**3. Dental-related stem cell initiation culture, culture systems and cryopreservation**

In dental related stem cell researches, the first step is to isolate cells from tissue sources. The next step is to expand the cells into sufficient number. In some occasion, it may be necessary to preserve the cells for future use since tissue sources would not be available on demand. In this section, we will describe the location of the certain teeth tissue from which the stem cell population can be derived and followed by description of common methods by which the isolation culture can be initiated. At the end, culture systems for the cell propagation as well as the main points regarding issue of cell cryopreservation will be explained.

#### **3.1. Anatomical location of teeth tissue from which DSCs can be derived**

**DPSCs SHED PDLSCs DFPCs SCAPs GSCs**

CD13 CD13 CD13 CD13 CD13 CD13

CD29 CD29 CD29 CD29 CD29 CD29 CD44 CD44 CD44 CD44 CD44 CD44

CD73 CD73 CD73 CD73 CD73 CD73 CD90 CD90 CD90 CD90 CD90 CD90 CD105 CD105 CD105 CD105 CD105 CD105 CD106 CD106 CD106 CD106 CD106

CD146 CD146 CD146 CD146

CD34 CD34 CD34 CD34 CD34 CD34

CD45 CD45 CD45 CD45 CD45 CD45

HLA-DR HLA-DR HLA-DR HLA-DR

CD40

CD80 CD86

**Table 1.** Cell Surface Marker Profiles of dental-related stem cells. DPSC; Dental pulp stem cell, SHED; Stem cells from human exfoliated deciduous teeth, PDLSC; Periodontal ligament stem cells, DFPC, Dental follicle precursor cells, SCAP;

In dental related stem cell researches, the first step is to isolate cells from tissue sources. The next step is to expand the cells into sufficient number. In some occasion, it may be necessary to preserve the cells for future use since tissue sources would not be available on demand. In this section, we will describe the location of the certain teeth tissue from which the stem cell population can be derived and followed by description of common methods by which the isolation culture can be initiated. At the end, culture systems for the cell propagation as well

**3. Dental-related stem cell initiation culture, culture systems and**

as the main points regarding issue of cell cryopreservation will be explained.

CD166 CD166 CD166 CD166

CD24

CD18

CD150

**CD (+)** STRO-1 STRO-1 STRO-1 STRO-1 STRO-1 STRO-1

CD26

CD10 CD10 CD10

CD59 CD59 CD59

CD117

100 Regenerative Medicine and Tissue Engineering

CD19 CD24

**cryopreservation**

**CD (-)** CD14 CD14 CD14

Stem cells from dental apical papilla, GSC; Gingival stem cells. [1-4]

Most of the human DSCs are come from teeth, which are subjected to the orthodontic treat‐ ments. Based on the studies, molars and premolars are mostly used for this purpose. Third molars (wisdom teeth) are the most common teeth for extraction in dental clinical practice. In addition, developing wisdom teeth during the adult life are the excellent candidates as the accessible source of developing tissue similar to those in embryonic development. There are a few studies considering the supernumerary teeth derived from other teeth, such as canine, for the isolation of DPSCs [12] In some cases, such as the isolation of DPSCs from inflamed dental pulps, endodontic treatments are used rather than orthodontics surgeries [46]. In any case, considering the precise location of the dental tissues in tooth anatomy is important to achieve certain types of DSCs with minimum cell contaminations. Hence, here is the brief description of the localization for the specific DSCs isolation.

#### *3.1.1. Tissues contained dental pulp-related stem cells (DPSCs, SHED & SCAP)*

Isolation of DPSCs following the pulp extraction could be achieved by either through the root or crown of the dental organ. In the case of SHEDs or DPSCs, which are derived from incom‐ pleted root teeth, the exposed pulp is accessible from the root without applying any specific procedure [21]. In other situation (extracted permanent or deciduous teeth), dental pulp extraction is accomplished through the dental crown by cutting the cementum-enamel junction (CEJ) using dental instruments, such as pliers (bone forceps), extirpation needle, Gracey curette, dental fissure burs, etc. [6] Moreover, in the case of inflamed teeth, pulp tissues are removed during the endodontic therapies [46] (Figure 2)

The isolation of SCAP is achieved by gentle separation of root apical papilla from the surface of the root with immature apex (i.e. located in the exterior of the root foramen area before the complete eruption of tooth in the oral cavity) during the extraction of third molars [47]. Root maturation results in the elimination of apical papilla; hence, the maturation period of teeth are important for isolation of SCAP. (Figure 2)

#### *3.1.2. Tissues contained periodontium-related stem cells (PDLSCs & DFPCs) & GSCs isolation*

PDLSC can be obtained from the middle third of the root surfaces of extracted PDL tissue, which is a soft connective tissue surrounded between the cementum and the inner wall of the alveolar bone socket. It's accomplished by scrapping surface of the middle third of the root [31].

