Keywords
Ex vivo Expansion; Mesenchymal Stromal Cells; Extracellular
Vesicles; stirred tank reactor; Feeding regime; continuous perfusion;
Fed-batch
INTRODUCTION
In the last decades, human mesenchymal stromal cells (MSC) have emerged
as a main player in the field of Regenerative Medicine. Up to 448
clinical trials with human MSC were running within phases I and II
including 48 for the treatment of SARS-CoV-2 Induced Acute Respiratory
Failure (www.clinicaltrials.gov, terms searched: “mesenchymal stromal
cells”, “mesenchymal stem cells” and “mesenchymal stem/stromal
cells”, December 2022). Human MSC have been defined as plastic
adherent, fibroblast-like cells, expressing cell surface markers such as
CD73, CD90, CD105, CD29, CD44, CD49a-f, while being negative for CD14,
CD19, CD34, CD45 and HLA-DR expression and can differentiate into
multiple mesoderm-type cell lineages. These multipotent cells are
present in several tissues including bone marrow (BM), adipose tissue
(AT), Wharton’s jelly (WJ), dental pulp, synovial fluid, placenta,
amniotic fluid and others. Due to their ability to home to injury sites
and promote a regenerative microenvironment, combined with their
multilineage differentiation potential, MSC have been widely employed in
clinical trials for blood-related diseases (Le Blanc et al., 2008;
Prasad et al., 2011), autoimmune diseases (Duijvestein et al., 2010;
Garcia-Olmo et al., 2005; Mazzini et al., 2010) and tissue engineering
(Horwitz et al., 2002; Horwitz et al., 1999), with encouraging results.
More recently, these beneficial effects of human MSC have been
attributed mostly to their paracrine activity, exerted through
MSC-secreted modulating factors, rather than their engraftment into
injured tissues (Kraitchman et al., 2005; Toma et al., 2009). Indeed,
emerging evidence associates these paracrine effects to MSC-derived
extracellular vesicles (EVs), which play a central role in cell-to-cell
interaction and communication in a pleiotropic way (Balbi et al., 2017;
Camussi et al., 2013; Zhang et al., 2014). The EVs released by the donor
cells can be uptaken by nearby cells or distant cells, subsequently
modulating recipient cells (Phinney & Pittenger, 2017). The universally
used expression “EVs” comprises all vesicle subtypes (exosomes,
microvesicles and apoptotic bodies) and is a highly heterogeneous pool
concerning size range (30-5000 nm), origin, content (proteins, lipids,
genetic material and organelles such as mitochondria), biochemical and
biophysical features, and biological functions. Compared to cell
therapies, the use of EVs as cell-free therapeutic products presents
several potential advantages namely: (i) EVs are relatively safer, as
they are completely non-replicative and not mutagenic (Elsharkasy et
al., 2020); (ii) EVs have also a low risk of inducing microvasculature
obstruction upon administration due to their smaller size; (iii) EVs
have long circulating half-life and the ability to cross the blood brain
barrier (BBB) (Cerri et al., 2015; Moon et al., 2019); (iv) EVs have a
simpler composition than parental cells, although still complex and with
a bioactive cargo; (v) EVs can used as delivery systems with increased
efficacy and homing capacity (de Almeida Fuzeta et al., 2022; Katsuda et
al., 2013; Pascucci et al., 2014); (vi) EVs as non-living biological
products are more resistant to manipulation than living cells; (vii) the
possibility of using reduced doses in vivo to achieve a
therapeutic response, as EVs can evade phagocytes (Baglio et al., 2015);
and (viii) EVs can be potentially stored with no need of potentially
toxic cryoprotectants at -20°C for six months without loss of their
biochemical activity (Alvarez-Erviti et al., 2011; Sun et al., 2010;
Webber & Clayton, 2013). Overall, the regulatory aspects for producing
EV-based products for therapeutic strategies is expected to be less
complicated than for any therapy based on in vitro expanded
cells. Concerning their therapeutic use, EVs can be used as
biomedicines, taking advantage of their natural intrinsic medicinal
effects, which can be further improved by manipulating the parental cell
towards the production of more specialized and efficient EVs (Fan et
al., 2020; Haraszti et al., 2018). Alternatively, EVs can be used as
drug delivery systems (DDS), by loading various types of therapeutics,
including genetic material and drugs (Malhotra et al., 2019).
