**2. Stem cells**

Stem cells are undifferentiated cells, with endless self-renewal sustained proliferation *in vitro* and multilineage differentiation capacity [3]. This *in vitro* multilineage differentia‐ tion capacity has targeted these cells with extreme importance for use in tissue and cellbased therapies.

The first stem cell appearance is in the early zygotic cells, which are totipotent and give rise to the blastocyst. They are capable to differentiate into all cell and tissue types. With differ‐ entiation, cells become less capable of self-renewal and differentiation in other cell type be‐ comes more limited [1]. Stem cells can be loosely classified into 3 broad categories based on their growth behavior and isolation time during ontogenesis: embryonic, fetal and adult.

Embryonic stem cells (ESCs) were first observed in a pre-implantation embryo by Bongso and colleagues in 1994 [4]. Since then, many cell lines and a multiplicity of tissues have been successfully derived from ESCs and tested in several animal disease models [5-7]. Neverthe‐ less, post-transplantation immune-rejection has been a major problem. Many studies are be‐ ing conducted to avoid this major issue. This could be resolved by personalizing tissues through somatic nuclear transfer (NT) or induced pluripotent stem cells (iPSC) techniques [8], but the teratoma development in animals is still a concern and a serious problem [9]. In order to overcome the limitations placed by ESCs and iPSCs, a variety of adult stem cell populations have been recently isolated and characterized for their potential clinical use. While still multipotent, adult stem cells have long been considered restricted, giving rise on‐ ly to progeny of their resident tissues [9]. *In vivo,* adult stem cells exist in a quiescent state, located in almost all tissues, until mediators activate them to restore and repair injured tis‐ sues. These cells are surrounded by mature cells that have reached the end line in terms of differentiation and proliferation [10]. Stem cell research focuses on the development of cell and tissue differentiation, so as characterization techniques, for tissue and cell identification with marker patterns. Such protocols are essential for regenerative therapies [11].

#### **2.1. Mesenchymal stem cells**

The development of cell-based therapies for cartilage [12] and skin [13] reconstruction marks the beginning of a new age in tissue regeneration. Mesenchymal stem cells (MSCs) have become one of the most interesting targets for tissue regeneration due to their high plasticity, proliferative and differentiation capacity together with their attractive immuno‐ suppressive properties. MSCs present low immunogenicity and high immunosuppressive properties due to a decreased or even absence of Human Leucocyte Antigen (HLA) class II expression [14]. Research in this field has brought exciting promises in many disorders and therefore in tissue regeneration. Currently the differentiation potential of MSCs in multiline‐ age end-stage cells is already proven, and their potential for treatment of cardiovascular [15], neurological [16], musculoskeletal [17, 18], and cutaneous [19] diseases is now well es‐ tablished. Fibroblast colony-forming units or marrow stromal cells, currently named MSCs, **2. Stem cells**

466 Advances in Biomaterials Science and Biomedical Applications

based therapies.

**2.1. Mesenchymal stem cells**

Stem cells are undifferentiated cells, with endless self-renewal sustained proliferation *in vitro* and multilineage differentiation capacity [3]. This *in vitro* multilineage differentia‐ tion capacity has targeted these cells with extreme importance for use in tissue and cell-

The first stem cell appearance is in the early zygotic cells, which are totipotent and give rise to the blastocyst. They are capable to differentiate into all cell and tissue types. With differ‐ entiation, cells become less capable of self-renewal and differentiation in other cell type be‐ comes more limited [1]. Stem cells can be loosely classified into 3 broad categories based on their growth behavior and isolation time during ontogenesis: embryonic, fetal and adult.

Embryonic stem cells (ESCs) were first observed in a pre-implantation embryo by Bongso and colleagues in 1994 [4]. Since then, many cell lines and a multiplicity of tissues have been successfully derived from ESCs and tested in several animal disease models [5-7]. Neverthe‐ less, post-transplantation immune-rejection has been a major problem. Many studies are be‐ ing conducted to avoid this major issue. This could be resolved by personalizing tissues through somatic nuclear transfer (NT) or induced pluripotent stem cells (iPSC) techniques [8], but the teratoma development in animals is still a concern and a serious problem [9]. In order to overcome the limitations placed by ESCs and iPSCs, a variety of adult stem cell populations have been recently isolated and characterized for their potential clinical use. While still multipotent, adult stem cells have long been considered restricted, giving rise on‐ ly to progeny of their resident tissues [9]. *In vivo,* adult stem cells exist in a quiescent state, located in almost all tissues, until mediators activate them to restore and repair injured tis‐ sues. These cells are surrounded by mature cells that have reached the end line in terms of differentiation and proliferation [10]. Stem cell research focuses on the development of cell and tissue differentiation, so as characterization techniques, for tissue and cell identification

with marker patterns. Such protocols are essential for regenerative therapies [11].