DFPCs can be isolated by dissecting dental follicle from the upside of the dental crown from impacted teeth. Human dental follicle is an ectomesenchymal tissue that is derived from cranial neural crest. This tissue surrounds developing tooth germ and involves in the coordination of tooth eruption and periodontium formation. This tooth germ's tissue can easily be isolated after wisdom tooth extraction by routine orthodontical related surger‐ ies. Impacted teeth, usually third molars, normally fail to erupt through the gum because of their encasement in the jawbone; therefore, routine surgical procedures are required for the extraction. [36, 37]. (Figure 2)

**Figure 2.** Overall view of dental-related stem cells based on different anatomical locations and stages during the hu‐ man lifetime in. a. tooth germ, b. primary teeth, c. permanent teeth.

GSCs could be isolated from clinically healthy gingiva, which are obtained as remnant or discarded tissues following routine dental procedures [43]. (Figure 2)

#### **3.2. DSCs culture initiation**

In general, dental-related stem cells could isolate by either (1) enzymatic digestion (ED) of tissues or (2) outgrowth (OG) from tissue explant. In the case of enzymatic digestion, after the extraction, tissues are placed into the digestion enzymes, (generally, collagenase type-I & dispase) for about 30-60 minutes at 37 °C to achieve single-cell suspensions. In order to purify DSCs, single-cell suspensions could be subjected into (1) size-sieved isolation (using 3 μm strainer followed by 20 μm strainer), (2) stem cell colony cultivation (single colony culture of stem cells) or (3) magnetic/ fluorescence activated cell sorting (sorting based on surface markers) [48].

In the outgrowth method (OG), tissues are minced into 1-2 mm pieces and placed into the culture dishes to outgrowth [18]. More recently, Lizier and his co-workers established a scaledup hIDPSCs culture system based on in vitro re-plating of pulp tissue explants followed by 3-4 days expansion [49].

There are some evidences, which suggested different behavior of DSCs according to the ED or OG isolation methods [41, 50, 51]. According to Huang et al. DPSC isolated by ED method (DPSC-ED) from permanent teeth showed higher proliferation rate than those isolated by the OG method (DPSC-OG) [50]. Moreover, STRO-1 & CD34 markers ex‐ pressed more in DPSC-ED in comparison with DPSC-OG. DPSC-ED derived from deciduous and permanent teeth has been reported to display higher mineralization rate in the defined osteo/odonto medium [51, 52].

#### **3.3. DSCs culture systems**

**Figure 2.** Overall view of dental-related stem cells based on different anatomical locations and stages during the hu‐

GSCs could be isolated from clinically healthy gingiva, which are obtained as remnant or

In general, dental-related stem cells could isolate by either (1) enzymatic digestion (ED) of tissues or (2) outgrowth (OG) from tissue explant. In the case of enzymatic digestion, after the extraction, tissues are placed into the digestion enzymes, (generally, collagenase type-I & dispase) for about 30-60 minutes at 37 °C to achieve single-cell suspensions. In order to purify DSCs, single-cell suspensions could be subjected into (1) size-sieved isolation (using 3 μm strainer followed by 20 μm strainer), (2) stem cell colony cultivation (single colony culture of stem cells) or (3) magnetic/ fluorescence activated cell sorting (sorting based on surface

In the outgrowth method (OG), tissues are minced into 1-2 mm pieces and placed into the culture dishes to outgrowth [18]. More recently, Lizier and his co-workers established a scaledup hIDPSCs culture system based on in vitro re-plating of pulp tissue explants followed by

There are some evidences, which suggested different behavior of DSCs according to the ED or OG isolation methods [41, 50, 51]. According to Huang et al. DPSC isolated by ED method (DPSC-ED) from permanent teeth showed higher proliferation rate than those isolated by the OG method (DPSC-OG) [50]. Moreover, STRO-1 & CD34 markers ex‐ pressed more in DPSC-ED in comparison with DPSC-OG. DPSC-ED derived from deciduous and permanent teeth has been reported to display higher mineralization rate in

man lifetime in. a. tooth germ, b. primary teeth, c. permanent teeth.

**3.2. DSCs culture initiation**

102 Regenerative Medicine and Tissue Engineering

markers) [48].

