Review (unsolicited)

Cell Transplantation
Volume 30: 1–12
Cryopreservation: An Overview ª The Author(s) 2021
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of Principles and Cell-Specific DOI: 10.1177/0963689721999617
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Considerations

David Whaley1, Kimia Damyar1, Rafal P. Witek2, Alan Mendoza2,

Michael Alexander1, and Jonathan RT Lakey1,3

Abstract
The origins of low-temperature tissue storage research date back to the late 1800s. Over half a century later, osmotic stress
was revealed to be a main contributor to cell death during cryopreservation. Consequently, the addition of cryoprotective
agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), or propylene glycol (PG), although
toxic to cells at high concentrations, was identified as a necessary step to protect against rampant cell death during cryo-
preservation. In addition to osmotic stress, cooling and thawing rates were also shown to have significant influence on cell
survival during low temperature storage. In general, successful low-temperature cell preservation consists of the addition of a
CPA (commonly 10% DMSO), alone or in combination with additional permeating or non-permeating agents, cooling rates of
approximately 1 C/min, and storage in either liquid or vapor phase nitrogen. In addition to general considerations, cell-specific
recommendations for hepatocytes, pancreatic islets, sperm, oocytes, and stem cells should be observed to maximize yields.
For example, rapid cooling is associated with better cryopreservation outcomes for oocytes, pancreatic islets, and embryonic
stem cells while slow cooling is recommended for cryopreservation of hepatocytes, hematopoietic stem cells, and
mesenchymal stem cells. Yields can be further maximized by implementing additional pre-cryo steps such as: pre-incubation
with glucose and anti-oxidants, alginate encapsulation, and selecting cells within an optimal age range and functional ability.
Finally, viability and functional assays are critical steps in determining the quality of the cells post-thaw and improving the
efficiency of the current cryopreservation methods.

Keywords
cryopreservation, cryoprotectants, low temperature banking, freezing

Introduction cooling and thawing rates. In addition, unique aspects of

hepatocytes, pancreatic islets, sperm, oocytes, and stem
The origins of low-temperature tissue storage research date
cells, as well as current methods in their preservation, and
back to the late 1800s. Since then, numerous advancements
some novel agents and potential biomaterials that can be
related to cryopreservation have been made and protocol
optimization continues to be an area of active research.
Cryopreservation allows the banking of a large number of
cells and tissues that can be utilized for scientific research 1
Department of Surgery, University of California Irvine, Orange, CA, USA
and medical applications including hepatocyte and pancrea- 2
Ambys Medicines, South San Francisco, CA, USA
tic islet transplantation, blood transfusion, bone marrow 3
Department of Biomedical Engineering, University of California Irvine,
transplantation, artificial insemination, and in vitro fertiliza- Irvine, CA, USA
tion. For these modes of treatment to be successful, however,
Submitted: January 28, 2021. Revised: January 28, 2021. Accepted: February
large quantities of the desired cell type must be kept on 12, 2021.
standby and be available for use on-demand which is made
possible through the process of cryopreservation. This Corresponding Author:
Jonathan RT Lakey, PhD, MSM, Professor, Department of Surgery and
review will discuss the history and basic physical principles Biomedical Engineering, University of California Irvine, 333 City Blvd
of cryopreservation, including: permeating, and non- West, Suite 1600, Orange, 92868, CA, USA.
permeating cryoprotecting agents, vitrification mixtures, and Email: [email protected]