The development of cell-based therapies for cartilage [12] and skin [13] reconstruction marks the beginning of a new age in tissue regeneration. Mesenchymal stem cells (MSCs) have become one of the most interesting targets for tissue regeneration due to their high plasticity, proliferative and differentiation capacity together with their attractive immuno‐ suppressive properties. MSCs present low immunogenicity and high immunosuppressive properties due to a decreased or even absence of Human Leucocyte Antigen (HLA) class II expression [14]. Research in this field has brought exciting promises in many disorders and therefore in tissue regeneration. Currently the differentiation potential of MSCs in multiline‐ age end-stage cells is already proven, and their potential for treatment of cardiovascular [15], neurological [16], musculoskeletal [17, 18], and cutaneous [19] diseases is now well es‐ tablished. Fibroblast colony-forming units or marrow stromal cells, currently named MSCs, were first isolated in 1968 from rat bone marrow [20]. These cells were clonogenic, formed colonies when cultured, and were able to differentiate *in vitro* into bone, cartilage, adipose tissue, tendon, muscle and fibrous tissue. Since then many other tissues have been used to isolate these cells. MSCs can be obtained from many different tissues, including bone mar‐ row, adipose tissue, skeletal muscle, umbilical cord matrix and blood, placental tissue, amni‐ otic fluid, synovial membranes, dental pulp, fetal blood, liver, and lung [21]. The concept of MSCs is based on their ability to differentiate into a variety of mesodermal tissues and was first proposed by Caplan in 1991 [22] and further validated by additional research in 1999 [23]. Due to the many different methods and approaches used for MSCs culture, the Mesen‐ chymal and Tissue Stem Cell Committee, of the International Society for Cellular Therapy (ISCT), recommended several standards do define MSCs [24]. Therefore, MSCs are defined as presenting: i) plastic adherent ability; ii) absence of definitive hematopoietic lineage markers, such as CD45, CD34, CD14, CD11b, CD79α, CD19 and class-II Major Histocompati‐ bility Complex (MHC) molecules, specially HLA-DR; and expression of nonspecific markers CD105, CD90 and CD73 iii) ability to differentiate into mesodermal lineage cells, osteocytes, chondrocytes and adipocytes. Along with mesodermal differentiation, it has been demon‐ strated the capacity of MSCs to differentiate into ectodermal cell lines, as neurons [25, 26], keratocytes [27] and keratinocytes [28], so as endodermal cell line, like hepatocytes [29, 30] and pancreatic β-cells [31]. Moreover, they also possess anti-inflammatory and immunomo‐ dulation properties and trophic effects [32, 33]. Increasing evidence now demonstrates that the therapeutic effects of MSCs do not lay only on the ability to repair damage tissue, but also on the capacity of modulating surrounding environment, by secretion of multiple fac‐ tors and activation of endogenous progenitor cells [34, 35]. Compared with ESCs and other tissue specific stem cells, MSCs are more advantageous. Moreover some studies have dem‐ onstrated that MSCs have a higher chromosomal stability and lower tendency to form tu‐ mors and teratomas, compared to other stem cells [36, 37].

Although they present similar biological characteristics, it cannot be ignored the existing of some disparities, as differences in, expansion potential under same culture conditions and age-related functional properties [38]. Compared to ESCs, MSCs isolated from the umbili‐ cal cord matrix (Wharton's jelly) have many advantages, such as shorter population dou‐ bling time, easy culture in plastic flasks, good tolerance towards the immune system, so that transplantation into non-immunesuppressed animals does not induce acute rejection, an‐ ticancer properties, [9] and most important absence of tumorigenic activity. As well as ESCs, these cells are originated from the inner cell mass of the blastocyst but with a major differ‐ ence: they do not raise ethical controversies, since they are collected from tissues usually dis‐ carded at birth [39].

#### *2.1.1. MSCs sources and validation of transport and processing protocols*

Bone marrow, adipose tissue, umbilical cord blood and umbilical cord matrix have been considered the main sources of MSCs for tissue engineering purposes. Among these sources, bone marrow represents the main source of MSCs for cell therapy. However, the prolifera‐ tive capacity [40-43], differentiation potential and clonal expandability [44] of MSCs derived from bone marrow decrease significantly with age, gender and seeding density, and the number of cells per marrow aspirate is usually quite low [3, 45]. It is still a mystery if MSCs ageing is due to factors intrinsic or extrinsic to the cells. Many possible reasons have been described in an attempt to explain MSCs ageing. Possible extrinsic factors include: reduced synthesis of proteoglycans and glycosaminoglycans reducing proliferation and viability [46], and production of glycosylated end products, inducing apoptosis and reactive oxygen spe‐ cies [47]. Intrinsic factors causing MSCs ageing might include: cell senescence-associated *β*galactosidase and higher expression of p53 and pathway genes p21 and BAX, resulting in blunted proliferation potential [43]. Regarding seeding density, many authors suggest that lower seeding densities induce faster proliferation rates [48, 49]. This has been explained by contact inhibition in higher seeding densities [49], and higher nutrient availability per cell in lower seeding densities [49]. Use of bone marrow MSCs has disadvantages; donors are sub‐ mitted to invasive harvest of bone marrow. This raises the need to find alternative sources of MSCs for autologous and allogenic use. Candidate tissue sources should provide MSCs dis‐ playing high proliferative and differentiation potency [50].

