Animal cell culture technique... Cryopreservation

1. CRYOPRESERVATION AND ITS APPLICATION
By:
Yash Shah
Anshu Gupta
Nikita Darekar
Shivam Renke
Shahu Paramjeet Kaur
Aditya Tambe
Guided By: Dr. Soumya Basu
Associate Professor
Dr. D.Y. Patil Biotechnology & Bioinformatics Institute
Tathawade , Pune - 411033

2. CONTENTS
✔ WHAT IS CRYOPRESERVATION
✔ BASIC PROTOCOL FOR CRYOPRESERVATION AND KEY CONSIDERATIONS IN
CRYOPRESERVATION
✔ DIFFERENT MODES OF CRYOPRESERVATION
✔ CRYOPROTECTANTS
✔ RECENT ADVANCEMENTS IN CRYOPRESERVATION METHOD USING HYDROGEL
TECHNIQUE
✔ THAWING OF CELLS
✔ EMBRYO CRYOPRESERVATION
✔ STEM CELL CRYOPRESERVATION
✔ EMBRYO CRYOPRESERVATION
✔ ADVANTAGES OF CRYOPRESERVATION
✔ DISADVANTAGES OF CRYOPRESERVATION
✔ REFERENCES

3. INTRODUCTION
WHAT IS CRYOPRESERVATION?
• Cryopreservation is a method in which cooling at very low temperatures keeps cells,
Tissues, organs, or, any other biological substance preserved for a long period of time (at -
196°C in liquid nitrogen).
• Cryobiology involves researching the effect of low temperatures on bioactivity and its
architecture.
• ‘Cryogenicists’ for the ones who are concerned with studies in the field of Cryobiology
• The First Mammalian Cells to be successfully Cryopreserved were Spermatozoa.
• The Basic Principle employed in Cryopreservation is that “The temperature is often
Hypothermic to Cryogenic. The Hypothermal storage of mammalian temperatures is often
more than 0°C, but below normal thermal temperatures (32°C to 37°C)”.
• There are various techniques of cryopreservation and different 63 cells and tissues can be
cryopreserved accordingly.

4. BASIC PROTOCOL FOR CRYOPRESERVATION
1.Harvesting or selection of material- Few important criteria should be followed while
selecting the biological materials such as - volume, density, ph morphology, and
without any damage
2.Addition of cryo-protectant - Cryoprotective agents such as glycerol, FBS, salts,
sugars, glycols are added to the samples as it reduces the freezing point of the
medium and also allow slower cooling rate which reduces the risk of crystallization
3.Freezing - Different methods of freezing are applied in this method of
cryopreservation to protect cells from damage and cell death by their exposure to the
warm solutions of cryoprotective agents.
4.Storage in liquid nitrogen- The cryopreserved samples are stored in extreme cold or
-80°C in a freezer for at least 5 to 24 hours before transferring it to the storage vessels
5.Thawing- The process of warming the biological samples in order to control the rate

5. KEY CONSIDERATIONS NEEDED FOR CRYOPRESERVING CELLS
• The success of cell freezing and preservation depends on many elements of the
cryopreservation workflow. Besides using an optimized protocol and choosing the
right cryopreservation media for the cell type of interest, researchers should keep the
following best practices in mind when freezing their cell samples:
⮚Aseptic techniques: Use proper aseptic techniques to maintain sterile conditions
while freezing your cells. It is a good practice to wipe down the outside of containers,
such as flasks and freezing media vials, with 70% ethanol or isopropanol before
opening them.
⮚Contamination-free cells: Prior to freezing, ensure that the cells are healthy and free
of any microbial contamination. It is recommended to include mycoplasma testing in
the pre-freezing workflow. Signs of culture contamination include media turbidity and
color change, or morphological changes to the cells.
⮚Confluent cells: for best results, cells should be harvested during their maximum
growth phase (or log phase) and should have greater than 80% confluency before

