N - CRYOPRESERVATION - OF - MESENCHYMAL - STEM - CEL

International Journal of Advanced Scientific and Technical Research Issue 7 volume 1, January –February 2017
Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
CRYOPRESERVATION OF MESENCHYMAL STEM

CELLS (MSCs): DIFFERENT APPROACHES AND
APPLICATIONS
Seema Tripathy*
Women Scientist-A
Post Graduate Department of Zoology
Utkal University, Vani Vihar
Bhubaneswar - 751 004, Odisha, India
Contact: 0 98535 87266
ABSTRACT
Cryopreservation is the most effective way to preserve biological samples for future use.
However, the cell often experiences cryogenic injuries due to formation of ice crystals,
osmotic shock and toxic effects of cryoprotectants (CPAs) during its cryogenic journey that
affects cell viability and function. Due to immunomodulatory and immunosuppressive
properties Mesenchymal stem cells (MSCs) create novel therapeutic opportunities currently
being used in area of frontier medicine, cell-based therapy and tissue engineering. Often, the
non-availability of donor, costly processing, tedious in vitro expansion and critical
manipulation techniques restrict the potential applications of MSCs in regenerative medicine.
Off-the-shelf remedial approaches of MSCs largely depend on novel preservation strategies.
So, an effective cryopreservation protocol is needed to develop for MSCs that will able to
maintain the cellular architecture and intrinsic characteristics of MSCs. Further,
establishment of allogenic and autologous cryo-banks of hMSCs facilitate their applications
in future to treat acute and chronic diseases. The present review describes various methods
followed to preserve MSCs derived from different sources by application of several
convectional and non-convectional approaches with their possible clinical utilities.
Key words: Mesenchymal stem cells, Off-the-shelf, Cryopreservation, Clinical utilities.
1. INTRODUCTION
Recently, a new acronym has been assigned to mesenchymal stem cells (MSCs) as
“medicinal signaling cells” [1]. Since, MSCs are considered to be store house of drugs for
sites of injury or inflammation and they are site-regulated multidrug delivery systems. Thus,
these may serve as modulatory or curative agents for a variety of human maladies. This may
due to special characteristics of MSCs. MSCs home to and engraft to injured tissues and
modulate the inflammatory response through synergistic down-regulation of pro-
inflammatory cytokines and up-regulation of both prosurvival and anti-inflammatory factors
(Fig. 1).
©2017 RS Publication, rspublicationhouse@gmail.com Page 435

Cell- or tissue- directed attraction or stimulation
Cytokine production with para and autocrine receptor activation
Differentiation Self-renewal
Immunosuppression No teratoma formation
Trophic function
Regeneration and repair
Migration
Fig 1: Characteristics of MSCs
These cells after therapeutic interventions influence the connective tissue

scaffolding and accelerate production of cytokines, chemokines and extracellular matrix
proteins that regulate hematopoietic homing and proliferation. Recently, it is reported that
genetic modification of MSCs to over-express antitumor genes has opened new prospects for
anticancer therapy [2]. However, the molecular mechanisms governing these key phenomena
are yet to be understood. Translating MSCs to treat many immune and non-immune disorders
requires a steady supply of viable and functional cells. Currently off-the-shelf use of MSCs
requires development of novel strategies which allow storing of MSCs for long term.
Cryopreservation is the most efficient approach to preserve cells for longer period [3]. But
are MSCs able to retain their immunomodulatory properties, multilineage differentiation
potential and immunosuppressive characters after cryopreservation? This review summarizes
current progresses conducted to cryopreserve MSCs by different approaches like formulation
of cryopreservation media, optimization of freezing protocol and thawing procedure that
ensure to maintain hMSCs therapeutic characteristics without raising biosafety concerns
following cryopreservation.
2. PRINCIPLE OF CRYOPRESERVATION
The mechanism of cryopreservation largely depends on temperature reaction kinetic,
colligative property and osmotic balance of the biological system. At ultra low temperature
(LN2, -196°C), all the cellular metabolism ceases, this enabled biological materials could be
store in inert condition for many years. The precise pathways of molecular interaction that

