Scalable expansion and harvesting of hiPSCs using
Synthemax-II dissolvable microcarriers
Authors: Rodrigues, A.L.; Rodrigues, C.A.V; Diogo, M.M
Abstract
Polystyrene microcarriers (PSM) have been exploited as 3D-platform for culturing hiPSCs. These micro-spherical beads can be
incorporated into xeno-free suspension cultures to achieve clinical-relevant cell numbers. However, equal importance should be given
to the downstream processing, which is subject of cell losses and reduced viability. Corning, Inc. developed a digestible polymer
based on polygalacturonic acid to prompt an efficient cell harvesting. Moreover, these dissolvable microcarriers (DM) are coated with
a xeno-free substrate, Synthemax-II (SII), to promote the expansion of hiPSCs under GMP-compliance. After the static screening,
the fully defined combination of mTeSR™1 and SII-coated DM was scaled-up using a spinner-flask, under dynamic condition. A
maximum fold increase of 3.76 was achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 25 rpm, which
generates a cell density of 9.39x105 cells/mL after 5 days of expansion. These results were found to be reproducible with another
hiPS cell line. Afterwards, this system was efficiently translated to a xeno-free platform by the replacement of mTeSR™1 for TeSR™2.
The downstream processing of the expanded hiPSCs was performed by the digestion of the DM-SII beads within the spinner flask.
The 97%-harvesting yield of DM-SII was considerably higher than what was obtained by the filtration of PSM cultured cells. After cell
harvesting, replated hiPSCs maintained their undifferentiated state, exhibiting pluripotency-associated markers. Moreover, their
differentiation capabilities were confirmed by their spontaneous differentiation through embryoid body formation and direct
differentiation towards neural progenitors and cardiomyocytes.
Introduction
Human induced pluripotent stem cells (hiPSCs) result from
a somatic cell reprogramed towards a more primitive state [1,
2]. Their long-term self-renewal and differentiation capabilities
lend themselves as a powerful tool for disease modelling and
drug screening [3, 4]. The use of such assets as cell-based
therapies is hampered by the lack of reprograming methods
that better balance efficiency and safety [5, 6]. On the other
hand, large number of cells are required to treat a specific
disease. For instances, 1-2x109 myocytes would be
necessary to treat a myocardial infarction [7], which can only
be achieved by suspension culture, such as in the case of
spinner-flask and bioreactors. Additionally, these cells have to
be obtained under Good Manufacturing Practices (GMP),
which implies a completely defined and xeno-free culturing
platform. Therefore, efforts have been made towards the
development of an expansion protocol, which can be scaled-
up to obtain the necessary number for clinical-grade hiPSCs.
Microcarriers have been used as 3D-culture platform that
can be further incorporated into dynamic conditions.
Moreover, there are studies reporting the use of polystyrene
microcarriers (PSM) under xeno-free configuration [8-11]. For
instances, Badenes et al developed a xeno-free platform
resorting to E8® medium and Vitronectin-coated PSM. This
protocol was also optimized in terms of agitation and initial cell
densities, through a three-level factorial design [10].
Yet, little focus has been given to the downstream
processing. The use of PSM envisages a filtration step, which
is subject to cell loss and reduced cell viability. As an
alternative, biodegradable matrices would prompt a reduction
in downstream processing steps, and thus reducing the
overall cost. For that purpose, Corning Inc. developed a new
type of microcarriers envisaging the scalability of the
harvesting process. These dissolvable microcarriers are made
of a polygalacturonic acid polymer coated with Synthemax-II
(SII), a chemically defined substrate containing the RGD-
sequence of the human vitronectin, which has been proven to
support the expansion of hiPSCs.
The aim of this study is to give preliminary results on the
use of SII-coated dissolvable microcarriers (DM-SII) for the
dynamic expansion of hiPSCs, under xeno-free conditions.
The hypothesis being tested is, if the expansion proved similar
for both types of microcarriers (PSM and DM-SII), the cell
harvesting yield would be higher for the newly developed DM-
SII. Moreover, it is intended to integrate the bioprocess –
expansion and harvesting – in one closed system.
Materials and Methods
Cell lines, Microcarriers and Culture media: The initial
static screening and dynamic expansion were performed
using the F002.1.13 hiPS cell line (TCLAB), which derived
from healthy fibroblasts (46, XX) through retroviral
transduction of the human genes OCT4, SOX2, C-MYC and
KLF4 2. The Gibco™ hiPS cell line (by Thermo Fisher
Scientific) was also used for the dynamic expansion. This is a
viral-integration-free human induced pluripotent stem cell
(iPSC) line generated using cord blood-derived CD34+
progenitors with seven episomally expressed factors (Oct4,
Sox2, Klf4, Myc, Nanog, Lin28, and SV40T). The hiPSCs were
routinely cultured on Matrigel-coated plates in mTeSR™1
medium in a humidified 5% CO2 incubator at 37ºC. The
medium was daily refreshed and when cells reached 80%
confluence, the EDTA method was used to passaged cells at
a split ratio of 1:4 [12]. Dissolvable microcarriers (Corning®,
Inc.), with 5,000 cm2/g of surface area, were used to support
cell growth. According to the manufacturer’s instruction,
Synthemax-II dissolvable microcarriers were hydrated for
1hour. In the static screening, uncoated DM were coated with
Matrigel™ in a proportion of 1:30(v/v) of culture medium. This
was performed for 2h, at room temperature and under
agitation. Polystyrene microcarriers, with 360 cm2 of surface
area were used as a control. Microcarriers were sterilized for
1h with Ethanol 70% at RT and washed 3 times with sterile
phosphate-buffered saline (PBS). Coating of microcarriers
was performed for 2h at RT with Vitronectin in sterile PBS,
using 0.5µg/cm2. Prior to cell inoculation, both DM and PSM
were incubated in culture medium for 30min at 37ºC.
Static Screening: The static screening was performed on
24-well ultra-low attachment plates. It was used 3 cm2 of
microcarrier surface are per well. Cells were collected from
2D-culture plates using the EDTA method, and inoculated on
the microcarriers with 1:1000 (v/v) ROCK inhibitor for the first
24h of culture. The culture media used were mTeSR™1,
TeSR™2 and E8® media. Cells were inoculated at initial cell
density of 5x104 cells/cm2. Vitronectin-coated PSM were
cultured on E8® medium, Matrigel- and SII-coated DM were
both tested on mTeSR™1. 80% of the culture media volume
was changed by fresh media for 5 days. The cell yield in total
cell number was calculated as the ratio Xday5/Xi, where Xday5 is
the number of viable cells, attached to the microcarriers, at
day 5, and Xi is the number of cells inoculated at day 0.