Extra-embryonic tissues are a good alternative to adult donor. This tissues, such as, amn‐ ion, microvillus, Wharton's jelly and umbilical cord perivascular cells, are routinely discard‐ ed at child-birth, so little ethical and religious controversy attends the harvesting of the resident stem cell populations. The comparatively large volume of extra-embryonic tis‐ sues increases the chance of isolating suitable amounts of stem cells, despite the complex and expensive procedures needed for their isolation. Some protocols use enzymatic diges‐ tion while others use enzyme-free tissue explant methods that require longer culture time [51]. There are also MSCs in cord blood (CB), but many studies report low frequency of these cells and unsuccessful isolation. However, Zhang and colleagues were able to iso‐ late MSCs from CB with a 90% successful rate when CB volume was ≥ 90ml and a trans‐ port time until storage was ≤ 2 hours [51].

In recent years, MSCs derived from umbilical cord matrix Wharton's jelly, have attracted much interest. Wharton's jelly is a mature mucous tissue and the main component of the umbilical cord, connecting the umbilical vessels to the amniotic epithelium. Umbilical cord derives from extra-embryonic or embryonic mesoderm; at birth it weighs about 40g and measures approximately 30-65cm in length and 1.5cm in width [52]. Anyway, individual differences are observed within newborn babies. Fong and colleagues characterized Whar‐ ton's Jelly stem cells and found the presence of both embryonic and MSCs, targeting this source as unique and of valuable use for clinical applications. MSCs from the Wharton's jel‐ ly can be cultured with little or even no major loss trough at least 50 passages [53].

CB and more recently, umbilical cord tissue (UCT) have been stored cryopreserved in pri‐ vate and public cord blood and tissue banks worldwide in order to obtain hematopoietic and MSCs and, although guidelines exist (Netcord – Foundation for the Accreditation of Cellular Therapy), standardized procedures for CB and UCT transport from the hospital / clinic to the laboratory, storage, processing, cryopreservation and thawing are still awaited. These may be critical in order to obtain higher viable stem cells number after thawing and limit microbiological contamination.

Our research group focused in determining whether UCT storage and transport from the hospital / clinical to the laboratory at room temperature (RT) or refrigerated (4-6°C) and im‐ mersed in several sterile saline solutions affects the UCT integrity in order to be cryopre‐ served. The umbilical cord contains two arteries and one vein, which are surrounded by mucoid connective tissue, and this is called the Wharton's jelly. The cord is covered by an epithelium derived from the enveloping amnion. The interlaced collagen fibers and small, woven bundles are arranged to form a continuous soft skeleton that encases the umbilical vessels. In the Wharton's jelly, the most abundant glycosaminoglycan is hyaluronic acid, which forms a hydrated gel around the fibroblasts and collagen fibrils and maintains the tis‐ sue architecture of the umbilical cord by protecting it from pressure [54].

One centimeter-long fragments of umbilical cords (N = 12) were collected from healthy do‐ nors after written informed consent and following validated procedures according to the clinical and technical guidelines of the Private Bank Biosckin, Molecular and Cell Therapies, SA (authorized for processing and cryopreserving CB and UCT units by the Portuguese Minister of Health, ASST – Autoridade para os Serviços de Sangue e de Transplantação). The 1 cm fragments were immersed for 168 hours in 4 different sterile saline solutions at RT (22-24°C) and refrigerated (4-6°C): NaCl 0.9% (Labesfal, Portugal), AOSEPT®-PLUS (Ciba Vision, Portugal), Dulbecco's Phosphate-Buffered Saline without calcium, magnesium and phenol red (DPBS, Gibco, Invitrogen, Portugal) and Hank's Balanced Salt Solution (HBSS, Gibco, Invitrogen, Portugal). The preservative-free, aqueous AOSEPT® PLUS solution con‐ tains hydrogen peroxide 3%, phosphonic acid (stabiliser), sodium chloride, phosphate (buf‐ fer system), and poloxamer (surfactant), and is usually used to transport and wash contact lenses. After 168 hours, the fragments were collected in 4% of paraformaldehyde and proc‐ essed for light microscopy. The samples were fixed in 4% paraformaldehyde for 4 hours and then washed and conserved in phosphate buffer saline (PBS) until embedding. The speci‐ mens were dehydrated and embedded in paraffin and cut at 10 μm perpendicular to the main umbilical cord axis. For light microscope analysis, sections were stained with haema‐ toxylin and eosin (HE) and observed with a Leica DM400 microscope equipped with a Leica DFC320 digital camera. The UCT integrity was evaluated through the following parameters:

tive capacity [40-43], differentiation potential and clonal expandability [44] of MSCs derived from bone marrow decrease significantly with age, gender and seeding density, and the number of cells per marrow aspirate is usually quite low [3, 45]. It is still a mystery if MSCs ageing is due to factors intrinsic or extrinsic to the cells. Many possible reasons have been described in an attempt to explain MSCs ageing. Possible extrinsic factors include: reduced synthesis of proteoglycans and glycosaminoglycans reducing proliferation and viability [46], and production of glycosylated end products, inducing apoptosis and reactive oxygen spe‐ cies [47]. Intrinsic factors causing MSCs ageing might include: cell senescence-associated *β*galactosidase and higher expression of p53 and pathway genes p21 and BAX, resulting in blunted proliferation potential [43]. Regarding seeding density, many authors suggest that lower seeding densities induce faster proliferation rates [48, 49]. This has been explained by contact inhibition in higher seeding densities [49], and higher nutrient availability per cell in lower seeding densities [49]. Use of bone marrow MSCs has disadvantages; donors are sub‐ mitted to invasive harvest of bone marrow. This raises the need to find alternative sources of MSCs for autologous and allogenic use. Candidate tissue sources should provide MSCs dis‐