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2 Cell Transplantation

used to enhance cellular viability beyond current reports are extracellularly, imposes the largest influence over harmful
discussed. biochemical, and structural changes that are thought to result
in unprotected freezing injury13. Two independent theories
exist that attempt to explain the harmful effects of freezing
History of Cryopreservation on cells: (1) ice crystals mechanically disrupt cellular mem-
Following the discovery of the microscope, Spallanzani branes thus making it impossible to obtain structurally-intact
observed that sperm could maintain mobility even when cells after thawing; and (2) deadly increases in solute con-
exposed to cold temperature conditions in 17761. Research centration occur to the remaining liquid phase as ice crystals
into the effects of cryopreservation on live tissue had its form intracellularly during cooling13. Whether the mechan-
roots in late 1800s when scientists used this technology to ical or osmotic effects of freezing dominate, the end result is
preserve both spermatozoa and red blood cells (RBCs). Dur- the same; unprotected cooling and thawing of cells is a pro-
ing this time, research demonstrated weaknesses in the pro- cess incompatible with life. To mitigate these effects, two
cess which caused inconsistent results and frequent protective actions must be carried out: use of a cryoprotectant,
infertility caused by early embryonic death. A breakthrough and selection of an appropriate cooling and thawing rate.
occurred in the 1950s when James Lovelock discovered that
the cryopreservation process caused osmotic stress in the cell
by instantly freezing the liquid which directly contributed to
Permeating Agents
the formation of ice crystals in RBCs2. In 1963, Mazur was A number of permeating agents (PAs) exist currently such
able to characterize that process when they demonstrated as glycerol (the first agent discovered), dimethyl sulfoxide
that the rate of temperature change within a cell-containing (DMSO), ethylene glycol (EG), and propanediol (propy-
medium controlled the movement of water across a cell lene glycol). The ability of each of these compounds to
membrane and thus the degree of intracellular freezing3. protect a cell from mechanical and osmotic effects of freez-
This together helped to improve the overall understanding ing depends on several properties. Permeating agents must
of the mechanism associated with the cryoprotective pro- be highly water soluble at low temperatures, able to easily
cess. During the 1980s, research surrounding the cryopreser- cross biological membranes, and ideally, be minimally
vation process revealed that the speed at which the freezing toxic13.
and thawing process occurred was the most important factor The structures of four common permeating agents of the
in determining the survivability of the cells4,5. It was demon- 100 that are known are represented in (Fig. 1). Their rela-
strated that small, slow increments in both the freezing and tively small size (typically less than 100 daltons), and some-
thawing processes prevented the rapid formation of ice crys- what amphiphilic nature allows them to easily penetrate cell
tals that increased membrane-bound solutes associated with membranes where they can exert their effects14. The struc-
early cell death6. Another initial advance in cryopreservation tures’ ability to hydrogen bond with water accounts for a
occurred in the late 1940s when researchers discovered that large portion of their protective effects. Normally, as water
the use of glycerol as a medium increased the survivability of freezes, the developing crystal structure excludes solutes as
spermatozoa in subfreezing (70 C) temperatures7. Using its lattice forms. Solutes are displaced to the diminishing
glycerol as a medium effectively served to protect the cells liquid phase which effectively increases the solute concen-
from rapid formation of ice crystal during the preservation tration to lethal levels within the cell. Because permeating
process. A commonly used cryoprotective agent currently cryoprotectants interact strongly with water through hydro-
employed is dimethyl sulfoxide (DMSO), which is added gen bonding, the freezing point of water is depressed, and
to cell media prior to the freezing process 8,9 . DMSO less water molecules are available to interact with them-
(10%) when added to the cell media, commonly at 2 M selves to form critical nucleation sites required for crystal
concentration, increases the porosity of the cellular mem- formation12. Formation of solid water with an irregular,
brane, which allows water to flow more freely through the amorphous structure is known as vitrification, and is
membrane 10,11 . Additionally, like glycerol, DMSO is achieved by utilizing a cryoprotectant accompanied by an
thought to help prevent the formation of water crystals by appropriate cooling rate15. To minimize toxicity, vitrifica-
increasing intracellular solute concentration, thus aiding in tion mixtures are often added in a stepwise fashion at tem-
the vitrification of water at low temperatures12. peratures near 0 C14. Thus, addition of permeating agents
under these conditions allows successful storage of cells in a
solid phase at the supercool temperatures required to halt
Principles of Cryopreservation biochemical processes without the formation of ice.
In order to fully understand the role of cryoprotective agents In addition to permitting vitrification, some PAs like
(CPAs), we must first understand the effects of subzero DMSO are thought to increase cellular permeability by
temperatures on otherwise healthy tissue. Exposing cells to affecting membrane dynamics in a concentration dependent
temperatures below 0 C without the aid of cryoprotectants is manner. At low concentrations (5%), evidence suggests
typically lethal. Since water constitutes approximately 80% DMSO decreases membrane thickness and, in turn, increases
of tissue mass, the freezing of water, both intra and membrane permeability. At commonly used concentrations
Whaley et al 3

Figure 1. Four common permeating cryoprotectants: glycerol (GLY), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propylene
glycol (PG).

(10%), water pore formation in biological membranes is

induced. Formation of pores can be advantageous as intra-
cellular water can be more readily replaced by cryoprotec-
tants that promote vitrification. At higher, toxic
concentrations (40%) however, lipid bilayers begin to disin-
tegrate16,17. It is evident then that selecting an appropriate
cryoprotectant concentration is critical to the structural
integrity, and therefore viability of cells after freezing.

