Cryopreservation: Anuja Kamath, M.SC Bangalore Assisted Conception Centre
Cryopreservation: Anuja Kamath, M.SC Bangalore Assisted Conception Centre
Cryopreservation: Anuja Kamath, M.SC Bangalore Assisted Conception Centre
Preservation of
◦ Gametes
◦ Embryos
◦ Gonadal Tissue.
donation
Ensure backup-for repeat ET
Aim of cryopreservation
AIM
To slow and/or shut down cellular activity
To preserve structure
Results:
Variable depending on the complexity
dehydration.
Cellular Destruction – Intra cellular
◦ 1,2 propanediol
◦ dimethyl sulfoxide DMSO
◦ ethylene glycol
◦ glycerol
Extracellular cryoprotectants (Non
Permeating)
◦ sucrose and raffinose
Cryoprotectant
Cryoprotectant - Non-permeating
DMSO
It has relatively low toxicity
Small size - penetrates membranes rapidly
glycerol
Also called 1,2,3-propanetriol
Antifreeze compound widely used for sperm
and blastocysts
It has low toxicity, but is slow to cross
membranes which can contribute to osmotic
shock
propanediol
1,2-Propanediol
Antifreeze compound - Used for human
embryos
It readily forms vitrifying solutions
Commonly abbreviated as PG or PRO
A potential problem is that one of its
Ice present
Warming rate – 4 to 100°C/min
Vitrification
State during Cooling
Glass like state without ice formation
Cooling rate usually 200 to 20,000°C/min
Cooling rate depends on the type of container
used
Slow Fast
Seeding No seeding
Not useful for small volume Very useful for small volume
(WASH)
◦ VIAL 4 : PBS
Warm for a minimum of 30 minutes at room temperature.
Remove straws from liquid nitrogen and keep at room temperature
minutes.
Transfer oocytes into medium from Vial 2 at room temperature for
1 for 5 – 10 mins.
Transfer the embryos to well 2 for 1 ½ min.
Finally transfer the embryos to well 3 for 30
1 min
Move the embryo to well 3 for 2 min.
Move the embryo to well 4 for 5 mins.
Transfer to pre equilibrated culture dish.
Cryodevices
McGill Cryoleaf
Rapid i
cryotop
Cryolock
Should be performed by trained embryologist
Embryos should be loaded on the device and
plunged in LN2 within 30 secs
Volume should be minimum
First step in thawing very important.
Temp should be maintained at 37 deg while
thawing.
Thank you