Extra-embryonic tissues are a good alternative to adult donor. This tissues, such as, amn‐ ion, microvillus, Wharton's jelly and umbilical cord perivascular cells, are routinely discard‐ ed at child-birth, so little ethical and religious controversy attends the harvesting of the resident stem cell populations. The comparatively large volume of extra-embryonic tis‐ sues increases the chance of isolating suitable amounts of stem cells, despite the complex and expensive procedures needed for their isolation. Some protocols use enzymatic diges‐ tion while others use enzyme-free tissue explant methods that require longer culture time [51]. There are also MSCs in cord blood (CB), but many studies report low frequency of these cells and unsuccessful isolation. However, Zhang and colleagues were able to iso‐ late MSCs from CB with a 90% successful rate when CB volume was ≥ 90ml and a trans‐

In recent years, MSCs derived from umbilical cord matrix Wharton's jelly, have attracted much interest. Wharton's jelly is a mature mucous tissue and the main component of the umbilical cord, connecting the umbilical vessels to the amniotic epithelium. Umbilical cord derives from extra-embryonic or embryonic mesoderm; at birth it weighs about 40g and measures approximately 30-65cm in length and 1.5cm in width [52]. Anyway, individual differences are observed within newborn babies. Fong and colleagues characterized Whar‐ ton's Jelly stem cells and found the presence of both embryonic and MSCs, targeting this source as unique and of valuable use for clinical applications. MSCs from the Wharton's jel‐

CB and more recently, umbilical cord tissue (UCT) have been stored cryopreserved in pri‐ vate and public cord blood and tissue banks worldwide in order to obtain hematopoietic and MSCs and, although guidelines exist (Netcord – Foundation for the Accreditation of Cellular Therapy), standardized procedures for CB and UCT transport from the hospital / clinic to the laboratory, storage, processing, cryopreservation and thawing are still awaited.

ly can be cultured with little or even no major loss trough at least 50 passages [53].

playing high proliferative and differentiation potency [50].

port time until storage was ≤ 2 hours [51].

468 Advances in Biomaterials Science and Biomedical Applications

It was concluded that the best transport solutions were HBSS or DPBS at a temperature of 4-6°C since those maintained the histological structure of UC evaluated through those 5 pa‐ rameters previously referred (Figure 1 and Figure 2).

**Figure 1.** Cross section of an umbilical cord transported immersed in DPBS at the refrigerated temperature of 4-6°C. Samples were stained with haematoxylin and eosin (HE). Magnification: 10X.

**Figure 2.** Cross section of an umbilical cord transported immersed in DPBS at the refrigerated temperature of 4-6°C. Samples were stained with haematoxylin and eosin (HE). The UCT integrity was quality evaluated through the follow‐ ing parameters: i) detachment of vessels and retraction of vascular structures; ii) loss of detail and integrity of the en‐ dothelium; iii) connective tissue degradation; iv) autolysis of fat (impossible to assess, due to histological technique); and v) loss of detail and integrity of the mesothelium. Magnification: 40X.

As a matter of fact, the UC immersed for 168 hours in DPBS and HBSS at refrigerated tem‐ perature presented integrity of the histological structure comparable to a UC collected and processed for histological analysis immediately after birth (Figure 3). With DPBS, a slight re‐ traction of the vessels was noted, which is advantageous since the vessels are stripped and discarded before cryopreservation of the UCT. It was concluded that the transport of the UC from the hospital / clinic to the cryopreservation laboratory should be performed with the UC immersed in DPBS or HBSS at refrigerated temperatures.

**Figure 3.** Cross section of an umbilical cord collected and processed for histological analysis immediately after birth under optimal conditions according to Netcord guidelines. Stained with haematoxylin and eosin (HE). Magnification: 40X.

**Figure 1.** Cross section of an umbilical cord transported immersed in DPBS at the refrigerated temperature of 4-6°C.

**Figure 2.** Cross section of an umbilical cord transported immersed in DPBS at the refrigerated temperature of 4-6°C. Samples were stained with haematoxylin and eosin (HE). The UCT integrity was quality evaluated through the follow‐ ing parameters: i) detachment of vessels and retraction of vascular structures; ii) loss of detail and integrity of the en‐ dothelium; iii) connective tissue degradation; iv) autolysis of fat (impossible to assess, due to histological technique);

As a matter of fact, the UC immersed for 168 hours in DPBS and HBSS at refrigerated tem‐ perature presented integrity of the histological structure comparable to a UC collected and processed for histological analysis immediately after birth (Figure 3). With DPBS, a slight re‐ traction of the vessels was noted, which is advantageous since the vessels are stripped and discarded before cryopreservation of the UCT. It was concluded that the transport of the UC

Samples were stained with haematoxylin and eosin (HE). Magnification: 10X.