Non-permeating agents. The second category of cryoprotec-

tant is non-permeating agents (NPAs). As the name suggests,
they do not permeate intracellularly and therefore exert their Figure 2. Naturally-occurring trehalose disaccharide. The unique
protective influence outside of the cell. They are typically a-1,1-glycosidic linkage prevents C-1 hydrolysis and increases sta-
bility under temperature extremes and low pH conditions.
larger, and covalently linked as either polymers, dimers, or
trimers. Some commonly-used agents in this class are:
Polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), mechanism, non-permeating agents can be added to solution
raffinose, sucrose, and trehalose14,15,18. Non-permeating to allow successful cryobanking but with lower concentra-
agents induce vitrification by the same mechanism as per- tions of permeating agents. This has the benefit of a reduc-
meating agents but extracellularly and to a lesser extent. tion in PA-induced toxicity and an increase in cellular
viability and yields post-thaw. Another method that showed
Trehalose: unique structure and consequences. Unlike the non- reduced CPA induced toxicity was demonstrated by Koja-
permeating polymers, the disaccharide CPAs are naturally yan et al. in which multi-molar combinations of reduced
occurring. Trehalose, a less-well-known sugar, is produced concentrations of EG, and DMSO were used to cryopre-
by a wide variety of organisms including bacteria, fungi, serve both human and murine islet cells21. They proposed
yeast, insects, plants, and some invertebrates. Although it that the reduction in adverse effects was likely due to
serves many functions, it has been demonstrated to act as a reduced exposure to each individual CPA even though
means to withstand freezing in these organisms 19,20 . overall osmolality of the different combinations was com-
The structure of trehalose gives it some useful properties. parable to a standard 2 M DMSO-only solution21. Other
It is a glucose dimer linked via an a-1,1-glycosidic bond. Its studies have shown combinations of sucrose, propanediol,
acetal link prevents reduction of C-1 in each monomer which and DMSO to be effective at supporting post-warming sur-
increases its stability under extreme temperatures and vival of porcine blastocysts15. Although segregated use of
reduces its susceptibility to acid hydrolysis in low pH con- EG and DMSO has been the paradigm in cryopreservation
ditions. The structure of trehalose is shown in (Fig. 2). since the mid-1900s, more current research suggests com-
binations of these and other CPAs offer the highest cell
Vitrification mixtures. Both permeating and non-permeating viabilities post-cryopreservation.
agents can prove toxic to cells at higher concentrations, Finally, to obtain consistency during cryopreservation,
although non-permeating agents in a lower degree. This exposure time of cells to permeating and non-permeating
undesirable feature of CPAs increases cell death and reduces agents during final packaging process have to be standar-
viable yields. Given that donor tissue supply for most organ dized. This is especially important when variable size of
types represents the limiting factor for transplant procedures, small (50 vials) or large (500 and more) lots of vials are
CPA induced toxicity should be minimized as much as pos- being packaged. For instance, during hepatocyte packaging,
sible. As such, cells should be exposed to those agents for the if a 30-minute exposure time is required to make a 50-vial lot
shortest amount of time that still allows for consistent packa- versus 2 hours to make a 500-vial lot, each lot will display
ging. Since both PAs, and NPAs share the same vitrification variable cell viability and overall health. This then can
4 Cell Transplantation