6. ⮚Concentration of cells in a vial:
oThe optimal concentration for freezing cells may vary depending on your cell type.
While freezing cell suspensions at a very low concentration could lead to low cell
viability after thawing1, a very high concentration could lead to undesirable cell
clumping.
o Typically, the concentration of cells in the cryogenic vial is within a general range
of 1x103 - 1x106 cells/ml.
⮚Cryopreservation media ingredients:
oCryopreservation media ingredients can have a significant impact on banked cells
and future experiments. Home-made freezing medium usually comprises a culture
media containing fetal bovine serum (FBS) with a cryoprotectant such as dimethyl
sulfoxide (DMSO).
oHowever, FBS contains undefined components and its use in freezing media raises
concerns about lot-to-lot variability and the risk of transmitting potentially infectious
agents.
oCommercially available options, dmso-containing, ready-to-use cryopreservation

7. ⮚Cooling rate:
o The rate at which cells are frozen can have a significant impact on their survival.
Controlled-rate freezing, a method that involves a gradual cooling rate of -
1°C/minute, before long-term storage can help maximize cell viability and integrity.
o Controlled-rate freezing can be achieved by using a controlled-rate freezer or by
placing cryogenic vials in an isopropanol freezing container (e.G. Nalgene® mr.
Frosty) or an isopropanol-free container such as corning® coolcell® and into a -80°C
freezer to cool slowly overnight.
o In general, slow freezing and rapid thawing is considered to be the basic rule to
follow in freezing and subsequently recovering cells.
⮚Proper storage:
o opt for single-use, sterile cryogenic vials for storing your cells. It is preferable to use
internal-threaded cryogenic vials to prevent contamination during filling or when
stored in liquid nitrogen.
o For optimal performance in the long-term, store cryogenic vials in liquid nitrogen
tanks (-135°C to -196°C).

8. FIG :The schematic of Cryopreservation Technique from Pre-treatment to Recovery.
A)Steps of Stem cell preservation via vitrification to liquid nitrogen and alternatively by
the slow freezing process.

9. DIFFERENT MODES OF CRYOPRESERVATION
⮚SLOW FREEZING:
▪ Slow freezing involves progressive cooling of sample over a period of 2–4 hrs automatically
using a semi programmable freezer.
▪ Cooling is sufficiently slow, cells efflux intracellular water rapidly enough to eliminate
supercooling and thus prevent intracellular formation of ice crystals. It substitutes the water
within the cytoplasm with CPAs which reduces cell damage and adjusts the cooling rate in
accordance with the permeability of the cell membrane.
▪ Slow-cooling protocols involve the use of <1.0 M of Cryoprotective agents (Cpas),which
have minimal toxicity at lower temperatures with the use of a high-cost controlled rate
freezer or a benchtop portable freezing container.
▪ Although CPAs show minimal toxicity due to low concerntration,if done properly,it can result
in less than 30 % of cell loss and therefore SLOW FREEZING is the most widely used
technique.

10. Concept Idea for Slow Freezing method
A B D
C
Slowly Freezed
Cell
Sample
Cell
“Main principle of Slow Freezing of Cells i.e. Extracelluar Crystal
Formation”
Cell subjected to Slow Cooling
▪ This method provides High Viabilitiy Post Thawing
▪ 1,2 Propanediol is the most commonly used CPA
▪ Low potential of contamination through pathogens
Pecularities:

11. VITRIFICATION
▪ Vitrification is a process by which cell suspensions are transformed directly from the
aqueous phase to a glass state after direct exposure to liquid nitrogen.
▪ The process requires cooling of the cells or tissues to deep cryogenic temperatures (i.e. with
liquid nitrogen) after their exposure to high concentrations of CPA (in the ratio of 40–60%,
weight/volume), with subsequent rapid cooling to avoid ice nucleation.
▪ It consists of the solidification of the aqueous material into a non-crystalline glass phase in
the cells and tissues. The cooling rate contains significantly greater cryoprotective agent
concentrations in relation to slow freezing
▪ The time taken by the cells to reach this glass state is approximately only 10 minutes.
▪ Vitrification is largely dependent on three factors:(a) Viscosity of the sample
(b) Cooling and Warming rates
(c) Sample volume(1-2 ul)