Dynamic Expansion: The expansion of hiPS cells in a
microcarrier stirred suspension culture was performed in pre-
siliconized (Sigmacote, Sigma) spinner flasks (StemSpanTM,
Stem- Cell Technologies), with a working volume of 30 mL.
Cells were seeded as small clumps, at an initial density of
5x104 cells/cm2 and 20g/L of microcarriers. For the first 24h
after inoculation, 15mL of medium were supplemented with
ROCK inhibitor for the first 24h. After day 0, the medium was
replaced and adjusted to 30 mL of fresh medium.
Subsequently, an intermittent stirring (3 min at 25 rpm every 2
h) was performed overnight to promote cell-cell and cell-
microcarrier contact. Thereafter, the culture was continuously
stirred at 25 rpm and feeding was performed daily by replacing
80% of volume with fresh pre-warmed medium. For spinner
flask cultures, cell attachment efficiency and maximum cell
yield were calculated accordingly to Badenes et al. [10]. The
protocol developed by Nienow et al was adapted to harvest
the expanded hiPSCs [13]. Herein, a harvest solution was
used to digest the PGA polymer. This solution was prepared
accordingly to the manufacturer’s instructions, by adding
EDTA (stock solution 0.5M, pH 8) to the protease solution,
ensuring a final pectinase concentration of 100 U/mL and
EDTA concentration of 10mM. The harvesting yield was
calculated as the percentage of Xday7/Xharvested, where Xday7 is
the number of viable cells attached to beads at day 7 and
Xharvested is the number of cells harvested from the dynamic
expansion.
Viability assay: LIVE/DEAD® viability/cytotoxicity Kit
(Thermo Fisher Scientific) was used to assess the viability of
expanded hiPScs. This was performed upon a sample of
500µL retrieved from the spinner-flask.
Immunocytochemistry and Flow cytometry: For
intracellular staining, the protocol used is described by
Miranda et al. [14]. For extracellular staining, the protocol
used is described by Badenes et al. [10]. For
immunocytochemistry, cells were examined using a
confocal/fluorescence microscope (LSM 710 confocal laser
point-scanning microscope (ZEISS) and fluorescence
microscope DMI 3000b (Leica)).
Antibodies: Primary antibodies used for the
immunocytochemistry and flow cytometry assays comprised
the intracellular OCT4 (1:150; Milipore), SOX2 (1:200; R&D
Systems) and the extracellular SSEA4 (1:100), SSEA4-PE
(1:10), TRA1-60 (1:100), TRA1-60-PE (1:10) (StemGent). The
secondary antibodies included goat anti-mouse IgG Alexa
Fluor– 488 or 546 (1:500 or 1:1000), goat anti-rabbit IgG Alexa
Fluor 546 (1:1000)–Invitrogen; and isotypes used for control
in flow cytometry tests included anti-mouse IgM-PE (Miltenyi
Biotec) and anti-mouse IgG-PE (1:10) (StemGent).
Immunocytochemistry against markers from the three germ
layers was performed using antibodies against alpha smooth
muscle actin (α-SMA; mouse: 1:1000; Dako), neuron-specific
class III β-Tubulin (TUJ1; mouse: 1:20 000; Covance) and
SOX17 (mouse: 1:1000; R&D Systems), for the mesoderm,
ectoderm and endoderm, respectively. Cardiomyocyte marker
was Troponin T cardiac isoform antibody (13–11) (cTNT;
mouse: 1:500; Thermo Scientific). Neural progenitor cell
markers were Sox2 (mouse: 1:1000; R&D Systems) and ZO-
1 (rabbit: 1:1000; Covance).
RT-PCR: Total RNA from cell samples of selected time
points was extracted using Invitrogen™ PureLink™ RNA Mini
Kit (Thermo Fisher Scientific) following the provided
instructions. RNA was treated with Invitrogen™ TURBO DNA-
free™ for total DNA digestion and then it was quantified using
a nanodrop. 1 µg of RNA was converted into cDNA with
Applied Biosystems™ High Capacity cDNA Reverse
Transcription Kit (Thermo Fisher Scientific) also following the
provided instructions. PCR reactions were run using 12.5 ng
of cDNA and 250µM of Applied Biosystems™ Taqman™
Gene Expression Assays (Thermo Fisher Scientific), along
with Applied Biosystems™ Taqman™ Gene Expression
Master Mix (Thermo Fisher Scientific). Reactions were run in
triplicate using Applied Biosystems™ ViiA™ 7 Real-Time PCR
Systems (Thermo Fisher Scientific) and data were analysed
using Applied Biosystems™ QuantStudio™ Real-Time PCR
Software. The analysis was performed using the ΔΔCt method
and values were normalized against the expression
of the housekeeping gene glyceraldehyde-3-phosohate
dehydrogenase (GAPDH).
Direct and Spontaneous differentiation: replated
hiPSCs were directly differentiated to neural progenitors and
cardiomyocytes according to the protocols developed by
Fernandes et al. [15] and Lian et al. [16], respectively. hiPSCs
differentiation potential was also evaluated in vitro via
embryoid body formation and spontaneous differentiation.
This was performed according to the protocol described by
Badenes et al. [10].
Statistical analysis: Error bars represent the standard
error of the mean (SEM). Unless otherwise stated, at least
three replicates were performed for every experiment. When
appropriate, statistical analysis was done using the Mann-
Whitney test for independent samples, and a p-value less than
0.05 was considered statistically significant.
Static expansion of hiPSCs using both polystyrene and
dissolvable microcarriers
Initially, it was performed a static screening to assess cell
adhesion and fold increase of hiPSCs onto this type of
microcarriers. Therefore, different combinations of coatings
and culture media were tried to evaluate the previous
parameters (Table 1). The system developed by Badenes et
al. was used as a comparable xeno-free condition.
Regarding the hiPSCs expansion with plastic
microcarriers (PSM), cells adhered to the vitronectin-coated
surface with a 73±6% yield, as it is possible to observe from
the bright-field microscopy images and direct cell
quantification, respectively (Figure 8). This was within
expectations, as vitronectin presents itself as an ECM-
glycoprotein, which promotes cell adhesion via αvβ5 integrins
[90]. Moreover, the adhesion yield was very similar to what
was reported by Rowland et al under 2D-configurations [90].
