Bioprocessing of cryopreservation for large-scale banking of human pluripotent stem cells.
ABSTRACT Human pluripotent stem cell (hPSC)-derived cell therapy requires production of therapeutic cells in large quantity, which starts from thawing the cryopreserved cells from a working cell bank or a master cell bank. An optimal cryopreservation and thaw process determines the efficiency of hPSC expansion and plays a significant role in the subsequent lineage-specific differentiation. However, cryopreservation in hPSC bioprocessing has been a challenge due to the unique growth requirements of hPSC, the sensitivity to cryoinjury, and the unscalable cryopreservation procedures commonly used in the laboratory. Tremendous progress has been made to identify the regulatory pathways regulating hPSC responses during cryopreservation and the development of small molecule interventions that effectively improves the efficiency of cryopreservation. The adaption of these methods in current good manufacturing practices (cGMP)-compliant cryopreservation processes not only improves cell survival, but also their therapeutic potency. This review summarizes the advances in these areas and discusses the technical requirements in the development of cGMP-compliant hPSC cryopreservation process.
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ABSTRACT: Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However, the current conventional method leads to poor recovery of hPSCs after thawing. Here, we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single-cell dissociation. After confirming hPSC survivability after freeze thawing, we found that hPSCs that were freeze thawed as colonies showed markedly decreased survival, whereas freeze-thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze-thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post-thawing. The improved method was also adapted to a xeno-free culture system. Moreover, the cell recovery post-cryopreservation was highly supported by coating culture surfaces with human laminin-521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high-quality hPSCs. © 2013 Wiley Periodicals, Inc.genesis 11/2013; · 2.58 Impact Factor
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ABSTRACT: Human pluripotent stem cells (hPSCs) hold great promise for revolutionizing regenerative medicine for their potential applications in disease modeling, drug discovery, and cellular therapy. Many their applications require robust and scalable expansion of hPSCs, even under settings compliant to good clinical practices. Rapid evolution of media and substrates provided safer and more defined culture conditions for long-term expansion of undifferentiated hPSCs in either adhesion or suspension. With well-designed automatic systems or fully controlled bioreactors, production of a clinically relevant quantity of hPSCs could be achieved in the near future. The goal is to find a scalable, xeno-free, chemically defined, and economic culture system for clinical-grade expansion of hPSCs that complies the requirements of current good manufacturing practices. This review provides an updated overview of the current development and challenges on the way to accomplish this goal, including discussions on basic principles for bioprocess design, serum-free media, extracellular matric or synthesized substrate, microcarrier- or cell aggregate-based suspension culture, and scalability and practicality of equipment.Annals of Biomedical Engineering 10/2013; · 3.23 Impact Factor
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ABSTRACT: Long term cryopreservation of tissue engineering constructs is of paramount importance to meet off-the shelf requirements for medical applications. In the present study, the effect of cryopreservation using natural osmolytes such as trehalose and ectoin with and without conventional Me2SO on the cryopreservation of tissue engineered constructs (TECs) was evaluated. MSCs derived from umbilical cord were seeded on electrospun nanofiberous silk fibroin scaffolds and cultured to develop TECs. TECs were subjected to controlled rate freezing using nine different freezing solutions. Among these, freezing medium consisting of natural osmolytes like trehalose (40mM), ectoin (40mM), catalase (100μg) as antioxidant and Me2SO (2.5%) was found to be the most effective. Optimality of the chosen cryoprotectants was confirmed by cell viability (PI live/dead staining), cell proliferation (MTT assay), microstructure analysis (SEM), membrane integrity (confocal microscopy) and in-vitro osteogenic differentiation (ALP assay, RT-PCR and histology) study carried out with post-thaw cryopreserved TECs. The mechanical integrity of the cryopreserved scaffold was found to be unaltered. The performance of the freezing medium towards cryopreservation of TEC was superior than the performance achieved using conventional Me2SO and similar to the non cryopreserved TEC. Overall we have formulated an efficient freezing medium that may pave the way of long term preservation of TECs with maintaining its integrity, MSCs viability and differentiation potentiality. It was observed that the performance of freezing medium for cryopreservation of TECs was better than the Me2SO.Cryobiology 04/2014; · 2.14 Impact Factor
Bioprocessing of Cryopreservation
for Large-Scale Banking of Human Pluripotent Stem Cells
Yan Li and Teng Ma
Human pluripotent stem cell (hPSC)-derived cell therapy requires production of therapeutic cells in large quan-
tity, which starts from thawing the cryopreserved cells from a working cell bank or a master cell bank. An optimal
cryopreservation and thaw process determines the efficiency of hPSC expansion and plays a significant role in the
subsequent lineage-specific differentiation. However, cryopreservation in hPSC bioprocessing has been a chal-
lenge due to the unique growth requirements of hPSC, the sensitivity to cryoinjury, and the unscalable cryopres-
ervation procedures commonly used in the laboratory. Tremendous progress has been made to identify the
regulatory pathways regulating hPSC responses during cryopreservation and the development of small molecule
interventions that effectively improves the efficiency of cryopreservation. The adaption of these methods in cur-
rent good manufacturing practices (cGMP)-compliant cryopreservation processes not only improves cell survival,
but also their therapeutic potency. This review summarizes the advances in these areas and discussesthe technical
requirements in the development of cGMP-compliant hPSC cryopreservation process.