From a manufacturing perspective, EV production comprises, sequentially:
culture of the parental cell line (i.e. MSC); their collection or
harvest from the conditioned medium (i.e. culture supernatant); and
purification. Until now, MSC-EV production have been performed using
serum-containing media, mostly of animal origin, in planar systems under
static conditions, which are limited in terms of cell productivity;
their non-homogeneous nature results in concentration gradients (e.g.
pH, metabolites); are difficult to monitor; and ultimately unsafe as
animal-origin material represent a risk of microbial/prion contamination
(Sotiropoulou et al., 2006). Moreover, since the concentration of MSC-EV
in the conditioned medium is typically low, scale-out to hundreds of
T-flasks or multiplate flask systems is required, involving an extensive
handling for feeding/harvesting procedures, to achieve a significant
final mass production. Additionally, several differences were observed,
in terms of the MSC-derived EVs cargo, between EVs isolated from culture
supernatants of MSC expanded under different culture conditions,
stressing the importance of controlling all culture process parameters
to obtain a consistent EV content (Hyland et al., 2020; Kay et al.,
2021). In this context, it turns necessary to move forward to fully
controlled reactors (i.e. stirred tank reactors (STR)) to establish a
reproducible and scalable process to produce MSC-EV. In addition, the
use of defined serum-/xenogeneic-free (S/X-free) culture medium
formulations could result in substantial improvements for MSC-EV
production in terms of reproducibility, stability and quality, while
ensuring the approval of regulatory agencies. Besides pH, dissolved
oxygen and temperature, another important bioprocess parameter that is
crucial to monitor and control during STR cultures is the
nutrient/metabolite concentration profiles in culture. For that reason,
it is important to design efficient feeding schemes able to maximize
cell densities in a cost-effective way. Different strategies can be
considered for human cell cultures: batch, where no culture medium is
added or withdrawn during culture; fed-batch, where fresh culture medium
is added discretely/continuously to the STR but no culture supernatant
is withdrawn; and continuous perfusion operation with cell retention,
where there is an automated continuous replenishment/removal of
fresh/exhausted culture medium. Fed-batch is the feeding operation mode
of choice in the pharmaceutical sector due to the high cell densities
and consequently high product titers (e.g. antibodies) attained, as well
as its simplicity, among other features. On the other hand, perfusion
cultures allow higher cell productivities and a steady state operation,
as well as better cell physiology control that is crucial when cells are
the target product. As main disadvantages, perfusion cultures are
generally more costly and sometimes difficult to implement, especially
due to the cell retention device (e.g. continuous centrifuges,
tangential flow membrane filters and spin-filters). In this work, a
scalable S/X-free process to produce MSC and MSC-EVs in a fully
controlled STR was successfully established. Two different feeding
operation modes were compared in what concerns MSC yields and identity
and EVs production: fed-batch (FB) and FB combined with continuous
perfusion (CP). The use of FB followed by CP is expected to combine the
advantages of the two processes, as no important autocrine factors are
withdrawn from the STR during the first days, when the cell number is
still low (FB process), and CP process, when the cell number increases
and the need for culture medium exchange in deemed needed.
The manufacturing platform established herein is expected to pave a new
way for the development of MSC-based therapies, eliminating time- and
labour-consuming procedures aiming at the scalable production of
well-defined MSC populations as well as their secreted EVs to boost
their medical uses.
MATERIALS AND METHODS
2.1 Human cells
The human MSC derived from bone marrow [MSC(M)] used in this study
are part of the cell bank available at the Stem Cell Engineering
Research Group (SCERG), iBB-Institute for Bioengineering and Biosciences
at Instituto Superior Técnico (IST). MSC were previously
isolated/expanded according to protocols previously established at
iBB-IST (Dos Santos et al., 2010). Bone marrow aspirates were obtained
from IPO Lisboa from healthy donors after written informed consent
according to the Directive 2004/23/EC of the European Parliament and of
the Council of 31 March 2004 on setting standards of quality and safety
for the donation, procurement, testing, processing, preservation,
storage and distribution of human tissues and cells (Portuguese Law
22/2007, June 29), with the approval of the Ethics Committee of the
respective clinical institution.
