixed bed
reactors: i) the fixed bed is separated from the conditioning vessel necessary for controlling
pH and pO2 of the culture medium, like for the CellCube system (Fig. S2), or ii) the fixed bed
and the conditioning vessel are integrated as for the Celligen packed bed reactor system (New
Brunswick Scientific) (Fig. S3), or the Icellis system, more recently developed by ATMI.
Both systems are characterized by the circulation of the culture medium through the fixed bed
reactor as shown in Fig. S3.
The main advantage is that under controlled conditions (pH, pO2, medium circulation) tissue
like cell mass can be achieved (up to 2x108 c/ml carrier) allowing a significant improvement
of reactor productivity and thus a considerable intensification of cell culture process. In this
context, in view of the production of monoclonal antibodies using hybridomas, Bliem et al.
(1990) reported that a fixed bed reactor of a bed volume of 21 L can produce 200-300 L of
culture supernatant per day which is comparable to a 1500 L stirred tank reactor. Further
advantages relate to the use of shear sensitive cells or biological production systems. The
production of retroviral vectors (MLV-based) using various anchorage dependent cell lines
(ψCRIP, PG13, TeFLY) was only possible using packed bed reactors due to reduced shear
stress which negatively impacted cell specific production rate in the case of microcarrier
based cultivation. 0.5 – 1.5 log differences in reactor productivity in favour of fixed bed
reactor have been observed (Merten 2004). Moreover, using human diploid fibroblast cultures,
only fixed bed reactors could be used for the production of hepatitis A virus for vaccine
purposes due to heterogeneous cell distribution and aggregation for microcarrier cultures
when cultures got confluent as well as during the virus production phase (Aunins et al. 1997,
Junker et al. 1992). For the same reason, the ‘large-scale’ CellFactory system (CF-40 system)
had been initially implemented for the production fibroblast interferon (Pakos & Johansson
1985) (see 4.1.). As for the production of various biologicals, packed bed reactors are also of
interest for the expansion of stem cells for different purposes.
SM3:
Hollow fibre reactor: Hollow fibre reactors, initially developed by Knazek et al. in 1972, are
culture systems allowing the production of tissue like cell concentrations (>108 c/ml). They
are characterized by a separation of the cell culture chamber (which is outside the hollow
fibres) and a medium compartment (inside the hollow fibres). In order to feed the cells with
nutrients and oxygen and to remove metabolic waste, the medium is circulated from a
conditioning vessel to the cartridge, passes through the fibres and circulates back to the
conditioning vessel which allows the control of pH and pO2 as well as a continuous or
discontinuous medium exchange (- perfusion culture)(fig. S4). This perfusion allows the
exchange of nutrients and waste across the hollow fibre membranes according to the chosen
cut-off. Further advantages are reductions in required materials such as serum, growth factors
or other additives. The main drawbacks are the limited scalability, formation of nutrient
gradients as well as the difficulties to harvest cells because initially, these devices have not
been developed for the expansion of stem cells but for the production of secreted proteins. A
particular system based on an interwoven four compartment capillary membrane technology
for 3D perfusion with decentralized mass exchange had been used for the expansion of hESCs
(Gerlach et al. 2010) and liver stem cells (Monga et al. 2005).
Rotating-wall perfused reactors: A second system of particular interest for the expansion of
clump and aggregate cultures (such as embryoid bodies (EBs)) or cells grown on carriers or
scaffolds (Martin & Vermette 2005) represents the rotating-wall perfused reactors from
Synthecon-EHSI (Fig. S5). Both reactor systems have been developed for use in space. They
are characterized by a very soft agitation and indirect oxygenation via a gas exchange
membrane. This membrane is either localized in the centre of the culture vessel as for the
STLV (Fig. S5A) or localized laterally on one side as for the HARV (Fig. S5B). The
membrane area to culture volume ratio is larger for the HARV allowing a reduction in the
rotation speed. Whereas in space, both devices can be run under micro-gravity regime, this is
less sure for using under ‘Earth’ conditions. These differences have an impact on the shear
level. For instance a 2 mm cancer-cell aggregate sees its Reynold’s number passing from 0.19
in space to 86 on Earth corresponding to an increase of the shear level from 0.0002 to 0.11
N/m2 (Martin & Vermette 2005). Although both devices are of interest for the cultivation of
various cells under very low shear fields and have shown their interest for the culture of
cancer-cell aggregates (Martin & Vermette 2005), hESCs/EBs (Zhao et al. 2006, Côme et al.
