Cell lines need to be routinely maintained and stored long-term to preserve their valuable characteristics. Routine maintenance involves periodic medium changes and subculturing depending on the growth rate of the specific cell line. Long-term storage is achieved through cryopreservation, where cells are frozen at temperatures below -100°C. This process aims to minimize ice crystal formation and associated cell damage through slow freezing in the presence of cryoprotectants like DMSO or glycerol. Proper freezing and storage methods help preserve cell lines for future use and distribution.

1. MAINTENANCE OF
CELL LINES-
Cryopreservation & Germplasm
Storage
MARIA K. JOHN
M.Sc. BIOTECHNOLOGY

2. Need for
Maintaining
Cell Lines?
 For the reliable and
reproducible recovery of
specifically selected and/or
manipulated cell lines with
unchanged defined
characteristics.
 Establishing these lines is
costly, and this, together with
their special characteristics
give them a significant value.

3. Routine Maintenance of Cell
Lines

4. • The primary culture or a subculture, once initiated, will need a periodic
medium change, followed eventually by subculture if the cells are
proliferating.
• In non-proliferating cultures, the medium will still need to be changed
periodically, as the cells will still metabolize and some constituents of the
medium will become exhausted or will degrade spontaneously.
• Intervals between medium changes and between subcultures vary from one
cell line to another depending upon the growth rate and metabolism.
• Rapidly growing transformed cell lines (HeLa) are usually sub cultured once
per week and the medium should be changed four days later.
• Non-transformed cell lines may need to be sub cultured only every two, three
or even four weeks and the medium should be changed weekly between
subcultures.

5. Factors
• Significance of cell morphology
 Culture should be free of contamination.
 Cells should be frequently checked for any deterioration like granularity around the
nucleus, cytoplasmic vacuolation, rounding up of the cells with detachment from the
substrate.
 Such signs indicate that there is need for medium change, inadequate or toxic
medium or serum, microbial contamination or senescence of the cell line.

6. • Replacement of the medium-It is required when there is
 A drop in pH:
-Most cells stop growing as the pH falls from pH 7 to pH 6.5
-Lose viability between pH 6.5 and pH 6
- If the medium goes from red through orange to yellow, the medium should be
changed.
 Cell concentration:
-Cultures at a high concentration exhaust the medium faster than those at a low
concentration.

7.  Cell type:
-Normal cells usually stop dividing at a high cell density because of cell
crowding, growth factor depletion, and other reasons.
-The cells block in the G1 phase of the cell cycle and deteriorate very
little, even if left for two to three weeks or longer.
-Transformed cells, continuous cell lines and some embryonic cells,
however deteriorate rapidly at high cell densities unless the medium is
changed daily or they are sub cultured.
 Morphological deterioration:
-Regular examination and familiarity with the cell line is needed.
-If deterioration is allowed to progress too far, it will be irreversible, as
the cells will tend to enter apoptosis.

8. Long Term Storage
• As cell culture develops within a
laboratory a number of cell lines will
be developed or acquired.
• The use of each cell line adds to its
provenance, and each one becomes a
valuable resource.
• If unique, the cell line might be
impossible to replace; at best
replacement would be expensive and
time-consuming.
• It is, therefore, essential to protect this
considerable investment by preserving
cell lines.
• The storage methods include:
 Freeze drying
 Freezing in a -20 °C freezer
 Low temperature freezing
 Freezing in the vapor phase of liquid
nitrogen (-130 °C and lower)
 Freezing in a -80 °C freezer
 Cryopreservation

9. CRYOPRESERVATION

10. • The process by which the cells are preserved in frozen state for future use.
• The storage is usually carried out using temperatures below -100°C.
• The word is derived from Greek word “Kryo” = cold, “bios” = life and “logos” =
science.
• Cryobiology is the branch of biology that studies the effect of low sub-zero
temperatures in biological activities.
• This stores cell stocks and prevents original cell from being lost due to unexpected
equipment failure or biological contaminations.
• It also prevents finite cells from reaching senescence and minimizes risks of changes
in long term cultures.

11. Reasons for freezing
• Genotypic drift due to genetic instability.
• Senescence and resultant extinction of the cell line.
• Transformation of growth characters and acquisition of malignancy associated
properties.
• Phenotypic instability due to selection and dedifferentiation.
• Contamination by microorganisms.
• Cross-contamination by other cell lines.
• Misidentification due to careless handling.
• Incubator failure.
• Saving time and materials by not maintaining lines other than those in current use.
• Need for distribution to others.

12. Steps involved
Cell harvesting
Media
preparation for
cell and tissue
cryopreservation
Temperature
Freezing
Thawing
cryopreserved
cells
Storage
Viability
assessment

13. Requirements before Cryopreservation
• Cell lines should be free of contamination.
• Authentic
• Proper validation should be carried out before major stocks are frozen.
• If it is a finite cell line, there should be less than five passages for freezing.
• Continuous cell lines should be cloned, complete characterization is required for
freezing.