Key words: cell banking; cryopreservation; human pluripotent stem cell
duced pluripotent stem cells (hiPSCs), can be expanded indef-
initely and differentiated into any cell type in three-germ
layers.1–3These unique characteristics make hPSCs attractive
as the cell source for tissue engineering, regenerative medi-
cine, and drug screening. Recently, hESC-derived oligoden-
drocyte progenitor cells (OPC) and hESC-derived retinal
pigment epithelial cells have been investigated in Phase I/II
clinical trials.4–6More clinical trials are expected for treatment
of other diseases, such as amyotrophic lateral sclerosis and
diabetes.7A critical requirement for hPSC’s clinical applica-
tions is the supply of a large quantity of hPSC-derived thera-
peutic cells. Production of these therapeutic cells starts from
thawing cells from qualified cell banks, such as the master
cell bank (MCB) or the working cell bank (WCB), under cur-
rent good manufacturing practices (cGMP). The quality of
the cell bank plays an important role in hPSC bioprocessing
as it impacts post-thaw hPSC expansion, the efficiency of
the subsequent lineage-specific differentiation, and process
reproducibility. A robust cryopreservation and thaw process
in generating MCB and WCB thus becomes the crucial first
steps that ensure the quality of hPSC-based cell production.
uman pluripotent stem cells (hPSCs), including
human embryonic stem cells (hESCs) and human in-
Cryopreservation has been a routine practice for hemato-
poietic stem cells (HSCs) for bone marrow transplantation
and cord blood banking since 1990s.8Other adult stem cell
banks, especially mesenchymal stem cells (MSCs), are also
being established for clinical application.9Besides the in-
creased flexibility and cell availability in laboratory practices,
cryopreservation has been an important bioprocessing step to
create large-scale cell banks that provide uniform cell popula-
tions for clinical study. Different from adult stem cell bank-
ing, the unlimited proliferation capacity of hPSCs enables
the banking scale in the order of greater than 109cells.10How-
ever, maintaining hPSC survival after cryopreservation and
thaw has been a significant challenge, and thus the main
focus of research efforts. The traditional cryopreservation
and thaw methods led to the diminishing Oct-4 expression
and poor hPSC survival.11Due to the high sensitivity to cryo-
injury, various groups have been working on optimizing
the protocols to efficiently cryopreserve hPSCs with different
formulations and procedures.12Although cell viability post-
thaw has been significantly improved, the feasibility of
these methods for large-scale banking has not been ade-
quately addressed. Some methods based on vitrification
may only be suitable for laboratory practices, while the scal-
ability of other methods, such as slow cooling, needs to be
further improved for large-scale cell banking.
Department of Chemical and Biomedical Engineering, Florida State University, Tallahassee, Florida.
BioResearch Open Access
Volume 1, Number 5, October 2012
ª Mary Ann Liebert, Inc.
This review started from cryopreservation principles and
adult stem cell banking that are currently used in clinical prac-
tice. The recent progress of hPSC cryopreservation bioprocess
was then summarized with an emphasis on slow cooling
methods due to their potential in large-scale application. The
regulatory mechanisms of apoptosis, reaction oxygen species
(ROS), and cell–cell and cell–matrix interaction on hPSC via-
bility and recovery were then discussed to highlight the role
of small molecule intervention in improving cryopreservation
efficiency. Finally, the current status and challenges of large-
scale hPSC banking under cGMP are discussed, and the im-
portance of integrated cell expansion and cryopreservation
process in cGMP cell banking is emphasized.
During cryopreservation and thaw, the main cause of cell
death usually is not the long-term storage at low temperature,
but rather the process that the cells travel across ?15?C to
?60?C, which happens once during cooling and once during
warming.13,14During slow cooling, ice formed in the extracel-
lular environment causes an increase of solution osmolality.
To compensate the osmolality imbalance, the intracellular
water passes through cell membrane to the extracellular solu-
tion, causing cellular dehydration, which may damage cell
membrane and organelles. During rapid cooling, however,
cellular damages can be resulted from intracellular ice forma-
tion due to insufficient water outflow and super cooling. As
both events are detrimental to the cells, the cell cryopreserva-
tion requires an optimal cooling rate and the presence of cryo-
protectant (CPA), which reduces cell shrinkage during
dehydration and inhibits intracellular ice formation until
the intracellular water achieves a ‘‘glassy state.’’15The typical
CPAs are low molecular weight organic compounds that ef-
fectively penetrate into the cells and prevent the intracellular
ice formation, which include glycerol, dimethyl sulfoxide
(DMSO), ethylene glycol (EG), and propylene glycol (PG).16
During warming, a rapid warming rate is preferred to pre-
vent ice recrystallization and the effective removal of CPA
is required to minimize the cytotoxicity.