3-4 days expansion [49].

the defined osteo/odonto medium [51, 52].

discarded tissues following routine dental procedures [43]. (Figure 2)

Following the isolation of dental-related stem cells, the next step is to culture-expand the cells into the multiple copies since in the most strategies related to the cell-based-treatment of tissue defects, the copious amount of regenerating cells is needed. Many researchers have been focused on optimizing effective conditions under which DSCs can efficiently be propagated. On the other hand, differentiation potential of the multiplied cells must be determined because discovering the potential commitments of the cells may lead to better selection of them for future organ-targeted treatments [27, 48, 53]. Due to these considerations, this section opens up a brief overview for different DSC culture systems designed for the cell expansion and differentiation.

#### *3.3.1. Serum free vs. serum rich culture systems*

Normally, the isolation and expansion of DSC have depend on a high concentration of serum culture media (10%), which provide better cell adhesion during the initial isolation of the cells. Unfortunately, in long-term cultivation, the high level of serum might lead to spontaneous differentiation or malignant transformation of cells. In addition, use of serum in culture may result in contamination of cell culture with bovine pathogen for instance bovine spongiform encephalopathy (BSE). For these reasons, serum free culture systems are highly recommended [54-57]. In this regards, many attempt has been made to optimize DSC cultivation in serumfree or low serum medium. For example, Karbanova et al. have reported that DPSCs cultivated in low-serum medium exhibit less proliferation rate and different expression of stem cell markers compared to those cultivated in serum rich medium [14]. In contrast, Hirata et al have cultivated the cells in serum free media and have found the same survival rate of the cells as those cultivated in the serum containing medium [58].

In the case of DFPCs, studies indicated that applying serum replacement media didn't affect the expression of connective tissue markers, such as collagen type I and type III, and also neural stem progenitor marker, nestin [59]; however, there is no information about the possible changes in other markers in this regards.

It's interesting to note that SHEDs & PDLSCs cultivated in defined serum free media have been reported to display higher proliferation rate than those cultivated in the medium containing serum. Moreover such cells have found to express comparatively higher "stem‐ ness" markers [60]. Applying serum free media is one of the critical requirements for the future clinical treatments; therefore, additional works are needed for optimizing conditions to achieve final conclusion.

#### *3.3.2. Neurosphere-forming vs. adherent culture systems*

Sphere-forming culture systems are generally applied for neurogenic differentiation of stem cells. This culture system has also been used for DSC cultivation. The idea of applying such a system came from the consideration of neural crest origin of DSCs. It has been well established that neural stem/progenitor cells which are isolated from variety of sources are grown as neurospheres in defined serum-free culture medium supplemented with EGF and ⁄ or bFGF [61-63].

DSC Suspension culture was first suggested by Morita et al. (2007). These authors have cultivated PDLSCs in the sphere culture and found that PDLSC have the ability to form neurospheres in serum-free culture containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) with the ability to differentiate into both neural and mesodermal progeny [64].

In 2008, Sasaki et al. have cultivated DPSCs from the rat incisor in the sphere-culture and found that under these conditions, the cells expressed neural and glial markers. They have also noticed that CD81 positive DPSCs that were localized in odontoblast layer of apical portion of the dental pulp may have more potential to form neurospheres [65] Later on, it was demon‐ strated that sphere form of SCAP had multi-differentiation potential into mineralized cells, adipocytes and also myocytes under the defined media in vitro. Furthermore, In vivo studies have indicated that sphere SCAP showed more potential to generate mineralized tissues in comparison with the non-sphere SCAP [47].

#### *3.3.3. Co-culture systems*

Site-specific tissue interactions are essential for orchestration of proliferation, differentiation and also homeostasis of cells during the tooth germ development. In particular, epithelialmesenchymal interactions are the most important developmental events, which are involved in reciprocal crosstalk between the ectodermal and mesenchymal tissues. These sequential interactions are critical for tooth morphogenesis and cell differentiation [66]. To imitate these in vivo interactions, co-culture systems have been developed.

Using co-culture systems, some attempts have been made to promote DSCs differentia‐ tion in vitro. In this regards different cell types have been examined as co-culture cells. For example, it has been demonstrated that co-culture of DFPCs/SCAP could lead to formation of bone-like structure in vivo and in vitro. This occurs since interaction be‐ tween the cells stimulate cementogenic/osteogenic differentiation of DFPCs leading to upregulation of bone intermediate proteins, such as bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), bone sialoprotein (BSP) and osteocalcin (OCN), as well as downregulation of receptor activator of nuclear factor κB ligand (RANKL) [67]. In other experiment, Arakaki et al. have co-cultured DPSCs with dental epithelial cells and found that in such a system, dental epithelial cells secret BMP2 and BMP4, thereby promote odontoblastic differentiation of DPSCs [68]. Interestingly, it has also been reported that coculture of DPSCs with epithelial cells lead to epithelium invagination, as well [69]. Moreover, Wang et al. have established a co-culture of hDPSCs with osteoblasts and demonstrated that a higher mineralization and up-regulation of osteogenic-related genes in hDPSCs compared with those cultured in the absence of osteoblasts [70]. Furthermore, co-culture of DPSCs with non-dental MSCs, such as amniotic fluid-derived MSCs (AF-MSCs), has been reported to leads to the bone differentiation of AF-MSCs indicating the pre-commitment of DPSCs to induce osteogenic differentiation [71].