repress metabolism at low temperature remain elusive [4]. However, during cryopreservation,
cells often experience cryogenic injuries due to temperature fluctuation, cytotoxicity and
intracellular edema. When a cellular system is forcibly exposed to lower temperature (below -
78.5°C), it experiences two most plausible events such as super cooling and ice nucleation.
Freezing induces the formation intracellular ice crystals. To combat this situation,
cryoprotective agents (CPAs) are often added to maintain cellular architecture.
These cryoprotectors also protect cellular proteins from denaturation especially those of
the plasma membrane by DMSO, through electrostatic interactions. These additives reduce
the formation of ice crystals by lowering freezing point. Many compounds including glycerol,
dimethyl sulfoxide (DMSO), ethanediol, propanediol, sucrose and trehalose possess such
properties. It is reported that when CPAs present at high concentrations in the extra and
intracellular media this increases viscosity and considerably reduces nucleation and
aggregation of crystals. However, CPAs at higher concentration cause cellular lesions via
direct toxic effects. This risk may be reduced by lowering the concentration, decreasing the
temperature and minimizing time of exposure of cells to CPAs before these cells proceeds to
cryopreservation. Ideal cooling parameters depend sometimes on contradictory factors and
much remains unknown. Experimentation often allows determining the best cooling
conditions (rate of cooling, levels, CPAs). The aim is to avoid thermal shock, increase of
saline concentrations to destructive levels and alteration of the colloidal state of the cellular
structure to glass state. Many mathematical equation models have been deduce that
enumerate different parameters such as gradient of water diffusion, formation of intracellular
aggregates and transfer of water as a function of the speed of cryoprotector transfer that
determine successful cryopreservation protocol.
3. DIFFERENT SOURCES OF MSCs
A noticeable pool of undifferentiated cells that reside within the ‘‘cell prosperous region’’
may serve as rich source of MSCs. The chief tissues utilized for retrieval of MSCs from
human are bone marrow (BM), peripheral blood (PB), dental pulp (DP) and adipose tissue
(AT) and neonatal birth-associated tissues including placenta (PL), Wharton’s jelly (WJ),
umbilical cord blood (UCB) and amniotic fluid [5]. MSCs were initially identified in BM in
late 1960s [6]. BM is the prime source used for MSCs based therapeutic interventions. MSCs
play a significant role in structural and functional aspects of BM niche. The major function of
these cells is to generate a tissue framework, which pledges a perfunctory support for
hematopoietic tissue organization [7]. However, MSCs constitute merely 0.002% of total
stromal cell population of BM. Currently, bone marrow-derived human MSCs (BM-hMSCs)
is considered as the safest tissue to use for the purpose of transplantation rather than other
convectional sources [8]. This may be due to source of collection of BM and harvesting of
BM is undertaken following a routine and standardized procedure. BM-hMSCs have been
extensively studied over many years and are used in the majority of hMSC clinical studies
and trials most notably, fracture non-union, musculoskeletal, vascular and reticulo-
endothelial and bladder diseases.
To date, the secondary tissues like UCB, placenta, amonotic fluid and WJ associated
with reproductive physiology is recognized as other potential sources of MSCs. The UCB
contains at least two distinct populations of stem cells i.e. hematopoietic stem cells (HSCs)
and non-hematopoietic stem cells including MSCs. In humans, the origin of amniotic fluid
cells is still debatable. Diversified stem cell population have been isolated and characterized
from amniotic fluid expressing both HSCs and MSCs markers [12, 13, 14]. A limited number
of MSCs is present in PB, as it is mixed with hematopoietic stem cells (HSCs) and other
immature progenitor cells [15]. It is clinically proven that MSCs derived from PB, an
effective cellular source to treat stroke and ischemia [16]. Adipose tissue is most derived
from the mesoderm during embryonic development. It contains stromal cell population that

includes microvascular endothelial cells, smooth muscle cells and stem cells.
Macroscopically, at least five different types of adipose tissue such as bone marrow, brown,
mammary, mechanical and white exist [17]. The lipoaspirate, collected from these tissues, are
rich source of MSCs. The net content of MSCs (ADSCs) present in stromal vascular fraction
(SVF) is 2%. However, painful surgical procedure is involved to collect these tissues from
donors. Development of effective procedure of isolation, expansion, characterization and
cryopreservation with a profound significance for therapeutic intervention of MSCs (Fig 2).
Fig 2: Therapeutic intervention of MSCs from bench to bed side
Dental tissues are different from other bony tissues as these do not undergo
continuous remodeling [18]. Interestingly, immature wisdom teeth, periodontal ligament, and
exfoliated deciduous teeth are emerged as a potential source of autologous MSCs that can be
used for tissue regeneration via tissue engineering and clinical research to treat neuronal
disorders, accidental brain injury, ischemic heart diseases, bone repair and cosmetology [19].
The long-term preservation of dental tissue after tooth extraction, creates a novel commercial
model in dentistry, as well as provides greater access to autologous MSCs for patients at the
time of need. The procedure followed for isolation, expansion and cryopreservation of DPSCs
from human (Fig. 3).

Collection of exfoliated deciduous tooth
Mechanical removal of dental pulp
Immersion in freezing medium
Isolation of cells (enzymatic treatment)
BA 35
in vitro expansion
Transfer to liquid nitrogen
Cryo-DPSCs Fresh-DPSCs
Thawing
Fig. (3). Isolation and cryopreservation of MSCs derived from dental pulp
4. MSCs IN CLINICAL RESEARCH

There are currently more than 500 clinical trials where MSCs are used for therapeutic
purposes and more than 35% are using cryopreserved cells [20]. An elegant review published
in 2013 [21] report that, MSCs become a successful component to treat of many diseases,
including both immune diseases and non-immune diseases (Fig.4).

Myocardial infarction
Diabetes
Spinal cord injury
Crobn's diseases
Aplastic anemia
Rhematoid arthritis
Brain injury
Graft versus host disease
Liver cirrhosis
Osteoarthritis
Multiple sclerosis
Systemic lupus
Parkinson’s disease
Other
Fig 4: Percentage of immune and non-immune diseases currently treated with MSCs [21]
5. CRYOPRESERVATION OF MSCS DERIVED FROM DIVERSE SOURCE

Cryopreservation of MSCs derived from different sources must follow regulations and
guidance set up by agencies such as the Food and Drug Administration (FDA) [3]. Briefly ,
translation of MSCs from bench side to bed side must involves the following steps Good
Manufacturing Practices (cGMP) monitoring, keeping backup, cGMP record-keeping
inventory and proper identification and cGMP in reuse of samples and rejecting of surplus
[8]. Many factors are taken into consideration while preserving MSCs. For development of an
optimize cryopreservation protocol of MSCs there is a need to standardize cell concentration,
type of CPAs, concentration of CPAs, freezing procedure and thawing.
5.1. RECORD KEEPING
It is noteworthy to keep records of some useful information such as (i) date and time of
collection of sample, (ii) donors’ name, and (iii) source of collection. The donors have to
satisfy certain conditions like (i) age (10-65 years), (ii) weight (> 35 Kg), and (iii) no severe
health complications. A successful transplantation results due to (i) safe tissue aspiration, (ii)
proper transportation of the tissue sample at appropriate temperature to cell therapy
processing facility centers, (iii) in vitro expansion, (iv) pre-cryostorage processing, (v)
freezing, (vi) storage duration, (vii) thawing procedure, and (viii) post-thaw processing [22].
When samples are collected from remote areas, these need a convenient transportation system
to increase cell retention. Even if samples are collected in medical center, these need to be
stored properly till processing. Lowering the temperature slows cellular metabolism and
minimizes the cell damage. The most recommended temperature for transportation of
samples to the site of manufacture and final product (MSCs and MSC-like cells) to the place
of administration is 4°C [23, 24]. This may be due to the fact that this temperature has
prevailing effect on cellular metabolism and specifically inhibits bacterial growth while
maintaining functional viability of MSCs. However, the number of mononuclear cells
gradually decreases during transportation in a time dependent manner.