impact metabolic qualities, thus further increasing variabil- expected that the cells will be stored and thawed for subse-
ity already observed between hepatocyte donors. quent use. Compared to cooling, however, cell storage and
thawing has been given less attention. Speculation as to why
Cooling and thawing rates. The second action that must be this is the case leads to several possible conclusions: theory
carried out during the cryopreservation process is the selec- surrounding storage conditions and thawing rates is already
tion of cooling and thawing rates. Since the success of cryo- well-entrenched within the scientific community, and it is
protection depends on the avoidance of intracellular ice expected that primary cells are stored long-term at
formation, careful consideration must be paid to the move- 140/196 C. It is also important to mention that type of
ment of water across membranes during this process. Mazur storage can impact cell health and recovery. For example,
demonstrated a dependence of survivability on the rate of storage of primary hepatocytes in vapor phase LN2 tank at
cooling of various cells. Specifically, he provided evidence 140 C is preferred over standard liquid LN2 tanks at
that the rate of cooling is proportional to the probability of 196 C. It has been shown that vapor phase LN2 storage
intracellular ice formation, and therefore inversely propor- allows for far greater recovery of viable hepatocytes and
tional to survivability3. To decrease the probability of intra- maintains hepatic metabolic function24–29.
cellular ice formation, water must leave the cell as its Thawing and warming of cells does not play as critical
temperature is decreased. The driving force behind the out- a role as cooling and therefore has not warranted the
ward movement of water is a pressure differential due to the same level of investigation historically. Common theory
supercooling of intracellular water. This water remains in holds that cells must be warmed rapidly to prevent recrys-
liquid phase below 0 C, a phenomenon enhanced by the tallization of ice30. The rationale for this idea is based on
freezing point depression induced by the presence of CPAs. thermodynamic principles and is as follows: vitrified
Supercooled water in the cytoplasm has a relatively high water exists in a higher-energy state compared to its crys-
vapor pressure compared to water in the external medium3. tallized form. It is only quasi-stable and therefore can
The resulting pressure difference would favor a net move- rearrange itself into a more-stable, lower-energy crystal
ment of water out of the cell, causing dehydration.
structure during thawing31. When measuring the effect of
A pressure differential is necessary for a net movement of
warming rate on viability, however, it is important to
water to occur, but it is not the only factor involved.
remember that two steps always precede thawing: addi-
The amount of water remaining in the cell at the time of
tion of cryoprotectant(s), and subsequent cooling of cells.
solidification is affected by the rate of water efflux during
Both of these have a significant effect on viability and
the cooling phase. Three variables that affect this rate are,
could confound warming survivability data. Nonetheless,
the cell’s surface area to volume ratio (SA: V), the mem-
current accepted protocols suggest cryovials be trans-
brane’s permeability to water, and the rate of cooling3.
ported to the lab on vapor LN2 (not on ice or dry ice)
The SA: V is a function of the inherent size of a cell and
and be warmed rapidly in a 37 C water bath for 90-120
cannot be modified. It is important to acknowledge, how-
ever, because it has significant consequences; namely that seconds to achieve maximum viability32–34. This trans-
dehydration of larger cells is inherently slow compared to lates to an approximate warming rate of 45–70 C/min
the dehydration of smaller cells. In light of this, adjustments between 140 C and 0 C although warming rate is more
during cryopreservation should be considered based on cell or less rapid outside of this window due to its non-linear
size to allow a sufficient volume of water to exit before nature30. At least one study, however, provided evidence
vitrification occurs. To make up for large cell size, for exam- that warming cells anywhere between 2 C/min to 100 C/
ple in hepatocytes which have a diameter of 20-30 mm, the min had no effect on viability post-thaw35. If true, sam-
two remaining variables can be modified22. First, membrane ples could be thawed at slower rates like those achieved
permeability can be increased, as discussed earlier, by the with air thawing (6.2 + 0.5  C/ min) 30 . With this
addition of certain permeating cryoprotective agents like approach, viability of samples would be equal to those
DMSO. Second, the rate of cooling can be slowed. subjected to rapid-thaw water bath protocols. However,
The relationship between dehydration and cooling rate is less reducing water bath use would also have the added ben-
evident but critical, nonetheless. As temperature decreases, efit of increasing sterility since they are one of the most
so too does intracellular kinetic energy. Rapid cooling rates common sources of contamination in the laboratory.
do not allow sufficient time for water to leave before vitri- Finally, it is important to note that no matter how well the
fication occurs, thus cellular water content remains high and cells are stored or how they are thawed, a decrease in post-
crystallization is more likely to occur23. In order to ensure thaw viability is inevitable. As such it is important to clean
maximum survival, a cooling rate of approximately 1 C/min recovered cells using increased density media (Percoll®,
is usually appropriate for most cells except exceptionally Ficoll®, or similar) to remove dead cells and maximize via-
large ones3. This is typically obtained by using controlled bility of cells prior to their use. Standardization of this
rate freezers that modulate chamber temperatures via elabo- approach will increase quality of recovered cells and reduce
rate cooling programs to maintain steady 1oC/min drop of variability when they are used for metabolic studies or for
vial content 5 . After successful cryopreservation, it is clinical treatment.
Whaley et al 5

