Lab Practical: Cryopreservation Flashcards
At what temperature are cryopresered gametes and embryos kept
-196 degrees Celsius
what is the purpose of cryopreservation
to maintain the viability of sperm, oocytes and embryos for manyyears
when was the first pregnancy resulting from cryopreserved sperm
1953
What are the main risks of cryopreservation to gametes/embryos
Internal ice crystal formation
Osmotic stress
Which cells are more likely to experience ice crystal formation and why
Oocytes as they are larger cells
What is used to help prevent the sperm being compromised during freezing/thawing
cryoprotectant agents
- evidence suggests that this helps to enhance sprerm cryosurvival rate
What are the main uses of cryopreserved sperm
Sperm donation for IVF treatment
Fertility preservation e.g. before cancer treatment
Describe how you would carry out the freeze process in the lab
- Measure the volume of the liquefied sample
- Make a wet-preparation of the liquefied semen sample to estimate the concentration and motility
- Slowly add the Sperm Freezing solution (Origio; add 1:1 ratio; pre-warm the freeze solution at room temperature for at least 15 mins prior to starting) to the liquefied semen. This should take a minimum of 5 minutes. Mix the sample by swirling the pot in hand through-out the procedure
- Label a cryovial and add semen to a maximum volume of 1 mL and put into the liquid nitrogen tank immediately
Describe how you would carry out the thaw process in the lab
- Take the cryovial out of the liquid nitrogen and immediately put in the 30 °C water bath for 2 minutes
- Over a minimum period of 5 minutes, add 4 mL of sperm wash solution to the semen in a 50 mL Falcon tube. Shake the sample through-out
- Perform density gradient centrifugation (DGC) using to separate gradients (2.5 mL thawed sample on each). Centrifuge 0.3 RCF for 20 minutes
- Pool 80% pellets into 4 mL sperm wash and centrifuge for 5 minutes at 0.3 RCF
- Aspirate the supernatant leaving 0.5 mL of sperm wash
- Resuspend the pellet
What are the two main methods of sperm cryopreservation and what is typically used now as it is shown to give superior results
slow programmable freezing
vitrification (often preferred these days)
Why does ice formation occur during cryopreservation
When tissues are cooled slowly, water migrates out of cells and ice forms in the extracellular space. Too much extracellular ice can cause mechanical damage to the cell membrane due to crushing
Some cells can tolerate extra cellular ice formation but any intracellular ice is usually fatal
how can osmotic stress cause injury to the sperm during freezing
Migration of water, causing extracellular ice formation, can also cause cellular dehydration. The associated stresses on the cell can cause damage directly