Although the cooling mechanism is universal for all types
of cells, cell type-dependent responses have long been ob-
served due to variations in several attributes: (1) cell surface
to volume ratio or cell size; (2) cell membrane permeability
to water and CPA and Arrhenius activation energy that
affects the temperature dependence of cell membrane perme-
ability; (3) osmotic limit of cells.13In general, a slower cooling
rate is necessary as cell size increases to avoid intracellular
freezing, because the fractional water loss during cooling is
proportional to the cell surface to volume ratio. For ESCs
(10–15lm), the optimal cooling rate is usually less than
3?C/min. In addition, the osmotically inactive cell volume
(Vb), which is occupied by inert materials with no osmotic
pressure, such as fats and proteins, is cell type-dependent.
ESCs have high nucleus-to-cytoplasm ratio and Vb was
found to be 49.8% of isosmotic cell volume, compared to
32% for cord blood CD34+cells and 20.5% for bone marrow
HSC.17–19The considerable variation in Vbis another factor
that influences the cooling rate. The cell membrane perme-
ability, including hydraulic conductivity and CPA permeabil-
ity coefficient, is a function of temperature and can be used to
predict the cell water change during cryopreservation in two-
parameter mass transport model.20However, the main limi-
tation of this type of theoretical analysis is the significant var-
iation in permeability and osmotic limit among different cell
types, donors, and CPA formulations. For the emerging cell
populations, such as hPSC, there is still lack ofquantitative in-
formation and analysis for cryobiological parameters and the
cryopreservation is mainly based on the empirical data and
Clinical application of adult stem cells over the last decades
has accumulated valuable knowledge for optimizing the
cryopreservation process and provides valuable experience
and prospects for future hPSC clinical application. In the
following sections, the current status of adult stem cell cryo-
preservation will be briefly summarized followed by the dis-
cussion of the advancements and challenges in large-scale
Cryopreservation of Adult Stem Cells
Cryopreservation has played an important role for HSC
collection and clinical transplantation. For example, more
than 250,000 human leukocyte antigen-typed cord blood
units are stored in public banks worldwide.21With the opti-
mal cooling rate and cryopreservation formulation, cryopres-
ervation of HSCs has become a routine practice in clinics.21
Recently, bone marrow-derived MSCs have emerged as an
important cell source for cell therapy and tissue regenera-
tion.22The standard MSC cyopresevation solution is 10%
DMSO plus 90% fetal bovine serum (FBS) with the recovery
efficiency around 80–90% after cryopreservation.23With
good cell recovery, the focus of adult stem cell cryopreserva-
tion has been on clinical safety. The two major research areas
are (1) elimination of animal serum in cell processing and
storage; (2) reducing the toxic DMSO level or developing its
nontoxic replacement. Serum residue after washing can trig-
ger adverse reactions in patients and DMSO residue has side
effects, including headache, nausea, and vomiting.9To reduce
animal serum, a defined serum-free freezing solution has
been developed, where FBS was replaced by serum albu-
min.23Recently, an animal product-free formulation CryoStor
has been used for adult stem cell cryopreservation with good
recovery.24,25In an effort to reduce DMSO, high molecular
weight polymers, such as polyvinylpyrrolidone (PVP) and
hydroxyethyl starch (HES) have been included in CPAs.26,27
Recently, ectoine, a natural osmoprotectant from micro-
organisms, has been tested as an alternative CPA to replace
DMSO in a serum-free cryomedium.28The clinical scale cryo-
preservation of adult stem cells usually requires large cryo-
containers (i.e., scale up) rather than hundreds of cryo units
(i.e., scale out).29The cryo unit for cord blood was around
15–30mL and 150mL for bone marrow, so cryobags in the
range of 50–250mL are sufficient for individual patients
and typically used in the banks.10,30,31Compared to unassoci-
ated adult stem cells, a unique aspect of hPSC is the intimate
cell–cell and cell–matrixinteractions, which possess new chal-
lenges in preserving hPSC viability and therapeutic potency
Cryopreservation of hPSCs
hPSCs usually grow as colonies of highly associated
adherent cells, which require tight gap junctions and adhe-
sion to extracellular matrix (ECM)-coated surface. hPSCs
206LI AND MA
are sensitive to traditional cryopreservation and thaw method
in large part due to the disruption of this cellular organization.