Cryopreservation of Hepatocytes, Pancreatic Islets, various rates of cooling ranging from 1 to 5 C/min up
Gametes, and Stem Cells to 40 C or 80 C before storing at 196 C in liquid nitro-
gen43. Rapid thawing at 37 C is recommended to avoid
Cryobiological responses can vary greatly depending on cell intracellular ice formation, minimize cellular damage, and
type due to variations in cell and tissue composition. There- enhance post-thaw viability.
fore, cell-specific biophysiological and biological character- After thawing, it is important to evaluate the viability and
istics should be considered during cryopreservation to functionality of cryopreserved hepatocytes. Attachment of
maximize post-cryo viability for each cell type36. The fol- hepatocytes post-thaw to collagen-coated plates provides a
lowing sections discuss cell-specific cryopreservation con- quick, qualitative measure of cell viability, and assays such
siderations, including optimal cooling and thawing rates, and as trypan blue exclusion, lactate dehydrogenase release,
selection of appropriate CPAs for hepatocytes, pancreatic mitochondrial function, and measurement of necrotic or
islets, sperm, oocytes, and stem cells. apoptotic markers allow for quantitative assessments of via-
bility and functionality. Assays that assess post-thaw
Hepatocytes. Hepatocyte transplantation, which involves the CYP450 enzyme functionality, like the 7-ethoxyresorufin-
infusion of mature adult hepatocytes in the portal system of a O-deethylase and testosterone hydroxylation assays, as well
recipient, is a novel therapeutic approach that can be used to as phase II metabolism through resorufin conjugation can be
treat various types of liver disease. Hepatocyte cryopreser- utilized as well. These assays can be used to establish
vation is a critical step in this treatment method since there is whether or not post-thaw hepatocyte drug metabolism cap-
often a necessary delay between the initial cell isolation and abilities remain intact. To further enhance yield and viabi-
the patient’s transplantation procedure. In addition, most lity, hepatocytes can also be encapsulated to guard against
patients require multiple cell transplantations over an mechanical stressors during cryopreservation24. One study
extended period making optimization of cryopreservation reported no significant difference between pre- and post-
techniques all the more important to help maintain cell cryopreservation viability when human hepatocytes were
functionality37,38. encapsulated with alginate/poly-L-lysine microcapsules44.
Several factors influence cryopreservation outcomes Encapsulation of hepatocytes also offers protection from
including: the quality of the tissue that hepatocytes are host immune defenses, an attractive feature for cells destined
derived from, the isolation procedure itself, preservation for transplantation after cryopreservation.
medium, and freeze/thaw rates. Hepatocyte quality is
adversely affected by the presence of steatotic or otherwise Pancreatic islets. Islet transplantation is an effective therapy
diseased tissue as well as prolonged warm ischemia times, for the treatment of patients with complicated type I diabetes
both of which could compromise the quality of the isolated mellitus. Advances in clinical islet isolation and transplanta-
hepatocytes25. Next, the cell isolation procedure itself can tion have been made since the development of the Edmonton
induce oxidative stress which, in turn, adversely affects protocol in 200045. It is reported that clinical islet transplan-
cryopreservation outcomes. It has been suggested that pre- tation can establish insulin independence for up to 5 years
incubation under non-attached culture conditions in media post-transplant with minimal complications46. However,
supplemented with 2 mM N-acetyl-cystein (an antioxidant) limited availability of donor pancreas and suitable islets for
and 15 mM glucose could provide antioxidant protection transplantation has remained a challenge45. Cryogenic bank-
after isolation and further optimize post-cryopreservation ing of islet cells from multiple donors allows for long-term
outcomes including cell viability, attaching capacity, and storage of islets and addresses the issue of donor availability
functionality, particularly GSH, glycogen levels, and drug- for islet transplantation47. Islets can be cryopreserved at
metabolizing cytochrome P450 enzymes39. Cryopreserva- 80 to 196 C with DMSO48,49. Freezing and thawing rates
tion medium can also influence storage outcomes, and the can affect islet morphology and function. An optimal freez-
University of Wisconsin (UW) is currently considered the ing rate should provide high yield and viability while keep-
standard medium for hepatocyte preservation. As an alterna- ing the immunostimulatory molecules low47. Foreman et al.
tive to UW solution, HypoThermosol (HTS) freezing solu- reported rapid freezing with the rate of 20  C/min and
tion supplemented with 10% DMSO at 4 C has been shown 70 C/min when islets were cultured in 1 M DMSO for
to produce high cellular viability, long-term hepatocyte 30 minutes at room temperature, followed by 2 M DSMO
function, and good-quality response to cytokine challenge exposure for 10 minutes at 0  C, improved in vitro insulin
post-thawing24,40,41. Furthermore, the combination of an secretory ability of the islets cells after thawing48. Current
NPA such as trehalose and a CPA like DMSO has been standard islet thawing protocols involve rapid thawing at
shown to significantly increase both human and rat hepato- 150-200 C/min in a 37  C water bath50. Evidence suggests
cyte viability post-thaw42. Thawing and freezing rates also that exposure to 50% oxygen during the thawing process could
play important roles in determining cryopreservation out- reduce the injury of cryopreserved human islets51. Glycerol or
come. The slow cooling rate is the best technique for cryo- DMSO can be used as CPAs for long-term storage of islet cells
preservation of mature isolated hepatocytes. Different at subzero temperatures (typically less than 100 C)52.
studies that utilized DMSO as their CPA have proposed Chandravanshi et al. have suggested supplementation with
6 Cell Transplantation

docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), process 64. Although the exact mechanism is unknown,
and metformin could provide a higher islet recovery from oxidative stress common during the cryo process is thought
196 C storage and allow for proper islet banking53. In addi- to underlie increased DNA fragmentation in human sperm
tion to the selection of CPAs and optimization of freezing and thereby resulting in reduced final yield and viability65. Yeste
thawing rate, other methods have been developed to improve et al. has suggested supplementing freezing media with vita-
islet cryopreservation outcomes. Alginate encapsulation of min E, hypotaurine, or other natural antioxidants to help
islets before cryopreservation has shown to be a promising mitigate oxidative effects58. While cryo-induced genetic
approach for future transplants. A study demonstrated that, alterations in sperm have been documented, data surround-
compared to non-encapsulated frozen islets, islets encapsu- ing potential epigenetic changes imparted by cryopreserva-
lated in 1.75% alginate had higher a survival and function tion are limited and warrant further study58.
post-thawing as well as an improved graft response54. In addition, pre-cryo sperm motility and viability can help
Post-thaw islet survival can be assessed by multiple meth- predict post-thaw outcomes and cryosurvival. Degl’Inno-
ods including fluorescein diacetate (FDA), calcein AM, or centi et al. assessed these characteristics in samples from
SYTO Green stain in conjunction with a propidium iodide patients with oligospermia, cancer, and other pathologies.
(PI), or ethidium bromide (EtBr) counter stain to measure They reported the lowest recovery rates occurred when
viability55–57. Additionally, post-thaw damage to general pre-cryo basal numbers, motility, and viability fell below the
baseline islet health can be monitored through PCR-based World Health Organization (WHO) fifth percentile reference
quantitation of mRNA transcripts including: HIF1A (acti- values (39  106 sperm per ejaculate, total motility: 40%,
vated in hypoxic conditions), SLC2A1 (GLUT1 transporter), progressive motility: 32%, and viability: 58%)66,67. In addi-
VEGFA (vascularization), and ACTB (b actin/ cytoskele- tion to the intrinsic characteristic and quality of the semen
ton)51. Impairment of metabolic ability and islet function sample, the holding period of the semen before freezing is
can be assessed through careful comparison of post-thaw critical. It is recommended that the sample should be pro-
oxygen consumption rate (OCR), and insulin production cessed within an hour post-delivery for optimal post-thaw
(GSIR) to pre-cryo baseline levels51. Ultimately, optimizing motility. Other factors that should be considered include
cryopreservation parameters could reduce the gap between initial sperm motility of at least 55% at one hour post-
transplantation from the donor to the recipient and further delivery with most cells in forward motility, less than 15%
advance the clinical transplantation of islet cells for the treat- decay in sperm motility, and no significant decrease in for-
ment of type I diabetes47. ward motility 4 hours after delivery68.

