This slide is just a basics of cryopreservation, it's uses and safety measures.

1. CRYOPRESERVATION

2. What’s is Cryopreservation??
 Cryopreservation is a process where cells, whole tissues, or any other
substances susceptible to damage caused by chemical reactivity or time
are preserved by cooling to sub-zero temperatures.
 Cryopreservation methods seek to reach low temperatures without causing
additional damage caused by the formation of ice during freezing.
 Cryoprotectants, such as dimethyl sulfoxide and glycerol, are used to
protect cells from freezing.
 The cryopreservation of stem cells is a crucial component of their
therapeutic use in hematologic disorders and regenerative medicine.

3. Collection and Cryopreservation
 Cord blood collection happens after the umbilical cord has been cut and is
extracted from the fetal end of the cord.
 After the collection, the cord blood unit is shipped to the lab and
processed, and then cryopreserved.
 The unit is processed, a cryopreservant is added to the cord blood to allow
the cells to survive the cryogenic process.
 After the unit is slowly cooled to −140 °C, it can then be added to a liquid
nitrogen tank which will keep the cord blood unit frozen at −196 °C.
 The slow freezing process is important to keep the cells alive during the
freezing process.

4. Cryopreservation Protocol
 Cryoprotectant: 10% DMSO
 Cooling rate: 1 C/min
 Controlled rate freezing
 Storage on LN 2
 Documenting the Location of samples in the Manual records as well as in
online applications.

5. Control Rate Freezer Procedure
 Cooling rate is known to have a most significant influence on cell survival.
 Controlled rate freezing before long-term storage maximizes viability for a
wide variety of cells.
 Programmed, uniform cooling rates are effective for a variety of freezing
applications.
 The initiation of steady state cooling starts at 1ºc.
 There are four stages while running a control rate freezer.
 Stage 1: -1ºc/m upto -3ºc
 Stage 2: -10º/min upto -20ºc
 Stage 3: -1º/min upto -40ºc
 Stage 4: -10º/min upto -140ºc

6. Storage of cryopreserved samples
 The samples are stored in the vapour phase of liquid nitrogen in LN2
vessels.
 After the samples reach -140ºc in the CRF, The samples are taken to a cryo
cart and stored in boxes according to the particular locations and kept
inside the LN2 vessels.
 We store Cord blood (Main bag and Pilot bag), cord tissue (Direct and
Passage), Dental(Direct and Passage) and menstrual blood(Direct and
Passage)(FEMME).

7. Liquid Nitrogen
 Liquid nitrogen is a cryogenic liquid. Cryogenic liquids are liquefied gases
that have a normal boiling point below –130°F (–90°C).
 Liquid nitrogen has a boiling point of –320°F (–196°C).
 Nitrogen is produced at air separation plants by liquefaction of
atmospheric air and separation of the nitrogen by continuous cryogenic
distillation.
 Liquid nitrogen is inert, colorless, odorless, noncorrosive, nonflammable,
and extremely cold. Nitrogen makes up the major portion of the
atmosphere (78.03% by volume, 75.5% by weight).

8. LN2 CYLINDERS
 LN2 cylinders are insulated, vacuum-jacketed pressure
vessels. They come equipped with safety relief valves
and rupture discs to protect the cylinders from
pressure buildup.
 Liquid product is typically removed through insulated
withdrawal lines to minimize the loss of liquid product
to gas.
 Insulated flexible or rigid lines are used to withdraw
product from storage tanks.
 The liquid nitrogen supply pressure at the inlet to the
refrigerator should be in the range of 10 psi (0.7
bar/69 kPa) to 22 psi (1.4 bar/152 kPa) for optimum
performance.

9. LN2 VESSELS
 The LN2 vessels provide unique solutions
to both short-term as well as long term
storage of large volumes of samples.
 With a capacity of approx. 80k 2ml vials or
40k 5ml vials or 11k cord blood 20ml bags,
a storage temperature near that of liquid
nitrogen in the vapour phase at the top of
the vessel and low nitrogen consumption.
 The bearing free easy-to-rotate turntable
with aluminium dividers permits convinent
access to the stored samples.

10. Transportation of Samples
 The samples are transported to the required location
using Dry shippers.
 The dry shippers are capable of maintaining the materials
in them at liquid nitrogen temperatures for nearly 2 weeks
without the risk of spilling liquid nitrogen.
 The samples are documented in an accountability sheets
which contains the list of samples which are going be
shipped.

11. LN2 Safety and Health Hazards
 Being odorless, colorless, tasteless, and nonirritating, nitrogen has no
warning properties.
 Inhalation of nitrogen in excessive amounts can cause dizziness, nausea,
vomiting, loss of consciousness, and death.
 Personnel, including rescue workers, should not enter areas where the
oxygen concentration is below 19.5%, unless provided with a self-
contained breathing apparatus or air-line respirator.
 Extensive tissue damage or burns can result from exposure to liquid
nitrogen or cold n
 Store and use liquid containers with adequate ventilation itrogen vapors.

12. Personal Protective Equipment (PPE)
 Personnel must be thoroughly familiar with properties and safety
considerations before being allowed to handle liquid nitrogen and/or its
associated equipment.
 The recommended personal protective equipment when handling or using
liquid nitrogen is a full face-shield over safety glasses; loose-fitting thermal
insulated or leather gloves; and long-sleeved shirts/Aprons and pants
without cuffs, especially whenever the possibility of exposure or a spill
exists.
 In emergency situations, selfcontained breathing apparatus (SCBA) must
be used.

13. First-AID
 People suffering from lack of oxygen should be moved to fresh air.
 For skin contact with cryogenic liquid nitrogen, remove any clothing that
may restrict circulation to the frozen area.
 DO NOT RUB frozen parts, as tissue damage may result. As soon as
practical, place the affected area in a warm water bath that has a
temperature not in excess of 105°F (40°C).
 If the eyes are exposed to the extreme cold of the liquid nitrogen or its
vapors, immediately warm the frostbite area with warm water not
exceeding 105°F (40°C) and seek immediate medical attention.
 Frozen tissue is painless and appears waxy with a possible yellow color.

14. CONCLUSION
 At this time, you should be able to preserve most cell types and some
selected tissue types by combining the following:
 Your knowledge of, and/or experience with, the chemical control of ice
formation.
 The selection process for state-of-the-art equipment that effectively
controls cooling and warming conditions.
 Selection and inventory configuration of reliable equipment to store your
preserved samples until they are needed .
 Availability of process procedures to facilitate the fulfillment of regulatory
requirements and improving product quality.

15. THANK YOU