12. Vitrification
Why do choose to Vitrify cells instead of Slow
Freezing ??
• No Ice formation.
• No Special Equipment Required.
• High Post Thaw Viability.
✔ Therofore, it is important to note that Depending on the sample type, an appropriate
method of Cryopreservation is choosen.
✔ DMSO(Dimethyl Sulphoxide),Glycerol are the CPAs frequently used in Vitrification

13. CRYOPROTECTANTS
▪ Cryoprotectants are the fluids used to minimize the cryoinjury from the cryopreservation
process.
▪ Cryoprotectants need to be biologically acceptable, can able to penetrate the cell
membrane, and less toxic.
▪ CPA concentration is a major factor influencing the success of the cryopreservation.
▪ Understanding how potential CPAs interact with cells is also important for optimising
treatments.
▪ Based on their penetrating capabilities through cell membrane, cpas are classified into two
categories:
1.Cell Membrane-Permeating Cryoprotectants:
• These have the capability to penetrate the cell membrane.
• They help in reducing the development of crystals and eliminating the rise in
intracellular solution concentration, therefore decrease cellular damage caused due to
cooling.
• These protective chemicals are neutral and low molecular weight.
• Example: 1,2 Propanediol; DMSO{Dimethyl Sulphoxide}; Glycerol

14. Cell Membrane-Permeating Cryoprotectants
⮚ 1,2 Propanediol :
o Preferred CPA for the Slow Freezing method of Cryopreservation.
o Extremely small molecular weight of 76.08g/mol.
o Amount added during protocol: 1.5 mol/liter.
o It has various advantages as low cell toxicity, and also helps in maintining the
viability of cells post thawing.
o Used extensively as a CPA for the Cryopreservation of Embryo and
Hepatocytes.
1,2 Propanediol

15. Cell Membrane-Permeating Cryoprotectants
⮚DMSO{Dimethyl Sulphoxide} :
oMost standard CPA used for the Cryopreservation of variety of cells.
oMolecular Weight: 78.13 g/mol.
oIt is an Organo-Sulphur compound and major CPA used for Vetrification Method.
oDMSO,“acts by reducing the electrolyte concentration in the residual unfrozen
solution in and around a cell at any given temperature.”
oAmount added during protocol : 5-10% v/v. It is often used in combination with
any other cryoprotectant.
oUsed extensively for the Cryopreservation of cultivated Mammalian Cells and
Hemopoitic Stem Cells.
DMSO Structure

16. Cell Membrane-Permeating Cryoprotectants
⮚GLYCEROL :
oSimple polyol compound. It is a sweet and non-toxic, colourless, odourless, thick
liquid. .
oMolecular Weight:92 g/mol.
oGlycerol has good osmotrophic characteristics; it produces water molecules
hydrogen bonding. This makes it Impossible to generate ice crystals by
combination, till and until the temperature is very low, such as -37.8°C (70%
glycerol and 30% water).
oGlycerol is less hazardous at high concentration compared to other
cryoprotective substances.
oGlycerol has, for years now, been employed as a Blood cell and Bull sperm
cryoprotectant in cryobiology to store in liquid nitrogen.
Glycerol Structure

17. 2.Non-Membrane-Permeating Cryoprotectants
• These are water-soluble cryoprotectant, but are unable to penetrate the
cell membrane.
• They can make the solution super-cooled, i.e. to reduce the solvent
(electrolytes) concentration in specific low temperatures, thereby
protecting them.
• During cooling, non-permeating cpas promote colligative freezing point
depression and vitrification by increasing cellular solute concentration
via dehydration, and by increasing carrier solution viscosity,
respectively.
• They are also believed to adsorb to the outer cell membrane, thereby
protecting the cell from extracellular crystal lattice ice formation.
• Less toxic to cells than permeating cryoprotectants.
• Example: Polymers(Polyampholytes), Trehalose, Sucrose