Cells were cultured in this platform until day 5. Bright-field
microscopy images show that the attached cells were able to
grow on the PSM-VTN surface. Additionally, direct
quantification shows a 4.95±0.5-fold increase (Figure 1A),
which demonstrated a significant growth in cell population.
According to the literature, this was expected as Badenes et
al. reported a 6.6±1.0-fold increase for the same platform
under static conditions. Despite the differences, the value
obtained in this experiment is not significantly different from
what was reported, which validates the suitability of this
method to expand hiPSCs [84].
Regardless of the previous results, the focus of this work
is the expansion of hiPSCs using dissolvable microcarriers.
Therefore, hiPSCs were cultured onto this matrix with different
coatings and culture media combinations. Cells were daily
monitored through bright-field microscopy, which
demonstrated cell adhesion to the beads, and its further
growth, regardless of the combination (Figure 1B). Direct cell
quantification shows that the highest adhesion yield was
observed in Matrigel-coated DM (93±8%) followed by DM-SII
(71±3%), both cultured on mTeSR™1. Nevertheless, the fold
increase for these two combinations was very similar
(4.42±0.6 and 4.39±0.44, respectively). There are no
references in the literature of DM being used as an expansion
scaffold for hiPSCs. However, these results were expected as
the use of these substrates and culture media has been
proven to support hiPSCs growth. For instances, Matrigel was
first used as a substitute for feeder-cells in 2D-cultures, since
it contains ECM components like laminin and collagen [59]. Its
use as a microcarrier coating is also presented in the
literature, with Bardy et al achieving cell densities close to
1.3x106cells/mL and a 7.7±0.2-fold increase, under static
conditions [81]. The results obtained for DM-Mat mT1
demonstrated that the cell density achieved ranged from 6.83
– 8.15x105 cells/mL, with an average of 6.69x105 cells/mL
over 5 days of expansion. The differences observed between
what is reported in the literature and these results may be due
to the expansion period rather than the microcarrier matrix or
cell line.
Regarding the DM-SII and mTeSR™1 combination, the
adhesion yield was similar to what was observed in the case
of PSM-VTN. This can be explained by the chemical nature of
SII. This synthetic peptide-copolymer contains the RGD-
sequence from the human vitronectin ECM-protein, which
promotes cell adhesion [17]. Additionally, the fold increase
was very similar to the previous cases without any significant
difference between each condition. In the literature, Silva et al
reported similar results using the SII-coated polystyrene
microcarriers, which demonstrates the versatility of such
substrate as a microcarrier coating [8].
Figure 1- Expansion of hiPSCs under static conditions using both polystyrene (PSM) and dissolvable microcarriers (DM). (A) From left to right: Cell adhesion yield and cell fold increase for all the tested combinations of microcarriers, coatings and culture media. (B) From left to right: Bright-field microscopy images from day 1 and day 5 of the previous combinations. Maximum intensity projection of confocal microscopy images of the pluripotency markers for the expanded cells. The nuclei were counterstained with DAPI. Scale bar: 132µm. Abbreviatures: vitronectin-coated polystyrene microcarriers and E8®medium (PSM-VTN E8); Matrigel-coated dissolvable microcarriers and mTeSR™1 medium (DM-Mat mT1); Synthemax-II coated dissolvable microcarriers and mTeSR™1 medium (DM-SII mT1); Synthemax-II coated dissolvable microcarriers and TeSR™2 (DM-SII T2).
The former combinations do not preclude the absence of
a GMP-compliant system, making them an unviable option for
the expansion of clinical-grade hiPSCs. Therefore, TeSR™2
was used as a xeno-free option for culturing these cells on
DM-SII. Through bright-field microscopy images on day 1, it is
possible to observe small aggregates being formed without
any attachment to the DM-SII beads (Figure. 1B). This is
translated into a slightly lower adhesion yield among all
combinations. However, the fold increase was not affected by
this event, since these small aggregates started to adhere to
the DM-SII over the expansion period. Interestingly, the
4.7±0.4-fold increase of such combination does not vary
significantly from the other options, which makes it a viable
option if GMP-compliant systems were ever to be considered.
Immunocytochemistry was used to assess the
pluripotency phenotypes. The results show that expanded
hiPSCs can maintain their pluripotency after 5 days of static
expansion, regardless of media, coating and microcarriers
combinations (Figure. 1B). Nevertheless, the expression of
stemness markers needs further validation with RT-PCR, flow
cytometry and differentiation assays to confirm the
pluripotency of the expanded cells.
Despite the promising results, the focus of this work is the
scalability of the expansion process using dissolvable
microcarriers. The DM-SII in combination with mTeSR™1
proved to be the chemically defined culture system with the
best cell adhesion yield. Therefore, this combination was used
a starting platform to expands hiPSCs under dynamic
conditions.
Dynamic expansion and characterization of hiPSCs with
DM-SII and mTeSR™1 culture medium
The results obtained for the expansion of hiPSCs under
static conditions using dissolvable microcarriers demonstrates
that this platform is suitable to expand hiPSCs at a laboratory
scale. The next step was to implement a dynamic
microcarrier-based culture system in spinner flasks,
envisaging the scalability of the expansion process using the
dissolvable matrices.
To achieve the established goals, TCLab cells were
previously expanded as a 2D-monolayer culture. Afterwards
they were transferred to a suspension culture device (spinner-
flask) with Synthemax II-coated dissolvable microcarriers
(DM-SII) and mTeSR™1 culture medium. A density of 55,000
cells/cm2 was used for the inoculation of the spinner-vessel.
Cells were cultured for a period of 7 days and two samples of
500 μL were daily retrieved for direct quantification of cell
number. After expansion, samples of cells attached to the
beads were retrieved for further pluripotency analysis. The
results are presented in Figure 2.
In Figure 2A, it is possible to observe the variation in the
total number of cells over the 7 days of dynamic expansion.
This graphic was obtained through direct cell quantifications.
