Alteration of the extracellular matrix and alpha‐gal antigens in the rat lung scaffold reseeded using human vascular and adipogenic stromal cells

Abstract

Regenerated organs are expected to solve the problem of donor organ shortage in transplantation medicine. One approach to lung regeneration is to decellularize the organ and reseed it with selected cells. An advantage of the procedure is reduced immunogenicity, because all cells can be theoretically replaced by autologous cells.However, little is known regarding the extracellular matrix (ECM) damage during decellularization and ECM reconstruction process in the organ regeneration. We aimed to evaluate ECM damage and reconstruction of the decellularizedrecellularized rat lung, including the removal of alpha-gal xenoantigens. Rat lungs were perfused with sodium dodecyl sulfate and Triton X-100 via the pulmonary artery, after which the decellularized scaffold was reseeded with rat or human endothelial cells and adipose-derived stem cell (ASCs). The ECM and alpha-gal antigen were evaluated using immunohistochemistry, western blotting, and a glycosaminoglycan assay. Alcian blue staining revealed increased production of proteoglycan following the addition of ASCs to the rat lung recellularized with rat lung microvascular endothelial cells. Glycosaminoglycan levels decreased in the decellularized lung and increased in the recellularized lung, especially in the ASC-treated group. Immunohistochemical expression of the alpha-gal protein was decreased to an undetectable level in the decellularized lung tissue and disappeared after recellularization with human cells. In western blot analysis, the bands of alpha-gal protein almost disappeared after recellularization with human cells. In conclusion, characteristics of the regenerated ECM might depend on the species and type of cells used for recellularization. Therefore, alpha-gal antigen might be eliminated after a prolonged culture, when using human cells.

Engineering a 3D Vascularized Adipose Tissue Construct Using a Decellularized Lung Matrix

“…Although the majority of immunogenic proteins are removed following detergent-based decellularization, there are some remaining extracellular matrix components that can cause adverse immune responses. For instance, the α-Gal antigen that is present in porcine scaffolds has been shown to induce an inflammatory reaction, which is now being addressed by the generation of α-Gal pigs [ 20 , 21 , 53 ]. Like any studies using human adipocytes, there are patient-to-patient differences, such as size, quantity, and metabolic function.…”

“…The lung is one of the most vascularized organs with high flow, low resistance, and a large blood–alveolar interface separated by a thin basement membrane (<1 µm [ 11 ]). Accordingly, the DLM offers the following unique advantages: (1) its large volume capacity within the alveolar compartment can be repurposed for high-density adipose cell filling; (2) it offers a preserved, acellular vascular bed allowing efficient graft perfusion and pre-vascularization [ 12 , 13 , 14 ]; (3) the alveolar compartment shields buoyant and fragile adipocytes [ 15 , 16 ] from high shear stresses [ 17 ] that compromise their viability by confining high flow rates to the vascular bed where it is required for proper endothelial functionality [ 18 ]; (4) it will not elicit an immune response by allogeneic or xenogeneic recipients [ 19 , 20 , 21 ]; and (5) the DLM is scalable, and can be manufactured from rat [ 12 , 14 ], pig [ 13 , 22 ], and donor human lungs [ 13 , 22 , 23 ] to meet the diverse size requirements for filling different defects. In the following studies, we use rats as our DLM as a proof of concept, with the long-term goal of scaling up in future studies to larger animal scaffolds.…”

Perpendicular implantation of porcine trachea extracellular matrix for enhanced xenogeneic scaffold surface epithelialization in a canine model

“…On the other hand, pigs organs have been used as a xenotransplant material for humans, and many experiments and applications have been conducted ( 31 , 32 ) Although xenotransplantation poses a problem of its antigenicity, decellularization process could eliminate its xenoantigens including alfa- gal epitopes. In addition, in the decellularization model of the lung, alfa- gal epitopes are said to decrease or disappear when the lung ECM is recellularized ( 33 ). Moreover, the use of organs from alpha 1,3-galactosyltransferase knockout pigs may aid the decellularization process, resulting in a more complete removal of alpha-gal antigens ( 34 ).…”

Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine

“…Moreover, ECM-antibodies interaction enhances macrophage activity and accelerates the degradation of dECM scaffolds, preventing cryptic peptides from releasing at a time sufficient for a good remodeling process [47]. Pre-implantation recellularization of xenografts with human stem cells seems a practical means to attenuate the host immune response against glycan antigens [147].…”