Advanced Biotechnology for Cell Cryopreservation

Abstract

Cell cryopreservation has evolved as an important technology required for supporting various cell-based applications, such as stem cell therapy, tissue engineering, and assisted reproduction. Recent times have witnessed an increase in the clinical demand of these applications, requiring urgent improvements in cell cryopreservation. However, cryopreservation technology suffers from the issues of low cryopreservation efficiency and cryoprotectant (CPA) toxicity. Application of advanced biotechnology tools can significantly improve post-thaw cell survival and reduce or even eliminate the use of organic solvent CPAs, thus promoting the development of cryopreservation. Herein, based on the different cryopreservation mechanisms available, we provide an overview of the applications and achievements of various biotechnology tools used in cell cryopreservation, including trehalose delivery, hydrogel-based cell encapsulation technique, droplet-based cell printing, and nanowarming, and also discuss the associated challenges and perspectives for future development.

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Transactions of Tianjin University
https://doi.org/10.1007/s12209-019-00227-6
REVIEW
Advanced Biotechnology forCell Cryopreservation
JingYang1,2,3· LeiGao1,2,3· MinLiu1,2,3· XiaojieSui1,2,3· YingnanZhu1,2,3· ChiyuWen1,2,3· LeiZhang1,2,3
Received: 29 October 2019 / Revised: 25 November 2019 / Accepted: 27 November 2019
© The Author(s) 2019
Abstract
Cell cryopreservation has evolved as an important technology required for supporting various cell-based applications, such
as stem cell therapy, tissue engineering, and assisted reproduction. Recent times have witnessed an increase in the clinical
demand of these applications, requiring urgent improvements in cell cryopreservation. However, cryopreservation technol-
ogy suffers from the issues of low cryopreservation efficiency and cryoprotectant (CPA) toxicity. Application of advanced
biotechnology tools can significantly improve post-thaw cell survival and reduce or even eliminate the use of organic solvent
CPAs, thus promoting the development of cryopreservation. Herein, based on the different cryopreservation mechanisms
available, we provide an overview of the applications and achievements of various biotechnology tools used in cell cryo-
preservation, including trehalose delivery, hydrogel-based cell encapsulation technique, droplet-based cell printing, and
nanowarming, and also discuss the associated challenges and perspectives for future development.
Keywords Cell cryopreservation· Biotechnology· Trehalose delivery· Hydrogel-based cell encapsulation· Droplet-based
cell printing· Nanowarming
Introduction
Cell cryopreservation is a technology used to preserve living
cells, while maintaining their cellular viability and func-
tions even at cryogenic temperatures (usually at − 80°C
or − 196°C). At such ultra-low temperatures, the chemi-
cal, biological, and physical processes normally occurring
at cellular level can remain suspended for a long time. In
recent times, cell cryopreservation has become an important
supporting technology for various cell-based applications
such as stem cell therapy, tissue engineering, assisted human
reproduction, and transfusion medicine [1]. The importance
of cryopreservation technology is correctly reflected by the
burgeoning demand of stem cell therapy, owing to which
approximately 400,000 units of umbilical cord blood have
been stored worldwide for public use and 900,000 units for
private use [2]. As per the report of Stem Cell Banking Mar-
ket, the global stem cell banking market had a current value
of ~ 18.2 billion dollars in 2017, and this will reach ~ 54.1
billion dollars by 2024. Red blood cell cryopreservation has
also gained importance in the past few years. Cryopreserva-
tion of red blood cells (RBCs) can extend the storage time
from 42 d (hypothermic preservation) to 10years, which will
ease the burden of short blood supply, especially in remote
areas [3].
However, during freezing–thawing cycles, cells inevitably
suffer from cryoinjuries, including solution injury and ice
injury. The freeze concentration-induced excessive dehydra-
tion can damage cells resulting in solution injury. Besides
this, ice formation and growth during cryopreservation can
mechanically damage the biological structure of cells result-
ing in ice injury [4] (Fig.1a). Cryoprotectants (CPAs) play
a pivotal role in protecting cells against these cryoinjuries
and allow their successful storage at cryogenic temperatures.
CPAs can be broadly classified into two main categories on
the basis of the permeability or non-permeability of CPAs
into the cellular membrane [5]. Permeating CPAs mainly
Jing Yang and Lei Gao have contributed equally to this work.
* Lei Zhang
lei_zhang@tju.edu.cn
1 Department ofBiochemical Engineering, School
ofChemical Engineering andTechnology, Tianjin
University, Tianjin300350, China
2 Frontier Science Center forSynthetic Biology andKey
Laboratory ofSystems Bioengineering (MOE), School
ofChemical Engineering andTechnology, Tianjin University,
Tianjin300350, China
3 Qingdao Institute forMarine Technology ofTianjin
University, Qingdao266235, China

J.Yang et al.
1 3
include organic solvents, such as glycerol and DMSO, which
can permeate phosphate bilayers. This permeability is usu-
ally driven by a concentration gradient and confers intracel-
lular protection to cells [6]. However, most of the organic
solvent CPAs exhibit toxicity or poor biocompatibility that
can cause serious side effects in patients like hemolysis,
neurotoxicity, cardiovascular failure, respiratory arrest, and
fatal arrhythmias [7, 8]. In comparison with this, non-per-
meating CPAs provide extracellular protection only. These
include natural non-toxic carbohydrates (such as trehalose
and sucrose) and biomacromolecules (such as proteins and
polymers) [6]. Generally, non-permeating CPAs are com-
bined with permeating CPAs to ensure both extracellular
and intracellular protection, where the latter is required for
the critical protection of cells from inside. This results in a
compromise between high cryopreservation efficiency and
CPA toxicity [9].