Day1 is presented as the timepoint with the lowest cell
number, with a mean of (6.8±0.7)x106 cells, which means
(2.26±0.2)x105 cells/mL. From day 0 to day 1, there is no
agitation to promote cell adhesion onto DM-SII. The attained
adhesion yield ranged from 49 – 75% with a mean of
56.6±6.2% (Figure 2B), which is comparable to what was
obtained under static conditions. The adhesion of hiPSC to the
DM-SII was expected due to the chemical nature of
Synthemax-II, which simulates the cell-ECM interactions [17-
19].
The total number of cells increased as they grew attached
to the available surface area. This correlated with the bright
field microscopy images at the final day of culture when
compared to the images obtained in the first day of the culture
(Figure 2C). At day 5, the total number of cells ranged from
1.99 to 3.8x107, with a total mean of (2.86±0.4)x107 cells in
the spinner-vessel. This day is presented as the timepoint with
the highest number of cells, after which it started to decline
(Figure 2A). In comparison, Bardy et al. achieved a higher cell
density (3.1±0.2x106 cell/mL), when using Matrigel-coated
microcarriers under dynamic conditions. However, this
platform precludes the expansion of hiPSCs under GMP-
compliant settings [20].
Regarding the cell fold increase, it varied in a proportional
manner, decreasing after day 5 (Figure 2B). This may be due
to the existence of cell-to-cell interactions, which leads to the
formation of large cell-bead aggregates (cluster), hampering
oxygen and nutrient diffusion (Figure 2C). In figure 2E, it is
presented the result of a viability assay performed on day 7.
The presence of a few dead cells within the cluster is
confirmed by the ethidium homodimer staining. This result
may be explained by the limitations of oxygen and nutrient
diffusion, which affect cell viability [21]. These observations
together with the direct cell quantifications, suggest that the
harvesting procedure should be performed on the 5th day of
culture. Nevertheless, at day 7 the cells attached to the DM-
SII beads expressed OCT4 and TRA-1-60 pluripotency
markers, which indicated the maintenance of pluripotency
characteristics in the cells cultured in the spinner-flask (Figure
2D).
At day 7, cells were harvested for further pluripotency
characterization. These assays comprised
immunocytochemistry, qRT-PCR and flow cytometry analysis
of the expression of pluripotency markers. After harvesting the
expanded cells, these were replated onto Matrigel-coated
plates. Human iPSCs maintained their capacity to form
colonies, since they stained positively for OCT4, SOX2 and
TRA1-60 pluripotency markers (Figure 2F). Flow cytometry
was used to confirm the previous results. As it is possible to
observe from figure 2G, more than 91% of the harvested cells
were positive for the expression of the pluripotency markers
SOX2, NANOG, TRA-1-60 and SSEA-4 after 7 days of
dynamic culture. At the beginning and at the end of the culture,
mRNA was isolated to evaluate the expression of pluripotency
genes by qRT-PCR (Figure 2I). It was observed the
expression of OCT4 and NANOG pluripotency genes, with
further downregulation of differentiation genes, such as PAX6,
SOX17 and T.
The pluripotency of the expanded hiPSCs was also
assessed by evaluating their ability to differentiate into
progeny of the three embryonic germ layers. The harvested
cells were replated onto Matrigel-coated plates and finally
inoculated in ultra-low attachment plates (ULA) as suspended
cell aggregates. Cells were able to from Embryoid Bodies
(EBs). After 4 weeks of culture, the EBs were replated onto
laminin-coated plates. The expression of the three germ
lineages was assessed through immunocytochemistry. In
figure 2H, it is possible to observe the expression of specific
markers for endoderm, ectoderm and mesoderm, such as
SOX17, TUJ1 and α-SMA, respectively.
The expanded hiPSCs were also directly differentiated
towards neural progenitors, based on the work developed by
Fernandes et al. [15]. For that, cells were replated onto
Matrigel-coated plates. When 90 – 100% confluence was
achieved, the dual-SMAD inhibition was used to induce neural
commitment. At day 12, neural progenitors were replated onto
laminin-coated plates and cultured in N2B27 medium, without
the chemical inhibitors SB and LDN. The bFGF was used to
enhance the viability and formation of neuroepithelial rosettes.
The structures obtained resemble in vitro the configuration of
the neural tube, from which neurons are derived. In figure 2J,
it is possible to observe a positive result for the
immunocytochemistry of SOX2 and apical ZO1 markers,
which demonstrate the polarization of the neuroepithelial cells
[15].
Figure 2 - Expansion of TCLab hiPSCs under dynamic conditions using Synthemax-II dissolvable microcarriers with mTeSR™1. (A) Total number of cells over 7 days of expansion. Results are presented as the mean average of n=4 experiments. The error bars represent the standard error of mean (SEM); (B) Graphic representation of the adhesion yield and fold increase attained on the first day and throughout the culture, respectively. This is the outcome of the mean of n=4 experiments, with the error bar representing the Standard Error of Mean (SEM). (C) Bright-field microscopy images of the cells attached to the beads on day 1 and on day 7, respectively. (D) Maximum confocal intensity projection of the immunocytochemistry analysis for expression of intracellular OCT4 and extracellular TRA-1-60 pluripotency markers. (E) Viability test of cells attached to the microcarriers cultured on day 1 and day 7 of the dynamic expansion. Green is the calcein metabolized by the living cells, whereas the dead cells (red) were stained by the ethidium homodimer; (F) Confocal microscopy images of immunocytochemistry for the pluripotency markers: SOX2, TRA-1-60 (Scale bar: 132 µm) and OCT4 (Scale bar: 66 µm) The nuclei were counterstained with DAPI; (G) Flow cytometry analysis of the hiPSCs harvested after 7 days of expansion in the spinner flask. Cells were stained for Oct4 and SOX2 intracellular markers and TRA-1-60 and SSEA-4 cell surface markers. The error bars represent the SEM of n=4 experiments; (H) Immunostaining showing the formation of cells expressing TUJ1 (ectoderm), SOX17 (endoderm) and α-SMA (mesoderm) after the EB formation and spontaneous differentiation assay with hiPSC cultured in spinner-flask. Scale bar: 100µm for TUJ1 and 50 µm for SOX17 and α- SMA; (I) Quantitative RT-PCR analysis of the pluripotency and differentiation genes of hiPSCs after seven days of culture. mRNA was isolated at the beginning and at end of the culture; (J) Confocal microscopy images for immunostaining for SOX2 and ZO-1. The nuclei were counterstained with DAPI. Scale bar: 33 µm.