5.2. OPTIMUM CONCENTRATION OF CELLS

Expanded MSCs derived from different sources must be harvested from log phase of culture
to achieve minimal cell concentration 0.5–1.0×106 cell/ml required for cryopreservation.
Several studies enumerate that if higher cell concentration is used at initial time of
cryopreservation, it always yields higher post-thawed cell survival percentage (%) with good
functional outcome and engraftment kinetics. Most of the reports suggest that harvesting the
cells from log phase may fulfill this requirement, which is most frequently achieved between
1st to 5th passages. Simultaneously, high cell concentration minimizes the total infusion
volume and may decrease the risk of dimethyl sulphoxide (DMSO) - related complications. Is
it due to distributive effect of DMSO on each cell? The paucity of MSCs in UCB led
Koliakos et al. [25] to develop a novel double-processing protocol for enrichment of MSCs
during transplantation. Recently, an effective method was developed to enrich PBMSCs from
PB [26].
5.3. CRYOPRESERVATION MEDIUM
Medium in which cells are going to cryopreserved is still an active area of research for MSCs
[27, 28]. A suitable cryoprotective medium should be nontoxic for cells and patients, non-
antigenic and chemically inert. Generally, DMSO at 10% concentration is used as standard
for preservation of stem cells [29]. The study by Liu et al. [30] confirmed that when 10%
DMSO is used as CPA for cryopreservation of ADSCs no adverse consequence was observed
on the phenotype, proliferation and osteogenic differentiation potential. A similar report was
obtained when BM-MSCs cryopreserved with 10% DMSO. The post-thawed viability was
72.95±/-6.14% and even BM-MSCs maintain the stemness after one year storage in nitrogen
liquid (LN2) [31]. When Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) were
cryopreserved using 10% DMSO, 10% polyvinylpyrrolidone (PVP) and a cocktail solution
comprising of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol, the post-thaw cell
viability was maximum (81.2±0.58%) with 10% DMSO in comparison to PVP
(62.87±0.35%) and cocktail solution (72.2±0.23%) respectively [32]. However, DMSO is
toxic to cells. Exposure to DMSO sometimes causes unexpected changes in cell fate. The
potent effect of DMSO on the epigenetic profile and to induce differentiation is well
documented in murine stem cells [33]. If DMSO is not thoroughly washed from cells before
administration, it can induce severe complications in patients like nausea, vomiting,
tachycardia, bradycardia, and hypotension. Nausea and abdominal cramping are observed
with an incidence upto 70% [34]. Exposure to DMSO induces genetic instability which may
further complicate the patient’s condition by forming tumor after transplantation. However,
toxicity of DMSO is concentration, time and temperature dependent.
Another limitation of current cryopreservation protocol is the use of fetal bovine
serum (FBS) and fetal calf serum (FCS). This is an essential supplement that needs to be
present in the formulation of freezing medium/vitrification solutions and thawing/warming
solutions. This highly proteinaceous agent FBS or FCS can protect cellular architecture and
function efficiently from cryogenic injury. On the other hand, it always accompanied with
risk of infection or allergic reaction in patients due to its xenobiotic origin. Thus, it is not
advisable to preserve the cells in FBS/ FCS used for transplantation and cell-based therapy.
So, development of a xeno-free medium containing non-toxic CPAs is highly desirable for
MSCs that has to use in regenerative medicine. Investigations continue, but the main focus is
to eliminate or reduce the concentration of DMSO and avoid use of animal serum in hMSC
cryopreservation protocols for clinical safety. hBM-MSCs cryopreserved alongwith
formulated cryopreservation medium containing 10% FCS and 5% DMSO has shown
approximately 90% viable MSCs after 0.3 to 37 months of cryo-storage [35]. Thus,
recovered cells exhibit similar characteristics with that of non-cryopreserved MSCs such as
(i) negative for hematopoietic cell markers such as CD14, CD34, CD45 and HLA-DR (ii)