For most cell types, conventional cryopreservation pro-
tocol involves stepwise freezing of sample at slow cooling
rates using 10–20% DMSO solution. In order to improve cell
cryopreservation efficiency, previous studies have mainly
focused on the optimization of CPA formulation, CPA intro-
duction, and freezing–thawing protocol suitable for different
cell types [10, 11]. Pollock etal. [12] reported the use of a
differential evolution algorithm to optimize cryopreserva-
tion protocols for Jurkat cells (300mmol/L trehalose, 10%
glycerol, and 0.01% ectoine at 10°C/min) and mesenchymal
stem cells (300mmol/L ethylene glycol, 1mmol/L taurine,
and 1% ectoine at 1°C/min), which resulted in post-thawing
cell viabilities of 95% and 96%, respectively. However, the
optimization of cryopreservation protocol still suffers from
two major challenges: (1) unfavorable post-thaw cell viabil-
ity or functions and (2) safety concerns induced by CPA
toxicity. For cryopreservation of hepatocytes, Mahler and
co-workers [13] reported only 61–75% survival of isolated
cells post-cryopreservation, and post-thawing cell attach-
ment efficiency of 30–39%. Besides this, there are reports
where the critical functions in some of the therapeutic cells
such as mesenchymal stem cells (MSCs), natural killer cells,
and dendritic cells (DCs) were compromised after use of
conventional cryopreservation protocol [14–17]. Organic
solvents glycerol and DMSO are widely used in intracellu-
lar protection; however, both lack biocompatibility. Glycerol
can induce severe hemolysis [18], while the use of DMSO
is found to be associated with many side effects in patients,
like neurotoxicity, cardiovascular failure, respiratory arrest,
fatal arrhythmias, and others [19]. Fetal bovine serum (FBS)
is frequently combined with organic solvents to supplement
Fig. 1 a Schematic diagram for
two types of cryoinjuries occur-
ring during cryopreservation
of cells; b schematic diagram
for various biotechnology tech-
niques used in cryopreservation
and their method of protection

Advanced Biotechnology forCell Cryopreservation
1 3
extracellular cryopreservation. However, FBS is derived
from animals and has risk of inducing viral infections and
immunogenicity inpatients [20, 21].
In recent years, the developments of biotechnology pro-
vided an opportunity to improve the final outcome of cell
cryopreservation. Generally, biotechnology refers to use
of scientific techniques to study and address the problems
associated with living organisms. In this review, we summa-
rize the applications and achievements of various advanced
biotechnology tools used in cell cryopreservation. These
include trehalose delivery, hydrogel-based cell encapsulation
technique, droplet-based cell printing, and nanowarming. All
these techniques broadly aim to enhance cell cryopreser-
vation and reduce or eliminate CPA toxicity. As shown in
Fig.1b, trehalose delivery is proposed to deliver non-per-
meable but biocompatible trehalose into cells as an alterna-
tive to organic solvents, with the aim to provide intracellular
cryopreservation. The other three techniques mainly enhance
extracellular protection during freezing or warming process
and thus aim to increase cryopreservation efficiency and
significantly reduce the concentration of organic solvents.
In addition to this, we discuss the challenges and provide
future perspectives for the development of biotechnology
tools used in cell cryopreservation.
Trehalose Delivery
Trehalose is a non-permeating disaccharide, which is used
as a bio-inspired CPA to protect cells or organisms against
cryoinjuries [22]. It facilitates the formation of a stable
glassy matrix and promotes preferential hydration in cellu-
lar biomolecules, stabilizing their functional conformations
[23–25]. However, trehalose generally provides extracellular
protection only owing to its lower permeability. For intracel-
lular cryopreservation, it is used in combination with organic
solvent CPAs (glycerol or DMSO) [26–28]. In order to avoid
the toxicity of organic solvents, several advanced biotechnol-
ogy tools have been used to deliver trehalose into cells to
provide both intracellular and extracellular cryopreservation.
These techniques help to achieve organic solvent-free cell
cryopreservation and high post-thaw cell survival efficiency
[29]. These biotechnology tools, including both physical and
chemical methods, can increase cellular membrane perme-
ability and thus transport non-permeable trehalose into cells
(Table1).
Physical Delivery Method
The physical delivery methods, including freezing-induced
membrane phase transition [30–32] and electroporation
technology [33–35], have been used to promote the per-
meation of trehalose into cells. These methods help to
achieve organic solvent-free cell cryopreservation. The
loading of trehalose can be easily controlled by manipu-
lating the concentration gradient. However, the increased
membrane permeability achieved by these methods suffers
from the issues of non-specificity, which results in uncon-
trolled influx and outflux of other molecules.
The use of freezing-induced membrane phase transition
method for intracellular delivery of trehalose into cells was
reported for the first time by Beattie etal. [36] in 1997, for
cryopreservation of pancreatic islet cells. During the cool-
ing process, changes occurring in the fluid-to-gel phase
transition result in reorganization of membrane lipid com-
ponents which increases the membrane permeability. A
concentration gradient then drives the intracellular move-
ment of trehalose to provide intracellular cryopreservation.