2008), MSCs (Chen et al. 2006) and hematopoietic stem cells (Liu et al. 2006), these culture
systems are not destined for large scale expansion of any cell type. Moreover, due the very
low shear fields, there is limited control of the aggregate size leading to the formation of
necrotic centres due to cell death inside the aggregates.
SM4:
Mechanosensitivity of stem cells – some background information:
MSCs: Mechanical stimuli (including fluid shear stress) are detected via ion channels and
integrins (both considered as mechanoreceptors involved in mechanotransduction for
activating downstream ERK1/2), glycocalyx (considered as primary sensor for mechanical
stimuli triggering two distinct cellular signalling pathways) and cytoskeleton (via actin
organization regulating cell stiffness, itself influenced by the substrate stiffness). For instance,
MSCs grown on stiff substrates spread out leading to cytoskeletal contraction generating high
level of tensile forces pulling on the surface (Patwari & Lee 2008). This promotes
differentiation towards the osteoblast lineage. In this context, Arnsdorf et al. (2009a)
demonstrated in mouse MSCs that the small GTPase RhoA and its effector protein ROCKII
regulate fluid flow induced osteogenic differentiation, and that the activation of RhoA and
actin tension are negative regulators of adipogenic and chondrogenic differentiation.
Specifically, non-canonical Wnt5a signalling involving Ror2 and RhoA as well as N-cadherin
mediated β-catenin signalling are required for mechanically induced osteogenic differentiation
(Arnsdorf et al. 2009b). Moreover, Dupont et al. (2011) could establish by comparing two
different elastic moduli (0.7 and 40 kPa) that stiffness is sensed via the modulation of
YAP/TAZ activity. Osteogenic differentiation is induced on stiff ECM when YAP/TAZ are
active, whereas depletion of YAP/TAZ is related to adipogenic differentiation normally
induced on soft ECM. The knockdown of YAP/TAZ enabled adipogenic differentiation on
stiff substances, thus mimicking a soft environment. Thus, a stiff culture surface (increased
matrix stiffness (11-30 kPa)) together with an adapted biochemical matrix such as
polyallylamine promotes spindle-like shape and osteogenic differentiation of MSCs (Huebsch
et al. 2010); a matrix with E=2.5-5 kPa leads predominantly to adipogenic differentiation,
whereas soft matrix (E<1 kPa) leads to neural differentiation (Engler et al. 2006, Lanniel et al.
2011, Shih et al. 2011). Finally, Doroudian et al. (2013) could show that applied mechanical
stresses tend to dominate over the scaffolds properties.
The reviews by Liu et al. (75) as well as Sart et al. (58) provide details on the mechanisms of
osteogenic differentiation of human MSCs and the impact of biomechanical stem cell fates on
MSC differentiation.
PSCs: As for MSCs, PSCs are able to sense matrix mechanics. Zoldan et al. (2011)
demonstrated that hESCs grown on hard surfaces promoted mesodermal commitment,
surfaces with intermediate elastic modulus promoted endodermal differentiation and soft
surfaces led to ectodermal differentiation. In addition, the self-renewal of hESCs was
promoted by hard surfaces (>6 MPa) which is in contrast to mouse ESCs. Chowdhury et al.
(2010) reported that mESCs could maintain their pluripotency under long-term conditions
(>15 passages) when cultured on soft polyacrylamide gels (E~500Pa), whereas hard
substrates favoured differentiation towards mesodermal and endodermal lineages.
Finally it is important to consider that the local microenvironment of aggregates is impacted
by the size of the aggregates. It modulates endogenous parameters influencing thus the
differentiation trajectories of PSCs (Bauwens et al. 2008). In this specific context, the choice
of the culture system whose fluid shear forces have a direct impact to aggregate size is highly
critical and agitation has to be carefully optimized for obtaining and maintaining the optimal
aggregate size also in order to direct differentiation into the desired direction.
More information on the effect of biomechanics on stem cell fate can be found in the review
by Sun et al. (2012).
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Supplementary figures:
Figure S1. WAVE reactor design (Singh (1999), reproduced with permission).
Figure S2. CellCube, lab version (module 25) (Corning).
Figure S3. Celligen – packed bed reactor perfusion system (New Brunswick Scientific).
Level control
Fresh medium
Waste, harvest
Base
Figure S4. Flow diagramme of a hollow fibre reactor system (placed inside an incubator at
37°C).
NNNNN
Figure S5. Rotating-wall perfusion reactor. A. Slow turning lateral vessel (STLV) reactor. B.
High aspect ratio vessel (HARV) reactor (Martin & Vermette (2005), reproduced with
permission).
A)
B)