18. Non-Membrane-Permeating Cryoprotectants
⮚ Polymers(Polyampholytes):
oPolymers can supply cells with a cryoprotective environment and have a large
ability to reduce the size of ice crystals generated
o Polymer substance preserves cells during freezing which results in the recovery
of more cells and the need for less solvent-based antifreeze.
oPolyampholytes are newly discovered cpas with balanced anion and cation side
chains.
o Are capable of stabilising cell membranes, therefore protecting cells against
freezing-induced damage.
Polyampholyte mechanism
and Post Thawing Viability

19. Non-Membrane-Permeating Cryoprotectants
⮚ Trehalose:
o This non-reducing sugar is produced by yeast and insects in large amounts to
survive freezing temperature and lack of water and hence it is used as a
cryoprotectant
o It can be utilized as a cryoprotectant because of its high-water retention
characteristics.
oTrehalose enhances after thawing cell survival in comparison to the normal
freezing technique.
✔ One of the main
peculiarities of using
sugars like Trehalose
and Sucrose is that they
also help in proving
nutrition if required

20. RECENT ADVANCEMENTS IN METHODS CRYOPRESERVATION
⮚HYDROGEL METHOD:
▪ Hydrogels are a three-dimensional network of
hydrophilic polymers that can expand in water
and contain a considerable quantity of water while
retaining their structure due to chemical or
physical cross-linking of the individual polymer
chains.
▪ Hydrogels are soft materials and has a unique
property such as outstanding biocompatibility with
live cells.
Graphical Abstract of
Hydrogel Method

21. HYDROGEL METHOD
o There are three hydrogel cryopreservation systems which are :
1. Natural Polymer Hydrogel Cryopreservation system (which includes alginate
hydrogel cryopreservation system and chitosan/alginate hydrogel cryopreservation
system)
2. Synthetic Polymer Hydrogel Cryopreservation system.
3. Supramolecular Hydrogel Cryopreservation system.
o The different hydrogel cryopreservation methods can confine ice crystal growth and
decrease the change rates of osmotic shock in cell encapsulation systems.
▪ Main Pecularity - The ice formation is confined in the
porous three-dimensional network structure, which
minimize the cell damage.
▪ Used extensively in Cell Therapy,Tissue Engineering
& Organ Transplantation

22. Thawing - Revival of cells
▪ The proper revival of cryopreserved cells is very
crucial to ensure the viability and functionality of
the cell, this process of revival of the cell is
called Thawing.
▪ It is the freezing process in reverse ,in this
technique the cryopreserved cells are warmed
at 37°C in water bath for 1-2 minutes. The cells
are not to be warmed up to 4°C as the
cryoprotectants quickly become toxic as the
temp.increases.
▪ But as the cryoprotectants concentration is
important for properly freezing it is often linked
with toxicity, which can be reduced by quicker
and feasible cooling and warming.
▪ Therefore, both rapid and uniform warming in a

23. Thawing - Revival of cells
▪ Thawing of cells can be done chiefly by two methods:
✔ Direct Plating Method -
▪ Remove cells from storage and thaw quickly in a 37°C water bath.
▪ Plate cells directly with complete growth medium.
- Use 10 to 20ml of complete growth medium per 1ml of frozen cells.
- Perform a viable cell count.
▪ Culture cells for 12 to 24hrs.
▪ Replace medium with fresh complete growth medium to remove the
cryopreservative
✔ Centrifugation Method –
▪ Remove cells from storage and thaw quickly in 37°C water bath.
▪ Place 1 to 2ml of frozen cells in ~25ml of complete growth medium, mix very gently.