Gläfke etal. [30] reported the use of high extracellular
concentrations of trehalose for freezing platelets. This
method resulted in 98% membrane intact platelets, 76%
of which were in non-activated resting state. This platelet
cryopreservation protocol avoided any use of DMSO.
For more than 40years, electroporation technology
has been widely used for intracellular delivery of xeno-
molecules such as saccharides, drugs, plasmids, DNA
vaccine, siRNA, and proteins. It offers several advantages
like controllability, reproducibility, and high efficiency.
Application of an external electric pulse assists in the for-
mation of hydrophilic pores on the membrane resulting
in an increase in membrane permeability. This pore for-
mation can be reversible or irreversible depending on the
electric pulse conditions [37–41]. In two separate studies,
Dovgan etal. [33] reported the use of electroporation for
efficient loading of trehalose into human adipose-derived
stem cells (hADSCs) and umbilical cord mesenchymal
stem cells (UC-MSCs). For electroporation in hADSCs,
the cells were incubated in 250mmol/L trehalose and
electroporated at the optimal conditions of 1.5kV/cm2,
at 8 pulses, 100μs, and 1Hz, prior to programmable
slow freezing. After thawing the cells, 83.8 ± 1.8% cell
recovery rate was observed, which was similar to that of
hADSCs (91.5 ± 1.6%) obtained using standard freezing
protocol (10% DMSO in 90% FBS). In comparison with
this, the electroporation of UC-MSCs under the optimal
electroporation conditions (430V, 8 pulses, 100μs, and
1Hz) resulted in 61% cell viability [34]. It has been pre-
viously shown that high voltage can result in great loss
of cell viability, while insufficient voltage compromises
the delivery of trehalose into cells. Therefore, to ensure
efficient trehalose delivery and favorable post-thaw cell
viability, it is important to optimize suitable voltage condi-
tions for different cell types.

J.Yang et al.
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Table 1 Different trehalose delivery methods used for cell cryopreservation
Delivery methods Cell type Main results Advantages Disadvantages
Physical delivery Freezing-induced lipid-phase
transition
Platelets Post-thawing, 98% platelets
showed/were having intact
membrane
Organic solvent-free cell
cryopreservation based on
trehalose
1. Trehalose loading into
cells easily controlled by
concentration gradient
2. Reversible based on exter-
nal stimuli
3. Simple processing
1. Non-specific membrane
permeability
2. Membrane injury concerns
induced by thermal and
electric shock
Electroporation hADSCs 1. 84% cell survival rate
post-thawing
2. Maintenance of normal
cell proliferation and dif-
ferentiation potential
UC-MSCs 61% cell survival rate post-
thawing
Chemical delivery PP50 Erythrocyte 1. 123mmol/L of intracellu-
lar trehalose was achieved
in erythrocytes
2. 83% post-thaw erythrocyte
survival
Loading of high amounts of
intracellular trehalose.
1. Non-specific membrane
permeability
2. Long incubation time
3. Cytotoxicity concerns
4. Tedious washing step
5. Complex material prepara-
tion
SAOS-2 The number of metabolically
active cells at 24h post-
thaw was between 91% and
103%
α-Hemolysin Fibroblasts
and
keratino-
cytes
Long-term post-thaw
survival rate was 80% for
fibroblasts and 70% for
keratinocytes
1. Reversible based on exter-
nal stimuli.
2. Trehalose loading into
cells easily controlled by
concentration gradient
Genipin-cross-linked
Pluronic-F127 nanoparti-
cles (GNPs)
hADSCs Approximately 90% cell
viability and normal dif-
ferentiation potentials, and
distinctive markers expres-
sion was maintained
1. Utilize natural endocytosis
process
2. Specific transport of high
amounts of trehalose
1. Long incubation time
2. Complex material prepara-
tion
Apatite nanoparticles RBCs Increasing RBC cryosurvival
up to 91%, which is com-
parable to FDA-approved
cryopreservation protocol
employing glycerol

Advanced Biotechnology forCell Cryopreservation
1 3
Chemical Delivery Method
Chemical delivery methods utilize cell membrane perforat-
ing agents like synthetic polymers [42, 43], α-hemolysin
[44–46], and nanoparticles [47–49] to achieve intracellu-
lar delivery of trehalose. These chemical materials usually
involve a complex preparation process. They can interact
with cell membranes and increase membrane permeability
temporarily. Trehalose has been used as sole CPA in combi-
nation with these chemical delivery methods to successfully
cryopreserve RBCs, fibroblasts, keratinocytes, and human
mesenchymal stem cells (hMSCs).
Mercado etal. [43] designed and synthesized a series of
biomimetic derivatives of PLP polymer to facilitate intra-
cellular delivery of trehalose. Particularly, co-incubation
of PP50 (composed of PLP grafted with l-phenylalanine)
with erythrocytes and trehalose suspension increased the
intracellular concentration of trehalose to 123 ± 16mmol/L.