positive for mesenchymal characteristics such as CD29 and CD105. These cells retain ability
to differentiate into mesodermal osteogenic lineages, characterized by alkaline phosphatase
(ALP) activity and in vitro mineralization. This study further suggests cryopreserved MSCs
could be applied for bone regeneration, facture non-union and to treat other orthopedics
complication like non-cryopreserved MSCs. Other investigators have also used similar type
composition for preservation of rat and human MSCs isolated from bone marrow [36, 37]. To
formulate a non-toxic or less toxic cryoprotective medium, the concentration of classical
CPA “DMSO” should be reduce or omitted from formulation. There are reports where
DMSO was replaced with sucrose or trehalose. The reduced concentrations of DMSO 7.5, 5
and 2.5% (v/v) were tried by Liu et al. [38] in a combination with polyethylene glycol (PEG)
or trehalose or 1, 2 propanediol to cryopreserve hBM-MSCs in serum-free condition. The
post-thawing viability of hBM-MSCs is 82.9 ± 4.3% in 7.5% DMSO (v/v), 2.5% PEG (w/v),
and 2% bovine serum albumin (BSA) (w/v) was comparable with that obtained in
conventional 10% DMSO (82.7 ± 3.7%), following a slow cooling rate (1°C/min). In
addition, this experiment inferred that combination of 5% DMSO (v/v) with 5% PEG (w/v)
and 7.5% 1, 2-propandiol (v/v) with 2.5% PEG (w/v) may be better alternative to 10%
DMSO to cryopreseve hBM-MSCs when 2% albumin (w/v) is present in cryopreservative
medium. Combination of mannitol, lactose, sucrose, trehalose or raffinose on cultivation and
cryopreservation results significant increase of viability of MSCs. The mechanism by which
disaccharides preserve the functional activity and archetypal structure of biological systems
during freezing and drying is not well understood. This may be due to the fact that they offer
protection from exterior part which further facilitates their removal after thawing. The
demerit of using these extracellular agents is that most of them do not prevent the formation
of ice crystals within the cells [39].
In an investigation polyvinylpyrrolidone (PVP), an extracellular cryoprotectant has
been used as an alternative to DMSO in cryopreservation medium and FCS [40]. Another
study conducted to cryopreserve unfractionated BM cells alongwith 5% DMSO and 6%
hydroxyethyl starch (HES). Significant yield of post-thawed cells and reduce number of
clumping after thawing was observed in DMSO/HES mixture in contrast to those in DMSO
alone [41]. In a study, cryopreservation of hBM-MSCs was performed with animal product-
free CryoStor medium containing 2%, 5%, and 10% dimethyl sulfoxide (DMSO) using a
programmable cell freezer [42]. The viability rate was 80% after freezing in CryoStor
solutions containing 10% DMSO after 5 months storage in liquid nitrogen. The recovered
cells were analyzed by measuring cell recovery, viability, apoptosis, proliferation rate,
expression of a broad panel of MSC markers, and osteogenic differentiation. It was observed
that cryopreservation did not alter intrinsic nature of BM-MSCs such as proliferation rate,
expression of MSCs markers and alkaline phosphatase (AP) activity. Development of a bio-
inspired ectoin solution helps to protect the functions of MSCs from cryo-damage [43].
Further, this study demonstrates the use of ectoin with serum-free cryo-medium to preserve
hMSCs by following a slow freezing protocol results 72% post-thaw viability. This DMSO-
free and serum-free approach of cryopreservation is suppose to be more suitable for clinical
therapy. Recently, in another study, when sugars like mannitol, lactose, sucrose, trehalose or
raffinose are added at concentration 300 mM into incubation medium for 24 hours prior to
cryopreservation and 200 mM sugars is present in cryoprotective solution, these combination
in medium increases the post-thaw viability and also store the inherent characteristic of
multilineage differentiation towards osteogenic and adipogenic lineages of MSCs [44].
Autologous serum can be taken as alternative to animal serum as it completely
eliminates the risk of transmission of infectious disease and also maintain the inherent
property of MSCs expansion and differentiation [45, 46]. However, cryopreservation of
MSCs with autologous serum carries its own limitation, for not being cost effective and not

patient friendly (requires a preoperative blood donation by the patient). Such limitations
restrict the use of autologous serum to small clinical studies and their off-the shelf use in
future stem cell therapy [47]. Human albumin (HA) and other proteins can be used in place of
serum to cryopreserve MSCs without loss of cell survival and proliferation potential.
Albumin having the advantage of being a single protein further reduces the complexity and
may eliminate the inaccessibility that occurs due to undefined factors present in FBS or FCS.
Derived from the same source (human), it further reduces the rejection or adverse conditions
arising due to transplanted cells.
The addition of neuropeptides (protein from vasoactive intestinal peptide/glucose-
dependent insulinotropic peptide/pituitary adenylate cyclase activating polypeptide family)
increases cell survival and proliferation rates of post-thaw MSCs [48]. “Sericin” a sticky
protein derived from the silkworm cocoon was able to replace FBS from the composition of
freezing medium of MSCs [49]. This study reported that 1% sericin and 10% DMSO in
freezing medium is suitable to cryopreserved primary cultured hMSCs. The sub-confluent
MSCs cultures obtained from UCB have been cryopreserved alongwith 10% human serum
(HS), 10% DMSO, 20% hydroxyethyl starch retain their functional activity and cytogenetic
status and morphologic characteristics of MSCs [50, 51]. On the contrary, Moll et al. [52]
reported that freeze‐thawed MSCs have shown impaired immunomodulatory and blood
regulatory properties. Freeze‐thawed MSCs responded to a lesser extent to pro‐inflammatory
stimuli and also produces less anti‐inflammatory mediators. These have increased triggering
of the instant blood mediated inflammatory reaction (IBMIR) and a strong activation of the
complement cascade compared to fresh cells. These changes seem to affect therapeutic
efficacy in treatment of immune ailments after hematopoietic stem cell transplantation.
As freshly collected adipose tissue is not always readily available, there will be a
need for improved cryopreservation methods to maintain the reproducibly and
multipotentiality of ADSCs after long-term storage. Cryopreservation of ADSCs conducted
by using 10% DMSO is able to preserve membrane integrity and colony forming ability [53].
Further, this study demonstrate that the post-thaw viability is a function of storage
concentration of cells and an optimal post-thawed viability was observed @ 0.5 x 106
cells/ml. ADSCs successfully cryopreserved using 10% DMSO as CPA without losing its
proliferative ability and multipotent nature after long term storage [30]. The cryopreserved
MSCs were differentiated to chondrocytes confirmed by Alcian Blue or Safranin O staining
[54]. However, in another study when ADSCs preserve in 10% DMSO and 90% FBS
displayed low adipogenic and osteogenic differentiation in contrast to fresh cells [55]. This
may be due to the fact that cryopreservation down-regulated the expression of adipogenic and
osteogenic genes. Therefore, before infusion in to patients, it is essential to determine and
compare the effects of cryopreservation on gene expression of differentiation capacity and
stemness among MSCs preserved in various CPAs. This will provide deep insight into
molecular changes that may occur in cells following cell freezing. However, to date 10% v/v
DMSO is used in all the cryopreservation protocol of ADSCs.Cryopreservation of amniotic
fluid-derived stem cells (AFSCs) was investigated by using disaccharides, antioxidants and
caspase inhibitors in combination with a reduced concentration 2.5% (v/v) of DMSO [56].
Here, the reduce concentration of DMSO than convectional used concentration did not affect
post-thawed cell viability and the cells retain their stemness as evaluated by expression of cell
surface antigens, mRNA expression of stem cell markers. This may be due to the fact that
addition of general caspase and calpain inhibitors can reduce the apoptosis rate thereby
achieving increased cell viability although the exact role of the natural biooxidant like
catalase is still not known [34]. In most of the published cryopreservation protocols for
DPSCs, the freezing medium contains 10% DMSO in FCS [57-59]. The specialty of DPSCs
is that these need to be isolated within 120 h after tooth extraction [60]. When human

