To date, the only definitive treatment for end-stage organ disease is orthotopic transplantation. Conventional surgical practices typically utilize organs from living or deceased donors for transplantation. However, the deficit between the number of patients waiting for a life-saving transplant and the number of organs available for transplantation has grown substantially in recent years. As a result, new options such as tissue engineering and regenerative medicine are gaining traction to overcome organ shortage.
Decellularization is a promising new approach in regenerative medicine that allows researchers to generate three-dimensional biological scaffolds by selective removal of all the cellular and nuclear components of an organ without significantly modifying the architecture and biochemistry of the vasculature, which can be used as an easy route to deliver cells throughout the entire organ [2,30,31]. Complete recellularization of the whole vascular network of an acellular scaffold represents a huge bottleneck towards the translation of bioengineered organs to the clinic. Blood vessels, such as arteries and veins, consist of three layers: tunica intima, mainly composed of EC; tunica media, which consists of connective tissue and SMC; and tunica adventitia, composed mainly of connective tissue. Capillaries and sinusoids are composed of a single layer of flattened EC and a basal lamina with few pericytes . Therefore, EC, SMC, and pericytes/MSC must be considered fundamental bricks to rebuild the vasculature while using the preserved native architecture of the acellular organ as a guiding blueprint. In this regard, and to thoroughly investigate this, the pig has been consistently used in the past years as one of the animal models of choice for regenerative medicine and transplantation research [17,33]. Thus, isolating porcine vascular cells for future organ bioengineering applications is paramount to advancing the field without increasing the experimental complexity by crossing xenogeneic immune barriers using human cells transplanted in pigs .
Hence, to define optimal cell sources for organ revascularization, we isolated the three main cellular components of porcine blood vessels, expanded under conventional bidimensional culture conditions, and characterized them by their immunophenotype and functionality. We found that all the three cell types isolated and cultured could maintain specific phenotypical features, as demonstrated by flow cytometry and immunofluorescence analysis and cell functionality, as shown through pBM-MSC trilineage differentiation and pUVEC DiI-Ac-LDL uptake assays. Additionally, we confirmed that our culture medium containing 10% FBS MSC Qualified induced robust cell proliferation providing adequate support for pBM-MSC expansion . However, it is essential to highlight that there is still a lack of consensus regarding the definition of the optimal markers and methods to verify the identity of pBM-MSC [21,35–37]. Thus, considering the existing literature, we tested a panel of markers to determine the phenotype of pBM-MSC. Many molecules involved in cell adhesion and ECM proteins, cytokines, and growth factor receptors are expressed by MSC and are all associated with their functions and cell interactions within the bone marrow [38,39]. We showed that cultured pBM-MSC were adherent to plastic, with characteristic spindle-shaped morphology and expressed specific mesenchymal surface markers (CD90, CD29, and CD44), while they did not express lineage markers (CD11b, CD14, CD31, CD34, CD45), as reported in the literature [21,40–43]. A general lack of consensus also exists about the optimal differentiation conditions for pBM-MSC . Thus, we performed in vitro differentiation of pBM-MSC into adipocytes, osteoblasts, and chondrocytes using commercial differentiation kits to obtain further information regarding the functionality of pBM-MSC. As these kits are formulated for human MSC, we followed the manufacturer's instructions, and we differentiated the porcine cells for 21 days. The multipotency of pBM-MSC has been demonstrated, as the cells could differentiate into the three lineages, confirming other reported results [20,21,34,40].
Moreover, it has been described that adipogenic differentiation kinetics of pBM-MSC are different from human MSC, and fat accumulates initially in the adipocytes but is secreted afterward. This behavior is observed in pBM-MSC but not in human MSC. Therefore, the measurement of adipogenic differentiation by Oil Red O staining may underestimate the actual fat formed by pBM-MSC . From our results, we could show that pBM-MSC can be successfully differentiated using these human MSC formulated commercial kits.