After cryopreservation of erythrocytes loaded with treha-
lose, the erythrocyte survival rate was 82.6±3.4%, which
was 20.4 ± 5.6% higher as compared to the unloaded eryth-
rocytes. PP50-mediated trehalose delivery method has been
also used in organic solvent-free cryopreservation of nucle-
ated human cell line SAOS-2. Although the post-thaw cell
viability was only 60 ± 2%, the number of metabolically
active cells at 24h post-thaw was in the range of 103±4 to
91±5%. This was comparable to the results observed for
cells frozen using DMSO [42]. Mechanically, PP50 adsorp-
tion onto the membrane contributed by its amphipathic char-
acteristic induced the appearance of thinner phospholipid
bilayer, which resulted in an increase in trehalose uptake
[42]. However, longer incubation times in cryopreserva-
tion of erythrocytes might result in undesirable hemoly-
sis of cells. Thus, PP50 must be removed to overcome the
safety issues, which generally involves a tedious washing
procedure.
α-Hemolysin is a genetically engineered endotoxin
derived from Staphylococcus aureus. It has been shown to
work via generation of pores in the lipid bilayers for both
fibroblasts and keratinocytes, allowing an influx of trehalose
[46, 50]. Buchanan etal. [46] used α-hemolysin to achieve
intracellular trehalose concentrations of up to 0.5mol/L.
Trehalose at concentration of only 0.2mol/L provided
cryopreservation in fibroblasts and keratinocytes, with tre-
halose as sole CPA. After long-term cryopreservation, the
post-thaw survival rates were 80% and 70% for fibroblasts
and keratinocytes, respectively. Despite being so promis-
ing, α-hemolysin being a bacteria-derived pore protein may
induce undesirable immune responses in patients. Therefore,
it must be removed from cells prior to their use in clinical
therapy. The safety concerns and necessary removal steps
associated with the use of α-hemolysin limit its clinical
application.
Nanomaterials are widely used in medical field espe-
cially as vehicles for the delivery of drugs, such as chemi-
cal molecules, DNA vaccine, and protein or peptide drugs,
to therapeutic target [51–54]. In recent years, several stud-
ies have reported efficient delivery of trehalose into cells
using nanomaterials. The nanoparticles generally utilize the
natural process of endocytosis to specifically deliver treha-
lose into cells without any harmful effects [29]. The use
of nanoparticles for trehalose delivery and cryopreserva-
tion of cells with trehalose as sole CPA has been reported
to maintain cell viability and functions. Rao etal. [48]
developed a pH-responsive genipin-cross-linked Pluronic
F127–chitosan nanoparticle (GNP), which efficiently encap-
sulated trehalose for intracellular delivery (Fig.2a). For
cryopreservation of hADSCs, the cells were incubated with
trehalose-loaded GNPs (nTre) for 24h and cryopreserved
in culture medium containing 200mmol/L free trehalose.
After rewarming, pre-incubation with nTre resulted in 90%
cell viability which was comparable to the cell viability
obtained post-cryopreservation with DMSO. Besides this,
the differentiation potential and the expression of distinctive
markers in hADSCs remained unchanged upon cryopreser-
vation. Another nano-vehicle used for delivery of trehalose
is a type of biomimetic (bone-like) apatite nanoparticle.
These apatite nanoparticles have been shown to efficiently
deliver drugs and nucleic acids into various types of cells.
Stefanic etal. [49] utilized colloidal bio-inspired apatite
nanoparticles to mediate intracellular delivery of trehalose
into RBCs. The local interactions between apatite NPs and
the bilayer enhanced the translocation of trehalose into the
cells (Fig.2b). Cryopreservation of trehalose-laden RBCs
demonstrated that the use of this glycerol-free cryopreser-
vation protocol tremendously increased survival of RBCs
to 91%, which was 42% higher as compared to the control
without NP treatment. These results were comparable to the
FDA-approved cryopreservation protocol that utilized glyc-
erol as CPA. Nanoparticles-mediated intracellular delivery
of trehalose has shown great potential to achieve clinical
cryopreservation of therapeutic cells without any use of
organic solvents. A possible limitation of this method could
be requirement of long incubation time to achieve sufficient
intracellular trehalose concentration. The incubation time
required for trehalose delivery was 24h and 7h for GNPs
and apatite nanoparticles, respectively.
Hydrogel‑Based Cell Encapsulation
Technology
Hydrogel-based cell encapsulation technology refers to
the encapsulation of living cells within semipermeable
capsules prepared using hydrogel materials. This technol-
ogy has been found to be highly promising for various

J.Yang et al.
1 3
cell-based studies and applications. This technology pro-
vides suitable3D microenvironment similar to the extra-
cellular matrix, blocks the immunogenicity of encapsu-
lated cells, and directs the differentiation of stem cells
[55–57]. In recent years, hydrogel-based cell encapsula-
tion technology has been widely used in cell cryopreser-
vation. The capsules not only protect the inner cells from
mechanical and osmotic stress during the freezing and
warming process, but also allow the bidirectional diffu-
sion of nutrients, oxygen, and waste products. Numerous
studies have established its positive effects on post-thaw
cell viability and functions [58–60].
The method used for cell encapsulation greatly influences
the final outcome of cell cryopreservation. These methods
can be broadly divided into three categories, namely emul-
sion/thermal gelation, extrusion (electrostatic spray, air flow
nozzle, and vibrating nozzle), and microfluidic method [58].
Choice of method depends on two main factors: capability
to maintain high cell viability/function and ability to control
the capsule phenotypic characteristic such as size, shape,
strength, and permeability [61].
