DISCUSSION
Over the last years, numerous basic and clinical studies have focused on
MSC due to their powerful regenerative potential and immunomodulatory
features [reviewed in (Zhou et al., 2021)]. More recently, most of
the potential beneficial effects of MSC therapies have been primarily
attributed to EVs, with several studies confirming that EVs recapitulate
many of the features of their parental cell line (MSC). Overall, MSC-EVs
have been being explored for the potential treatment of spinal cord
injury (Wang et al., 2021), acute kidney injury (Bruno et al., 2012),
atherosclerotic cardiovascular disease (Badimon et al., 2022),
myocardial ischemia (Charles et al., 2020; Ma et al., 2017; Zhu et al.,
2018), among other diseases. To support all these clinical applications,
large doses of EVs will be required and the limited manufacture capacity
of traditional static culture systems will be a major hurdle towards the
implementation of EVs-based therapies. In this context, the large-scale
production of therapeutic MSC and their EVs will require the development
of a cost-effective manufacturing platform based on fully controlled
bioreactor systems for the expansion of well-characterized MSC
populations and the production of their MSC-CM, as well as a robust
downstream process to isolate and purify MSC and MSC-EVs [reviewed in
(Mawji et al., 2022; Syromiatnikova et al., 2022)]. In this work, a
S/X-free microcarrier-based culture system was successfully established
for the expansion of MSC(M) and the production of MSC-EV using a 2
L-scale controlled STR as it represents a scalable, robust,
cost-effective, and well-characterized platform widely used to produce
biotherapeutics. In our previous work, MSC(M) and umbilical cord-derived
Wharton’s jelly MSC [MSC(WJ)] were expanded on CellStart-coated
plastic and Cultispher S microcarriers, respectively, using
S/X-free StemPro culture medium and a bench-scale STR (1L) operated
under repeated medium exchange mode (Dos Santos et al., 2014;
Fernandes-Platzgummer et al., 2016). Based on those works, the
operational process parameters were set to pH 7.2, dissolved oxygen (DO)
20%, temperature 37ºC and agitation rate 60 rpm (40 rpm during cell
adhesion to the microcarriers). The entire culture was carried out in
the STR, representing an advance compared to our previous protocol,
where cell adhesion and initial culture (4 days) were performed in a
spinner flask and subsequently transferred to the reactor (i.e.
cell-containing microcarriers plus culture medium) (Dos Santos et al.,
2014). After 24h, MSC(M) successfully adhered to the microcarriers, with
adhesion efficiencies between 30-50%, and with a very homogeneous cell
distribution, a factor identified to be crucial for a successful
microcarrier-based culture (Carmelo et al., 2015). Despite losing more
than 50% of the inoculated cells, carrying out the entire process
inside the reactor has the advantages of having the whole protocol
controlled from the first day of culture, while minimizing the risk of
contamination associated to transferring the cell suspension between
culture systems. Additionally, the adhesion efficiency can be maximized
by optimizing the culture conditions in the first 24h, including the use
of other agitation regimes or alternative microcarriers [reviewed in
(Tsai & Pacak, 2021)]. For example, in previous studies from our
group under S/X-free culture conditions, adhesion efficiencies of
MSC(WJ) on gelatin-based Cultispher S microcarriers around 75%
were attained in a 2L bioreactor culture (Mizukami et al., 2016),
whereas initial adhesion efficiencies of 71±7.4 and 74±0.3% were
obtained for MSC(M) using Low Concentration
SynthemaxTM II and CellBIND® microcarriers,
respectively, in spinner flask cultures (Carmelo et al., 2015).
Since culture medium represents one of the major costs in the
manufacturing of human MSC, the delineation of feeding strategies able
to maximize cell densities in a cost-effective way is of utmost
importance. The feeding scheme adopted in most dynamic
microcarrier-based culture systems used to expand MSC, is the repeated
medium exchange strategy, where the cell-containing beads are allowed to
settle before changing the culture medium once or twice a day (Cunha et
al., 2015; de Almeida Fuzeta et al., 2020; Dos Santos et al., 2014;
Lembong et al., 2020). This procedure requires stopping the reactor
operation (i.e. discontinuous operation), which has the disadvantage of
increasing the probability of aggregation between the cell-containing
microcarriers during stagnant periods, potentially impairing cell growth
and impacting cell identity. In this work, we hypothesized that an
automated feeding protocol would potentially result in fewer
fluctuations in cell proliferation/metabolism patterns and that a
continuous operation would minimize cell-containing microcarrier
aggregation. Therefore, two feeding operation modes were compared: FB,
where the fresh culture medium was added discretely to the STR and
FB/CP, where there was an automated continuous removal/replenishment of
the medium with retention of the cell-containing microcarriers through a
spin-filter. Growth curves of MSC(M) expanded under the two feeding
regime strategies were quite similar during the first 6-7 days of
cultivation, diverging upon this time point to reach considerable
different maximal cell densities of (2.0±0.51)×105 and
(4.1±0.90)×105 cells/mL at days 8 and 12 of
cultivation for FB and FB/CP cultures, respectively. This difference
could be explained by the accumulation of toxic by-products such as
lactate, although it never reached values described as inhibitory to
cell growth (35 mM (Schop et al., 2009)), and/or by the lack of
replenishment of other important nutrients, as glucose concentration in
the culture medium was always ranging from 2-6 mM, above the
non-limiting value (over 1 mM (Schop et al., 2009)). Indeed, the
concentrated glucose pulses added throughout culture, allowed us to
maintain the glucose concentration within the desire range without
adding volumetric capacity to the bioreactor.