2.2 MSC(M) expansion under static conditions
Prior to bioreactor inoculation, cryopreserved MSC(M) were thawed and
expanded at a cell density of 3000 cells/cm2 with
StemProTM MSC SFM XenoFree supplemented with 1% (v/v)
GlutaMAXTM and 1% (v/v) Antibiotic-Antimycotic
(10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin and 25
µg/mL of Amphotericin B) (StemPro culture medium) on pre-coated T-flasks
with CELLstartTM Substrate [diluted 1:100 in
phosphate buffered saline (PBS)]. At 70-80% cell confluence, MSC were
detached from the flasks by adding TrypLETM Select
Enzyme (diluted 1:10 in PBS) for 7 min at 37ºC. Cell number and
viability were determined using the Trypan Blue exclusion method. All
reagents were acquired from GibcoTM, Thermo Fisher
Scientific, USA.
2.3 MSC(M) expansion under stirred conditions
Non-porous SoloHill® plastic microcarriers (Sartorius, Germany) were
prepared according to manufacturer’s instructions. Briefly, 20 g of
plastic microcarriers were autoclaved at 121ºC for 20 minutes, washed
once with PBS and coated with CELLstartTM Substrate
(diluted 1:100 in PBS) for 2 h at 37ºC, using a 250 mL Bellco spinner
flask (Bellco Glass, Inc., USA), equipped with 90º paddles and a
magnetic stir bar, with an agitation of 40 rpm. After the coating
process, microcarriers were washed once with StemPro culture medium.
50x106 MSC(M), previously expanded under static
conditions with StemPro culture medium for 2 passages, were mixed with
the pre-coated plastic microcarriers and the microcarrier-cell
suspension (100 mL) was transferred to a 2 L STR (F0 BABY Bioreactor;
Bionet, Spain), equipped with a three-blade pitched impeller, and
dissolved oxygen (DO), pH, and temperature probes, already containing
500 mL of StemPro culture medium. For process monitoring and control,
the F0 BABY was coupled to ROSITA software (Bionet). Process parameters
were set to pH 7.2, temperature at 37ºC and DO of 20% of air saturation
(Dos Santos et al., 2014; Fernandes-Platzgummer et al., 2016). pH
control was performed by adding CO2 through overlay (50
ccm), DO concentration was maintained through overlay (100 ccm) with a
mixture of gases (air, CO2, N2) and
temperature was controlled via thermal jacket. The cell seeding on the
microcarriers (first 24 h) was performed under continuous stirring at 40
rpm and afterwards the agitation was increased to 60 rpm. STR cultures
were operated under FB and FB/CP mode. For FB cultures, after 3 days
with 600 mL of volume, fresh culture medium was added at days 3, 6 and 7
(final volume of 1750 mL). For the FB/CP cultures, from day 3 to day
5.5, 700 mL of fresh culture medium was added at a constant rate of 0.2
mL/min and after reaching a volume of 1300 mL, the STR started to
operate under continuous perfusion at the same flow rate (0.22
day-1 dilution rate) until day 11. Cell-containing
microcarriers were maintained inside the reactor, by using a Spin Filter
mounted in the agitation shaft. To maintain glucose concentration
between 2-6 mM, pulses of concentrated glucose (100 g/L, Sigma) were
added to the STR daily (days 3-8) or every 2 days (days 4-12) for FB and
FB/CP cultures, respectively. At days 9 and 13, the conditioned medium
(MSC-CM) from the STR cultures operated under FB and FB/CP,
respectively, was separated from the cell-containing microcarriers,
streamed through a 0.22 µm filter and stored at -80ºC for EVs processing
and characterization.
2.4 Monitoring of cell culture in the
STR
Cell Counts and Viability
STR culture sampling was performed using a previously established
protocol (Fernandes-Platzgummer et al., 2014). Briefly, every day,
duplicate samples of evenly mixed culture were collected from the STR
and transferred to 2 mL tubes. After the microcarriers settle, the
supernatant was collected for glucose/lactate analysis and the
cell-containing beads were washed with PBS and incubated with TrypLE
solution for 7 min in the thermomixer (Eppendorf AG, Germany) at 37º C
and 750 rpm. Afterwards, cells were separated from the microcarriers
with a 100 µm cell strainer and the cell number and viability were
assessed by using the Trypan Blue exclusion method.