exfoliated deciduous teeth (SHED) were preserved in freezing medium containing 10%
DMSO and 90% FBS and cells were recovered after two years. The recovered cells retained
stem cell properties including clonogenicity, self-renewal activity, stem cell marker
expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory
function. These cryo-recovered cellular modules (SHED) able to improve the
pathophysiological condition of bone defect model-immunocompromised mice, SLE-like
disorder and nephritis-like renal dysfunctions upon transplantation [61]. A study was
conducted in rat model to estimate whether transplanted DPSCs are able to ameliorate
diabetic polyneuropathy lesion. It was noticed that transplantation of cryo-DPSCs is able to
improve nerve conduction velocity and nerve blood flow in diabetic rats [62]. This may be
due to the fact that DPSCs express angiogenic genes, such as bFGF and VEGF.
Transplantation of DPSC into the hind limb of skeletal muscles causes increased micro
vasculatures in the skeletal muscles accompanied with an increase in the sciatic nerve blood
flow at the transplanted side. Isolation of DPSCs is laborious, time-consuming and expensive,
especially while employing for clinical use of the cells. Recently, Nd:YAG laser beam (laser
piercing) is being used for digging micro-channels into the tooth to extract dental pulp for
cryopreservation study [63].
The addition of Rho-associated kinase (ROCK) in the freezing medium enhances
viability of hESCs [64, 65]. Recently, it was observed that MSCs also behave similarly like
hESCs in presence of ROCK inhibitor Y-27632 [37]. The supplementation of ROCK
inhibitor Y-27632 had no significant effect on the immediate post-thaw viability. However, it
is observed that it could enhance adherent viable cells (MSCs) when added to post-thaw
culture media and differentiated easily towards neuronal cells. Both these results explain that
ROCK inhibitor plays significant role in inhibiting apoptotic pathway mediated by caspase 8
and caspase 9 without inhibiting their differentiation capacity.
5.4. DIFFERENT FREEZING PROTOCOLS
Rate of freezing plays decisive role in estimation of post-thaw viability of all biological
samples. Many groups tried to preserve MSCs derived from BM and CB by using slow
freezing protocols utilizing DMSO as cryoprotectant, following same protocols which were
primarily used for haematopoietic analogues [3]. Cellular recovery and differentiation
capacity of MSCs has been studied in a number studies following different freezing
approaches. The freezing rate of 1°C/min is suitable to preserve large number of MSCs in one
vial with the required CPA at low concentrations (<1.5M). Further, there is no direct contact
between cells and nonsterile liquid nitrogen during the slow freezing process which
significantly reduces the potential risk of contamination during clinical application [66]. A
slow freezing method has been employed to cryopreserved in vitro expanded BM-MSCs
using DMSO as cryoprotectant (CPA). The freezing program was the cells were incubated at
4°C for 10 min, then cooled to −80°C at a rate of 1°C/min in a controlled-rate freezer and
finally stored at −196°C. After 12 months, the cells were recovered and the viability at the
time of thawing ranged from 84.6% to 100%. The recovered cells are resuscitated and
cultured for 15 passages. The post-thawed cultured cells retain the MSCs–like properties as
verified by differentiation ability to adipocytes and neurocytes. Further, these differentiated
cells are characterized basing on morphological evidences, immuno-cytochemistry and RT-
PCR techniques. This study inferred that BM-MSCs cryopreserved with DMSO following
slow freezing protocol retain their biological properties and can be used as therapeutic tools
[67]. It was demonstrated that application of faster cooling rate (10°C/min and 100°C/min)
with increased concentrations of DMSO significantly drop-off viability [68]. To improve
cryopreservation efficiency, different programmable freezing protocols are employed using
costly controlled-rate freezer [34]. A study was conducted by Fuller and Devireddy to
compare the effect of two different freezing methods (controlled rate freezing and directional