470 Advances in Biomaterials Science and Biomedical Applications

and v) loss of detail and integrity of the mesothelium. Magnification: 40X.

The isolation and culture of MSCs from the Wharton's jelly was performed by our research group in order to obtain undifferentiated MSCs and *in vitro* differentiated into neural-like cells to be tested in axonotmesis and neurotmesis lesions of the rat sciatic nerve. The isola‐ tion has been performed by enzyme-free tissue explant and enzymatic isolation. Despite our standard approaches, we are aware that there are still significant variations that exist be‐ tween laboratory protocols, which must be taken into account when comparing results us‐ ing other methodologies. There is a wide range of individual differences among donor tissues also and our protocols usually use 15 - 20 cm of UC. While most UC samples will provide a reasonable number of MSCs using the provided protocols, some samples may re‐ sult in sub-optimal cell isolation and expansion. The reasons behind this phenomenon still remain to be clarified, but as we have previously mentioned, the temperature and the time of transport from the hospital / clinic to the cryopreservation laboratory is crucial.

Irrespective of the specific protocol, the washing procedure of the umbilical cord fragments is crucial in order to avoid microbiological contamination of the cultures. After obtaining the writ‐ ten informed consent from the parents, fresh human umbilical cords are obtained after birth and collected in HBSS or DPBS at 4-6°C, as it was previously described. After washing the um‐ bilical cord unit 4 times in rising DPBS, disinfection is performed in 75% ethanol for 30 sec‐ onds. Finally, and before the dissection step, umbilical cord unit is washed in DPBS. The vessels are usually stripped with UC unit still immersed in DPBS. Once washing step in MSCs isola‐ tion and culture is essential to achieve good UCT units for cryopreservation and future clini‐ cal use, washing protocol was validated. DPBS from the first washing step (used immediately after collection for transportation of the unit to the laboratory – *washing step 1 solution*) and DPBS used in washing step after disinfection in 75% ethanol (*washing step 6 solution*) from 14 umbilical cord units (N = 14) collected from healthy donors and transported from the hospital/ clinic at 4-6°C in less than 96 hours were tested for microbiological contamination using BacT/ ALERT® (bioMérieux). Each unit was tested for aerobic and anaerobic microorganisms and fungi using 10 ml of the *washing step 1 solution* and *washing step 6 solution* which were asepti‐ cally introduced into the BacT/ALERT® testing flasks. All procedures were performed in a lam‐ inar flow tissue culture hood under sterile conditions. All the units that presented microbial contamination in DPBS obtained from the first washing step (*washing step 1 solution*) present‐ ed no contamination in the analysis performed to DPBS from the last washing step immediate‐ ly performed before MSCs isolation or UCT cryopreservation (*washing step 6 solution*). The following microorganisms were identified in the DPBS solution from the first washing step: *Staphylococcus lugdunensis* (N = 2); *Staphylococcus epidermidis* (N = 1); *Staphylococcus coagulase* (N = 2); *Escherichia coli* (N = 4); *Enterococcus faecalis* (N = 1); and *Streptococcus sanguinis* (N = 1). The DPBS solution from the first washing step (*washing step 1 solution*) from 3 units was neg‐ ative for microbial contamination (N = 3). These results permitted us to conclude that the wash‐ ing protocol was 100% efficient in what concerns microbiological elimination (including aerobic and anaerobic bacteria, yeast and fungi).

Once the transport and washing protocols were validated, it was important to isolate and expand *in vitro* the MSCs from the UCT units for pre-clinical trials.

**Figure 4.** MSCs isolated from Wharton's jelly using the "enzymatic protocol" exhibiting a mesenchymal-like shape with a flat polygonal morphology. Magnification: 100x.

In the "enzymatic procedure" we use collagenase type I (Sigma-Aldrich). With the written informed consent from the parents, fresh human umbilical cords were obtained after birth and stored in HBSS (Gibco, Invitrogen, Portugal) for 1–48 hours before tissue processing to obtain MSCs. After removal of blood vessels, the mesenchymal tissue is scraped off from the Wharton's jelly with a scalpel and centrifuged at 250 g for 5 minutes at room temperature and the pellet is washed with serum-free Dulbecco's modified Eagle's medium (DMEM, Gibco, Invitrogen, Portugal). Next, the cells are centrifuged at 250 g for 5 minutes at room temperature and then treated with collagenase (2 mg/ml) for 16 hours at 37°C,washed, and treated with 2.5% trypsin-EDTA solution (Sigma-Aldrich) for 30 minutes at 37°C with agita‐ tion. Finally, the cells are washed and cultured in DMEM (Gibco, Invitrogen, Portugal) sup‐ plemented with 10% fetal bovine serum (FBS), glucose (4.5 g/l), 1% (w/v) penicillin and streptomycin (Sigma), and 2.5 mg/ml amphotericin B (Sigma) in 5% CO2 in a 37°C incubator (Nuaire). Around 2 × 105 cells are plated into each T75 flask in 10 ml culture medium. Cells are allowed to attach and grow for 3 days. To remove the non-adherent cells or fragments, the flasks are gently washed using pre-warmed DPBS after which 10 ml of pre-warmed cul‐ ture medium is added. The culture medium is changed every third day (or twice per week). Confluence (80-90%) is normally reached at day 12–16, and the cells are removed with prewarmed trypsin-EDTA solution (4 ml per flask), for 10 min at 37°C. The cells are plated onto poly-l-lysine coated glass coverslips (in 6- or 24-well tissue culture plates) or on biomaterials used in the nerve reconstruction. Normally, 5000 cells/cm2 are plated on the coverslips or on the membranes (Figure 4).