Vascular SMC are present in distinct phenotypic states in blood vessels. Indeed, they exhibit different morphological and functional properties within the same blood vessel [44,45] and different blood vessels, e.g., arteries and veins . SMC show a high degree of plasticity and the ability to switch from a contractile to a synthetic phenotype in response to environmental stresses and vascular injury. This plasticity creates a complex diversity among SMC and is associated with changes in morphology, proliferation and migration rates, and the expression of different marker proteins. Contractile SMC express SM22𝛼, 𝛼SMA, Caldesmon, smooth muscle calponin, smooth muscle myosin heavy chain, and smoothelin, among other markers, while synthetic SMC express osteopontin, collagen I, moesin, platelet-derived growth factor A and other markers . Cell morphology represents an essential parameter for the definition of SMC phenotype. Contractile SMC are elongated, spindle-shaped cells, while synthetic SMC are epithelioid or rhomboid with a cobblestone-like morphology [47,48]. In this study, we have efficiently isolated vascular SMC from the porcine aorta, which retained important phenotypic features of contractile SMC when in culture. Indeed, pASMC showed a characteristic spindle-shaped morphology and expressed specific muscle markers (SM22𝛼, 𝛼SMA, Caldesmon) and mesenchymal markers (CD90, CD29, CD44) while the expression of lineage markers were absent (CD11b, CD14, CD31, CD34, CD45, CD144). In addition, the cells expressed CD56, which is considered a natural killer, neuronal, and muscle marker but has also been described as a marker of a subset of MSC derived from human bone marrow [49–51]. 𝛼SMA is the most general marker of SMC lineage, and it is highly expressed in spindle-shaped SMC, while SM22𝛼, smoothelin, calponin, and Caldesmon are late differentiation markers abundantly expressed in spindle-shaped SMC [52–54]. CD44 is a cell-surface receptor of hyaluronate, which has been described on various cell types that play a role in ECM binding, cell adhesion, cell-cell interactions, and cell migration [55,56]. The expression of this mesenchymal marker is reported in vascular cells derived from the porcine aorta  and rat aortic SMC . Also, our results are consistent with the results showed by Zaniboni et al. that reported that vascular cells derived from the tunica media of porcine aorta expressed CD56 and CD90, while conversely, the cells did not express the hematopoietic markers CD34 and CD45 . Our results show that pASMC express CD29 is also consistent with several published reports, demonstrating that integrin beta 1 (i.e., CD29) is predominant in vascular SMC in vivo and cultured SMC [59,60]. Therefore, we have shown that pASMC isolated from the aorta's tunica media showed specific mesenchymal surface markers. Moreover, the cells exhibited an elongated, fusiform, spindle-shaped morphology and expressed CD29, SM22𝛼, 𝛼SMA, and Caldesmon, which are marker proteins of the contractile phenotype of SMC.
EC are the main component of the endothelium, which serves as a permeable barrier for blood vessels and regulates blood flow. EC are present in the whole vascular system, from the heart to capillaries, and control the exchanges of materials and the transit of white blood cells in and out of the bloodstream. Furthermore, EC are involved in regulating blood flow, coagulation, endothelial permeability, vascular tone, and vascular remodeling in responses to physiological and pathological stimuli. In terms of recellularization of decellularized scaffolds, a complete re-endothelialization of the acellular vascular network is required to avoid thrombosis after transplantation, induced by the interaction between ECM proteins and the plasma clotting factors and platelets. In this study, we were able to isolate vascular EC from the porcine umbilical vein and expand them under conventional bidimensional culture conditions. The isolated primary cultures were composed of heterogeneous cell populations with different morphology, similar to the morphology reported for their human equivalents  and Chrusciel and colleagues' work . After that, we characterized the cells by flow cytometry and immunofluorescence analysis to define the phenotypical identity of expanded pUVEC. We showed that pUVEC expressed specific endothelial markers (CD31, CD105, CD144, Tie-2) and mesenchymal markers (CD29, CD44, Vimentin), while the expression of CD34 was absent. The expression of endothelial markers such as CD31, CD105, and CD144 has been previously reported for EC isolated from the porcine umbilical vein [26,62], aorta [18,63], heart , as well as for EC isolated from the human umbilical vein .
Moreover, the expression of vimentin, an intermediate filament of the cytoskeleton which plays a critical role in the physiological endothelial mechano-response and inherent to the endothelial phenotype [27,28], has been reported for human umbilical vein EC . Further, we assessed the functionality of pUVEC by DiI-Ac-LDL uptake assay and showed that cultured pUVEC were able to internalize the fluorescent-labeled-DiI-Ac-LDL with success. These results suggest that pUVEC could maintain specific endothelial immunophenotypical features and functionality when isolated and cultured under these defined conditions.