freezing) on the membrane integrity of ADSCs by using 10% DMSO [69]. The survival % of
post-thawed cells was significantly lower in the directional freezing in contrast to controlled
freezing. This observation hold strongly the fact that alongwith thermal conditions imposed
during freezing, the choice of CPA, the storage concentration and the method of freezing are
important factors in the development of optimal cryopreservation protocol of ADSCs. Use of
‘‘Cell Alive System’ (CAS) further reduce the risk of freeze injury to MSCs is an additional
technical advantage. This magnetic cryopreservation has been successfully employed in tooth
banking with satisfactory implantation outcomes, suggesting that the method preserves
human periodontal ligament cells and dental pulp stem cells [70].
Vitrification is another way followed to preserve MSCs using high cooling rate and
high concentration of cryoprotectants. Another study compared vitrification and programmed
freezing cryopreservation conducted via an optical-differential scanning calorimetry (DSC)
system for cryopreservation of UCB derived MSCs using DMSO-free CPA solutions [71].
Here, the freezing medium contains CPAs ethylene glycol (EG), 1, 2-propylene glycol (PG)
and sucrose are used as basic CPAs, supplemented with addition of polyvinyl alcohol (PVA).
It was observed that the viability of thawed UCB-derived MSCs is enhanced from 71.2% to
95.4% in the presence of PVA for vitrification, in contrary to < 10% to 45% of viability
found for programmed freezing. This may be due to the fact that most UCB-derived MSCs
shrink and lost their membrane integrity after freezing thawing of cryopreservation by
programmed freezing, which was suggested as major cause of the ice crystals formation
during freezing. PVA seems to alleviate cell injury by inhibiting ice crystal formation. These
results suggest the vitrification in combination with the dimethyl sulfoxide free CPA
solutions supplemented with PVA is an effective protocol for the cryopreservation of UCB-
derived MSCs. Other contemporary studies recommend hydroxylethyl starch (HES) may be a
better substitution of DMSO in freezing medium [72]. There is scanty amount of MSCs
present in PB, to avail for clinical practices. Studies using a 6% HES + 5% DMSO solution
without using rate-controlled freezing, usually show superior cryopreservation in comparison
to 10% DMSO when cryopreserved at -80°C [73]. Another study was undertaken by using
controlled rate freezer where comparison is made between the convectional freezing medium
(FHCRC cryopreservation medium) with non-convectional preservation solution (CryoStor)
and Cryomed 1010 for PBSC samples. Here the samples are cooled @ 1°C/minute until heat
of fusion, then @ 1°C/minute until −40°C is achieved and then @ 10°C/minute until −90°C
then transferred to vapor phase of LN2. It was observed that MSCs derived from
cryopreserved PBSC products able retain the MSCs like properties in CryoStor medium
significantly high compared to the standard well recognized FHCRC cryopreservation
medium [74]. Three-dimensional culture of MSCs, encapsulated in alginate microbeads is a
perspective direction of cell biology, tissue engineering and regenerative medicine. An
investigation was undertaken to compare the post-thaw viability of cryopreserved MSCs in
non-encapsulated cell suspension condition and within alginate microbeads. It was observed
that post-thawed viability of MSCs in non-encapsulated system was significantly higher than
of within alginate microbeads in presence of 5% DMSO. Whereas, no significant difference
observed in viability among these two insisted cellular systems with increasing DMSO
concentration up to 10% [75].
However, vitrification is seen to be a reliable and effective method for
cryopreservation of human amnion-derived mesenchymal stem cells (HAM) The study
followed a two-step vitrification protocols. Two types of freezing medium were used such as
equilibration and vitrification solution. The equilibration solution contains 20% ethylene
glycol (EG) and the vitrification solution contains 40% EG, 18% Ficoll 70 and 0.3M sucrose.
The post-thaw viability of HAM after vitrification was 84.3 ± 3.2%. This may be
significantly attributed by components of vitrification solution. EG is the most commonly

used cryoprotectant in vitrification solution due to its low molecular weight and also less
toxic to cells. In addition, sucrose can significantly reduce the toxicity by decreasing the
concentration of penetrating CPAs required to constitute vitrificaiton solution. Ficoll 70, is a
macromolecule that promote permeation of cryoprotectants, seems to confer lower toxicity,
higher solubility and lower viscosity. The post-thawed HAM showed similar characteristic of
MSCs like fresh MSCs (control) [76]. Since, HAM is an easily accessible and ethically
acceptable source of cells so cryopreserved HAM may be a suitable source for regenerative
medicine and cell-based therapy. A recent study demonstrates that significant (p < 0.05)
higher cell survivability of Wharton’s jelly mesenchymal stem cells (WJMSCs) was achieved
using cocktail cryoprotectants (10% DMSO + 0.05 M glucose + 0.05 M sucrose+1.5 M
ethylene glycol) using programmed freezing protocol than sole DMSO and conventional
method of freezing [77].
The studies have shown that MSCs derive from diverse sources can be
cryopreserved in different ways using different cryoprotectants and employing various
cooling rates, storage periods, and temperature without compromising their proliferation
potential, phenotypic characteristics and capacity for differentiation (Table 1).
Table 1. Studies on cryopreservation of MSCs derived from various sources of human
Tissue CPA used Cooling protocol Storage Duration Significant finding References
Source temperature of storage
Bone 10% DMSO Unspecified -196°C 24h The growth kinetics, self-renewing capacity and Bruder et al. [78]
marrow (liquid N2) the osteogenic potential of purified MSCs did not
affect by cryopreservation.
10% DMSO -1°C/min in -70°C -196°C 7 days Cryopreservation had no effect on osteogenic Rust et al. [79]
freezer (24h) (liquid N2) differentiation or growth.
5% DMSO Controlled rate Indefinite MSCs can either be frozen in cryovials or in Haack-Sorensen et al.[80]
freezing (1°C/min -196°C freezing bags together with cryopreserve solutions
from 4°C to 0°C; (liquid N2) containing dimethyl sulfoxide (DMSO) using
2°C/min to -45°C; controlled rate freezing.
5°C/min to -
100°C)
10% DMSO 1°C/min -196°C Not The cooling rate of 1°C/min helps to maintain cell Xu et al.[81]
(liquid N2) specified morphology and attachment, integrity and
uniformity of filamentous actin, and leads to better
cell recovery after cryopreservation.
10% DMSO 1°C/min in -196°C Not Cryopreservation affects cells attached to different Xu et al.[82]
controlled rate (liquid N2) specified substrates and they respond differently to cells in
freezer from 4°C suspension.
to -80°C
Amniotic DMSO or In the -196°C 3 or 6 DMSO gave best result. Time programmed and Janz et al.[83]
fluid glycerol (5% nonprogrammed (liquid N2) months nonprogrammed freezing methods could be used
or time freezing for successful AFSCs cryopreservation for 6
10%);sucrose protocol, (−20°C) months.
(30 or 60 mM); for 20 min
trehalose (60 followed by
or 100 mM) −80°C freezing
for 12–16 h and
programmed time
freezing protocol
1°C/min at −60°C,
then 3°C/min at
−100°C.
Teeth 10% DMSO or 4°C for 1h; -20°C -196°C 6 months Cryopreservation does not affect the biological Ding et al. [84]
10% glycerol for 2h; -80 °C (liquid N2) and immunological properties of stem cells from
or 10% overnight apical papilla (SCAP), supporting the feasibility of
ethylene glycol SCAP cryopreservation in nitrogen.
Adipose Different -20°C for 30 min - -196°C 1 or 6 or Cells stored using slow cooling in 6% threalose, De Rosa et al. [85]
tissue combination of 80°C for 1h (liquid N2) 12 months 4% dimethyl sulfoxide, and 10% fetal bovine
DMSO and serum retain all the functional properties.
trehalose or
10% DMSO