To prove the standardization of this culture platform, the
expansion of another hiPS cell line was performed under the
same conditions (data not shown). When comparing these
results with the expansion of TCLab cell line, the Mann-
Whitney statistical test did not present any significant
differences between cell adhesion and maximum cell yields.
Likewise, the harvesting procedure did not affect the ability of
cells to form undifferentiated colonies, neither their
differentiation capabilities.
Overall, these results proved that the use of DM-SII for the
expansion of hiPSCs is cell line-independent. This is in
agreement with previous results described in the literature, as
Synthemax-II has been proven to support the proliferation of
hPSCs under static conditions [17-19, 22]. Despite the
differences between hESCs, Jin et al. demonstrated that
hiPSCs could be expanded onto Synthemax-II coated
surfaces as efficiently as hESCs. The authors took advantage
of the same combination of substrate and medium (SII and
mTeSR™1) to expand cells in 2D-culture. In this work, cells
could maintain their undifferentiated state up to 10 passages,
with the cell-SII interactions mediated via αvβ5 integrins [23].
However, this study entailed a static platform which is not
easily scalable and devoid from shear stress of the dynamic
cultures, which have been proven to improve homogeneity of
the culture environment and regulate stem cell fate.
Regarding expansion methods for hPSCs, microcarriers
have been used as a 3D-platform that can be further
incorporated into suspension cultures. Oh et al. developed a
protocol for the expansion of hESCs, with Matrigel-coated
microcarriers. In this study, the two cell lines tested achieved
cell densities close to 3.5x106 cells/ml, which demonstrated
the robustness and efficiency of such system [24].
Analogously, Bardy et al. also reported the use of Matrigel as
a microcarrier coating for the expansion hiPSCs, which
yielded a cell density similar to the previous study [20]. In both
cases, the harvested cells exhibited a phenotype consistent
with a pluripotent stem cell, as they expressed pluripotency
markers and were able to generate progeny derived from the
three germ layers. On the other hand, other animal-derived
substrates have been reported to support hPSCs growth onto
microcarriers. Chen et al. observed that shear-resistant hES
cell lines would exhibit a comparable growth when cultured
onto microcarriers coated with both Matrigel and mouse-
derived laminin. Nevertheless, shear-sensitive cells would
exhibit a reduced cell growth, viability and pluripotency when
propagated on laminin-coated microcarriers. The authors
postulated that the gelatinous thick nature of the Matrigel
substrate would offer a shear protective element [25].
Despite the results, such platforms were not GMP-
compliant, which hampers the clinical translation of the
expanded hPSCs. Therefore, other alternatives were
developed to counteract such disadvantage. For instances,
Badenes et al reported the use of SII-coated polystyrene
microcarriers for the development of an expansion protocol for
hiPSCs. In this work, the authors highlighted the possibility of
integrating this platform into a fully controlled bioreactor
configuration [9]. Within this context, Silva et al used similar
microcarriers and mTeSR™1 media for the dynamic
expansion of hESCs. The authors were able to achieve 5x105
cells/ml over five days. At the end of the culture, the harvested
cells retained their undifferentiated phenotype [8]. In
comparison, the cell density attained by DM-SII was higher
than the one reported by Silva et al. Therefore, the use of DM-
SII promises to be an efficient alternative for the expansion of
hESCs under defined conditions.
Metabolic profile of expanded hiPSCs with DM-SII and
mTeSR™1 culture medium
The direct measurement of glucose, lactate and glutamine
concentrations was performed to assess the metabolism of
the TCLAB cell line, during the 7 days of dynamic expansion.
At each daily culture medium change, a sample of fresh and
exhausted medium was retrieved to establish the typical
concentration profile for each nutrient and metabolite (Figure
3).
Glucose concentration decreased thoroughly due to an
increased consumption by the growing cell population (Figure
3A). Human PSCs require large amount of glucose to fulfill
their metabolic needs, namely cell growth. Consequently,
lactate concentration increases over time, as a waste product
(Figure 3C). From day 5 until day 7, lactate concentration
raises above 15mM. Chen et al. observed that hPSCs growth
was hampered by lactate concentrations above 20mM [26].
On the other hand, Horiguchi et al. demonstrated that lactate
concentration higher than 15mM would exert an inhibitory
effect upon cell growth [27], which might explain the decrease
in cell density after day 5. Overall, these observations explain,
at least partially, why the hPSCs culture media must be
changed on a daily basis.
The apparent yield of lactate from glucose (Y´qLac/qGlu)
was also calculated (Figure 3E). This parameter gives an
estimation of the glucose fraction converted into lactate. The
theoretical maximum yield is equal to 2, as one molecule of
glucose can only give rise to two molecules of lactate via
Figure 3 - Metabolic profile of TCLAB cell line during expansion under dynamic conditions with DM-SII and mTeSR™1. The culture media was daily changed for newly mTeSR™1 culture media. The results are the mean of n=4 experiments, with the error bars standing for the standard error of mean (SEM) (A) Concentration of glucose (mM) over seven days of expansion. (B) Specific rate of glucose consumption over seven days of expansion (µM.cell-1.day-1). (C) Concentration of lactate (mM) over 7 days of expansion. (D) Specific production rate of lactate per day over seven days of expansion (µM.cell-1.day-1). (E) Apparent yield of lactate produced from glucose over seven days of expansion.
glycolysis. Overall, the attained Y´qLac/qGlu ranged from 1.5
to 2 (Figure 3E). Kropp et al. also obtained similar results for
hiPSCs cultured as cell aggregates in a repeated batch
strategy [28]. This is consistent with the majority of glucose
being converted into lactate, rather that entering the
tricarboxylic acid cycle (TCA).
In the presence of oxygen, somatic cells direct glucose-
derived pyruvate for oxidative phosphorylation, where electron
transfer to oxygen is catalyzed to produce ATP and CO2. In
contrast, hPSCs are highly proliferative cells with immature
mitochondria that need to synthetize proper intermediates for
cell growth, namely nucleic acids, proteins and substrates for
membrane biosynthesis. Instead of metabolizing all glucose
into CO2, pyruvate is deviated from entering the mitochondria
which slows the rate of TCA cycle. Therefore, hPSCs rely on
glycolysis to produce ATP even if there is oxygen available to
conduct the alternative metabolic path. This is known as the
Warburg effect, where cells exploit a less profitable ATP
metabolic pathway in order to channel this energy for the
biomass formation [29-31]. As this analysis was only
performed for the expansion of TCLab cell line, it would be
interesting to see if the same effect would be observed on
Gibco cell line expansion.
