Cryobiology

• Cryobiology


  Study of the effect of subzero temperatures on biological materials
• Reasons for cryopreservation


  • Increasing longevity of sample
  • Food storage
  • Research applications - stores stocks of stem cells to keep research consistent between different stem cell and tissue culture lines
  • Agricultural/medical applications - preservation of tissues or cells like sperm, oocytes, embryos
• Freezing


  Conversion of water to ice, prevents chemical reactions from occurring, cells become quiescent
• Cell membranes


  • Lipid bilayer interspersed with proteins and carbohydrates
  • Semi-permeable, regulates movement of ions, molecules, solutes and water in and out of cell
  • Damage to the cell membranes causes cell death (cell must be intact and functional)
• Causes of cell membrane damage during cryopreservation

  • Physical ice damage
  • Oxidative stress from lipid peroxidation
  • Changes to fluidity in the membrane
• Osmolarity


  Concentration of a solution (also referred to as osmols/L)
• Tonicity


  The osmotic pressure of a solution, often expressed as a relative to an intercellular environment (e.g. hypo, iso, or hypertonic)
• pH buffer


  Solution which reduces the pH upon addition of small amounts of acid or base
• Cooling rates


  • Too slow - intracellular ice formation will explode cells
  • Too fast
  • Just right
• Optimal freezing rate


  Depends on cell size and shape, strongly related to surface area:volume ratio, also depends on what is in the cryodiluent (medium, extender) and what cryoprotectant is used
• Components of cryopreservation solution

  • Nutrients for metabolic processes
  • pH buffer
  • Osmotic balance
• Penetrating cryoprotectants


  Small water soluble molecules that stay in solution at low temps, slow water loss in instances of slow cooling, less of tonicity difference between intracellular and extracellular environments
• Nonpenetrating cryoprotectants


  Large water soluble molecules that can't get into lipid biolayer, hasten water loss in fast cooling, causes dehydration in the cell, modify membrane properties
• Glycerol in sperm preservation


  Impacts cooling rate and is displayed as peaks
• Thawing rate


  Faster freezing requires faster thawing, avoid recrystallisation of ice, avoid dehydration of cell, avoid long term exposure to cytotoxic compounds
• Stages of a thawing blastocyst

  By 5hrs post thawing, the blastocyst begins to grow
• Vitrification


  Freezing in the absence of ice OR Solidification of liquid without crystallisation (instead, with formation of glass)
• Glass


  Much less rigid than crystal, less likely to form damaging surfaces that could injure the cell and would be less likely to fracture
• Effect of penetrating cryoprotectants

  • No cryoprotectant + slow freezing - extracellular ice formation squashes cells
  • Cryoprotectant + slow freezing - added cryoprotectant resists ice formation, leaving more space for cells
  • High concentration cryoprotectant + ultra fast freezing (vitrification) - entire volume resists freezing and transitions to a solid (glass) instead of ice
• Advantages and disadvantages of vitrification

  • Improved survival rates
  • Application to whole tissues not cells (hearts and livers)
  • Technically difficult
  • Exposure of cells to high conc. Of cryoprotectant will make cells cytotoxic
  • Questionable biosecurity - unpackaged (this is changing)
• Applications of cryopreservation


  • Agriculture
  • Wildlife preservation and breeding programs
  • Gene banking
  • Service animal programs
  • Selective breeding programs
  • Humans
• Agricultural applications


  • 232,347,071 frozen doses of bull spermatozoa produced in 2000
  • ~145,000 frozen-thawed cattle embryos transferred worldwide in 2020
  • >85% dairy cows bred with frozen-thawed semen in the USA at an average dose cost of $10-120 (record is $8000/straw!)
  • Sheep, goat and horse frozen semen also commercially available and widely used
• Vital for Australia - enables transport across vast geographic distances and import of biological material in spite of strict quarantine laws and border security
• Species specificity


  Sperm (and embryos) from different species have different freezing tolerance
• Gene banking applications


  • Storage of tissues (not just spermatozoa and embryos) to aid in conservation
  • Requires technologies such as creation of stem cells and cloning
• Service animal programs using cryopreservation

  • Guide dogs - $50,000
  • PTSD dogs - $10-30,000
  • Police dog - $12-15,000
  • Drug detection dog - $55,000
• Other breeding programs using cryopreservation

  • Pets - Dogs, Cats, Birds, Ferrets, Rabbits
  • Lab animals - Mice, Rats, Rabbits, Guinea pigs
• Human cryopreservation applications in Australia and New Zealand in 2021

  • 41,131 frozen embryo transfer cycles performed
  • 96.6% of these were vitrified embryos
  • Live birth rate from frozen embryos was 31.5% (higher than fresh embryos; 25.3%)
• Cryodamage to cells


  • Lethal damage (loss of membrane integrity and death)
  • Sub-lethal damage (still viable, but compromised)
• Causes of cryodamage


  • Reactive oxygen species (ROS)
  • pH changes – deamination, depurination and depyrimidination
  • Temperature change – denaturation (protein destruction)
  • Hyperosmotic stress – DNA strand breaks, misfolded proteins
• Effects of cryoprotectants on DNA

  • Ethylene glycol – chromatin damage
  • DMSO – DNA methylation, conformational DNA changes
  • Propylene glycol – DNA methylation
  • Glycerol – Conformational DNA changes
• Oocyte freezing


  • Oocyte freezing has been a much more difficult process and was only deemed out of the experimental phase for human oocytes in 2012
  • Success rates now in the range of 40% live births, but is highly dependent on age at which oocytes are frozen
  • Lots of debate around the ethics and social considerations of planned oocyte cryopreservation (for social reasons)
• Oocyte freezing success rates in animals

  • Cow ~30% blastulation
  • Ewe <10% blastulation
  • Mare <50% pregnancy
  • Pig – largely unsuccessful
  • Bitch – very little work done, largely unsuccessful
  • Queen >10% blastulation
  • Mouse doe ~40% live birth rate