**3. Properties of MSCs for cellular therapy**

Although MSCs first attracted attention due to their ability to differentiate into various cell types, current data suggest that MSCs, as a result of their peculiar biological features, may not only replace damaged tissues, but may be also capable of secreting several bioactive molecules with paracrine and autocrine properties. Such functional secretions of factors are responsible for trophic [20], antiapoptotic, angiogenic, and antiscar effects [21, 22]. MSCs have a further interesting characteristic, related to the capacity to exert immunoregulatory effects on cells of adaptive and innate immunity, such as T and B lymphocytes, dendritic cells, natural killer cells, and monocytes [23]. These immunomodulatory properties that have been extensively demon‐ strated by several *in vitro* and *in vivo* studies, seem to permit MSC‐allogenic transplantation.

## **4. Sources of MSCs**

**1. Introduction**

40 Cryopreservation in Eukaryotes

they are banked for future therapeutic purposes.

**2. Mesenchymal stem cells**

cells [11], and cardiomyocytes [19].

The aims of regenerative medicine are to renew cells, regenerate fully functional tissues, and organs or structures that are lost or damaged after disease, injury, or aging [1]. In recent years, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have attracted much attention due to their potential use in regenerative medicine and tissue engineering as shown by the main applications described in the literature and the noteworthy progress that has been made toward their better understanding and characterization [2]. Those cells display a significant therapeutic plasticity as reflected by their advantageous characteristics: the ability to enhance tissue renovation, the immunomodulatory, and anti-inflammatory effects [3, 4] and the possibility to be used for both autologous and allogeneic therapies [5]. For these reasons, MSC-based cell therapies have been investigated for several years in human medicine and, more recently, the same approach has been considered in veterinary medicine as a novel potential therapy for animal diseases [6–9]. While most studies using animal models and even small clinical trials have utilized fresh MSC cultured on-site, cryopreservation of MSC is essential to the widespread application of MSC-based therapies. Cryopreservation allows for MSC to be prepared by specialized facilities, in large batches under the application of accepted quality control measures to ensure their safety. Currently, much information concerning the effects of cryopreservation on MSCs is difficult to interpret because MSCs are frequently isolated from different tissue sources and stored for variable periods of time. The capability of MSCs to survive to storage, maintain their phenotype, and differentiate along multiple lineage pathways upon thawing is of paramount importance if

MSCs were first described as a specific cell population by Friedenstein's research group in the late 1960s [10]. Previously, stem cell populations were supposed to reside solely in adult tissues with a high turnover rate, such as blood, skin, hair, gastrointestinal epithelium, and bone. Indeed, these cells are present in variable amounts in specific stem cell "niches" (organs), in almost all the body tissues and even if the exact locations of these niches are poorly understood, there is growing evidence suggesting a close relationship with pericytes [11]. Generally, these cells remain in a quiescent state until activated by significant events, such as during tissue repair after injury or following transplantation, to regain tissues' homeostasis [12, 13]. MSCs are undifferentiated, self-renewable, multipotent adult stem cells originated from the mesoderm germ layer during the embryonic development, characterized by the ability to evolve both *in vitro* and *in vivo* along multiple lineage pathways [14]. Furthermore, MSCs have shown evidence of plasticity by trans-differentiating into a broad range of cell types of mesodermal origin (osteocytes, chondrocytes, adipocytes, and myocytes) [15, 16], but also deriving from other germ layers including ectodermal neurons [17] endodermal hepatocytes [18], endothelial In veterinary medicine, the first source reported to contain MSCs was the bone marrow (BM) that in the past was also the most widely used [15]. Nevertheless, more recent studies have identified MSCs with similar properties in almost all mammalian tissues such as skeletal muscle [24, 25], tendon [26], skin [27, 28], adipose tissue [29], periosteum [30], synovial membrane [31], dental pulp [32], peripheral blood [33], umbilical cord blood [34], amniotic fluids [35], and cornea [36].
