(M-487) Irreversible electroporation-assisted decellularization of intestinal tissues for use as a potential graft in bladder reconstruction therapy

Introduction:: Irreversible electroporation (IRE) is a clinical technique used for tumor ablation therapy by delivering microseconds-long electric pulses using needle electrodes. The working principle of IRE allows killing of cells in the treated tissue while sparing the extracellular matrix (ECM) and stromal structures (such as blood vessels, bile duct etc). We rationalize that this feature of IRE enables its use as decellularization technique under both in vivo and in vitro conditions. Here, we developed a workflow and investigated the application of IRE for ex vivo decellularization of rat small intestine, interrogated the molecular and mechanical properties of the ECM, and tested it for long-term culture of primary cells.


Materials and Methods:: Sprague Dawley rats were sacrificed to extract the small intestine (SI, 1-inch long). IRE of SI was performed in a 4mm gap cuvette (treatment parameters:1500 V/cm, 100 µs, 50 pulses at 1 Hz) with a treatment time of 1 minute, then transferred to media and incubated at 37 °C for 4h, followed by PBS 4x wash and DNase wash. Detergent wash (DT; positive control) was performed by washing the SI in triton X-100, sodium deoxycholate, DNase, and saline solution. H&E and Masson’s Trichrome (MT) stains were used for assessing morphological status and collagen in SI samples. Villin, alpha SMA, and CD34 stains were used to test decellularization efficiency. Colorimetric quantitative assays were used to measure residual DNA, glycosaminoglycan and collagen levels, and ultimate tensile testing was performed to quantify mechanical properties of IRE treated SI. IRE treated SI was lyophilized and digested with pepsin-acetic acid to produce a ECM gel then mixed with human bladder smooth muscle cells (HBdSMCs; 2 x 10⁵ cells/well) from which 50 µL aliquots were cultured up to 14 days. Cell titer glo assay and DNA quantification were performed to assess the cell growth and proliferation at different timepoints (1, 3, 7 and 14 days). SMC differentiation was studied using alpha-SMA staining and imaged through Nikon A1R25 confocal microscope. The decellularized SI tissue was mounted on a trans-well insert, seeded with 5637-bladder epithelial cells (2 x 10⁵ cells/well) and cultured for 7 days. The tissue was fixed and processed for H&E staining.


Results, Conclusions, and Discussions:: Post-IRE incubation was found necessary to complete the cell death process where washing with PBS/DI water produced decellularization (~41 hrs) comparable to detergent treatment (~82 hrs). Histological analysis (H&E, MT, villin, alpha SMA, CD34 and DAPI) of IRE samples demonstrated complete removal of cellular components with the superior preservation of ECM (collagen and GAG) including blood vessel architecture compared to detergent treatment (Fig 1 A-D & F). Total residual DNA in IRE (30 ng/mg) group was below the 50 ng/mg level that elicits immunological response. Mechanical stiffness of IRE and DT groups was higher than native tissue, with no significant difference when comparing IRE to sham (Fig 1E). ECM gel produced with IRE treated IS demonstrated growth profile comparable to collagen gels (positive control) during initial days and 1.5 mg/mL IECM gel group surpass the growth rate at day 14 compared to other groups (2A, 2C & 2D). This could be due to the presence of other ECM proteins and GAG that mimic native tissue-like environment helping in growth and differentiation. The SMCs were stained for alpha-SMA to check for differentiation (Fig 2B) and showed increased expression in IECM gels compared to collagen at day 7 & or above. Finally, 5637 cells cultured on decellularized SI showed proper monolayer formation on the apical side of the tissue. Cumulatively, our results show that IRE is an efficacious decellularization technique that is quick, spares ECM proteins. Gels and scaffolds produced from IRE treated SI demonstrated excellent ability to support the growth of smooth muscle and epithelial cells, demonstrating suitability for use with regenerative applications or reconstructive surgery.


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