In addition to necrosis caused by ice crystallization or osmotic
shock, several other cryopreservation-induced events that af-
fect the hPSC survival include activated apoptosis, disruption
of cell–cell and cell–matrix adhesions, and the elevated ROS
Induction of apoptosis and anoikis
Apoptosis was found to be the major cause of cell loss after
hESC cryopreservation rather than necrosis.32Apoptosis may
not happen immediately post-thaw, but 12–24h later. The
main inducers of apoptosis during hESC cryopreservation in-
clude caspase activation and cytokine interaction. Analysis
revealed that progression of apoptosis coincides with upregu-
lation of caspase-8 and -10 genes, which activate caspase-3
and lead to apoptosis.33Caspase-8 and -10, components of mi-
togen-activated protein kinase family, are activated through
the transforming growth factor (TGF)-b and interleukin (IL)-
1b pathways, in which TGF-b and its receptor ACVR1C
along with IL-1b and its receptor IL-1R are upregulated fol-
lowing cryopreservation.33Because of the roles of TGF-b and
IL-1b in initiating the apoptotic events,34,35Rho-associated ki-
nase (ROCK) inhibitor Y-27632, which suppresses the caspase
activation through IL-1b/IL-1R and TGF-b/ACVR1C interac-
tions, has been used to reduce the apoptosis.33,36Other apo-
ptosis inhibitors, including caspase inhibitor Z-VAD-VMK
and p53 inhibitor pifithrin-l have also been shown to im-
prove hPSC cryopreservation.37,38
Cell detachment and dissociation during hPSC cryopreser-
vation also contribute to cell death through a process known
as ‘‘anoikis,’’ meaning ‘‘homelessness,’’ which is a subtype of
apoptosis induced by the loss of cell adhesion or inappropri-
ate cell adhesion.39In the cascade of cellular events leading to
anoikis, Bcl-2 family, caspase-3, and Fas signaling play the
key roles.40For example, loss of anchorage to ECM leads to
an increase in Fas expression, which then activates caspase
activity.40While, in general, the adherence on ECM proteins
prevents hPSC to enter anoikis, addition of a growth factor,
such as the basic fibroblast growth factor (bFGF), has been
found to prevent anoikis by activation of extracellular
signal-regulated kinase and inhibition of Bcl-2-interacting me-
Anoikis can be avoided by preserving ECM attachment and in-
tact cell–cell interactions during cryopreservation. Anoikis in-
duced by detachment and dissociation of hPSC can also be
protected by ROCK inhibitor, which disrupts extracellular
cues that would normally induce apoptosis and increases the
cell–cell interactions through modulating cadherins and gap
junctions to enable the cell reaggregation.42
Disruption of cell–cell adhesion and cell–matrix adhesion
and F-actin through the G13 signaling pathway and functional
cell-to-cell gap junctions, such as connexin 43 and connexin 45
caused hESC-death after freeze–thawing.43–45E-cadherin was
found to be essential for hESC survival because E-cadherin-
mediated cell–cell adhesion regulates Rho activity and en-
hances cell attachment.46As such, small molecules that target
Rho-ROCK signaling and stabilize E-cadherin, such as Thi-
azovivin and Tyrintegin have been used to promote cell sur-
vival by enhancing cell–ECM adhesion, activating integrin
signaling, and synergizing with growth factors, such as
bFGF and insulin-like growth factor.46hPSCs treated by
ROCK inhibitor Y27632 clustered rapidly and maintained
E-cadherin and F-actin distribution, improving the recov-
ery.45Similarly, preserving endogenous ECM during cryo-
preservation has also been shown to improve cell survival
by maintaining cell–ECM and cell–cell adhesion, supporting
the strategy of in situ hPSC cryopreservation.47
Production of ROS
ROS is typically resulted from the transfer of one, two, or
three electrons to O2to form, respectively, a superoxide rad-
ical (O2?), hydrogen peroxide (H2O2), or a hydroxyl radical
(HO?) in mitochondria.48Cell cryopreservation is associated
with elevated intracellular ROS as a result of mitochondrial
leakage and damage.49The high levels of O2?and H2O2
after cryopreservation lead to the release of cytochrome C
from mitochondria to cytosol that activates caspase activity.
Production of HO?can also be induced via Fenton reaction
(Fe/H2O2) as a consequence of elevated iron release from
ferritin by NADPH or from iron–sulfur clusters by O2?
under cold temperature.50,51Since the scavengers for the
free radicals and oxidative stress became less active at low
temperature, the elevated ROS is more potent for inducing
apoptosis. The approach to reduce the oxidative stress and
prevent ROS production is to add antioxidants and radical
E, or its analog Trolox, and polyethylene glycol (PEG).38,52,53
pounds have been shown to prevent injury from free radicals
and improve the storage of islet cells, MDCK cells, and hepa-
tocytes.53–55Adding glutathione to the CPA and the post-
thaw solution also improved the survival of mouse ESC.56
Unknown events that impact the
To date, the main efforts in hPSC cryopreservation have fo-
cused on improving hPSC survival and the maintenance of
pluripotency markers, but the impact of different freeze–
thawing methods on hPSC lineage-specific differentiation
has been inadequately investigated.57In the development of
hESC-derived OPC and cardiomyocyte production process
at Geron, the author (Y.L.) has observed a large variation of
differentiation potential from different hESC banks, although
they all demonstrated comparable survival and pluripotency
(unpublished results). One hypothesis is that cryopreserva-
tion and thaw generate selective pressure that influences
the lineage-specific propensity during hPSC differentiation.
Although the prediction and detection of cryopreservation-
induced differentiation preference have been challenging,
elucidating the mechanism involved in the lineage-specific
differentiation post-cryopreservation beyond the cell survival
is crucial in refining hPSC cryopreservation methods.