Sperm. Sperm cryopreservation is an effective method of

fertility preservation in humans, and is particularly useful Oocyte. Oocyte cryopreservation is a reliable method for the
to patients prior to cancer treatment and in other mammals maintenance of fertility in women undergoing treatment for
including endangered species58–60. Sperm are so amenable medical conditions such as cancer or autoimmune diseases.
to this method of storage that one study reported a batch of Treatment of these diseases could lead to ovarian insuffi-
cells kept for 21 years was used to successfully fertilize an ciency, which indicates the importance of fertility preserva-
oocyte ultimately resulting in a live birth61. Long-term cold tion. Ethical and legal concerns regarding embryo
storage of these cells usually involves glycerol and/or egg cryopreservation have led to research advancements in
yolk as CPAs, and an increase in the concentration of gly- oocyte cryopreservation during the past few years as an
cerol at a constant cooling rate has been shown to increase alternative approach. Freezing eggs provides autonomy for
sperm survival rates62. For cryopreservation of human sperm women since it allows for elective fertility preservation com-

cells, an initial cooling rate of 0.5–1 C/min is recommended pared to embryo preservation. Moreover, oocyte cryopre-

when freezing the cells from room temperature to 5 C. servtation provides women who have concerns about

Afterwards, an increase in the freezing rate to 10 C/min age-related reproductive decline with the option to delay

when cooling from 5 to 80 C, is recommended to maxi- pregnancy69,70.
mize sperm cryosurvival. When thawing samples, rates of Cryostorage of human oocytes is almost exclusively per-
43.5 C/min in 20 C air or 55.2 C/min in 35 C air on a dry formed at the Metaphase II stage when both nuclear and
surface are suggested to result in optimal sperm viability and cytoplasmic maturation has completed. The large size, high
motility post-thaw63. water content, and unique intracellular composition of
In general, post-thaw damage common to sperm include oocytes makes cryopreservation challenging, however.
reduced acrosome integrity, motility, fertilization capability, Damage to the meiotic spindles, actin filaments, and DNA
and an overall decrease in viability regardless of the species in addition to chromosome dispersion, microtubule depoly-
of origin.58 More specifically, chromatin disruption through merization, and increased polyspermy rate are among spe-
protamine translocations, DNA fragmentation, and lesions to cific challenges that that arise with oocyte preservation71.
genes involved in fertilization capability and embryonic Like sperm, oocytes also appear to be susceptible to epige-
development (ADD1, ARNT, PEG1/MEST, SNORD116/ netic changes as a result of low-temperature storage,
PWSAS) are known consequences of the cryopreservation although research in this area is ongoing70.
Whaley et al 7