Emulsion Method
Emulsion is a generic method used to encapsulate cells
within bulk hydrogels. In this method, pre-gel solutions are
first prepared by suspending cells and gel materials. When
the dispersion reaches an equilibrium state, gel formation
is triggered by adding an initiator or changing the physical
conditions, such as UV light. Although emulsion method
is simple and easy to scale up, the process of gelation may
result in cell death and loss of functions owing to the toxicity
of initiator, chemical cross-linking, and unfavorable reaction
conditions [58, 62].
In recent years, numerous novel cross-linking approaches
have been used in the preparation of cell-loaded hydrogels
to avoid the negative effects of conventional methods.
PVA-based hydrogel including PVA–gelatin cryogels and
Fig. 2 Nanomaterials-mediated
trehalose transportation into
cells. a The encapsulation of
trehalose in genipin-cross-
linked Pluronic F127–chitosan
nanoparticles (GNPs) to pro-
duce nanoparticle-encapsulated
trehalose (nTre). b Interactions
of biomimetic (bone-like) apa-
tite nanoparticles with the lipid
bilayer and enhanced delivery
of trehalose into RBCs

Advanced Biotechnology forCell Cryopreservation
1 3
PVA–carrageenan (Car) scaffold was prepared via freeze-
gelation technique. The encapsulation process only involved
a freezing step without requirement of any external cross-
linking agents. After cryopreservation, cell viability and
functions were observed to be unaffected [63–66]. Zeng
etal. [67] developed a supramolecular gel that also involved
a cooling process to trigger the gel formation. For cryo-
preservation of encapsulated PC12 cells and RSC96 cells, a
mixture of cell suspension, gelator, and DMSO was prepared
and cooled in ice-water bath. Gelator self-assembled to form
supramolecular gel at 8.2°C and then programmed freez-
ing of the system was carried out at − 80°C (Fig.3a). The
post-thaw viability of PC12 and RSC96 cells increased sig-
nificantly owing to the protection provided by the hydrogel
during the freezing and thawing process. In addition to this,
the thermo-reversible supramolecular gel could be removed
easily by centrifugation. Jain etal. [68] reported a two-
component molecular recognition gelation method that was
Fig. 3 Schematic diagram for cell encapsulation in hydrogel cap-
sules by different methods. Emulsion method: a cooling and thaw-
ing process for cells encapsulated in supramolecular gel, b insitu
hydrogelation via SPAAC click chemistry for cell encapsulation and
cryopreservation, c preparation and cryopreservation process for
hASCs–K-carrageenan hydrogel construct. Extrusion method: d a
two-fluidic electrospraying method for encapsulation of cells in core–
shell capsules. Microfluidic method: e electrohydrodynamic atomi-
zation (EHDA) method for fabrication of cell-laden microcapsules
with uniform size, f encapsulation of individual rat islets into alginate
hydrogel using a droplet microfluidic device at room temperature,
and g preparation, vitrification and warming process for cell-laden
alginate-based core–shell hydrogel produced using a double emulsion
flow-focusing tube-in-tube capillary microfluidic device

J.Yang et al.
1 3
adopted to develop a dextran-based polyampholyte hydrogel
having cryoprotective properties. The gelation process was
initiated by mixing of azide-Dex-PA and DBCO-Dex, and
this gelation mainly depended on the reactant concentration
(Fig.3b). In the absence of any CPA, L929 cells encapsu-
lated within these hydrogels showed a recovery rate of > 90%
(in optimum gelation condition) after thawing. As a natural
thermo-sensitive polymer, K-carrageenan could form stable
hydrogel via ionic gelation process. Potassium chloride was
used for the cross-linking of K-carrageenan, allowing further
stabilization of the hydrogels (Fig.3c). HMSCs encapsulated
with K-carrageenan hydrogel showed improved proliferation
and chondrogenic potential post-cryopreservation [69].
Extrusion Method
Extrusion methods majorly include two methods: electro-
static spray and air-jet encapsulation technology. These are
commonly used for cell encapsulation owing to their high
throughput and production of evenly sized beads [61].
Wolters etal. [70] developed an air-jet droplet generator
and used it to produce small, uniform, and smooth alginate
beads while maintaining high throughput. In 2010, Mal-
pique etal. [71] investigated for the first time cryopreserva-
tion of brain cell neurospheres by encapsulating the cells
within alginate hydrogel using the air-jet two-channel drop-
let generator. The results showed that the cell viability and
metabolic activity were significantly higher in encapsulated
neurospheres as compared to the non-encapsulated ones. It
might be contributed by reduced fragmentation and better
maintenance of spherical shape of aggregates upon encap-
sulation in alginate hydrogels. However, air-jet technology
applied during microcapsule formation has several limita-
tions such as use of harsh shearing forces and formation of
air bubbles and “tails.”
Cell encapsulation using electrostatic spray method
involves generation of droplets containing cells and poly-
mers from the nozzle, followed by spraying into a container
with gelling bath to form hydrogel beads. The hydrogel
beads formation is assisted by the electrostatic force between
the gelling bath and the nozzle [71]. Zhang etal. [72] encap-
sulated mouse MSCs into small (~ 100μm) Ca-alginate
microcapsules generated by electrostatic spray method. The
vitrification of cell-loaded microcapsules with low concen-
tration of DMSO maintained high post-thaw cell viability
in encapsulated cells. The Ca-alginate microcapsules pro-
vided great protection to the cells during cryopreservation.
