Preface
Cryopreservation, vitrification, and freeze-drying can be used to ensure off-the-shelf availability of cells and tissues that are used for research purposes or for applications in medicine
or agriculture. Whereas cryopreserved and vitrified samples can only be stored at ultralow
temperatures in energy-consuming cryostorage devices, freeze-dried specimens can be
stored at room temperature, which has clear advantages for storage and transport.
Despite the fact that nowadays many types of cells and tissues can be cryopreserved
using relatively easy and standardized protocols, cryopreservation is not a routine procedure, and there is no unified freezing protocol that works for all specimens. The various steps
involved in cryopreservation, including the introduction of protective agents into the cell or
tissue and the freezing and thawing conditions, all need to be optimized. Knowledge of the
permeation rate of protective agents into cells or tissues can be used to estimate the time
needed to load specimens with protective compounds. The optimal cooling rate can either
be determined experimentally or, when cell-specific membrane permeability parameters are
known, can be predicted using mathematical transport models. In vitrification approaches,
the challenge is to minimize the exposure time during the introduction of high concentrations of protective agents to avoid toxicity effects. Protective agents that need to be used for
freeze-drying are typically larger than those used for cryopreservation or vitrification, and
hence the main challenge is to introduce them into cells. Cryopreservation and dry preservation of cells and tissues are highly interdisciplinary fields of research requiring insights
from biologists, chemists, physicists, medical scientists, as well as engineers.
In this edition of Cryopreservation and Freeze-Drying Protocols, we provide standardly
used cryopreservation, vitrification, and freeze-drying protocols for specimens that are used
for research purposes, conservation of genetic reserves, and applications in agriculture and
medicine. In addition, experimental and mathematical modeling approaches are introduced
that can be used to rationally design the different steps involved in cryopreservation and
freeze-drying. The book is divided in three parts. Part I describes the fundamental principles
of cryopreservation, vitrification, and freeze-drying, as well as the use of mathematical
modeling to solve cryobiological problems. In Part II, technological aspects of freezing
and drying are discussed, and various analytical methods are presented to study: protectant
loading of cells and tissues, cell behavior during freezing and drying, and thermodynamic
properties of preservation solutions. In Part III, cryopreservation, vitrification, and freezedrying protocols are presented for a wide variety of samples and different applications
including sperm, oocytes, blastocysts, ovarian tissue, blood cells, stem cells, megakaryocytes,
cell monolayers, cardiovascular tissues, marine invertebrates, fern spores and pollen, algae,
bacteria, plant cell lines, plant shoot tips, seeds, and seed embryos.
The book serves as a practical guide that can be used without the need of other reference
sources. In addition to protocols that require the use of specialized equipment, practical and
cheaper alternatives are also described. Our intended readers are researchers and technical
vii
Cryopreservation, vitrification, and freeze-drying can be used to ensure off-the-shelf availability of cells and tissues that are used for research purposes or for applications in medicine
or agriculture. Whereas cryopreserved and vitrified samples can only be stored at ultralow
temperatures in energy-consuming cryostorage devices, freeze-dried specimens can be
stored at room temperature, which has clear advantages for storage and transport.
Despite the fact that nowadays many types of cells and tissues can be cryopreserved
using relatively easy and standardized protocols, cryopreservation is not a routine procedure, and there is no unified freezing protocol that works for all specimens. The various steps
involved in cryopreservation, including the introduction of protective agents into the cell or
tissue and the freezing and thawing conditions, all need to be optimized. Knowledge of the
permeation rate of protective agents into cells or tissues can be used to estimate the time
needed to load specimens with protective compounds. The optimal cooling rate can either
be determined experimentally or, when cell-specific membrane permeability parameters are
known, can be predicted using mathematical transport models. In vitrification approaches,
the challenge is to minimize the exposure time during the introduction of high concentrations of protective agents to avoid toxicity effects. Protective agents that need to be used for
freeze-drying are typically larger than those used for cryopreservation or vitrification, and
hence the main challenge is to introduce them into cells. Cryopreservation and dry preservation of cells and tissues are highly interdisciplinary fields of research requiring insights
from biologists, chemists, physicists, medical scientists, as well as engineers.
In this edition of Cryopreservation and Freeze-Drying Protocols, we provide standardly
used cryopreservation, vitrification, and freeze-drying protocols for specimens that are used
for research purposes, conservation of genetic reserves, and applications in agriculture and
medicine. In addition, experimental and mathematical modeling approaches are introduced
that can be used to rationally design the different steps involved in cryopreservation and
freeze-drying. The book is divided in three parts. Part I describes the fundamental principles
of cryopreservation, vitrification, and freeze-drying, as well as the use of mathematical
modeling to solve cryobiological problems. In Part II, technological aspects of freezing
and drying are discussed, and various analytical methods are presented to study: protectant
loading of cells and tissues, cell behavior during freezing and drying, and thermodynamic
properties of preservation solutions. In Part III, cryopreservation, vitrification, and freezedrying protocols are presented for a wide variety of samples and different applications
including sperm, oocytes, blastocysts, ovarian tissue, blood cells, stem cells, megakaryocytes,
cell monolayers, cardiovascular tissues, marine invertebrates, fern spores and pollen, algae,
bacteria, plant cell lines, plant shoot tips, seeds, and seed embryos.
The book serves as a practical guide that can be used without the need of other reference
sources. In addition to protocols that require the use of specialized equipment, practical and
cheaper alternatives are also described. Our intended readers are researchers and technical
vii