Ice-free cryopreservation or vitrification employs high cooling rates
(>100
C/min) and high CPA concentrations (30–50% (v/v), up
to ~9 M) to completely avoid ice formation. The challenge with
slow freezing cryopreservation approaches is to find the cell-specific
optimal cooling rate resulting in maximal cryosurvival [18],
whereas the challenge with vitrification procedures is to ensure
homogenous mass and heat transfer while minimizing CPA toxicity
[19]. Slow freezing cryopreservation is routinely done for maintenance of cell culture lines and sperm, although large variation exists
in cryosurvival among cell types and lines as well as individuals.
Vitrification is routinely used for embryos and many types of small
mammalian and plant tissues. Currently, vitrification is probably the
only method that holds promise to preserve whole organs, but
many hurdles need to be overcome to make this happen [20].
Freeze-drying is commonly used in pharmaceutics for preserving, e.g., proteins and liposomes, because thereafter samples can be
stored and shipped in a stable dried state at room temperature.
Freeze-drying is not frequently used to preserve cells or tissues,
because it is a much more damaging process for cells compared to
freezing only. In contrast to freezing, during drying water normally
surrounding biomolecules is also removed [21]. For freeze-drying,
lyoprotectants like sucrose and trehalose need to be used to protect
during both freezing and drying. The challenge is to load cells with
lyoprotectants, for which cellular membranes typically are impermeable. Freeze-dried cells may retain specific properties for use in
applications that do not require fully functional cells. Freeze-dried
platelets, for example, can be used for topical wound healing [22]
and freeze-dried sperm for fertilization via intracytoplasmic sperm
injection [23]. It is unlikely that cells in tissues survive freezedrying, but freeze-dried acellular matrices can be used as biological
scaffolds in regenerative medicine [24].
3 Freezing and Drying Injury
3.1 Freezing Injury
Cryopreservation of cells or tissues relies on the use of ultra-low
temperatures to suspend chemical reactions, biological processes,
and physical intra- and extracellular activities. However, the processing pathways cells have to traverse to reach safe storage temperatures and to return back to physiological temperatures can be very
damaging. First of all, cooling results in the conversion of liquid
water to crystalline ice causing many different types of damage to a
biological sample. Original theories of freezing injury envisaged ice
crystals piercing cells and intracellular structures, destroying them
by direct mechanical action [25]. Also intracellular ice formation
causes damage in the structure and organization of plasma and
organelle membranes, eventually leading to cell death
[26, 27]. Upon extracellular ice formation, presence of high salt
Principles of Cryopreservation and Freeze-Drying
7
(>100
C/min) and high CPA concentrations (30–50% (v/v), up
to ~9 M) to completely avoid ice formation. The challenge with
slow freezing cryopreservation approaches is to find the cell-specific
optimal cooling rate resulting in maximal cryosurvival [18],
whereas the challenge with vitrification procedures is to ensure
homogenous mass and heat transfer while minimizing CPA toxicity
[19]. Slow freezing cryopreservation is routinely done for maintenance of cell culture lines and sperm, although large variation exists
in cryosurvival among cell types and lines as well as individuals.
Vitrification is routinely used for embryos and many types of small
mammalian and plant tissues. Currently, vitrification is probably the
only method that holds promise to preserve whole organs, but
many hurdles need to be overcome to make this happen [20].
Freeze-drying is commonly used in pharmaceutics for preserving, e.g., proteins and liposomes, because thereafter samples can be
stored and shipped in a stable dried state at room temperature.
Freeze-drying is not frequently used to preserve cells or tissues,
because it is a much more damaging process for cells compared to
freezing only. In contrast to freezing, during drying water normally
surrounding biomolecules is also removed [21]. For freeze-drying,
lyoprotectants like sucrose and trehalose need to be used to protect
during both freezing and drying. The challenge is to load cells with
lyoprotectants, for which cellular membranes typically are impermeable. Freeze-dried cells may retain specific properties for use in
applications that do not require fully functional cells. Freeze-dried
platelets, for example, can be used for topical wound healing [22]
and freeze-dried sperm for fertilization via intracytoplasmic sperm
injection [23]. It is unlikely that cells in tissues survive freezedrying, but freeze-dried acellular matrices can be used as biological
scaffolds in regenerative medicine [24].
3 Freezing and Drying Injury
3.1 Freezing Injury
Cryopreservation of cells or tissues relies on the use of ultra-low
temperatures to suspend chemical reactions, biological processes,
and physical intra- and extracellular activities. However, the processing pathways cells have to traverse to reach safe storage temperatures and to return back to physiological temperatures can be very
damaging. First of all, cooling results in the conversion of liquid
water to crystalline ice causing many different types of damage to a
biological sample. Original theories of freezing injury envisaged ice
crystals piercing cells and intracellular structures, destroying them
by direct mechanical action [25]. Also intracellular ice formation
causes damage in the structure and organization of plasma and
organelle membranes, eventually leading to cell death
[26, 27]. Upon extracellular ice formation, presence of high salt
Principles of Cryopreservation and Freeze-Drying
7