6. THAWING AND POST-THAW VIABILITY ASSESSMENT

Available literatures suggest that rapid thawing rate (>100°C/min) can prevent damaging ice
crystals during re-crystallization. So rapid thawing is the optimal choice and generally results
best post-thaw recovery and viability of cells [3]. The standard method is warming in a water
bath at 37°C until all ice crystals disappear. Cryopreservation studies with early passage
MSCs, however, indicate no significant variation in immediate post-thaw viability when the
frozen samples are thawed at 10°C/min in controlled rate freezer or in waterbath at 37°C
(>100°C/min). However, according to guidelines of cGMP, the thawing should be performed
under defined aseptic conditions. Because of the potential contamination of the water-bath
with microorganisms and risk of subsequent transmission to patients, it might be safer to
thaw cell suspensions in a dry environment. So Röllig et al.[86] invented a new method of
thawing for cryopreserved mobilized peripheral blood by using an electric dry-warming
device containing warmed gel pads (Sahara, Transmed). The post-thaw viability of the cells
was same as that of when thawing was performed in a water bath (37°C). This thawing
procedure may potentially decrease the risk of bacterial contamination caused by waterbath.
Post-thaw viability assessment is a critical parameter in clinical transplantation. So
choosing an appropriate viability measurement assay and tool is essential to evaluate the
quality and quantity of post-thawed cells. Dye exclusion, apoptosis/necrosis
(annexin/propidiumiodide staining) and clonogenic potential (CFU-E plus BFU-E, CFU-GM)
of the cells are some of the viability assessment tests used to determine survival percentage
after thawing. Trypan blue dye exclusion is the most commonly used test owing to its ease
and quickness. However, this test is observer dependent and sometimes overestimates the
viable population. The transfusion medicine should not rely on such observation. According
to some reports fluorescent dyes are more accurate and reliable indicators of cell viability.
Fluorescent dyes clearly discriminate between apoptotic, necrotic and live cells present in
post-thaw cell population. A significant question is whether it is possible to revive the
apoptoptic and necrotic cells. However, it is now possible to isolate pure MSCs from
particular fraction of post-thaw samples using fluorescence activated cell sorting (FACS)
technique, that can be used for the purpose of transplantation [87, 88].
7. CONSIDERATION OF INFECTIOUS AGENTS

Beside all these, the contamination of stem cell products with infectious pathogens can occur
at several points during the stem cell processing. The marrow harvesting, cord blood
collection or apheresis, the transport of the product to the cryopreservation facility, the pre-
thaw processing, thawing and washing process as well as the infusion of the final product are
all points with the potential for microbial contamination. Contamination during these
processes is observed in varying degrees, generally 0-4.5% for PBSCs and upto 26% for bone
marrow, even if strictly aseptic protocols are followed [89]. Alongwith this, bacteria, fungi
and viruses are able to survive in LN2 storage phase and cross contamination between
different units, stored in the same container have been observed [90]. If microbial
contamination present in other blood products, such as red blood cells, platelet concentrates
or plasma products are detected, these are usually discarded. However, in case of stem cell
concentrates, this decision is complicated since the total amount of stem cell is limited.
However, contaminating agents should be avoided by implementing cGMP and handling the
cells with experts when used for therapeutic purposes.

8. TRANSPORTATION AND BANKING OF MSCS

MSCs are developed therapeutic agents for many regenerative approaches [91]. The current
medicine requires surplus of MSCs for the purpose of autologous and allogenic
transplantation. So, there is serious need to set up MSCs banks for future use (Fig. 5).
MSCs CRYOBANK
Consent letter
I or we, Asha Singh
is signing this consent
Asha
Singh
Transportion to stem cell
Consultation Signature of donor Collection laboratory
Off-The-Shelf use in future
MSCs CRYOPRESERVATION STEM CELL LABORATORY