14. Principles of Cryopreservation
THEORETICAL BACKGROUND TO CELL FREEZING
• Optimal freezing of cells for maximum viability depends on- minimizing
intracellular ice crystal formation, reducing cryogenic damage due to high
salt concentration.
• This can be controlled by:
 Freezing slowly for allowing water to leave the cell, but not too slowly.
 By using a hydrophilic cryopreservant to help sequester water.
 By storing cells at lowest possible temperature to minimize the effects of
high salt concentration on protein denaturation.
 By thawing rapidly to minimize ice crystal growth.

15. CELL CONCENTRATION
• Cells appear to survive best when frozen at a high cell
concentration.
• If cell number is less, freezing should not be done.
• If less concentration is used, use in 1:10 or 1:20 dilution on
thawing.
FREEZING MEDIUM
• Mainly contains serum + cryoprotectant
• Cell suspension is frozen in the presence of a cryoprotectant
such as glycerol or dimethyl sulfoxide (DMSO).
• DMSO penetrates the cell better than glycerol.
• Cells should be kept at 4°C after DMSO is added to the medium
and before freezing.
• If glycerol is used, it should be not more than one year old.
Demerits of DMSO
^Neurotoxin.
^Cytotoxic in some cell types
^Can be directly absorbed
through the skin.
^Induce cells to differentiate.
^Combustible.
Merits of DMSO
^Effective.
^Colorless.
^Powerful solvent.
Requirements
As DMSO is a powerful
solvent, it needs to be stored
in glass or polypropylene.

16. Cryoprotective Agents (CPA)
• Chemicals that minimize injuries to
the cell due to ice formation or it
suppresses ice formation.
• Reduce the freezing point of the
medium.
• Allows slower cooling rate.
• Criteria for choosing a CPA
i. Least toxic to cells.
ii. Should be permeable to cells.
iii. Should be soluble in water during
freezing.
• Commonly used cryoprotectants
o Dimethyl sulfoxide (DMSO)
o Glycerol
o Polyvinylpyrrolidone (PVP)
o Polyethylene glycol (PEG)
o Hydroxyethyl starch (HES)

17. COOLING RATE
• Most cultured cells survive best if they are cooled at
1°C/min.
• This is probably a compromise between fast freezing
minimizing ice crystal growth and slow cooling
encouraging the extracellular migration of water.
CRYOFREEZER
• Storage in a liquid nitrogen freezer is currently the most
satisfactory method of preserving cultured cells.
• The frozen cells are transferred rapidly to the cryofreezer
when they are at or below −70°C.
• Cryofreezer differ in design depending on size of the
access neck, storage system employed, and location of
liquid nitrogen.

18. • Neck size: Canister storage systems tend
to have narrow necks, which reduces the
rate of evaporation of the liquid N₂. Wide-
necked freezers are chosen for ease of
access and maximum capacity, but tend to
have a faster evaporation rate.
• Storage system: There are two mains types
of storage used for 1-mL ampoules for cell
culture work. They include: the cane
system and the storage in rectangular
drawers.
• Ampoules: - Plastic ampoules are
preferred as they are safer and more
convenient, but some repositories and cell
banks prefer glass ampoules for seed
stocks, because the long-term storage
properties of glass are well characterized.
• Location of liquid N₂: In some freezers,
liquid N₂ is located in the main body of
the freezer, whereas, in certain others,
have the liquid nitrogen located within the
wall of the freezer and not in the storage
compartment.
Narrow-necked
freezer
Wide-necked
freezer

19. Why Liquid Nitrogen?
• Chemically inert.
• Relatively low cost.
• Non-toxic.
• Inflammable.
• Readily available.

20. FREEZER RECORDS
• Records should provide
(a) an inventory showing what is in
each part of the freezer
(b) an indication of free storage spaces
(c) a cell strain index, describing the
cell line, its designation, its origin,
details of maintenance and freezing
procedures, what its special
characteristics are, and where it is
located.

21. THAWING STORED AMPOULES
• When required, cells are thawed and
reseeded at a relatively high
concentration to optimize recovery.
• The ampoule should be thawed as
rapidly as possible, to minimize
intracellular ice crystal growth during
the warming process.
• This can be done in warm water, in a
bucket or water bath.
• The cell suspension should be diluted
slowly after thawing as rapid dilution
reduces viability.
• Then some cells must be centrifuged
after thawing.

22. Methods of Cryopreservation
• SLOW FREEZING METHOD
 Here, cells in a medium, with cryoprotectant
are cooled to below freezing point.
 At certain stage, water molecule is converted
to pure water crystal and unfrozen fraction of
cells and their solutes remains.
 Upon cryopreservation unfrozen fraction of
sample decreases while concentration of
salts, sugar and cryoprotectant increases.
 This osmotic change in cell cause outward
movement of water.
 The rate of freezing is slow(0.1-10°C/min).
 Commonly used for animal germplasm.
Advantages
 Easy to perform.
 Does not need continuous operator
intervention.
 The process increase the reproducibility
of the freezing operations.