Two-fluidic electro-co-spraying technique was developed
and adopted to continuously produce core–shell alginate
capsules, which had better mass transfer and were used to
encapsulate organoids (Fig.3d). The core–shell structure
of the capsules provided better cell recovery after cryo-
preservation of organoids, probably through prevention of
intracellular ice formation [73–75]. Electrohydrodynamic
atomization (EHDA) is an attractive approach that immobi-
lizes living cells into biomaterials permitting localized and
minimally invasive delivery. It minimizes cell leakage and
maintains viability during the delivery process. Naqvi etal.
[76] combined alginate and EHDA technique to fabricate
bone marrow stomatal cells (BMSCs)-encapsulated micro-
capsules (Fig.3e). The results of cryopreservation showed
that micro-encapsulation of BMSCs within alginate main-
tained their cell viability and potential to synthesize sGAG
and collagen. Electrostatic spray method offers several
advantages including cytocompatibility, ease of operation,
and high efficiency. Besides this, the manufacturing process
of capsules could be performed in a sterile environment.
Therefore, the electrostatic spray method is promising and
suitable to encapsulate cell resources or CPTs for long-term
storage purpose.
Microuidic Method
Rapid development of micro- and nanotechnologies has
allowed operation of cell encapsulation procedures on-chip
[1]. Built on flow focusing, miniaturized devices are to
encapsulate cells into capsules. Microfluidic methods permit
a high degree of control over the morphological and dimen-
sional properties. The experimental platforms are physically
smaller than the macro-encapsulation systems. In addition
to these, microfluidic method offers several advantages over
macro-encapsulation systems, such as low cost, ease to scale
up, disposability, specific designs, and rapid implementation
[57, 76].
Many groups investigated the cryopreservation of cells
encapsulated within hydrogel beads produced by microflu-
idic methods. In a droplet microfluidic platform, individual
rat pancreatic islets were encapsulated with FOSD function-
alized hydrogel microcapsules. This study aimed to establish
single-islet-based quality control assay for assessing qual-
ity and functionality of individually cryopreserved islets
(Fig.3f). Hydrogel membrane surrounding the encapsulated
islet effectively enhanced the insulin secretion after thawing.
The unique microstructure of the hydrogel was character-
ized by the presence of a compact 3D porous network and
considerable amount of non-freezable bound water, which
may alleviate the cryoinjury to the cells, playing a role simi-
lar to the CPAs [77]. Large-volume low-CPA cell vitrifica-
tion was achieved by microfluidic-based alginate hydrogel
micro-encapsulation system (Fig.3g) [78, 79]. HASCs in
low concentration of CPA medium (2mol/L) were encap-
sulated into core–shell microcapsules using an elaborate
microfluidic system and then loaded into 0.25mL conven-
tional plastic straw (PS). These were directly plunged into
liquid nitrogen to realize/induce vitrification. After thawing,
hASCs liberated from the microcapsules showed no changes

Advanced Biotechnology forCell Cryopreservation
1 3
in viability and differential capacity. In terms of mechanism,
the microcapsule effectively inhibited the ice formation and
further propagation during cooling and warming process.
The IRI activity of microcapsules especially protected the
cells against severe mechanical injuries.
Droplet‑Based Cell Printing
Droplet-based techniques find wide applications in various
fields, such as inkjet printing, emulsion polymerization, and
DNA arraying, owing to their high efficiency and low cost
[80–84]. Introduction of this advanced technology into cell
cryopreservation created a series of novel vitrification proto-
cols that are characterized by lower CPA concentration and
higher cooling and warming rates. This is mainly applied
to smaller volumes of cryo-system [85]. Droplet-based cell
vitrification process not only solved the problem of high
CPAs concentration required for conventional vitrification
procedure, but also conferred significant protection to cells
by reducing the time required for ice crystal formation and
alleviating the osmotic shock. Besides this, the whole pro-
cess is easy and quick, allowing a possibility of large-scale
automation [86–88]. Therefore, this innovative approach
may contribute significantly in the development of cell
cryopreservation.
Fig. 4 Schematic diagram for preparation of cell-laden droplets using
different devices. a Cell-CPA solution was loaded into valve-based
droplet injector, and the resulting droplets were directly injected into
liquid nitrogen. The ejected cells were collected in a cell strainer and
were rapidly transferred (in nitrogen vapor) to the thawing media, fol-
lowed by a step-by-step thawing process. b RBC-CPA droplets were
printed onto a cryo-paper as nitrogen gas flowed through a droplet
ejector, which transformed the bulk of the RBC-CPA mixture into
nanoliter droplets. Vitrification was achieved by submerging the cryo-
paper into liquid nitrogen. Warming process was performed by thaw-
ing the cells on a cryo-paper in phosphate-buffered saline at 37°C.
c Cell-laden droplets were rapidly ejected onto a freezing film using
a cell printer with high throughput and precise spatial controllabil-
ity. Vitrification/thawing process was achieved by pouring the liquid
nitrogen/warm water onto the other side via boiling heat transfer that
helped to maintain high cooling/warming rate

J.Yang et al.
1 3
Demirci etal. [84] reported successful vitrification of
many cell types by means of optimal droplet-based proce-
dure. This technique involved generation of droplets contain-
ing cell and CPAs solution using a modified jet device, which
were further received/transferred in a container filled with
liquid nitrogen and warmed in 37°C water bath (Fig.4a).