Currently, two approaches to oocyte cryopreservation cryogenic banking protocols should be followed that are
dominate. They include slow cooling (equilibrium freezing suitable for each stem cell type. Cryopreservation protocols
protocols) and vitrification (ultra-rapid cooling or non- for HSCs and MSCs follow traditional slow cooling meth-
equilibrium protocols). Briefly, slow cooling involves gra- ods, whereas ESC protocols follow a rapid cooling/ vitrifica-
dual cell dehydration by adding low concentrations of a tion approach. The standard approach for cryopreservation
permeating agent such as DMSO (typically  1.5 M) and of HSCs includes the use of DMSO as a CPA, controlled rate
a non-permeating agent (commonly sucrose or trehalose, of freezing at 1 to 2  C/min, and rapid thawing77,81. MSCs
 0.3 M) with controlled slow cooling rates. After cooling can be cryopreserved similarly using slow freezing protocols
to 150 oC, the cells are stored in liquid nitrogen at 196oC and DMSO as a CPA82.
until they are needed for use. To thaw the cells, solutions Conventional methods (DMSO and slow cooling rate),
with decreasing concentrations of NPAs are used to obtain however, have shown low efficiency for cryopreservation
gradual rehydration70. Cryosurvival of oocytes has increased of ESCs. As a result, vitrification protocols have been devel-
with the improvement in slow cooling protocols, especially oped for cryopreservation of ESCs. A higher recovery rate
with the introduction of sucrose concentrations of greater has been reported for vitrified ESCs compared to the cells
than 0.1 M during pre-freeze dehydration. However, evi- cryopreserved by slow cooling protocols (75% compared to
dence from widespread practice indicates survival rates pla- about 5%)83–85. A detailed protocol for the vitrification of
teau around 70%–80% with this method72. human ESCs has been described. Briefly, human ESCs are
Vitrification protocols, however, are currently considered exposed to the stepwise addition of two vitrification solu-
the best method for cryopreservation of oocytes. The most tions of increasing concentration of CPA. The common com-
common method of vitrification involves the stepwise addi- ponents of both solutions include DMSO and EG.
tion of CPAs in cryomedia. In the first equilibrium phase, The composition of the vehicle solution can vary with dif-
oocytes are added to a solution containing 7.5% v/v EG and ferences in the concentration of sucrose and the presence or
7.5% v/v DMSO for 5 to 15 minutes. The cells are then absence of serum and the buffer used. Human ESC colonies
exposed to a vitrification solution with 15% v/v EG and are exposed to the two vitrification solutions sequentially for
15% v/v DMSO, plus 0.5 M sucrose. After a brief incubation 60 and 25 seconds respectively at room temperature or
( 1 minute), the cells are t stored in liquid nitrogen at 37  C. In addition, it has been suggested that the higher
196oC. Warming should be performed rapidly to avoid ice cooling rate in non-equilibrium vitrification processes could
crystal formation, and after gradual removal of the CPA, the allow a lower total concentration of CPA to be used, and a
cells should be incubated in culture medium until use70,73,74. combination of CPAs could help to reduce their toxic effects.
A systematic review reported that the rates of oocyte cryo- Reubinoff et al. suggested a mixture of 20% DMSO,
survival and fertilization were higher in vitrified oocytes 20% EG, and 0.5 M sucrose with rapid cooling rates72,83,86.
than slow-cooled oocytes. Vitrification also resulted in a Like fully-differentiated cells, non-or partially committed
higher top-rate quality embryo (22.4% vs 8.0%) and a higher stem cells are subject to adverse alterations in structure and
cleavage rate (day 2: 64.6% vs 47.7%) when compared to function after cryopreservation. A recent systematic review
slow rate cooling. Moreover, the rates of ongoing pregnancy, compared post-thaw assessments of bone marrow-derived
top-quality embryo, embryo cleavage, and fertilization were MSCs across ten species from 41 separate studies 87 .
not significantly different between vitrified and fresh The authors reported an overall consensus that morphology,
oocytes. These findings suggest vitrification is the better immunophenotype, differentiation and proliferation poten-
procedure for the cryopreservation of oocytes75. tial were largely unaffected by cryopreservation, but that two
thirds of experiments reported decreased metabolic activity
Stem cells. Stem cells’ ability to differentiate into various cell post-cryopreservation. In addition, reported changes in
phenotypes provides the foundation for regenerative cell viability varied with approximately 43% of studies reporting
therapies including treatment of degenerative diseases and no change and 27% reporting significantly decreased
traumatic injuries. Stem cell-based therapies allow the viability87. Post-cryo decreases in viability and metabolic
restoration of tissue structure and functional recovery. More- activity are often the result of physical and molecular cell
over, the biomolecules synthesized by stem cells can aid in damage. Physical injuries include intracellular ice formation,
tissue repair76. Hematopoietic stem cells (HSCs) can be used solution effects, cryo solution toxicity, and molecular damage
for the treatment of hematological as well as non- that manifests as alterations in gene expression, stress response
hematological diseases77 . The immunomodulatory and induction, and epigenetic alterations88,89,90. The molecular
immunosuppressive properties of mesenchymal stem cells mechanisms behind genetic responses to cryopreservation in
(MSCs) make this cell type ideal for allogenic transplanta- stem cells, much like gametic responses, are not completely
tion.78,79 In addition to their clinical applications in tissue understood and are still an area of active research87.
regeneration and transplantation, human embryonic stem Finally, several assays exist to assess cell viability after
cells (ESCs) are used in research to study basic developmen- cryopreservation which includes membrane integrity test,
tal processes and cell signaling pathways80. In order to fur- metabolic and mechanical activity assays, and mitotic activ-
ther extend the clinical applications of stem cells, long-term ity assays such as “plating” tests. To determine the in vivo
8 Cell Transplantation