Methods of hPSC Cryopreservation
With the goal of preserving cell viability and multilineage
potential, strategies that reduce ROS production and pre-
serve cell–cell and cell–matrix contacts during cryopreserva-
tion have been actively pursued together with the traditional
HUMAN PLURIPOTENT STEM CELL CRYOPRESERVATION 207
cryobiological parameters. In general, there are two types of
cryopreservation method: vitrification and slow cooling.58
Vitrification has been extensively applied in the cryopres-
ervation of embryos for animal reproduction, including
sheep, mouse, and bovine.59–61Some early studies have sug-
gested that vitrification could also be beneficial for the cryo-
preservation of human blastocysts,62the stage at which
hESCs were derived. In the subsequent studies, vitrification
has been shown to be effective for hESC cryopreservation
with significantly improved recovery compared to the slow
cooling method (100% vs. 16% colony recovery).63,64The ini-
tial vitrification method was performed in open pulled straw,
which drew small volume of cell clusters resuspended in vis-
cous CPA (20% DMSO, 20% EG, and sucrose). The straw
was then quickly immersed into the liquid nitrogen (N2) to
achieve the vitrification and avoid intracellular ice formation.
The thawing of vitrified hESCs was also very rapid to avoid
ice recrystallization. Table 1 summarizes the modifications
that have been made for ease of operation, fast processing,
and safe clinical applications.
Improvements to avoid contamination.
rification, where the straw was closed by heat-sealing after
the cells were taken, has been developed to avoid direct con-
tact with liquid N2as occurred in the open pull straw vitrifi-
cation.65In addition, human serum albumin has been used to
replace FBS to achieve serum-free cryopreservation. Toward
xeno-free formulation, a DMSO-free vitrification solution
using 40% EG, 10% PEG, and Eurocollins solution (dextrose
and salts) has been tested for hPSC cryopreservation.66This
solution improved the recovery rate to about 30% compared
to 18–19% with the DMSO and serum replacement medium.
Although the cooling rate was about ?150?C/min in this
study, it was sufficient to achieve vitrification.
Improvement of process efficiency.
method has been developed using a cell strainer to hold
hESC clusters instead of straw.67In this method, each cell
strainer was able to hold about 100–150 clumps compared
to 5–10 clumps in one open pulled straw. Later, this bulk vit-
rification method was modified using the custom-made vitri-
A bulk vitrification
fication cryovials.68The stainless-steel mesh with 70-lmmesh
size was assembled to the upper half of the cryovial. hESC
clumps were transferred to the modified cryovial with the vit-
rification solution and immersed into liquid N2.
Adherent cell vitrification.
was proposed to achieve rapid cooling and long-term storage
in liquid N2by using the cell culture plates with detachable
culture wells.69Recently, a surface vitrification method has
been developed to vitrify the adherent cell colonies.70The
cells were immobilized on modified Thermanox?coverslips
with feeder cells before the vitrification, and the vitrified col-
onies showed better vital residual area compared with slow
frozen colonies (89% vs. 51%).
Vitrification of adherent cells
methods include the difficulty to scale up and the possibility
of contamination in liquid nitrogen. Vitrification is a tedious
and operator-dependent process, and requires strict timing
with a very low capacity. The bulk vitrification method has
increased the processing capacity with about 100–150 cell
clumps loaded in each cryocarrier. However, the cryocarriers
need to be individually handled, which is a significant limit in
large-scale cell banking. Additionally, direct exposure to the
liquid phase of liquid nitrogen for rapid cooling and long-
term storage raises the concern of pathogen transmission.
Closed-straw vitrification has been developed to address
this issue, but this method is operator-dependent and the
heat-sealing process is not scalable.
The inherent problems of vitrification
Despite the poor initial performance, advances in slow
cooling of hPSC have improved its scalability. Cryobiological
parameters and CPAs have been extensively studied for freez-
ing hPSC clusters and the new strategies to freeze hPSC as ei-
ther adherent or single cells have also improved the recovery
and efficiency. Currently hPSCs can be frozen in three
different organizations: cell clusters, adherent cells, and single
cells (Table 2).
Cryopreservation of hPSCs as cell clusters.
hPSCs are not amenable to be passaged and cryopreserved
as a single-cell suspension because failure to maintain
Table 1. Summary of Different Vitrification Methods for Human Pluripotent Stem Cell Cryopreservation
Method descriptionCryoprotectanthESC/hiPSC Colony recoveryReferences
Open pulled straw
Closed sealed straw,
20% DMSO, 20% EG,
sucrose, SR medium
20% DMSO, 20% EG,
20% DMSO, 20% EG,
sucrose, SR medium
20% DMSO, 20% EG,
sucrose, SR medium
40% EG, 10% PEG,
20% DMSO, 20% EG,
sucrose, SR medium
HES-2, HES-3, HES-4
All colonies recoveredReubinoff et al.;63
Li et al.64
Richards et al.65
a-ES-C 95–99% of frozen
95–99% of frozen
Li et al.67
b-HES-2, a-ES-C Li et al.68
hiPSC 253G4 Nishigaki et al.66
H1184% vital residual area
Beier et al.;70
Heng et al.69
hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; DMSO, dimethyl sulfoxide; EG, ethylene glycol; SR, serum
replacement; HES, hydroxyethyl starch; HSA, human serum albumin; PEG, polyethylene glycol.