Dynamic expansion and characterization of hiPSCs
under xeno-free conditions
From the previous results, it was possible to conclude that
hiPSCs were able to grow onto the DM-SII, maintaining their
phenotype. It was also proven that this expansion is cell line
independent, as two hiPSC lines were tested. Despite the
positive results, the previous combination of DM-SII and
mTeSR™1 did not comply with GMP conditions. This culture
medium contains bovine serum albumin. Therefore, the use of
TeSR™2 in combination with DM-SII was evaluated for the
expansion of clinical-grade hiPSCs, as this culture media is
free of animal proteins [32]. The results of this expansion
experiment are presented in Figure 4.
In Figure 4A, it is possible to observe the total number of
cells over 7 days of expansion using DM-SII along with
TeSR™2. As in the previous cases, an initial cell density of
55,000 cells/cm2 was used to inoculate the spinner-flask. At
day 1, the attained 61±4.2%-adhesion yield proved to be
higher than what was observed for the expansion with
mTeSR™1. Despite the results, Mann-Whitney statistical test
demonstrated a p-value of 0.3429, therefore, no significant
difference was observed between the two conditions (data not
shown). The chemical nature of Synthemax-II explains the cell
adhesion to the DM surfaces, as it simulates the cell-ECM [17-
19].
As cells grew attached to the DM-SII surface, the 4th day
was proven to be the timepoint where the highest number of
cells was achieved. On this day, the value ranged from 1.36 –
2.26x107, with a mean of (1.87±0.2)x107cells in the spinner-
flask. Interestingly, the total number of cells started to
decrease thoroughly only after day 6, with a cell density of
(5.56±0.8)x105cells/mL being achieved at the end of the
culture (day 7). The previous combination of DM-SII and
mTeSR™1 yielded a higher cell density over the same period,
with the Mann-Whitney statistical test providing a p-value
lower than 0.05, which demonstrates significant differences
between the two expansion conditions. Despite the results,
the use of one platform over the other depends on the
biomedical applications that is intended for the expanded
hiPSCs. If clinical use as cell therapies is to be considered,
then the use of TeSR™2 over mTeSR™1 is preferable as the
former avoids the use of xenogeneic components.
Characterization assays demonstrated the maintenance of
a pluripotent phenotype for the hiPSC while being expanded
on DM-SII and TeSR™2. Both nuclear (OCT4) and surface
(SSEA-4) markers were expressed by cells attached to the
beads (Figure 4D). After the harvesting procedure and
replating, cells formed undifferentiated colonies that positively
stained for OCT4, SOX2 and SSEA-4 pluripotency markers
(Figure 4F). Flow cytometry analysis confirmed the
maintenance of a pluripotent phenotype, as the results
showed more than 92% of expanded cells expressing SSEA-
4 (92±2%) and TRA-1-60 (95±2%) extracellular markers.
Additionally, the expression of intracellular markers was also
measured, with 89±4% of cells expressing SOX2 marker
whereas 75±10% of cells expressed OCT4 marker (Figure
4G).
At the beginning and end of the culture, mRNA was
isolated for further RT-PCR analysis. In Figure 4I, it is possible
to observe the downregulation of differentiation genes, such
as PAX6 and T, at the end of the culture. The maintenance of
a pluripotency core network was also confirmed by the
upregulation of OCT4 and NANOG genes. Nevertheless,
SOX17 gene was found to be slightly upregulated in expanded
hiPSCs, which is consistent with hPSCs-derived endodermal
progeny. In the literature, the differentiation towards definitive
endoderm is coupled with the decrease in NANOG expression
[33-35]. Therefore, the results hereby presented are not
consistent with a differentiation state of expanded hiPSCs.
The pluripotent phenotype of the expanded hiPSC was
also assessed through spontaneous differentiation in EB’s.
After the formation of EB’s, these were replated onto laminin-
coated plates for further immunocytochemistry analysis. In
Figure 4H, it is possible to observe the expression of SOX17,
TUJ1 and α-SMA, which confirmed the ability of harvested
hiPSCs to differentiate into cells originated from the three
germ layers. Neural progenitors were also obtained through
direct differentiation. The Dual-SMAD inhibition protocol led to
the formation of polarized cells, which expressed SOX2 and
apical ZO-1 markers (Figure 4K). As it was previously
mentioned, these structures correspond to neuro-epithelial
rosettes, which recapitulates the neural tube formation in vitro
[15, 36]. Likewise, expanded cells were also directly
differentiated towards cardiomyocytes (Figure 4J). Expanded
hiPSCs were replated onto Matrigel-coated plates for further
cardiac differentiation. At day 12, cells started to
spontaneously contract, which is the first indicator of a
successful differentiation protocol. Other studies using to the
same protocol reported the first beating cells between day 8
or 10, which may be due to the different cell lines used [16].
On day 15, cells were fixed for further immunocytochemistry
analysis, which positively stained for the cardiac troponin T
marker (Figure 4J). Overall, the characterization assays could
prove the maintenance of the phenotype of the expanded
hiPSCs, under xeno-free conditions.
The preliminary results hereby obtained, demonstrated
that hiPSCs can be expanded on DM-SII under xeno-free
conditions. Moreover, expanded cells would maintain their
phenotype throughout the culture, being able to differentiate
into progeny of the three germ layers.
According to the literature, there are two xeno-free
methods reported for the dynamic expansion of hiPSCs. Fan
et al. used polystyrene microcarriers coated with cation poly-
L-lysine and vitronectin, in combination with TeSR™2 media.
The authors attained a 38.7±6.6%-yield in terms of cell
adhesion (n=3), which was even lower when culturing hiPSCs
clumps at the same cell-to-bead ratio. When comparing these
results to the use of dissolvable microcarriers, the authors
could achieve higher cell densities (2x106cells/ml).
Nevertheless, this was the outcome of five microcarrier
passages, where the authors removed the clusters from the
spinner and added new microcarriers under static conditions
in the presence of mTeSR™1 culture medium [37]. On the
other hand, a different hiPSC line was used, which may also
influence the outcome of such experiments.