Growth Rate and Doubling Time
Calculation
The growth kinetics of MSC(M) cultured under stirred conditions on
microcarriers was also characterized. For the exponential phase
(considering the death rate constant negligible), the maximum specific
growth rate, \(\mu\)max (day-1), was
calculated as dXv/dt=\(\mu\)max×Xv,
where Xv is the viable cell number for a given time (t).
After calculating \(\mu\)max, the doubling time, td
(day), was calculated as
td\(\ =\ \)ln(2)/\(\mu\)max.
Glucose/Lactate Analysis
Glucose and lactate concentrations in the samples retrieved form the STR
cultures were determined by membrane-bound immobilized enzyme
quantification in a YSI 2500 Biochemistry Analyzer (YSI, USA)
DAPI staining (Nuclear integrity)
Every 2 days, samples of cell-containing microcarriers were stained with
DAPI (1 mg/mL) (4’,6-diamidino-2-phenylindole dihydrochloride, Sigma)
for 5 min at room temperature (RT) and protected from light, to observe
cell adhesion and distribution on the microcarriers.
Immunophenotypic analysis
MSC(M) were analysed by flow cytometry before and after expansion under
stirred conditions, using a panel of mouse anti-human monoclonal
antibodies for the expression of CD90 (PE-conjugated), CD73
(phycoerythrin (PE)-conjugated), CD14 (PE-conjugated), CD19
[PE-conjugated], CD34 [fluorescein isothiocyanate
(FITC)-conjugated], CD45 (FITC-conjugated) and HLA-DR (PE-conjugated)
(all from Biolegend, USA); anti-CD105 (PE-conjugated,
InvitrogenTM, Thermo Fisher Scientific, USA) was also
used. Briefly, cells were incubated with each antibody for 20 min in the
dark and at RT, washed with PBS and then fixed using a solution of 1%
(v/v) PFA in PBS. Samples were analysed in a FACScalibur (Becton
Dickinson, USA) flow cytometer and CellQuestTMsoftware (Becton Dickinson) was used for acquisition. A minimum of 10
000 events were collected for each sample. Analysis was performed using
the FlowJo software (Tree Star, USA).
2.5 MSC-EVs separation by
precipitation
MSC-EVs were isolated from the MSC-CM (thawed on ice) using the Total
Exosome Isolation Kit (InvitrogenTM) according to
manufacturer’s instructions (de Almeida Fuzeta et al., 2020). Briefly,
50 ml of MSC-CM were incubated with half the volume of Total Exosome
Isolation reagent overnight at 4ºC. Afterwards, samples were centrifuged
for 1h at 10,000×g and 4ºC, the supernatant was removed, and the
MSC-EV enriched pellet was resuspended in 1 ml of cold RNase-Free PBS
(InvitrogenTM) diluted in
UltraPureTM DNase/RNase-free Distilled Water
(InvitrogenTM). MSC-EV were aliquoted and stored at
-80ºC in Eppendorf Protein Lowbind Tubes (Eppendorf AG) until further
use.
2.6 MSC-EV
characterization
Nanoparticle Tracking Analysis
(NTA)
NTA was performed using NanoSight LM14C instrument equipped with a 405
nm laser (Malvern Panalytical, United Kingdom) and the software
NanoSight 3.1 (Malvern Panalytical, United Kingdom). Silica 100 nm
microspheres (Polysciences, Inc.) were routinely analysed to check
instrument performance (Gardiner et al., 2013) and NTA acquisition and
post-acquisition settings were optimized and kept constants for all
samples. Particle concentration and size distribution on MSC-EVs diluted
samples (1:50) were determined according to protocols optimized at iBB
described elsewhere (de Almeida Fuzeta et al., 2020).
Protein Quantification
Protein concentration on MSC-EVs samples was measured using Micro
BCATM Protein Assay Kit (Thermo
ScientificTM) following manufacturer’s guidelines.
Briefly, bovine serum albumin (BSA) standards and samples were diluted
1:2 with an equal volume of reagent solution and incubated for 2 h at
37ºC. Absorbance was measured at 562 nm using the plate reader (BioTek
Synergy NEO, software Gen5 version 2.09). Three replicates were
quantified for each sample. A linear fit was applied to the BSA
standards, and the resulting equation was used to determine each sample
concentration from its absorbance measurement.
Purity Assessment
The purity of the MSC-EV samples was given by the particle to protein
ratio (PPR), which is the ratio between the particle concentration
determined by NTA and the protein concentration measured by MicroBCA
protein quantification.