24. Thawing - Revival of cells
▪ Centrifuge the cells at ~200*g for 2 to 3
minutes.
▪ Discard supernatant.
▪ Gently resuspend the cells in complete growth
medium and perform a viable cell count.
▪ Plate the cells.
Culture Media :
Appropriate culture media Examples include:
1)Iscove’s Modified Dulbecco’s Medium
(IMDM) with 10% fetal bovine serum (FBS)
2)DMEM with 4500 mg/L D-Glucose with 10%
FBS

25. CRYOPRESERVATION OF EMBRYOS
• Cryopreservation of embryos is the process of preserving an embryo at sub-zero
temperatures, generally at an embryogenesis stage corresponding to pre-implantation,
that is, from fertilization to the blastocyst stage.
• Embryo cryopreservation is useful for leftover embryos after a cycle of in vitro
fertilization, as patients who fail to conceive may become pregnant using such
embryos without having to go through a full ivf cycle. Or, if pregnancy occurred, they
could return later for another pregnancy.
• Spare oocytes or embryos resulting from fertility treatments may be used for oocyte
donation or embryo donation to another woman or couple, and embryos may be
created, frozen and stored specifically for transfer and donation by using donor eggs
and sperm. The cryopreservation of embryos was first successfully attempted in 1984
in the case of zoe leyland, the first baby to be born from a frozen embryo.

26. WHY FREEZE EMBRYO ?
• IVF TREATMENT INVOLVES STIMULATED CYCLES, TO PRODUCE MANY EGGS WHICH MAY
RESULT IN MANY EMBRYOS. IN ORDER TO AVOID THE RISK OF MULTIPLE PREGNANCIES,
ONLY FEW EMBRYOS ARE TRANSFERRED (D3 – TWO TO THREE EMBRYOS; D5 – ONE TO TWO
BLASTOCYST) AND THE REST SURPLUS EMBRYOS CAN BE STORED. EMBRYOS ARE USUALLY
FROZEN IN THE FOLLOWING SITUATIONS:-
• OVARIAN HYPER STIMULATION SYNDROME (OHSS) – WHERE TRANSFERRING OF EMBRYOS
CAN BE LIFE THREATENING FOR THE PATIENT.
• TO REDUCE THE RISK OF MULTIPLE PREGNANCIES.
• IN CASE OF SURPLUS EMBRYOS (CAN HELP TO INCREASE THE CUMULATIVE PREGNANCY
RATE).
• FERTILITY PRESERVATION – SOCIAL OR MEDICAL REASONS.
• EMBRYO DONATION.

27. PROCEDURE FOR CRYOPRESERVATION IN EMBRYOS
• Preserving the embryo for later use is the primary objective of embryo freezing. Therefore, the embryo has
to be maintained in such an environmental condition that it does not die.
• The steps involved in freezing or cryopreservation of the embryo are:-
• STEP ONE: EMBRYO SELECTION
• As part of the IVF process, the woman is medically stimulated to make more eggs. The man also gives a
higher number of sperm cells. When both are mixed, several embryos often tend to develop. The doctor
usually transfers one to four embryos into the woman while the rest is left over. Here, the healthiest
embryo or embryos are selected so that it can be stored for future use
• STEP TWO: REMOVAL OF THE WATER CONTENT
• THE SELECTED EMBRYO CANNOT BE FROZEN DIRECTLY WITHOUT REPLACING THE WATER
CONTENT WITHIN THE CELLS. THIS IS BECAUSE WHEN IT IS KEPT FOR FREEZING THE WATER
CONTENT INSIDE THE CELLS ALSO FREEZES AND CRYSTALIZES. THE CRYSTAL FORMATION CAN
CAUSE EXPANSION WHICH CAN BURST THE CELL OPEN, CAUSING IT TO DIE. TO PREVENT THIS
FROM HAPPENING, CRYOPROTECTANTS ARE USED TO REPLACE THE WATER CONTENT INSIDE THE
CELLS.

28. • STEP THREE: EMBRYO FREEZING
• It involves the use of programmable freezers and relatively slow rates of cooling (0.3°–
0.5°C/min).
• The cryoprotectants in widespread use include 1,2 Propanediol and
Dimethylsulfoxide(DMSO) in the presence of sucrose for zygotes or early-stage
embryos.
• Glycerol for uterine-stage embryos (blastocysts).