Another reported method consisted on the use of
vitronectin-coated polystyrene microcarriers (PSM-VTN) in
Figure 4 – Dynamic Expansion of TCLab hiPS cell line using Synthemax-II dissolvable microcarriers with TeSR™2. (A) Total number of cells over 7 days of expansion. This graphical representation is the mean average of n=4 experiments, one of which was performed by Sara Vieira. The error bars stand for the standard error of mean (SEM) (B) Graphic representation of the adhesion yield and fold increase attained on the first day and throughout the culture, respectively. This is the outcome of the mean of n=4 experiments, with the error bar standing for the Standard Error of Mean (SEM). (C) Bright-field microscopy images of the cells attached to the beads on day 1 and on day 7, respectively. (D) Maximum confocal intensity projection of the immunocytochemistry results for intracellular OCT4 and extracellular SSEA-4 pluripotency markers. (E) representative images from cell-viability assays (Calcein, live cells in green; Ethidium homodimer, dead cells in red). Scale bar: 100 µm. (F) Confocal microscopy images of the immunocytochemistry for SOX2, OCT4 intracellular markers and SSEA-4 extracellular marker.The nuclei were counterstained with DAPI. Scale bar: 94 µm (G) Flow cytometry analysis of the hiPSCs harvested after 7 days of expansion in the spinner flask. Cells were stained for Oct4 and SOX2 intracellular marker and TRA1-60 and SSEA-4 extracellular marker. The error represents the SEM of n=4 experiments. (C) Quantitative RT-PCR analysis of the pluripotency and differentiation genes of hiPSCs after seven days of culture. mRNA was isolated at the beginning and end of the culture. The error bars represent the SEM of n=4 experiments for the pluripotency genes, where the differentiation genes were the outcome of n=3 experiments. (D) Immunostaining showing the formation of cells expressing TUJ1 (ectoderm), SOX17 (endoderm) and α-SMA (mesoderm) after the EB formation and spontaneous differentiation assay with hiPSC cultured in spinner-flask. The nuclei were counterstained with DAPI. Scale bar: 100µm (E) Confocal microscopy images for the immunostaining of cells expressing SOX2 and apical ZO-1 neural progenitor markers. The nuclei were counterstained with DAPI. Scale bar: 33 µm. (F) Immunostaining of cells expressing cTnT cardiac marker of hiPSCs differentiated into cardiac marker, after seven days of dynamic expansion. The nuclei were counterstained with DAPI. Scale bar: 50 µm.
combination with E8 culture medium. Badenes et al. optimized
the use of this type of microcarriers through a three-level
factorial design. The highest cell density reported by the
authors was of 1.4x106 cells/mL after ten days of culture [10].
Nevertheless, both DM-SII and PSM-VTN platforms achieved
almost 20x106 cells in the spinner flask, after 4 days of culture.
The differences between the results obtained and these
two platforms, must be due to the different cell lines used, as
well as the lack of an optimized protocol that better exploits
the use of dissolvable microcarriers. Most importantly, the
same three-level factorial design carried by Badenes et al.,
should be performed for the system here presented, using
DM-SII and TeSR™2, to obtain the optimal expansion
conditions, namely in term of agitation and seeding cell
densities.
The previous studies show a feasible protocol for the xeno-
free expansion of hiPSCs. However, the authors did not study
the process of cell harvesting from the microcarriers. This
crucial aspect of stem cell bioprocessing will be discussed
further on.
Growth kinetics and cell harvesting.
The results hereby obtained, demonstrate the efficiency of
using DM-SII to expand hiPSCs under xeno-free conditions.
Growth kinetic parameters were also analyzed, such as the
specific growth rate (µ, day-1) and the doubling time (t2, day),
which entailed five days of exponential growth phase. The
results can be observed in table 1.
Table 1 - Growth kinetic parameters analyzed for the expansion of two hiPS cell
line, resorting to DM-SII. The results are the mean average of n=4 experiments,
with the error representing the SEM.
The Mann-Whitney statistical test showed no significant
statistical differences between the parameters of the different
studied conditions, as the p-values were higher than 0.05
(data not shown). Therefore, the use of DM-SII proved to be a
suitable microcarrier type for efficient incorporation under
xeno-free conditions.
After being cultured in the spinner-flask, the hiPSCs were
recovered using a harvesting procedure adapted from Nienow
et al. [13]. In this study, the authors used a short period of
intense agitation, coupled with an enzyme to detach the
expanded cells from the microcarriers. In the case of DM-SII,
the harvesting solution was a mixture of EDTA and Pectinase
to promote the respective destabilization and further
dissolution of the PAG-matrix. Accutase was used as a
detaching agent to promote cell dissociation. In the case of
polystyrene microcarriers (PSM), the harvesting protocol was
similar with the difference that only accutase was used.
Moreover, a filtration mesh was used to physically separate
the cells from the beads. To quantify the recovery yield, cells
were counted before and after the harvesting protocol. The
results can be observed in figure 5.
Figure 5 - Graphical representation of the Harvesting yield for hiPSCs cultured on
dissolvable and polystyrene microcarriers. The protocol developed by Nienow et
al. was adapted for the downstream processing of expanded cells. The results for
the harvesting yield resorting to polystyrene microcarriers are the outcome of a
single experiment, whereas for DM is the mean of n=7 experiements. The error
bar represents the standard error of mean.
Suspension cultures proved to yield relevant numbers for
disease modelling, drug screening and even cell therapies.
Consequently, several groups have been developing PSM-
based platforms for the xeno-free expansion of hiPSCs [84,
85, 97]. However, current protocols often give little focus to the
downstream processing. Nienow et al. highlighted the equal
importance of cell proliferation, as well as a harvesting
procedure that can be effectively scaled-up. The authors
defined harvesting as a two-step process. The first step
consists on the cell-bead detachment, whereas the second is
the separation technique (centrifugation/filtration) that leaves
cells in suspension without the presence of microcarriers.
Enzymatic dissociation has been reported to efficiently detach
cells from the beads [92]. Regarding the use of polystyrene
microcarriers, the prior cell dissociation envisages the use of
a filtration unit proceeding the bioreactor unit. From the results
obtained, this unit operation yielded 36% of the cell content
inside the spinner-flask (Figure 5). Regarding the use of DM-
SII, the harvesting procedure was integrated into the spinner-
flaks, which ensure the scalability of the downstream
processing. Moreover, the harvesting yield for all cell lines was
above 90%, being considerably higher than what was attained
by the alternative method. It should be noticed the need for
further experiments, as the filtration yield was the result of an
isolated experiment.