Overall, continuous medium perfusion operation allowed MSC expanded
under S/X-free conditions to reach densities in the STR that had
previously only been achieved on small scale stirred culture systems
(≤400 mL) operated under S/X-free
conditions (Cunha et al., 2015), or often employing culture media
containing human serum components (de Almeida Fuzeta et al., 2020;
Lembong et al., 2020). The specific growth rates estimated were 0.6±0.1
and 0.5±0.1 for FB and FB/CP cultures, respectively, which are in
agreement with the literature for MSC cultures cultured under S/X-free
conditions (Carmelo et al., 2015; Cunha et al., 2015; Heathman et al.,
2018). Other studies have compared different feeding modes of operation.
In a previous work by our group, two different feeding regimes were
compared for MSC(M) cultivation using a combined system employing a
spinner flask transferred thereafter to a 1L-scale controlled STR: daily
medium renewal every day or every 2 days versus fed-batch
addition of concentrated nutrients and growth factors every 2 days. No
significant differences were observed in terms of MSC(M) proliferation,
although the fed-batch approach led to a faster accumulation of
metabolites, namely lactate, as expected, as no culture medium was
withdrawn from the STR. Moreover, a continuous perfusion process was
tested in a smaller 400 mL STR with a dilution rate of 0.25
day-1 (starting from day 3) throughout the whole
process. A maximal cell density of 5.0x105 cells/mL
was reached at day 11, demonstrating the advantage of working under
perfusion operation mode (Dos Santos et al., 2014). Cunha and colleagues
also compared two different culture operation modes for expanding MSC(M)
in a STR, 50% medium renewal every 2.5 days versus continuous
perfusion cultures at a dilution rate of 0.2 day-1.
The results attained showed that MSC achieved higher cell concentrations
(3.7x105 cell/mL) and maximum growth rate (0.38
day-1) in continuous perfusion cultures when compared
to the repeated medium exchange strategy (2.9 x 105cell/mL and 0.26 day-1), respectively. Although
continuous perfusion processes led to higher cell densities when
compared to fed-batch or repeated medium exchange operation modes, it
utilizes larger quantities of culture media, which increases the
operation costs. Moreover, the greater logistic of implementing it
(especially in what concerns the cell retention device) and higher
probability of technical failures have been hindering the use of this
feeding operation mode for the production of MSC. In the present work, a
spin-filter was used to retain the cell-containing microcarriers inside
the STR. This system consists in a cylinder cage with a porous mesh
wall, normally mounted on the impeller shaft. Perfusate (bleed) is
pumped out from inside the spin-filter at the same rate at which fresh
culture medium is pumped into the bulk of the STR (i.e. outside the
spin-filter). Minimum fouling and an optimum cell retention at the
necessary medium perfusion rate are crucial parameters for a successful
operation of a spin-filter-based STR (Castilho & Medronho, 2002). No
fouling phenomenon was observed in the mesh of the spin-filter in the
present work and the cell-containing microcarriers were efficiently
retained inside the STR, as no microcarriers were detected in the STR
bleed. Another important parameter in continuous perfusion cultures is
the dilution rate, as low perfusion rates result in growth inhibition
due to nutrient exhaustion and/or accumulation of metabolites, and high
perfusion rates results in wasting valuable medium components and over
dilution of autocrine factors promoters of cell growth. In this context,
as it has been previously demonstrated for human hematopoietic
stem/progenitor cells (Madlambayan et al., 2005), the expansion of stem
cell populations can be boosted by removing inhibitory factors produced
by their more differentiated progeny. For those reasons, it would be
interesting in the future to study different medium residences times in
the STR platform operating under a continuous perfusion mode and their
impact on MSC attributes.