#### **3.4. DSCs cryopreservation**

grown as neurospheres in defined serum-free culture medium supplemented with EGF

DSC Suspension culture was first suggested by Morita et al. (2007). These authors have cultivated PDLSCs in the sphere culture and found that PDLSC have the ability to form neurospheres in serum-free culture containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) with the ability to differentiate into

In 2008, Sasaki et al. have cultivated DPSCs from the rat incisor in the sphere-culture and found that under these conditions, the cells expressed neural and glial markers. They have also noticed that CD81 positive DPSCs that were localized in odontoblast layer of apical portion of the dental pulp may have more potential to form neurospheres [65] Later on, it was demon‐ strated that sphere form of SCAP had multi-differentiation potential into mineralized cells, adipocytes and also myocytes under the defined media in vitro. Furthermore, In vivo studies have indicated that sphere SCAP showed more potential to generate mineralized tissues in

Site-specific tissue interactions are essential for orchestration of proliferation, differentiation and also homeostasis of cells during the tooth germ development. In particular, epithelialmesenchymal interactions are the most important developmental events, which are involved in reciprocal crosstalk between the ectodermal and mesenchymal tissues. These sequential interactions are critical for tooth morphogenesis and cell differentiation [66]. To imitate these

Using co-culture systems, some attempts have been made to promote DSCs differentia‐ tion in vitro. In this regards different cell types have been examined as co-culture cells. For example, it has been demonstrated that co-culture of DFPCs/SCAP could lead to formation of bone-like structure in vivo and in vitro. This occurs since interaction be‐ tween the cells stimulate cementogenic/osteogenic differentiation of DFPCs leading to upregulation of bone intermediate proteins, such as bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), bone sialoprotein (BSP) and osteocalcin (OCN), as well as downregulation of receptor activator of nuclear factor κB ligand (RANKL) [67]. In other experiment, Arakaki et al. have co-cultured DPSCs with dental epithelial cells and found that in such a system, dental epithelial cells secret BMP2 and BMP4, thereby promote odontoblastic differentiation of DPSCs [68]. Interestingly, it has also been reported that coculture of DPSCs with epithelial cells lead to epithelium invagination, as well [69]. Moreover, Wang et al. have established a co-culture of hDPSCs with osteoblasts and demonstrated that a higher mineralization and up-regulation of osteogenic-related genes in hDPSCs compared with those cultured in the absence of osteoblasts [70]. Furthermore, co-culture of DPSCs with non-dental MSCs, such as amniotic fluid-derived MSCs (AF-MSCs), has been reported to leads to the bone differentiation of AF-MSCs indicating the

and ⁄ or bFGF [61-63].

104 Regenerative Medicine and Tissue Engineering

*3.3.3. Co-culture systems*

both neural and mesodermal progeny [64].

comparison with the non-sphere SCAP [47].

in vivo interactions, co-culture systems have been developed.

pre-commitment of DPSCs to induce osteogenic differentiation [71].

According to the diversity of dental-related stem cells and their remarkable features for cellmediated therapies and tissue engineering, developing a reliable method for cell banking have become a priority for future use. In this regard, cryopreservation could be established at the levels of teeth (organs), dental tissues or cells. In the case of teeth or dental tissues cryopre‐ servation, minimal processing may needed for banking. There are some evidences which are demonstrated the successful cryopreservation of healthy and diseased teeth as well as dental tissues [72-78]. On the other hand, cryopreservation of DSCs still has been considered as an active area of the researches. There are several parameters which need to be considered prior to an establishment of a successful and more efficient protocol. The parameters which need to be determined include (1) DSCs-cultured passage(s), which leads to high-efficiency recovery post thaw, (2) concentration of cryoprotective agent, (3) cell concentration for high-efficient cryopreservation, (4) storage temperature, (5) the process of cryopreservation, and (6) evalu‐ ation of growth, surface markers and differentiation properties of DSCs after post thaw. So far, there have been many researches considering the comparative cryopreservation methods for optimizing the best protocols. More recently, it has been suggested that magnetic cryopreser‐ vation of DSCs was much better than conventional slow-freezing procedure in terms of cytotoxicity [76, 79, 80].