208 LI AND MA
cell–cell contact usually leads to significant cell death and
spontaneous differentiation.12The cells are usually har-
vested by mechanical scraping and cryopreserved as cell
clusters in the slow cooling process. Since the initial at-
tempt to freeze hESC clusters by the slow cooling method,
various improvements have been made with emphasis
on two aspects: (1) cryobiological variables, including the
seeding process, the cooling rate, and step-wise loading
and unloading CPA; (2) CPA formulations.
Seeding, a process to introduce ice crystals in a super cool
solution, causes quick formation of extracellular ice that min-
imizes the detrimental effect of phase change from liquid to
ice in the course of cooling and preserves adhesive ECM.
Seeding should be performed at the temperature above spon-
taneous intracellular ice formation, which is in the range
between ?7?C and ?12?C. Introducing seeding during
hESC cryopreservation has achieved about 80% survival
rate.71A similar study has been performed to optimize the
cooling rate with seeding at ?10?C and the optimal cooling
rate at 0.5?C/min.72As another cryobiological variable,
step-wise CPA addition can minimize the mechanical dam-
age due to osmotic shock.73More than 50% hESC recovery
has been observed with a two-step equilibration compared to
20% using one-step equilibration. A four-step CPA addition
Table 2. Summary of Different Slow Cooling Methods for Human
Pluripotent Stem Cell Cryopreservation
Method descriptionCryoprotectanthESC/hiPSC RecoveryReferences
Frozen as cell clumps
Cooling program and
Synthetic serum and
Adding EG as
10% DMSO, 25% FBS
10% DMSO, 90% SR,
2M DMSO, SR
5% DMSO, 10% EG,
5% DMSO, 5% HES
H1 79%, 55%Ware et al.;71Yang
Lee et al.;73Valbuena
H9, CHA-hES3, VAL-3,
30%SNUhES-3Ha et al.75
HS293, HS306About 90%a
Holm et al.78
Frozen as adherent cells
hESCs grown on
hESCs grown on clinical
cell culture cassette
hESCs grown on
hESC immobilized on
Frozen as dissociated single cells
Dissociated hESCs on
feeders or feeder-free
Dissociated hESCs on
VUB01, H1, H9, 181,
T’joen et al.77
10% DMSO, FBS,
H1 and H9About 80%
71% at day 1
Ji et al.47
10% DMSO, 90% FBSShef 4, Shef 5, Shef 6,
H1 and H9
Amps et al.79
10% DMSO, 30% FBS,
10% DMSO, 90% SR,
Nie et al.80
SCEDTM461 Serra et al.82
10% DMSO, 90% SRCA1, CA2, H1, H9 About 50%b
Li et al.83,87
10% DMSO, 90% SR
10% DMSO, 90% FBS,
10lM ROCKi; 20%
DMSO, 80% SR
7.5% DMSO, 2.5% PEG,
90% SR medium
10% EG, 90% SR
10% DMSO, 90% FBS,
HS207, HS401 About 50%a
Martin-Ibanez et al.85
hiPSCs in feeder-free
Royan H5, H6, hiPSC1,
hiPSC4; H9, BG01V,
About 85%a; Use
Xu et al.38
hiPSC derived from
Katkov et al.91
alternative to ROCKi
Shef 4, Shef 5, Shef 7, H7About 85%a
Barbaric et al.90
aViability determined by Trypan blue immediately post-thaw.
bViability determined by PI/Annexin staining.
cViability determined by 7AAD staining.
The methods using PI/Annexin or 7AAD staining are more sensitive than Trypan blue method.
FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; PI, propidium iodide.
HUMAN PLURIPOTENT STEM CELL CRYOPRESERVATION 209
has also been tested with the improved post-thaw survival
comparable to the vitrification method.74
CPA formulation has been extensively studied in hPSC
cryopreservation during the past 10 years. Similar to adult
stem cell cryopreservation, eliminating DMSO and animal
serum without compromising cell recovery become two
major tasks. To reduce DMSO level, EG was tested and the
most favorable combination was found to be 5% DMSO, 5%
EG, and 50% FBS.75Disaccharide trehalose has also been
added into the formulation and showed beneficial effect in
the elution buffer and freezing medium.76However, the ben-
eficial effect of trehalose was small in a separate study and
only observed when serum concentration exceeded 50%.47
Adding nonpenetrating polymers accelerates the vitrification
of extracellular solution by reducing the water diffusion out
of cells and preventing extracellular ice propagation.16High
molecular weight HES has recently been tested in hESC cryo-
preservation and a combination of 5% DMSO and 5% HES
was found to be optimal.77To eliminate animal serum, a com-
STEM-CELLBANKER, containing a high molecular weight
polymer (not disclosed), was used for hPSC cryopreserva-
tion.78More than 90% cells remained alive after thawing com-
pared with 50% for control condition.