Before vitrification, droplet generation process did not affect
the call viability and cell survival rate was maintained to
approximately 90%. Propanediol and trehalose were used as
CPAs instead of toxic DMSO for cell vitrification. Even after
thawing, the viability of cells was maintained well.
Assal etal. [89] developed a novel cryo-printer that could
transform a bulk volume of human blood into nanoliter cryo-
inks on a cryo-paper, which was immersed into liquid nitro-
gen for rapid vitrification (Fig.4b). After rewarming, the
recovered human RBCs showed normal characteristic fea-
tures. In addition to this, there was no effect on the essential
functions of recovered RBCs, including phosphorylation of
band 3 protein, expression of complement receptor 1, and
maintenance of intracellular nitric oxide and reactive oxygen
species levels. Besides these intrinsic advantages offered by
droplet-based vitrification, the cryo-ink containing CPAs
medium such as ectoine, trehalose, and PEG also reduced
the injuries suffered by RBCs during the cooling and warm-
ing processes.
High-throughput non-contact vitrification of cell-laden
droplets was reported by Shi etal. [90] in 2015. Cell printing
generated droplets containing cell CPAs onto an ultra-thin
freezing film. Vitrification/thawing process was operated by
pouring liquid nitrogen/warm water onto the other side via
boiling heat transfer. This ensured maintenance of high cool-
ing/warming rate and avoided direct contact between cells
and liquid nitrogen/water, preventing chances of potential
contamination (Fig.4c). The use of this novel method pro-
vided successful vitrification in NIH 3T3 cells and hASCs.
After thawing, both cell viability and differentiation poten-
tial remained unaffected.
Recently, a CPA-free cryopreservation method-based
inkjet cell printing technology was developed by Akiyama
etal. [91]. It was successfully used for the vitrification of
several mammalian cell types such as 3T3 cells, C2C12
cells, and rat MSCs at ultra-rapid cooling rates. The drop-
lets containing cells and culture medium were printed onto a
glass substrate cooled with liquid nitrogen to realize/induce
solid-surface vitrification. Immediately after thawing, the
viability of 3T3 cells for 40-pL droplets on thick substrates
(thickness: 150μm) was comparable to the cell viability
obtained using conventional freezing method. The ultra-
rapid cooling and warming rates significantly inhibited ice
formation and ice recrystallization and also protected the
cells against cryoinjuries during the freezing and warming
processes.
Nanowarming
Conventional warming method (37°C water bath) fails to
provide sufficient warming rates and uniform warming effect
with the increasing scale of bio-specimen. During rewarm-
ing process, once the warming rates go below the critical
warming rates, ice recrystallization/devitrification occurs,
which is one of the major causes of cell suffered injuries.
However, advances in bio-specimen cooling for cryopreser-
vation have not been matched well by similar developments
in rewarming procedure [92]. Nanomaterials-mediated
nanowarming technology has a potential to be established as
a new approach to allow ultra-rapid and uniform rewarming
[93]. Nanowarming generally involves use of some nano-
materials such as Fe3O4 nanoparticles (Fe3O4 NPs) or gold
nanorods (GNRs) that can rapidly convert electromagnetic
or light energy into heat energy. Thus, rapid and uniform
rewarming of bio-specimens can be realized/achieved by
incorporating these nanomaterials into CPA solution and
heating with external electromagnetic, radiofrequency (RF)
or laser fields [94–96]. Numerous studies have affirmed that
nanowarming technology is significantly effective and can
be used for improving cryopreservation of cells, tissues, and
organs.
RF Inductive Warming Process
Magnetic iron oxide nanoparticles are capable of transform-
ing external electromagnetic energy into heat energy rapidly.
Modified iron oxide nanoparticles (msIONPs) character-
ized by compatibility, colloidal stability, and capability to
remain in solution at higher concentration were synthesized
by Manuchehrabadi etal. [93]. Human dermal fibroblasts
(HDFs) were vitrified using VS55 cryo-solution loaded with
msIONPs and warmed by different methods. In the 1mL
system, the viability of nanowarmed HDFs was statistically
similar to the fresh control sample and higher than the via-
bility of slow-warmed sample.
Liu etal. [97] successfully achieved low-CPA vitrifica-
tion of stem cell-alginate hydrogel constructs by combining
nanowarming and micro-encapsulation technology (Fig.5a).
Fe3O4 NPs were mixed with low-CPA solution for RF induc-
tive warming process. After nanowarming, porcine adipose-
derived stem cells (pADSCs) showed viability > 80%, while
the attachment efficiency improved by three times as com-
pared to the pADSCs treated with slow warming process.
Besides these, the expression of surface markers and multi-
lineage potentials of pADSCs after nanowarming remained
unaffected. Mechanically, in addition to the cryopreservation
provided by cryo-solution and alginate hydrogel, Fe3O4 NPs
uniformly present outside of hydrogel further suppressed
devitrification and recrystallization during nanowarming

Advanced Biotechnology forCell Cryopreservation
1 3
process. Nanowarming was the primary reason for promoted
attachment of thawed pADSCs [97]. This technology was
also found to be efficient in human UC-MSCs and resulted
in an improved vitrification outcome [98] (Fig.5b).