DPBS used in washing step after disinfection in 75% ethanol (*washing step 6 solution*) from 14 umbilical cord units (N = 14) collected from healthy donors and transported from the hospital/ clinic at 4-6°C in less than 96 hours were tested for microbiological contamination using BacT/ ALERT® (bioMérieux). Each unit was tested for aerobic and anaerobic microorganisms and fungi using 10 ml of the *washing step 1 solution* and *washing step 6 solution* which were asepti‐ cally introduced into the BacT/ALERT® testing flasks. All procedures were performed in a lam‐ inar flow tissue culture hood under sterile conditions. All the units that presented microbial contamination in DPBS obtained from the first washing step (*washing step 1 solution*) present‐ ed no contamination in the analysis performed to DPBS from the last washing step immediate‐ ly performed before MSCs isolation or UCT cryopreservation (*washing step 6 solution*). The following microorganisms were identified in the DPBS solution from the first washing step: *Staphylococcus lugdunensis* (N = 2); *Staphylococcus epidermidis* (N = 1); *Staphylococcus coagulase* (N = 2); *Escherichia coli* (N = 4); *Enterococcus faecalis* (N = 1); and *Streptococcus sanguinis* (N = 1). The DPBS solution from the first washing step (*washing step 1 solution*) from 3 units was neg‐ ative for microbial contamination (N = 3). These results permitted us to conclude that the wash‐ ing protocol was 100% efficient in what concerns microbiological elimination (including aerobic

Once the transport and washing protocols were validated, it was important to isolate and

**Figure 4.** MSCs isolated from Wharton's jelly using the "enzymatic protocol" exhibiting a mesenchymal-like shape

In the "enzymatic procedure" we use collagenase type I (Sigma-Aldrich). With the written informed consent from the parents, fresh human umbilical cords were obtained after birth and stored in HBSS (Gibco, Invitrogen, Portugal) for 1–48 hours before tissue processing to obtain MSCs. After removal of blood vessels, the mesenchymal tissue is scraped off from the Wharton's jelly with a scalpel and centrifuged at 250 g for 5 minutes at room temperature and the pellet is washed with serum-free Dulbecco's modified Eagle's medium (DMEM,

expand *in vitro* the MSCs from the UCT units for pre-clinical trials.

and anaerobic bacteria, yeast and fungi).

472 Advances in Biomaterials Science and Biomedical Applications

with a flat polygonal morphology. Magnification: 100x.

In our "enzyme-free tissue explant protocol" for isolation of MSCs, enzymatic digestion is not employed. The mesenchymal tissue (Wharton's jelly) is diced into cubes of about 0.5 cm3 and the remaining vessels are removed by dissection. Using a sterile scalp, the cubes are diced in 1-2 mm fragments and transferred to a Petri dish pre-coated with poly-l-lysine (Sigma) with Mesenchymal Stem Cell Medium (PromoCell, C-28010) supplemented with 1% (w/v) penicil‐ lin and streptomycin (Sigma), and 2.5 mg/ml amphotericin B (Sigma) and cultured in 5% CO2 in a 37°C incubator (Nuaire). Some tissue fragments will allow cell migration from the ex‐ plants in 3-4 days incubation. Confluence is normally obtained 15-21 days after.

The laboratory's processing and cryopreservation protocols of the UCT units following the technical procedures of Biosckin, Molecular and Cell Therapies S.A. (BSK.LCV.PT.7) were validated for the ability of isolating and expanding *in vitro* MSCs after cryopreserved UCT thawing. The protocols of processing and cryopreservation of the UCT are protected by a Confidentiality Agreement between Biosckin, Molecular and Cell Therapies S.A. and all the involved researchers. Briefly, the UCT collected from healthy donors (N = 60), and according to Netcord guidelines and following the Portuguese law 12/2009 (*Diário da República, lei 12/2009 de 26 de Março de 2009*) is diced into cubes of about 0.5 cm3 and the remaining vessels are removed by dissection. In order to ensure the viability of the UCT after parturition and limit the microbiological contamination of the samples, the umbilical cords were transported from the hospital / clinic to the laboratory at refrigerated temperatures monitored by a data‐ lloger in less than 72 hours. The UCT units from 15-20 centimeters-long umbilical cords and after the blood vessels dissection are treated and processed for cryopreservation using a cry‐ oprotective solution (freezing medium). The UCT units are transferred to a computer-con‐ trolled slow rate freezer (Sylab, Consensus, Portugal) and a nine-step freezing program is used to set up the time, temperature, and rates specifically optimized for the human umbili‐ cal cord-MSCs cooling. To thaw frozen cells, the cryovials are transferred directly to a 37°C water bath. Upon thawing in less than a minute, the cell suspension is centrifuged at 150 × g for 10 min, and the supernatant is gently removed and the cell pellet is resuspended in cul‐ ture medium. It was possible to obtain MSCs in culture from 52 out of 60 thawed UCT units. In some UCT cryopreserved units (N = 8) it was not possible to isolate MSCs due to increase number of erythrocytes' lysis or microbiological contamination during cell culture. The MSCs morphology was observed in an inverted microscope (Zeiss, Germany) at different points of expansion. The MSCs exhibited a mesenchymal-like shape with a flat and polygo‐ nal morphology. The MSCs obtained were characterized by flow cytometry (FACSCalibur®, BD Biosciences) analysis for a comprehensive panel of markers, such as PECAM (CD31), HCAM (CD44), CD45, and Endoglin (CD105). In the presence of neurogenic medium, the MSCs were able to, became exceedingly long and there was a formation of typical neuro‐ glial-like cells with multi-branches and secondary branches. These results permitted to con‐ clude that the processing and cooling protocols used for UCT units' cryopreservation were adequate to preserve the UCT viability since it was possible to isolate and expand MSCs af‐ ter appropriate thaw and in presence of adequate cell culture conditions.

