Cryopreservation

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Cryopreservation
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The Dilemma

• must remove water from blastomeres
• ice crystal formation within embryo is lethal
• if remove too much water, cell damage will result
• must remove just enough water but not too much,
  and not too fast

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Cryoprotectants

• protect cells from ice crystal damage by
  increasing solute
• Intracellular cyroprotectants
• 1,2 propanediol
• dimethyl sulfoxide DMSO
• ethylene glycol
• glycerol
• Extracellular cryoprotectants
• sucrose and raffinose

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Vitrification-quick freeze

• increased cryoprotectant - no ice crystal
  formation
• plunge into liquid nitrogen after equilibration

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Serial dilutions- slow freeze

• add cryoprotectants in steps to minimize
  shrinkage
• 0, 3, 6, 10 gylcerol with sucrose added the last
  step (10 sucrose)
• slow, to allow water to leave cells

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Seeding

• forceps dipped in LN2, and touch straw
• at -5oC cause media to become solid before
  freezing pt.
• normally freezes at -8o to -10oC.
• avoids super-cooled state/heat of fusion
• no fracture planes to reduce viability

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Advantages

• not enough recipients
• shipping
• health concerns
• embryo testing
• dont need to synchronize recips
• human storage - cancer, finances, age

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Disadvantages

• decrease viability 50 survival
• cost
• time and management