Immunophenotype analysis before and after STR cultures revealed that
MSC(M) cultured in S/X-free culture medium under stirred conditions
maintained the high expression of CD73, CD90, and CD105, whereas the
expression levels of hematopoietic cell markers (CD34, CD45, CD14 and
CD19), and HLA-DR molecules were very low in all conditions, satisfying
the minimal phenotypic criteria for describing human MSC (Viswanathan et
al., 2019). The expression of CD105 decreased after the STR cultures,
which was expected, as this event has been reported previously by our
group and others (de Soure et al., 2016; Dos Santos et al., 2014), and
may be attributed to longer exposure times to the enzymatic agent for
cell detachment, which is known to affect surface receptors (Brown et
al., 2007; Tsuji et al., 2017).
Overall, the results obtained in this work are in line with previous
results from our group and others showing that MSC main features are
well maintained upon cultivation under S/X-free stirred conditions
(Carmelo et al., 2015; Cunha et al., 2015; Dos Santos et al., 2014).
At the end of STR cultures, the MSC-CM was collected and the EVs were
successfully isolated and characterized by different techniques proposed
by the International Society of Extracellular Vesicles (Witwer et al.,
2017). TEM and Western blot techniques confirm the “cup-shaped”
morphology of EVs and the presence of their characteristic markers,
respectively and no significant different were found between EVs mean
and mode sizes [(163±5.27) nm vs (162±4.44) nm and (134±4.23)
nm vs (137±6.92) nm] for FB and FB/CP cultures, respectively
(P>0.05). In what concerns the concentration of EVs and
their purity in the MSC-CM retrieved from the STR cultures, average
concentrations of (2.4±0.35)x1011 and
(3.0±0.48)x1011 EVs/ml and similar PPRs of
(1.7±0.21)x108 and (2.0±0.22)x108particle/µg protein were estimated by NTA and protein quantification for
FB and FB/CP cultures, respectively. The MSC-EV densities obtained
herein and in agreement with the results reported on the production of
MSC-EVs using microcarriers in a Vertical-Wheel bioreactor (VWBR) by our
group (de Almeida Fuzeta et al., 2020) in spinner flasks by others
(Haraszti et al., 2018). To the best of our knowledge, this is the first
work describing a fully controlled process that maximizes the
proliferation of MSC(M) under S/X-free conditions in a 2L STR and the
subsequent isolation of EVs from the enriched MSC-derived conditioned
medium.
Conclusions
The bioreactor-based platform developed herein will allow to transform
laboratory-based protocols into robust MSC and MSC-EVs manufacturing
processes, with a tight control over the culture process and significant
reduction of the production times. By addressing the manufacturing
challenges of cell-based products, this technology is expected to
facilitate translation of MSC therapies and likely to impact the
development of therapeutic strategies employing MSC-EVs, which could
rapidly progress towards clinical studies exploiting their potential as
intrinsic therapeutics or as drug delivery systems (de Almeida Fuzeta et
al., 2022; Syromiatnikova et al., 2022). In addition, this platform
could be applied to the production of EVs from other parental cells
lines (i.e. dendritic cells, natural killer cells) in therapeutic
settings as cancer.
Author Contributions : Ana Fernandes-Platzgummer:
Conceptualization, Investigation, Methodology, Writing - Original Draft,
Funding Acquisition. Raquel Cunha: Investigation, Methodology; Marta
Carvalho: Investigation; Sara Morini: Investigation; Juan Moreno-Cid:
Methodology; Joaquim M.S. Cabral: Funding acquisition and Writing -
Review & Editing; Cláudia Lobato da Silva: Conceptualization, Writing -
Review & Editing and Funding Acquisition. All authors have read and
agreed to the published version of the manuscript.
Acknowledgments : We would like to acknowledge Instituto
Português de Oncologia Francisco Gentil and Clínica de Todos-os-Santos
for their kind donations of BM samples for this study.
Funding : This research was financed by national funds from
FCT—Fundação para a Ciência e Tecnologia (FCT), I.P., within the scope
of the projects UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit
Institute for Bioengineering and Biosciences‐iBB, LA/P/0140/2020 of the
Associate Laboratory Institute for Health and Bioeconomy—i4HB, and
PTDC/EQU‐EQU/31651/2017 of the EXOpro project. This research was also
funded by the project Bioprocessing Strategies for high productivity in
the Culture of Human Stem Cells in Stirred Tank Bioreactors and their
downstream. CELLS4ALL H2020-INNOVOUCHER-2018-0039.
Conflicts of Interest : J.M.-C. is employee of Bionet company.