An established and ready-to-use Human MSC cell line was also employed for promoting ax‐ onotmesis and neurotmesis lesions regeneration. Human MSCs from Wharton's jelly umbili‐ cal cord were purchased from PromoCell GmbH (C-12971, lot-number: 8082606.7). Cryopreservated cells are cultured and maintained in a humidified atmosphere with 5% CO2 at 37°C. Mesenchymal Stem Cell Medium (PromoCell, C-28010) is replaced every 48 hours. At 80-90% confluence, cells are harvested with 0.25% trypsin with EDTA (Gibco) and passed into a new flask for further expansion. MSCs at a concentration of 2500 cells/ml are cultured on poli-D-lysine coverslips (Sigma) or on biomaterials membranes and after 24 hours cells exhibit 30-40% confluence. Differentiation into neuroglial-like cells is induced with MSC neurogenic medium (Promocell, C-28015). Medium is normally replaced every 24 hours during 3 days. The formation of neuroglial-like cells can be observed after 24 hours in an inverted microscope (Zeiss, Germany) (Figure 5 and Figure 6).

**Figure 5.** MSC cell line from Wharton's jelly (PromoCell) exhibiting a mesenchymal-like shape with a flat polygonal morphology. Magnification: 100x.

In some UCT cryopreserved units (N = 8) it was not possible to isolate MSCs due to increase number of erythrocytes' lysis or microbiological contamination during cell culture. The MSCs morphology was observed in an inverted microscope (Zeiss, Germany) at different points of expansion. The MSCs exhibited a mesenchymal-like shape with a flat and polygo‐ nal morphology. The MSCs obtained were characterized by flow cytometry (FACSCalibur®, BD Biosciences) analysis for a comprehensive panel of markers, such as PECAM (CD31), HCAM (CD44), CD45, and Endoglin (CD105). In the presence of neurogenic medium, the MSCs were able to, became exceedingly long and there was a formation of typical neuro‐ glial-like cells with multi-branches and secondary branches. These results permitted to con‐ clude that the processing and cooling protocols used for UCT units' cryopreservation were adequate to preserve the UCT viability since it was possible to isolate and expand MSCs af‐

An established and ready-to-use Human MSC cell line was also employed for promoting ax‐ onotmesis and neurotmesis lesions regeneration. Human MSCs from Wharton's jelly umbili‐ cal cord were purchased from PromoCell GmbH (C-12971, lot-number: 8082606.7). Cryopreservated cells are cultured and maintained in a humidified atmosphere with 5% CO2 at 37°C. Mesenchymal Stem Cell Medium (PromoCell, C-28010) is replaced every 48 hours. At 80-90% confluence, cells are harvested with 0.25% trypsin with EDTA (Gibco) and passed into a new flask for further expansion. MSCs at a concentration of 2500 cells/ml are cultured on poli-D-lysine coverslips (Sigma) or on biomaterials membranes and after 24 hours cells exhibit 30-40% confluence. Differentiation into neuroglial-like cells is induced with MSC neurogenic medium (Promocell, C-28015). Medium is normally replaced every 24 hours during 3 days. The formation of neuroglial-like cells can be observed after 24 hours in

**Figure 5.** MSC cell line from Wharton's jelly (PromoCell) exhibiting a mesenchymal-like shape with a flat polygonal

ter appropriate thaw and in presence of adequate cell culture conditions.

474 Advances in Biomaterials Science and Biomedical Applications

an inverted microscope (Zeiss, Germany) (Figure 5 and Figure 6).

morphology. Magnification: 100x.

**Figure 6.** MSC cell line from Wharton's jelly (PromoCell) after 72h of incubation in neurogenic medium. The cells be‐ came exceedingly long and there is a formation of typical neuroglial-like cells with multibranches. Magnification: 100x.