functionality of the cryopreserved cells, fertilization and high throughput alternative to enrich post-thaw cell viability
development assays as well as transplantation assays can in an array of cell types.
be performed91.
Conclusion
Novel agents, and biomaterials to improve viability. More recent
approaches to cell cryopreservation demonstrate increased Significant improvements in our understanding of the cryo-
post-thaw viability and yield when cells are encapsulated preservation principles and techniques for long-term storage
prior to freezing. One example used alginate encapsulation and cryobanking of the cells have been made since the late
of hepatocytes to increase cryopreservation success. In com- 1800s when the research on the effect of cryopreservation on
bination with University of Wisconsin (UW) solution þ 10% live tissue first began. Long-term storage of the cells at
DMSO þ 5% glucose, encapsulated cell microbeads showed 196 C halts cellular metabolism which results in inevitable
better viability post-thawing than non-encapsulated cells, alternations in lipids and proteins that could impair cell func-
and better than the standard cryopreservation solution tion and structure. Ideally, a higher concentration of CPAs
(Bambanker®). The same approach could be tested on other could allow the cells to be preserved perfectly. However,
cells types to improve cryopreservation yields. This effect is increasing the concentrations of CPAs could also be dama-
further improved when using pan-caspase inhibitor (benzy- ging to the cells. DMSO has remained the gold standard CPA
loxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone for many different cell types. New techniques such as a
[ZVAD])92. A more recent study bypasses the need for algi- combination of CPAs or the use of new CPAs has been
nate, instead, freezing the hepatocytes as droplets in the CPA investigated to address the toxicity effect of some known
mixture (UW supplemented with 2 mg/mL BSA, 32.5% v/v agents99–101. In addition, alginate encapsulation of cells prior
DMSO, 32.5% v/v EG, and 800 mM sucrose). The droplets to freezing has been shown to improve preservation yields
were frozen rapidly in liquid nitrogen, unusual in compari- through non-chemical means. Regardless of cell type, the
son to the slow freezing normally used for cryopreservation. success of any cryopreservation protocol is dictated by care-
Bulk-droplet-vitrified hepatocytes had significantly higher ful selection of a few common variables: cryoprotecting
viability, better morphology, and higher metabolic activity agent type including permeating and non-permeating agents
than non-droplet frozen hepatocytes93. Aside from changing or a combination of both, as well as appropriate cooling and
the cryopreservation techniques, addition of cell-survival thawing rates. More recent approaches such as alginate
signal, such as myricetin (inhibitor of mitogen-activated pro- encapsulation and nanotechnology-based cell sorting may
tein kinase kinase 4 (MKK4) in culture after thawing cryo- offer enhanced post-cryopreservation viability and could
preserved hepatocytes aids both the cell survival in vitro and eventually become a part of standard cryopreservation
after transplantation in immunodeficient mice94. milieu. Understanding the principles behind the chemistry
and biology of freezing and thawing processes could allow
Novel agents to improve post-cryo cell recovery. Great effort is the development of more efficient procedures for cryopre-
made to preserve cell functionality and viability throughout servation of cells and further expand their clinical
the cryopreservation process since each step, primary cell applications.
isolation, initial purification, culture, CPA selection, and
freeze-thaw rates carries with it the potential to damage cells Statement of Human and Animal Rights
and decrease overall cell function. In spite of these efforts, This article does not contain any studies with human or animal
cryopreserved cells invariably experience a decrease in via- subjects.
bility post-thaw. Current strategies for identifying cells that
Statement of Informed Consent
remain viable after preservation utilize organic fluorophores,
and dyes95. And, as discussed previously, density gradients There are no human subjects in this article and informed consent is
not applicable.
can be utilized to increase viable cell density although this
method often involves exposing cells to additional, Acknowledgments
potentially-harmful centrifugation. More involved methods
The authors would like to acknowledge the support of the Univer-
to separate viable and non-viable cells include magnetic
sity of California, Irvine Department of Surgery, and Ambys Med-
affinity cell separation (MACS), and fluorescence- icines, for providing their expertise and support throughout the
activated cell sorting (FACS) although the cost and practi- writing of this manuscript.
cality sometimes limit their utility95,96. The emerging field
of nanoscience could provide the next step forward in cryo- Declaration of Conflicting Interests
biology by offering alternative non-destructive cell sorting The author(s) declared no potential conflicts of interest with respect
and live cell imaging methods95. One recent study utilized to the research, authorship, and/or publication of this article.
annexin V-conjugated magnetic nanoparticles to enrich
viable sperm content in fresh boar semen via selective bind- Funding
ing to the exposed phosphatidylserine present in apoptotic The author(s) received no financial support for the research, author-
cells97,98. This sorting scheme could be utilized as a simple ship, and/or publication of this article.
Whaley et al 9

ORCID iD dimethyl sulfoxide. J Phy Chem B. 2007;111(35):

Jonathan RT Lakey https://orcid.org/0000-0001-8553-4287 10453–10460.
17. He F, Liu W, Zheng S, Zhou L, Ye B, Qi Z. Ion transport
through dimethyl sulfoxide (DMSO) induced transient water
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