Western Blot
The detection of Calnexin, Synthenin, CD63, CD81 and GAPDH through
Western Blot was performed according to the protocol optimized at SCERG
and described elsewhere (de Almeida Fuzeta et al., 2020). Primary
antibodies included anti-Calnexin (1:1000, BD, USA), anti-Synthenin
(1:1000, Abcam, UK), anti-CD63 (1:1000, Genetex, USA), anti-CD81 (1:500,
Abcam, UK) and anti-GAPDH (1:1000, Santa Cruz Biotechnology, USA).
Secondary antibodies included Goat anti-Mouse IgG (H + L) Cross-Adsorbed
Secondary Antibody, HRP (1:5000, InvitrogenTM) and
Goat anti-Rabbit IgG HRP-conjugated (1:1000, R&D Systems, UK). Image
acquisition was performed on iBrightTM CL1500 Imaging
System (InvitrogenTM).
Transmission Electron Microscopy
(TEM)
TEM Imaging was performed following negative staining protocol. Briefly,
100 Mesh copper grids were coated with formvar/carbon and glow
discharged right before use. MSC-EV samples were mixed (1:1) with
formaldehyde 4% in 0.1 M PBS (final concentration 2% formaldehyde in
0.05 M PBS) and incubated for 5 min at RT. Then samples added to the
grids and were incubated for 5 min to promote adhesion of EVs to the
grids. Next, washing in 10 drops of distilled water was performed and
samples were stained in 2 drops of uranyl acetate 2% by incubation for
5 min at RT in the dark followed by sample imaging. A
TecnaiTM G2 Spirit BioTWIN
Transmission Electron Microscope (TECNAI Tecnogroup, Italy) operating at
120k was used, and data was collected with an Olympus-SIS Veleta CCD
Camera (OLYMPUS, Japan).
2.7 Statistical Analysis
Results are presented as mean ± standard error of the mean (SEM) of the
values obtained from different MSC donors (i.e., biological replicates).
When appropriate, comparisons between experimental results were
determined by the non-parametric Mann–Whitney U test and statistically
significance was assessed with a p-value less than 0.05.
RESULTS
3.1 Influence of the feeding strategy on MSC(M)
proliferation
Based on previous work from our group (Dos Santos et al., 2014), a
S/X-free microcarrier-based culture system was successfully established
for MSC(M) proliferation using a 2 L-scale fully controlled STR operated
under FB and FB/CP modes. 50×106 MSC(M) from 3 donors
were expanded on CellStart-coated plastic microcarriers with S/X-free
StemPro culture medium, which varied over culture time as shown in
Figure 1A. During the adhesion stage (first 24 h of cultivation), a
minimum agitation speed of 40 rpm was used, which simultaneously allowed
to keep the microcarriers in suspension, maximizing the
cell-microcarrier contact necessary for cell adhesion, and minimized
cell death due to agitation (Hewitt et al., 2011). Subsequently, the
agitation speed was increased to 60 rpm to avoid/minimize the formation
of aggregates. After 24 hours, cell adhesion efficiencies of (34±5.8)%
and (40±5.9)% corresponding to (1.9±0.19)×107 and
(2.1±0.23)×107 cells were attained for FB and FB/CP,
respectively. Throughout the STR experiments, for both feeding operation
modes, cell viabilities were always above 90%, and the fraction of
live/dead cells found in suspension was (5.8±1.3)%, after day 2. The
growth curves depicted in Figure1B display an exponential phase leading
to maximal cell numbers of (3.0±0.12)×108 and
(5.3±0.32)×108 corresponding to cell densities of
(2.0±0.51)×105 and (4.1±0.90)×105cells/mL (Figure1B) and fold increase (FI) values in total cell number
of 16±2.1 and 24±5.5 (Figure1C), obtained at days 8 and 12 of
cultivation for FB and FB/CP cultures, respectively. No major
differences were observed in terms of cell number until day 7 for the
two feeding operation modes tested. After this day, cell growth ceased
in the STR operating under FB mode. Specific growth rates
(µmax) and doubling times (td) were also
calculated for the two STR feeding operation modes according to
“Materials and Methods” and were 0.6±0.1 day-1 and
1.3±0.2 days; and 0.5±0.1 day-1 and 1.5±0.2 days,
respectively, for FB and FB/CP cultures.