29. STEM CELL CRYOPRESERVATION
• An undifferentiated cell of a multicellular organism which is capable of giving rise
to indefinitely more cells of the same type, and from which certain other kinds of cell
arise by differentiation.
• Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of a
blastocyst, an early-stage pre-implantation embryo
• Hematopoietic Stem Cells (HSCs) are multipotent primitive cells that can develop into
all types of blood cells, including myeloid-lineage and lymphoid-lineage cells.
• HSCs can be found in several organs, such as Peripheral blood (PB), Bone marrow
(BM), and Umbilical Cord blood (UCB).
• We mainly preserve these cells for certain clinical uses like Gene therapy,
Transplantation and Research purposes.

30. HAEMOPOIETIC STEM CELL CRYOPRESERVATION PROCEDURE
▪ The cryopreservation process entails the following general components:
⮚ HARVESTING OF THE DONOR CELLS, WHICH ENTAILS THE ACTUAL
COLLECTION OF THE SPECIMEN AND THE REDUCTION OF BULK.
▪ Hematopoietic Progenitor Stem cells in a minimally manipulated fashion as
defined by the foundation for the Accreditation of Hematopoietic Cell Therapy
(FAHCT) with a minimal cell dose of at least 2.5 × 106–5.0 × 106 cells/kg body
weight, as currently considered standard.
▪ The specimen is then centrifuged to develop the cell rich pellet. In autologous
transplants donor plasma is used.
⮚ Addition of Cryoprotectants
▪ The supernatant out of this cryoprecipitation process is used for the
reliquidification of the pellet cells, after the addition of a solution of Heparinized
Plasmalyte solution and 10% DMSO (Dimethylsulfoxide) along with normal Saline
and Serum Albumin.
▪ This usually eventuates into a cellular concentration of 500 × 10−6 cells in the

31. HAEMOPOIETIC STEM CELL CRYOPRESERVATION PROCEDURE
⮚ THE ACTUAL FREEZING PROCEDURE:
▪ In controlled rate freezing, the concentrated stem cells are frozen down at a rate
of 1–2°C/min up to a temperature point of about −40°C.
▪ Then, the freezing process down to a target of −120°C is performed at a faster
pace, about 3–5°C/min. For umbilical cord stem cells, bone marrow, and PBSCs
the controlled rate freezing process is considered standard
▪ Then the sample is frozen down to the target temperature of −156°C (when stored
in the vapor phase) to −196°C (when stored in the liquid phase), depending on
where in the container the specimen is stored.
⮚ THE THAWING PROCEDURE:
▪ The standard method is warming in a water bath at 37°C until all ice crystals
disappear.
▪ The removal of DMSO is crucial due to its toxic effect.

32. ⮚WASHING OF CELLS:
▪ For stem cells of cord, peripheral blood, and bone marrow origin, the process of washing out the
cryopreservative after the thawing can still be considered standard.
▪ The current standard washing protocol follows the New York Blood Center Protocol, in which the
two step dilution of the thawed stem cell unit with 2.5% Human Serum Albumin and 5% Dextran 40
is used .
▪ It is followed by centrifugation at 10°C for 10 min. The supernatant is then removed and HSA and
Dextran solution is again added twice to a final DMSO concentration of less then 1.7%.
⮚STORAGE:
▪ The International Society for Cellular Therapy (ISCT) described on its supplier information
website for cryocontainers nine different cryostorage container products as :
Cryocyte/baxter
Cellflex/maco pharma
Cell freeze™ charter medical
Pall medical freezing bag 791-05
Cryostore EVA/Origen Biomedical Inc.
Thermogenesis corp./Freezing bag.