Laser Radioactive Warming Process
Gold nanoparticle-based laser warming has the potential to
provide a platform for both extra- and intracellular heating
of vitrified biomaterials, ranging in size from nm to mm
in µL-sized droplets. Khosla etal. [99] mixed GNRs with
cryo-solution for rapid cooling and nanowarming of HDFs
(Dcell = 10µm) in droplet volume. The method of nanowarm-
ing used was laser warming technology irradiated with a
1064nm laser pulse for 1ms (Fig.5c). A cell viability of >
90% was maintained in HDF cells post-laser warming. Simi-
lar warming method was used to rewarm vitrified zebrafish
embryos. Before vitrification, biocompatible PEGylated
GNRs were microinjected directly into zebrafish embryos
with 2.3mol/L PG, thereby helping to distribute the laser
energy throughout the embryo during warming (Fig.5d). As
compared to the conventionally warmed control group, the
GNRs-mediated laser warming of embryos resulted in 31%
viable embryos with consistent structure at 1h, 17% viable
embryos continuing development at 3h, and 10% viable
embryos showing movement at 24h post-warming [100].
Two-dimensional (2D) graphene oxide (GO) and molyb-
denum disulfide (MoS2) nanosheets (NSs) were used to
improve warming process of bio-samples owing to their
photothermal effects. Human umbilical vein endothelial cells
Fig. 5 Schematic diagram for different cell vitrification techniques
and nanoparticles-mediated nanowarming process. RF field-mediated
warming process: a Nanowarming procedure for vitrified PS loaded
with cell-alginate hydrogel constructs and NPs in CPA solution. b
Vitrification and nanowarming of hUCM-MSC-laden PS with mag-
netic induction heating. Laser field-mediated warming process: c
Sample droplet consisting of biomaterial(s), CPA, and gold nanorods
(GNRs) with a maximum volume of 1.8 µL was loaded onto a cus-
tomized cryotop. For rapid cooling, cryotop was directly immersed
into liquid nitrogen. Laser warming was achieved by pulsed laser irra-
diation yielding ultra-rapid rewarming at rates up to 2×107 °C/min.
d Overview of zebrafish embryo cryopreservation and laser GNRs
rewarming. Micro-injection consisting of 1064 nm resonant GNRs
and PG was injected into the space of the embryo, between yolk and
chorionic. Rapid cooling was achieved by immersing the modified
cryotop into liquid nitrogen. Laser irradiation of the embryo taken
from liquid nitrogen was used to achieve nanowarming. e Multistep
addition of vitrification solution and loading in PS, followed by rapid
cooling and photothermal rewarming with GO and MoS2/NSs at
37°C water bath

J.Yang et al.
1 3
(HUVECs) were chosen to study this novel NSs-mediated
spatial heating approach. For rewarming, cryopreserved
HUVECs were placed into warming solution (37°C) under
a near-infrared laser field and photothermal effect was
achieved at 5000mW/cm2 for 8–10s (Fig.5e). This warm-
ing technology significantly improved the cell viability as
compared to the conventional rewarming method and also
maintained normal cell function and subcellular ultrastruc-
ture. Further investigation showed that near-infrared laser
irradiation effectively decreased ice formation and restricted
recrystallization growth via micro- and macro-effects during
rewarming [101].
Conclusions andFuture Perspectives
With the advancement of cell-based applications, conven-
tional cell cryopreservation clearly failed to keep pace with
current and emerging needs. This review summarized the
recent advances in biotechnology tools, including trehalose
delivery, hydrogel-based cell encapsulation, droplet-based
cell printing, and nanowarming technology, used in cell
cryopreservation. Trehalose delivery technology helped to
overcome the major limitation of ultra-low permeability
of trehalose, and its application resulted in favorable post-
thaw cell survival rates without need of any organic solvent.
Trehalose delivery method provided both extracellular and
intracellular cryopreservation with trehalose as sole CPA
[29]. Hydrogel-based cell encapsulation technology created
a new platform for efficient cell transportation and preserva-
tion. It has promoted long-term storage of cell resources and
banking “off-the-shelf” cell-based therapy products at large
scale. Since hydrogel capsules supplement excellent cryo-
preservation, satisfactory outcomes of cell cryopreservation
can be also achieved at reduced DMSO concentrations [59,
102, 103]. Droplet-based cell printing can improve cell vitri-
fication and can be helpful to achieve high efficiency at low-
CPA concentration and reduce CPA toxicity and osmotic
stress during CPA loading and unloading process [86].
Nanowarming technique has revolutionized the progress in
warming method. Its use can achieve ultra-fast and uniform
rewarming, while avoiding the adverse effects of devitrifica-
tion on cells [93].
Remarkable progress has been made in the application of
advanced biotechnology tools to improve cell cryopreser-
vation. However, a serious challenge associated with cell
cryopreservation has been identified that needs to addressed.
Cryopreservation-induced delayed onset cell death has
been reported to result in significant loss (> 50%) in the
total cell population and compromises cellular functional-
ity [104–106]. In order to further explore the true poten-
tial of biotechnology for cell cryopreservation, future work
should focus not only on the advancement of the existing
applications, but also discover new “binding domain” to
introduce other innovative biotechnology techniques.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
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Dr. Lei Zhang is a professor in
Department of Biochemical
Engineering, School of Chemical
Engineering and Technology,
Tianjin University. He received
his Ph.D. degree in chemical
engineering from University of
Washington in 2012. He was a
postdoctoral fellow at the Univer-
sity of Washington between 2012
and 2013. His research interests
are mainly focused on the devel-
opment of polymer/protein based
functional biomaterials, preser-
vation of living cells/tissues, and
marine anti-biofouling coatings.