Preface

Genetic resources represent the genetic storehouse of information, which needs to be explored, evaluated, cataloged, and conserved so that no genotype is lost from the treasury of genome diversity. Plant genetic resources (PGRs) are essential components of agro-biodiversity and are defined as the genetic material of plants having value as a resource for present and future generations [1]. With no limitation to plants, animal genetic resources (AnGRs) can be referred to as genetic diversity in domesticated animal species with economic or other socio-cultural values and found among species, among animal breeds within the species, and in cryopreserved material in the form of embryos and semen [2].

Posterity is essential and it is critical that all available methods including cryopreservation applications are explored to maintain the available scope of diversity. Cryopreservation, which is the cooling of cells to sub-zero temperatures, at which all metabolic systems are preserved indefinitely, may be an illusion due to the associated difficulties in revitalizing cells after cooling. However, when successful, this method holds the future for cell preservation.

Major parameters that require attention for a successful cryopreservation procedure include the method of cooling, type of cryoprotectant, recovery and revitalization after preservation, and type of cells or tissues being preserved (e.g., plant parts, germlines, etc.). Cryoprotection is the process during which cells to be cryopreserved are treated with substances that will protect the tissues from freezing damage. Freezing damage occurs when inter- and intracellular fluids form ice crystals that perforate membranes and organelles thus damaging their viability. The formation of glass in cells instead of crystals prevents this damage, and this can be facilitated using cryoprotective agents (CPAs). The formation of glass instead of ice crystals is referred to as vitrification. CPAs are thought to inhibit ice formation by interacting with hydrogen bonding among water molecules [3], resulting in the production of nonspecific toxic effects [4].

The most known CPAs are dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG), 2-Methyl-2,4-pentanedol (MPD), propylene glycol, sucrose, and trehalose. The CPAs can be categorized as either membrane permeating, such as glycerol, ethylene glycol, and DMSO, which can freely diffuse the membrane, and non-membrane permeating, such as sugars, which cannot permeate the cell membrane. Although useful, CPAs may be toxic to cells, causing biological material to compromise their genetic makeup, metabolic systems, and functionality. Factors that must be controlled when using CPAs include the concentration of the cryoprotectant, duration of exposure, and combination of two or more cryoprotectants.

This book is a review of research outputs, proposals, and challenges associated with using cryopreservation techniques to conserve germplasm for posterity. It considers various types of plant parts and germlines used for conservation. Successful preparation of cells and tissues prior to cooling is critical to ensure restoration after cooling as is the appropriate use of cryoprotectants.

This book includes two sections. Section 1, "Gametes and Embryos Cryopreservation", includes 5 chapters.

Chapter 1, "Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality Controls for Commercial Applications", sheds light on the cryopreservation of sperms of various fish species, namely, carp, sturgeon, eel, salmonid, and catfish, some of which are endangered species. The chapter reviews the conservation of germ cells and embryos. It also discusses contamination that may be associated with fish gamete cryopreservation and the thawing process. It presents a thorough review of successful procedures, disease transmission via gametes to the embryo, elimination of non-cellular disease agents from gametes, antibiotics, sperm washing and cell separating methods, and disinfection of gametes and embryos.

Chapter 2, "The Current Status of Semen and Oocytes Cryopreservation", reviews challenges associated with the cryopreservation of gametes with specific references to oocytes and sperms. Adequate reference is made to the effect of these challenges on in vitro fertilisation (IVF). It also discusses the use of cryoprotectants, antioxidants, and various freezing methods.

Chapter 3, "Female Fertility Preservation: Different Interventions and Procedures", discusses cryoprotectants, particularly permeable cryoprotective agents (CPAs) such as glycerol, dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PG). It also discusses non-permeable cryoprotectants as well as cooling methods such as controlled slow-rate freezing and vitrification. The chapter discusses the use of permeable and non-permeable CPAs, their mode of action, and their toxic effects. It is suggested that a mixture of cryoprotectants is less toxic and more effective for successful cryoprotection. The non-permeable CPAs are presented as remaining in extracellular space and having the ability to reduce the formation of extracellular ice formation. Ovarian tissue cryopreservation is reported as an evolving technique that requires more standardised applications.

Subsequently, Chapter 4, "Ovarian Tissue Cryopreservation Guidelines", highlights ten systems that could serve as guidelines for successful cryopreservation and discusses their associated challenges.

Chapter 5, "Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes", addresses critical topics associated with CPAs, the dimension and complexity of cells, ice formation, and cooling rate. A thorough review is provided on cryopreservation of larger structures like the ovaries, thymus, and biopsies. The chapter also presents ways to overcome practical challenges associated with CPA loading and unloading, diffusion of heat and intracellular water, ice nucleation and direct ice damage, and control of ice structure.

Section 2 includes two chapters that focus on the prospects of cryopreservation as applied in blood and plants. Chapter 6, "Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells (PBMCs)", highlights cooling

methods and makes recommendations based on research activities conducted by the authors. Three cooling methods are discussed: -1° C/min cooling rate that requires only isopropyl alcohol, a cooling rate of -1° C/min solely, and a user-predefined programmable controlled rate of freezing. The method using isopropyl alcohol is not recommended, whereas the two other cooling methods are recommended for cryopreservation of peripheral blood mononuclear cells (PBMCs).

As the world grapples with the threats of climate change and food insecurity, the fate of clonally propagated crops is crucial and cryopreservation techniques must be considered.

Chapter 7, "Plant Cryopreservation Importance, Approaches and Future Trends", reviews various affordable cooling systems with attention to the removal of freezable water and proposes a way forward that includes the development of required technical expertise.

This book provides a comprehensive overview of cryopreservation in cell therapies, tissue-engineered constructs, and other larger tissues.

> **Dr. Marian Dorcas Quain** Biotechnology Laboratory, Council for Scientific and Industrial Research, Crops Research Institute, Kumasi, Ghana

#### **References**

[1] Qualset CO, McGuire PE, Warbuton M.L. (1995). 'Agrobiodiversity' key to agricultural productivity. Calif. Agric. 1995;49:45-49.

[2] Kantanen J, Løvendahl P, Strandberg E, Eythorsdottir E, Li M-H, Kettunen-Præbel A, Berg P and Meuwissen T (2015) Utilization of farm animal genetic resources in a changing agro-ecological environment in the Nordic countries, Frontiers in Genetics. Vol. 6 article 52. doi: 10.3389/fgene.2015.00052

[3] Towey JJ and Dougan (2012). Structural Examination of the Impact of Glycerol on Water Structure *J. Phys. Chem. B* 2012, 116, 5, 1633–1641 https:// doi.org/10.1021/jp2093862

[4] Fahy GM, Wowk B, Wu J, and Paynter S (2004) Improved vitrification solutions based on the predictability of vitrification solution toxicity. Cryobiology 48 pp. 22–35

Section 1