Cryopreservation of hPSCs as adherent cells.
anoikis can be minimized when cell–cell gap junctions and
cell–ECM interactions are preserved, cryopresevation of ad-
herent hESC colonies has been investigated.47The hESCs
were cryopreserved on a tissue culture plate directly with a
thin layer of CPA covering the cell layer. The post-thaw sur-
vival was significantly improved compared to the cell clusters
frozen in a suspension (80% vs. 2% colony recovery). Another
in situ cryopreservation of hESCs has been performed in a gas
permeable culture cassette.79A range of 8–115-fold higher
post-thaw proliferation ratio was achieved compared to a tis-
sue culture flask. However, freezing hESCs in the tissue cul-
ture plate or cassette is not feasible for future scale up. To
address this issue, the method of freezing adherent hPSC has
been adapted on microcarriers to increase the scalability.80
The recovery of the microcarriers with cells, however, was
still performed on the tissue culture surface with mouse em-
bryonic fibroblasts. The future development of this method
should allow the direct recovery of hESC-microcarrier con-
structs in a suspension, which has been demonstrated for
bone marrow MSC.81Cells grown on microcarriers have also
been encapsulated by alginate and cryopreserved in compari-
son with encapsulated cell suspension.82Similarly, hESCs
ery compared to the suspended cells.
Cryopreservation of hPSCs as single cells.
preservation as single cells has become possible in the pres-
ence of ROCK inhibitor. Addition of ROCK inhibitor
Y27632 in the post-thaw medium has been shown to increase
hESC survival from 5% to 53%, because Y27632 enhances
cell–cell adhesion and cell aggregation by modulating gap
junctions, thereby blocking the pathway to apoptosis.83,84
The mechanism that ROCK inhibitor improved cell survival
may not only be due to blocking the apoptotic pathway,
but also the ability to avoid anoikis.42Cryopreservation of
single cells has been further optimized by adding Y27632 in
the preservation solution or Matrigel? in addition to the
post-thaw medium.85,86The effect of Y27632 on feeder-free
culture is similar to feeder culture both for hESCs and
hiPSCs.87–89However, adding Y27632 during cryopreserva-
tion did not change the survival of CHiPS-A cells, which
may be due to the effect of the solution STEM-CELLBANKER
or a cell line-dependent response.78An alternative chemical
Pinacidil has recently been found to have similar effect com-
pared to Y-27632.90Since Y-27632 has a higher cost and is as-
sociated with patent issues, Pinacidil has been suggested to
be more suitable for commercial applications.
The CPA of single hPSC has been optimized at reduced
DMSO concentration. A combination of 7.5% DMSO and
2.5% PEG has improved hESC recovery by 30% compared
to 10% DMSO alone.38Introducing PEG in the preservation
solution could inhibit ROS production and reduce apoptosis.
The presence of Y27632 and p53 inhibitor in the subsequent
culture medium further improved cell recovery. Another
study completely replaced DMSO by EG during hiPSC cryo-
preservation because DMSO was found substantially more
toxic to hPSC than EG.91In this study, similar recovery of
freezing dissociated single cells and adherent cells with EG
has been reported.
Large Scale hPSC Banking Under cGMP
For therapeutic cell production, large-scale hPSC banks
provide the starting materials for a new bank (in the case
of MCB) or the differentiated therapeutic cell products (in
the case of WCB). A general MCB or WCB should contain
at least 300–400 vials with each vial containing 5–10·106
cells. In practice, the banking process requires the capacity
to handle up to 4·109cells in a single day, in which the pro-
longed exposure to CPA must be minimized. This scale out
approach requires efficient cell processing, where the vitrifi-
cation methods appear unfeasible. The alternative approach
of hESC lyophilization for long-term storage has been pro-
posed, but still requires proof-of-concept data.10To date,
the slow cooling method for dissociated single cells appears
to be the best choice for making MCB and/or WCB. With the
development of bioreactor systems and the integration of
cryopreservation and hPSC expansion, freezing adherent
cells on microcarriers or as aggregates is another important
development that has the potential to address the limitations
for large-scale cryopreservation.92,93The further develop-
ment of microcarrier or aggregate-based cell preservation
depends on understanding the role of cell–cell and cell–
matrix interactions and the impact of spatial temperature
and cryogen gradients on cell properties.
Different from laboratory protocols, a production process
should be a well-defined procedure with controlled inputs,
outputs, and operational parameters that ensure reproduc-
ibility and regulatory compliance.94For large cGMP banking,
the process should be simple, robust, and easily performed by
different operators. However, many current cryopreservation
methods have been developed using a variety of hPSC lines
with large variations in procedure, making it difficult to eval-
uate their feasibility for MCB/WCB production. In future
development, both process efficiency and regulatory compli-
ance should be considered and implemented in the early
stage of development. At a minimum, a desired process for
cGMP hPSC banking must meet the following criteria: (1)
210 LI AND MA
Cell recovery and expansion post-thaw is fast enough to
achieve *4·109cells within five passages; (2) Xeno-free
formulation is preferred with a simple procedure capable of
processing a large number of cells in one operational day;
(3) The thawed cells maintain pluripotency; (4) The thawed
cells maintain the ability to produce high purity of the tar-
geted cell product; (5) vial-to-vial variability is minimized
for process consistency.