In the literature, Fan et al. specified that the use of
biodegradable matrices would be advantageous as a
microcarrier scaffold, since it would reduce steps of
downstream separations of cells from the beads, and thus
decreasing the overall cost [85]. Regarding the expansion of
hMSCs, other studies resorted to thermo-responsive polymers
that need further validation on hPSCs model. Nevertheless,
the use of DM-SII proved to be advantageous over
polystyrene microcarriers, not only because similar cell
densities were achieved under defined conditions (data not
shown), but also, because the majority of cells were recovered
without losing their core properties.
Conclusion
In the field of regenerative medicine, hiPSCs lend
themselves as extremely valuable assets. Not only they are
suitable for human disease modelling and drug screening, but
also, they could potentially serve as cell-based therapies.
However, the latter application of hiPSCs is hampered by the
lack of a reproducible method for scalable production of
Cell line TCLAB GIBCO
Culture medium mTeSR™1 TeSR™2 mTeSR™1
Specific growth
rate (day-1) [3.3±0.4]x10-1 [2.4±0.5]x10-1 [2.7±0.3]x10-1
Doubling time (day) [2.16±0.2]x100 [3.39±0.6]x100 [2.66±0.3]x100
Productivity
(cells.ml*1.day-1) [9.5±1]x104 [7.9±1]x104 [6±0.9]x104
clinically-relevant cell numbers, under xeno-free conditions.
Several approaches have been developed over the years. The
use of polystyrene microcarriers proves to be an efficient
platform. However, little focus has been given to the
downstream processing, which often leads to cell losses and
reduced viability.
Regarding the use of dissolvable microcarriers, the static
screening demonstrated that different combinations of
coatings and culture media, would prompt a good cell
adhesion yield and fold increase as efficiently as previously
established platforms. Additionally, expanded cells could
maintain their pluripotency and differentiation potential, as it
was confirmed by immunocytochemistry assays.
The dynamic expansion with DM-SII and mTeSR™1 yield
56±5% in terms of cell adhesion. Moreover, as cells grew
attached to the available surface are, high cell densities were
achieved, with (2.86±0.4)x107 cells being present after 5 days
of culture. The results were found to be reproducible with
another hiPSC line. Regarding the metabolic pathway
followed by expanding hiPSCs, further analysis is needed,
namely the direct measurement of glutamine and ammonium
for both cell lines. Nevertheless, envisaging the clinical
applications of expanded hiPSCs, mTeSR™1 was replaced
by the xeno-free alternative, the TeSR™2 culture media. The
results demonstrated the effective translation of DM-SII into a
xeno-free culture system. When compared to the previous
conditions, cell expansion yielded a lower cell density, which
may be explained by the lack of an optimized expansion
protocol.
In terms of the downstream processing, the cells
harvested from polystyrene microcarriers were subjected to a
filtration step. This unit operation achieved a 36%-harvesting
yield, which is considerable lower than the 95±2% of cells
recovered from DM-SII. Moreover, the latter harvesting
protocol was integrated into the spinner-flask, which
suppresses downstream processing steps, such as filtration,
and thus reducing the overall costs. From the characterization
of the expanded hiPSCs, pluripotency maintenance was
confirmed by immunocytochemistry, flow cytometry and qRT-
PCR. The differentiation capabilities were also demonstrated
by the spontaneous differentiation into cells derived from the
three germ layers – ectoderm, mesoderm and endoderm.
Additionally, for both dynamic conditions using mTeSR™1
and TeSR™2, the replated cells were directly differentiated
towards neural progenitors and cardiomyocytes.
Overall, the use of DM-SII under defined and xeno-free
conditions achieved rather similar results to the platform
resorting to polystyrene microcarriers. Nevertheless, the use
of DM-SII prove to be advantageous over the established
platform, as it presented an efficient and integrated bioprocess
for the harvesting of expanded hiPSCs. Other advantages
comprise, the relatively easy manipulation of such
microcarriers, as they only need a hydration step prior to
utilization, and its transparency, which facilitates the
observation of attached cells.
Future work
The preliminary results hereby presented, demonstrated
the feasible expansion of hiPSCs resorting to SII-coated
dissolvable microcarriers. When compared to polystyrene
microcarriers, the harvesting procedure yield higher cell
numbers in a cost-effective manner, as it eliminated the
filtration step.
Nevertheless, to establish a reproducible and xeno-free
expansion, this protocol would benefit from further
improvements. For instances, Badenes et al. performed a
three-level factorial design [84], which should also be
performed for the use of DM-SII beads. With this analysis, the
expansion protocol would be optimized in terms of initial
seeding densities and the agitation throughout the culture.
Regarding the metabolic profile of the expansion method,
further analysis is in need to confirm which pathway is carried
by the growing cell population. Within this context, the
concentrations of glutamine and ammonium should also be
directly measured, as these are nutrients and waste products
of the hPSCs cell metabolism, respectively. Moreover, this
analysis should be performed for all the dynamic conditions
tested in this work, namely the culture of GIBCO and TCLab
using mTeSR™1 and TeSR™2, respectively.
The harvesting protocol would also benefit from an
optimized agitation that promoted an efficient detachment of
expanded cells from the DM-SII beads. In parallel, a protease-
free method should be developed, as it would decrease the
overall cost for the downstream processing. Regarding the
harvesting procedure for the polystyrene microcarriers, the
results obtained should be further validated, as these were the
outcome of a single experiment. It would also be important to
complement the characterization panel of expanded cells with
further analysis, namely the alkaline phosphatase,
karyotyping and the formation of teratomas in
immunocompromised mice.
Another important aspect is the scalability of the expansion
platform. Kropp et al demonstrated the use of single-use
instrumented stirred-tank bioreactors for the expansion of
hPSCs as cell aggregates [90]. In this study, the perfusion
feeding strategy achieved higher cell densities [90]. Within this
context, the use of DM-SII could be incorporated in the same
type of bioreactors, coupled with a comparison between
repeated batch and perfusion feeding strategies. Analogously,
it should be envisaged the incorporation of a differentiation
stage proceeding the expansion of hiPSCs. This would reduce
the risk of contamination and labor-intensive tasks, as media
exchanges can be fully automated in bioreactors. Ultimately,
the integrated bioprocess resorting to DM-SII – expansion,
differentiation and harvesting – should be automatically
performed in one closed system and, most importantly, in
compliance with GMP-guidelines.
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