UNIT
Processing Expansion
BA 55 BA 55 BA 55 BA 55
Testing Manipulation
Fig 5:. Establishment of MSCs cryobank.
These banks have well equipped facilities, skilled personnel and device for
processing, storing and shipping of cellular products to end users. Shipping cryopreserved
samples plays another important role in the successful application of MSCs. The quality and
utility of the product must be maintained during transfer from cell banks to clinics. Shipment
tracking is essential, knowing where the sample is at any given time. Adult stem cells that are
stored by using slow freezing are generally shipped on dry ice [3]. Vitrified cells need to be
transported at temperature below its devitrification temperature. The transported cells must
carry all the documents giving the necessary information regarding how they are processed,
passage history, its origin and provenance for traceability, actual cell number delivered,
cryopreservation method employed, compatibility with the device used for shipping,
confirmation of authenticity and free from mycoplasma and biohazard information. These
further facilitate the delivery of stem cells to patients and improve the efficacy of translation..
Thus, establishment of cryo-banks not only provide suitable cell source to recipients and also
open new financial opportunities which certainly improve the economy of developed and

developing counties. Currently Obama administration to enhance stem cell research has been
recommended its activity as one of the pillars of his health agenda. The U.S. Army is
investing over $250 million in stem cell research to treat injured soldiers in a project called
Armed Forces Institute for Regenerative Medicine [60]. Thus, cryopreservation of MSCs
may be the alternative answer to many unsolved aliments of clinical research in next decade.
9. CONCLUSION
A large number of studies have been performed during recent decades to design
cryopreservation protocols. The allogenic and autologous therapeutic aspects of MSCs are
realized however, MSC based therapies require to fulfill standard and stringent regulations
declared by Government prior to clinical transition. Although, yet there is a lack of consensus
protocol to preserve MSCs. So, efforts are being taken worldwide to preserve this valuable
cellular product for future. Further research is suggested to understand the molecular
mechanisms associated with physical changes of cells and elevated chemical reactions that
cause damage during cryopreservation, so that it will be easier to nullify them.
10. ACKNOWLEDGEMENTS
Author is thankful to the Department of Science and Technology, Government of India, for
providing financial support under vide reference number SR/WOS-A/LS-13/2016 dated
06.09.2016 under Women Scientist Scheme to carry out this work. Author extends her thanks
to Ms Swati Singh for critical reading and editing of the manuscript. Author owes her thanks
to the Head, Post-Graduate Department of Zoology, Utkal University, Vani Vihar,
Bhubaneswar- 751 004 for providing the facilities.
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International Journal of Advanced Scientific and Technical Research Issue 7 volume 1, January –February 2017

Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954

CRYOPRESERVATION OF MESENCHYMAL STEM

Key words: Mesenchymal stem cells, Off-the-shelf, Cryopreservation, Clinical utilities.

©2017 RS Publication, rspublicationhouse@gmail.com Page 435

Cell- or tissue- directed attraction or stimulation

Cytokine production with para and autocrine receptor activation

Immunosuppression No teratoma formation

Fig 1: Characteristics of MSCs

These cells after therapeutic interventions influence the connective tissue

©2017 RS Publication, rspublicationhouse@gmail.com Page 436

©2017 RS Publication, rspublicationhouse@gmail.com Page 437

Fig 2: Therapeutic intervention of MSCs from bench to bed side

©2017 RS Publication, rspublicationhouse@gmail.com Page 438

Collection of exfoliated deciduous tooth

Mechanical removal of dental pulp

Immersion in freezing medium

Isolation of cells (enzymatic treatment)

Transfer to liquid nitrogen

4. MSCs IN CLINICAL RESEARCH

©2017 RS Publication, rspublicationhouse@gmail.com Page 439

5. CRYOPRESERVATION OF MSCS DERIVED FROM DIVERSE SOURCE

©2017 RS Publication, rspublicationhouse@gmail.com Page 440

5.2. OPTIMUM CONCENTRATION OF CELLS

©2017 RS Publication, rspublicationhouse@gmail.com Page 441

©2017 RS Publication, rspublicationhouse@gmail.com Page 442

©2017 RS Publication, rspublicationhouse@gmail.com Page 443

©2017 RS Publication, rspublicationhouse@gmail.com Page 444

©2017 RS Publication, rspublicationhouse@gmail.com Page 445

©2017 RS Publication, rspublicationhouse@gmail.com Page 446

6. THAWING AND POST-THAW VIABILITY ASSESSMENT

7. CONSIDERATION OF INFECTIOUS AGENTS

©2017 RS Publication, rspublicationhouse@gmail.com Page 447

8. TRANSPORTATION AND BANKING OF MSCS

I or we, Asha Singh

is signing this consent

Off-The-Shelf use in future

MSCs CRYOPRESERVATION STEM CELL LABORATORY

Fig 5:. Establishment of MSCs cryobank.

©2017 RS Publication, rspublicationhouse@gmail.com Page 448

©2017 RS Publication, rspublicationhouse@gmail.com Page 449

[11]. S. Karahuseyinoglu, O. Cinar, E. Kilic, F. Kara, G. G. Akay, D. O.

©2017 RS Publication, rspublicationhouse@gmail.com Page 450

[25]. G. Koliakos, D. H. Alamdari, N. Tsagias, K. Kouzi-Koliakos, E

©2017 RS Publication, rspublicationhouse@gmail.com Page 451

cryopreservation procedure efficiency. Transplantation Proceedings., Vol.

©2017 RS Publication, rspublicationhouse@gmail.com Page 452

[49]. M. Verdanova, R. Pytlik, and M. H. Kalbacova, Evaluation of sericin

©2017 RS Publication, rspublicationhouse@gmail.com Page 453

©2017 RS Publication, rspublicationhouse@gmail.com Page 454

cells using combinations of hydroxyethyl starch and dimethylsulfoxide,

©2017 RS Publication, rspublicationhouse@gmail.com Page 455

[85]. A. De Rosa, F. De Francesco, V. Tirino, G. A. Ferraro, V. Desiderio,

©2017 RS Publication, rspublicationhouse@gmail.com Page 456

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