Successful production of MCB/WCB under cGMP requires
the integration of expansion and cryopreservation process.
On the cryopreservation day, all the expanded cells need be
harvested, formulated, and distributed into hundreds of freez-
ing containers, such as cryovials, which are frozen in the
tion process need to be streamlined and validated for cGMP
production. Well-trained personnel, the detailed documenta-
tion, such as batch production record, the qualified raw mate-
rials, and the well-monitored cGMP facility are required to
ensure the successful MCB/WCB production.95Quality assur-
ance and quality control also need to be implemented to meet
the regulatory requirement for cell bank release. Figure 1A
illustrates the overall process flow for cell banking, demon-
strating the need for process integration by various functional
groups. Integrated cell expansion and cryopreservation pro-
cess would improve efficiency and facilitate automation.82
Due to the rapid progress in hPSC expansion, MCB/WCB
are expected to be thawed and expanded in different culture
conditions, which could potentially impact cell properties
post-cryopreservation (Fig. 1B). From the feeder cell culture
to feeder-free culture, from the conditioned medium to the
defined medium with known essential components, such as
the E8 medium,96–98from the undefined ECM to the synthetic
polymerase substrate,99the research cell bank made years
back may need to be thawed into a completely different cul-
ture system to make cGMP banks.100Recent advances of
hPSC expansion on microcarriers and as aggregates also re-
quire the compatible cryopreservation process with the cul-
ture process.82,92,101During the culture process transition
from bank to bank, care needs to be taken for validating the
banking process and maintaining the bank history. To ad-
dress these issues, a bridging or compatibility study is re-
quired to use different MCB/WCB for therapeutic cell
production. In particular, the impact of expansion and
cryopreservation process on lineage-specific differentia-
tion is critical and needs to be addressed before clinical
To date, the primary focus of hPSC characterization post-
cryopreservation remains on the pluripotency rather than
the lineage-specific differentiation. Cryopreservation and
thaw procedure may lead to a selection process and affect
the differentiation capacity to the desired lineage even
panded, and cryopreserved as MCB in current good manufacturing practices facility. This operation requires the cooperation
of materials management, facility monitoring, personnel training, and documentation, quality control, and quality assurance.
(B) Example of different hPSC banks expanded in different growth conditions, including feeder-culture and feeder-free cul-
tures in a conditioned medium on ECM, a defined medium on ECM, and a defined medium on a synthetic polymer. The
cells are thawed from the research cell bank to manufacture MCB and the cells are thawed from MCB to make WCB. Thus,
the cells in WCB may have been histologically cultured in different growth conditions. hPSC, human pluripotent stem cell;
MCB, master cell bank; WCB, working cell bank; ECM, extracellular matrix.
(A) Illustration of bioprocess for hPSC MCB manufacturing. The cells are thawed from the research cell bank, ex-
HUMAN PLURIPOTENT STEM CELL CRYOPRESERVATION211
when they maintain the pluripotent markers. Thus, the capac-
ity for lineage-specific differentiation is another set of criteria
for choosing the cell banking process in clinical application.
While studies have led to the development of ROCK inhibitor
with significantly improved cell survival and maintenance of
pluripotency, mechanistic insight is required to develop scal-
able and GMP-compatible strategy that better preserves
hPSC’s capacity for lineage-specific differentiation. In addition
to optimizing traditional cryobiological and process parame-
ters, identifying the regulatory pathways associated with cryo-
preservation will play a critical role to improve process
efficiency. Although the focus of this article is the cryopreser-
vation of undifferentiated hPSC banks, cryopreservation of
hPSC-derived cells for final product storage is another impor-
tant task in hPSC therapy.102
Significant progress has been made in hPSC cryopreserva-
tion during recent years. Strategies to reduce ROS production
and apoptosis and to preserve cell–cell and cell–matrix adhe-
sions have played important roles to improve the large-scale
hPSC banking process. Experience in hPSC cryopreservation
as cell clusters, adherent cells, and single cells using the slow
cooling method suggests its suitability for large-scale biopro-
cessing. To translate the protocols to industrial bioprocess,
detailed characterization of cryopreservation and thaw pro-
cess using lineage-specific differentiation as output criteria
will be a necessary next step. Identification of novel markers
and assays to predict the ability of MCB/WCB to produce
high purity of therapeutic cell products will also play an im-
portant role in improving process efficiency and ensuring
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Yan Li, PhD
Department of Chemical and Biomedical Engineering
Florida State University
2525 Pottsdamer Street
Tallahassee 32310, FL
214 LI AND MA