Impact of Porcine Pancreas Decellularization Conditions on the Quality of Obtained dECM

Abstract

Due to the limited number of organ donors, 3D printing of organs is a promising technique. Tissue engineering is increasingly using xenogeneic material for this purpose. This study was aimed at assessing the safety of decellularized porcine pancreas, together with the analysis of the risk of an undesirable immune response. We tested eight variants of the decellularization process. We determined the following impacts: rinsing agents (PBS/NH3·H2O), temperature conditions (4 °C/24 °C), and the grinding method of native material (ground/cut). To assess the quality of the extracellular matrix after the completed decellularization process, analyses of the following were performed: DNA concentration, fat content, microscopic evaluation, proteolysis, material cytotoxicity, and most importantly, the Triton X-100 content. Our analyses showed that we obtained a product with an extremely low detergent content with negligible residual DNA content. The obtained results confirmed the performed histological and immuno-fluorescence staining. Moreover, the TEM microscopic analysis proved that the correct collagen structure was preserved after the decellularization process. Based on the obtained results, we chose the most favorable variant in terms of quality and biology. The method we chose is an effective and safe method that gives a chance for the development of transplant and regenerative medicine.

Impact of various detergent-based immersion and perfusion decellularization strategies on the novel caprine pancreas derived extracellular matrix scaffold

“…On the contrary, Milan et al reported that SDC alone showed no success in retaining collagen, but in our study, it has retained the collagen on tissue edges (Milian et al, 2021). Besides this study, numerous other xenogeneic tissues have also exhibited superior results with TX-100, such as porcine aortic valves (GRAUSS et al, 2005), porcine pancreas (Klak et al, 2021;Zhu et al, 2021), and porcine liver (Mirmalek-Sani et al,…”

“…On the contrary, Milan et al reported that SDC alone showed no success in retaining collagen, but in our study, it has retained the collagen on tissue edges ( Milian et al, 2021 ). Besides this study, numerous other xenogeneic tissues have also exhibited superior results with TX-100, such as porcine aortic valves ( GRAUSS et al, 2005 ), porcine pancreas ( Klak et al, 2021 ; Zhu et al, 2021 ), and porcine liver ( Mirmalek-Sani et al, 2013 ), etc. Whereas in other investigations, SDS has been favored over TX-100 for decellularization of densely fibrous tissues such as kidney and liver, as TX-100 has demonstrated poor outcomes ( Hussein et al, 2016 ).…”

“…This increased proportion of collagen following decellularization can be ascribed to the revelation of previously concealed ECM structure revealed after cell removal, raising the overall collagen content in the quantitative analysis. Excessive sterilization procedure is also known to diminish these components, and we did not undertake rigorous sterilization procedure, which explains the surplus amount of Collagen and sGAG achieved after decellularization ( Klak et al, 2021 ). Another study involving the decellularization of bovine cartilage yielded similar results showing higher collagen content after decellularization with SDS ( Utomo and Sari, 2019 ).…”

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

“…TX100 remove GAGs and decreases laminin and fibronectin content, but it does not affect collagens, showing its detrimental effects on ECM are milder than their ionic counterparts [75,84]. Moreover, considerable research indicates TX100 is cytotoxic, necessitating TX100-derived ECM safety control before clinical translation [85]. Retention of TX100 within dECM should be minimized by applying lower concentrations and different washing steps to achieve scaffolds with less than 15mM remaining TX100 concentrations, which was previously shown to get tolerated well by human cell lines [86].…”

Preparation and assessment of an optimized multichannel acellular nerve allograft for peripheral nerve regeneration

“…This method was confirmed to clear cells and myelin sheaths effectively, and the yielded allografts achieved good nerve regeneration after transplantation. 18 Although nonionic detergent Triton X‐100 is described to cause minimal damage to the native tissues, 19 SDC, an ionic detergent, is considered to be one of the most potent and disruptive chemical agents. Subsequently, Hudson et al proposed a milder chemical treatment consisting of several steps repeated twice as well.…”