This established human MSC cell line is preferred for *in vivo* testing in rats, since the number of MSCs obtained is higher in a shorter culture time, it is not dependent on donors availabil‐ ity and ethic committee authorization, and the protocol is much less time consuming which is advantageous for pre-clinical trials with a large number of experimental animals. As a matter of fact, there is no need of administrating immunosuppressive treatment to the ex‐ perimental animals during the entire healing period after the surgical procedure. The phe‐ notype of MSCs was assessed by PromoCell. Rigid quality control tests are performed for each lot of PromoCell MSCs isolated from Wharton's jelly of umbilical cord. MSCs are tested for cell morphology, adherence rate and viability. Furthermore, each cell lot is characterized by flow cytometry analysis for a comprehensive panel of markers.

The MSCs isolated with the two protocols described (from fresh and the cryopreserved UCT units) and from the established Promocell cell line exhibited a mesenchymal-like shape with a flat and polygonal morphology. During expansion the cells became long spindle-shaped and colonized the whole culturing surface. After 96 hours of culture in neurogenic medium, cells changed in morphology. The cells became exceedingly long and there was a formation of typical neuroglial-like cells with multi-branches and secondary branches. Giemsa-stained cells of differentiated MSC cell line at passage 5 were analyzed for cytogenetic characteriza‐ tion. However, no metaphases were found, therefore the karyotype could not be established. The karyotype of undifferentiated HMSCs was determined previously and no structural al‐ terations were found demonstrating absence of neoplastic characteristics in these cells, as well as chromosomal stability to the cell culture procedures [55, 56]. The differentiated MSCs karyotype could not be established, since no dividing cells were obtained at passage 5, which can be in agreement with the degree of differentiation. The karyotype analysis of undifferentiated MSCs previously determined, excluded the presence of neoplastic cells, thus supporting the suitability of our cell culture and differentiation procedures. This con‐ cern also resulted from our previous experience with N1E-115 neoplastic cell line and the negative results we obtained in the treatment of axonotmesis and neurotmesis injuries [57-59]. Nevertheless, undifferentiated MSCs from the Wharton's jelly culture (obtained from either protocol or from the Promocell cell line) showed normal morphology when in‐ spected with an inverted microscope (Figure 7).

The differentiation was tested based on the expression of typical neuronal markers such as GFAP, GAP-43 and NeuN by neural-like cells attained from MSCs. Undifferentiated MSCs were negatively labeled to GFAP, GAP-43 and NeuN. After 96 hours of differentiation the attained cells were positively stained for glial protein GFAP and for the growth-associated protein GAP-43. All nucleus of neural-like cells were also labeled with the neuron specific nuclear protein called NeuN showing that differentiation of MSCs in neural-like cells was successfully achieved for MSCs obtained from UCT (fresh and cryopreserved) and for the Promocell MSC cell line (Figure 8) [55].

#### *2.1.2. Differentiation into neuroglial-like cells*

MSCs express nestin, a maker for neural and other stem cells [60, 61] and can be differentiat‐ ed in adipose tissue, bone, cartilage, skeletal muscle cells, cardiomyocyte-like cells, and neu‐ roglial-like cells [54, 55, 60, 62], presenting great potential to biomedical engineering applications. These cells fit into the category of primitive stromal cells and because they are abundant and inexpensive, they might be very useful for regenerative medicine and biotech‐ nology applications.

By employing neuron-conditioned media, sonic hedgehog and fibroblast growth factor 8, MSCs isolated from the Wharton's jelly can be induced toward dopaminergic neurons. These cells have been transplanted into hemiparkinsonian rats where they prevented the progressive degeneration/behavioral deterioration seen in these rats [63]. Rat MSCs isolated from the Wharton's jelly when transplanted into brains of rats with global cerebral ischemia significantly reduced neuronal loss, apparently due to a rescue phenomenon [64]. Neuronal differentiation of human MSCs could also provide cells to replace neurons lost due to neuro‐ degenerative diseases. Recent studies showed that transplanted MSCs-derived neurons be‐ come electrophysiologically integrated within the host neural tissue [65]. However, all these therapeutic applications need uniform and reproducible regulation.

A consequence of cell metabolism during *in vitro* expansion is that culture conditions are constantly changing. The comprehension and optimization of the expansion and differentia‐ tion process will contribute to maximization of cell yield, reduced need of cell culture, and a decrease in total processing costs [66, 67]. Elucidation of regulatory mechanisms of MSCs differentiation will allow optimization of *in vitro* culture and their clinical use in the treat‐ ment of neural-related diseases. Research is being performed to optimize expansion process parameters in order to grow MSCs in a controlled, reproducible, and cost-effective way [68]. Metabolism is certainly one of these parameters.


*3.2. Participant Recruitment*

Participants were recruited using a convenience sampling approach where students and employees from the University of Skövde were recruited. Participants with education or work experience in cybersecurity were excluded from the study. All participants were invited with a direct email that they were asked to reply to in order to participate. Upon registration, participants were randomly assigned to one of the three groups and provided with a description of the experiment, a description of the persona, and an informed consent form. The three groups were the following:

• Game: Participants in this group were prompted to play an educational game before arriving for the experiment. The game is called Jigsaw (https://phishingquiz. withgoogle.com/) (accessed on 6 March 2022) and is developed by Google. It is an example of game-based training that is implemented as a quiz and was selected for use

in this research because it is readily available for users. It also covers all the identifiers of phishing previously described. Jigsaw takes about five minutes to complete.

**Figure 3.** Demonstration of CBMT-based training.