23. • RAPID FREEZING METHOD
Here, freezing is done quickly so that there should be least change or
development of intracellular crystals.
Technique between the slow freezing and vitrification.
Faster than the slow freezing technique.
Requires low concentration of cryoprotectant.

24. • VITRIFICATION
Samples are solidified to form a glass like structure and avoid the development of
intracellular and extracellular ice.
High concentration of cryoprotecting agents and/ or very high cooling rates are used
to accomplish the cryopreservation.
Cost effective as well as time effective process.
Does not involve any expensive instruments.
Take quite a few minutes as compared to slow freezing.

25. ADVANTAGES
a) No ice crystal formation is occurred
during this process.
b) Rapid equilibrium is achieved.
c) Absence of water leak after equilibrium.
d) A very short time period is required for
the cryopreservation.
e) Minimum damage of the membrane
lipids will be occurred during this process
of cryopreservation.
f) This procedure of cryopreservation is
very simple as compared to the other
methods of cryopreservation.
g) As compared to the other methods in
this method do not require any equipment
and machine.
DISADVANTAGES
a) This method of cryopreservation
requires a well-established protocol.
b) Ultra rapid thawing is required for
this method of cryopreservation.
c) Ice crystal formation may be occurred
during hesitating thawing procedure.
d) A test for the genetic damage is
needed to carry out for the survivability
and viability of the samples.

26. Factors affecting Cryopreservation
• Water- Crystallization of water inside the cell increases the volume. It causes
physical damage to the cell membrane and other cellular organelles which resulted in
cell death.
• Cryoprotectant- It is important to add cryoprotective agents to minimize the injury
due to freezing and thawing.
• Membrane permeability- Permeability of the cell membrane to the solution is an
important factor in cryopreservation. The extent to which a cell can shrink and re-
swell is depending on the permeability of cell membrane and on the concentration of
cryoprotectant.
• Seeding temperature- The cell’s viability is significantly affected by the ice seeding
temperature. The intracellular ice formation interrelated with the extracellular ice-
seeding temperature.

27. • Buffer- Buffers resists the change in pH during freezing. pKa value of a buffer closer
to optimum freezing pH is more stable during temperature changes in cooling.
• Salt concentration- High concentration of solutes in an unfrozen fraction affects the
cell’s stability.
• Cooling rate- Every biological system and cells has a specific optimal cooling rate.
Below or above this rate the survival of the cell is decreased during the slow cooling
damage or fast cooling damage.
• Cryodamage- It is the damage of the cells, tissues and biological entity due to
freezing at low temperature. There are two types of damages occurs during freezing
of samples, one is physical damage and the other is chemical damage. The physical
damage is caused by rapid dehydration and hydration, sudden fall in temperature and
shrinkage of cell. The chemical damage is caused by the cryoprotectants.

28. Applications
• In the preservation of the animal cells, tissues and organs etc.
• In prevention of endangered species of animals.
• Genome resource banking, in assisting reproductive technologies, preservation of
embryo, oocytes, ovarian tissue, semen, testis tissues etc.

29. Germplasm Storage

30. • A germplasm is a collection of genetic resources for an organism.
• For plants, the germplasm may be stored as a seed, stem, callus, whole plant in
nurseries.
• In case of animals, it is stored in the form of genes, body parts stored in gene
bank/cryobank.
• The main objective of germplasm conservation is to preserve the genetic diversity
of selected stock for its utilization at any time in future.
• Its main aim is to provide essential support for collection, conservation and
utilization of plant and animal genetic resources all over the world.

31. Mainly 2 approaches:
1. In situ conservation- Maintained in their natural environment by establishing
biosphere reserves (or national parks/gene sanctuaries). Particularly useful for
preservation of animals in a near natural habitat without the human interference.
High priority germplasm preservation programme. Includes wild life sanctuaries,
national parks, biosphere reserves, gene sanctuary, and on-farm conservation.
1. Ex situ conservation- Also known as off-site conservation, where, conservation
occurs away from its natural habitat. The genes or genotypes are conserved outside
their natural occurrence for current or future use. They include seed storage, field
gene bank, botanical/ zoological gardens, pollen storage, DNA storage and
cryopreservation.

32. References
• Ian FR. Culture of animal cells. A manual of basic technique, 5th ed. Wiley-Leiss;
2005.
• Introduction to animal tissue culture science, Saurabh Bhatia, Tanveer Naved and
Satish Sardana(2019).
• ePG-pathshala (Animal cell biotechnology –Cell cryopreservation and animal
conservation)
• Cell culture basics- Invitrogen.
• www.onlinebiologynotes.com/germplasmconservation
• www.atcc.org/
• www.slideshare.com/

33. THANK YOU