Perfusion-decellularization of vascularized pig stomachs

Background Total-partial gastrectomy, congenital gastric malformations and other causes of permanent gastric size reduction may result in severe side effects and poor life quality. Organ transplantation is a treatment of choice in end-stage chronic organ failure but is limited by the shortage of organs and the problems related to lifelong immunosuppression. A bioengineered stomach transplant could thus restore a physiological transit and a digestive function, while being fully compatible. This work aim was to apply, in piglet stomachs, the “decellularization-recellularization” approach, as previously reported by our team in human and porcine composite tissues. Methods/Materials Seven neonatal porcine stomachs were surgically harvested along with their vascular pedicle and decellularized by sequential perfusion of demineralized water, sodium dodecyl sulfate, Triton X-100, finalized by DNASe and PBS solutions. Cell clearance was evaluated by H&E and DAPI stainging,and DNA quantification. Extracellular matrix preservation was assessed by Masson’s Trichrome (MT), type I and type IV collagens, and laminin stainings, elastin and GAG quantification. The vascular network was evaluated with angio-CT. Finally, matrix samples were sterilized and cultured with a fibroblastic cell line and analyzed by H&E and Live/dead stainings. Native and decellularized stomachs were perfused through the esophagus with air to assess the preservation of the tightness of the gastric wall and then with saline serum to evaluate the mechanical properties during the filling. Results Stomachs were successfully decellularized, with a fast bleaching during the SDS perfusion while preserving their morphology. The air perfusion after decellularization also confirms a total preservation of the tightness of the gastric and no absence of air leakages (bubbles) in the water tank (during the perfusion). Nuclei were absent on H&E, MT and DAPI staining; DNA reduction was significant (p0.01). MT showed a well-preserved microscopic architecture of the mucosa, submucosa, muscularis and serosa layers. Type I, type IV collagens, and laminin were positively stained in both native and decellularized stomachs. Matrix proteins quantification revealed an increase for the collagen but a decrease for elastin and GAG. The angio-CT showed a well preserved and accessible vascular bed. Seeded fibroblasts were viable after 7 and 14 days, as shown by the Live/dead and the H&E stainings. The pressure-volume curve during the saline perfusion had a similar morphology before and after decellularization but we observed an increase of the accumulated volume of 1,4-2,1x reaching the same gastric lumen pressure than inside a native stomach. Conclusion We demonstrated the ability to produce porcine stomach extracellular matrix with a preserved vascularization. This could offer broad new perspectives in organ tissue engineering at this anatomical level