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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