33. Stem Cell
Cryopreservation
Critical Steps

34. Advantages of Cryopreservation
• Cryopreservation is an effective way to preserve the germplasms of endangered
plant species and helps to maintain their fertility.
• It is beneficial in storing a large range of disease-free biological samples for long
periods of time.
• The national and international transportation of samples has become easier with
cryopreservation
• Since this technique freezes the biological product at a particular stage, the sample
remains viable for an indefinitely long period of time.
• It also saves a lot of research time since scientists do not have to wait for fresh
viable cells.
• To study about the adapting nature of plant and animals at low temperature.
• Preserving the genetic nature of species that are on the edge of becoming extinct.
• Microbial contamination, cross-contamination with other cells, genetic drift, and
morphological alterations are all reduced.
• The cryopreservation of embryo can be used to store good quality excess embryos
that are produced during IVF treatment cycles.

35. Disadvantages of Cryopreservation
▪ One of the major disadvantages of this technique is that ice crystals can form
inside the cells thereby causing cell damage.
▪ The use of unsuitable cryoprotectants also affects cell viability.
▪ Water migration can cause extracellular ice formation and cellular dehydration.
Such stresses can damage the cells directly.
▪ During eggs storing procedure as the patients have to undergo a conventional
IVF practice, which have known side effects related to fertility medication such as
the risk of ovarian hyper stimulation syndrome which leads to swollen and painful
ovaries.
▪ There is no standard protocol for every biological sample, and thus expertise and
time are required to store precious samples.
▪ The risk increases in some cases resulting in weight gain, abdominal pain,
vomiting, breathing problems.
▪ Moreover, the prolonged process of slow-rate freezing and long-term storage of
these cells require costly equipment and higher maintenance cost overall.
▪ Cryopreservation techniques work on extremely low temperature like -90°C which
can result in formation of ice crystals particularly of needle-shaped like structures

36. ⮚ REFERENCES
1) JANG TH, PARK SC, YANG JH, KIM JY, SEOK JH, PARK US, CHOI CW, LEE SR, HAN J. CRYOPRESERVATION AND ITS
CLINICAL APPLICATIONS. INTEGR MED RES. 2017 MAR;6(1):12-18. DOI: 10.1016/J.IMR.2016.12.001. EPUB 2017
JAN 10. PMID: 28462139; PMCID: PMC5395684.
2) J. WANG, X. SHI, M. XIONG, W.S. TAN, H. CAI, TREHALOSE GLYCOPOLYMERS FOR 1 CRYOPRESERVATION OF
TISSUE-ENGINEERED CONSTRUCTS, CRYOBIOLOGY. (2021). HTTPS://DOI.ORG/10.1016/J.CRYOBIOL.2021.11.004.
3) J. YANG, L. GAO, M. LIU, X. SUI, Y. ZHU, C. WEN, L. ZHANG, ADVANCED BIOTECHNOLOGY 1588 FOR CELL
CRYOPRESERVATION, TRANSACTIONS OF TIANJIN UNIVERSITY(2019) HTTPS://DOI.ORG/10.1007/S12209-019-00227-
6.
4) PRESERVATION OF EMBRYONIC STEM CELLS, METHODOLOGICAL ADVANCES IN THE CULTURE, MANIPULATION AND
UTILIZATION OF EMBRYONIC STEM CELLS FOR BASIC AND PRACTICAL APPLICATIONS. (2011).
HTTPS://DOI.ORG/10.5772/13860
5) L. JI, J.J. DE PABLO, S.P. PALECEK, CRYOPRESERVATION OF ADHERENT HUMAN EMBRYONIC STEM CELLS,
BIOTECHNOL BIOENG. 88 (2004) 299–312. HTTPS://DOI.ORG/10.1002/BIT.20243.
6) HTTPS://WWW.STEMCELL.COM/CRYOPRESERVATION-BASICS-PROTOCOLS-AND-BEST-PRACTICES-FOR-FREEZING-CELLS
7) ZHANG C, ZHOU Y, ZHANG L, WU L, CHEN Y, XIE D, CHEN W. HYDROGEL CRYOPRESERVATION SYSTEM: AN EFFECTIVE
METHOD FOR CELL STORAGE. INT J MOL SCI. 2018 OCT 25;19(11):3330. DOI: 10.3390/IJMS19113330. PMID:
30366453; PMCID: PMC6274795.