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Cryopreservation

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ice crystal formation within embryo is lethal. if remove too ... forceps dipped in LN2, and touch straw. at -5oC cause media to become solid before freezing pt. ... – PowerPoint PPT presentation

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Title: Cryopreservation


1
Cryopreservation
2
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

3
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

4
Vitrification-quick freeze
  • increased cryoprotectant - no ice crystal
    formation
  • plunge into liquid nitrogen after equilibration

5
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

6
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

7
Advantages
  • not enough recipients
  • shipping
  • health concerns
  • embryo testing
  • dont need to synchronize recips
  • human storage - cancer, finances, age

8
Disadvantages
  • decrease viability 50 survival
  • cost
  • time and management
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