Narciso Maria, Ulldemolins Anna, Júnior Constança, Otero Jorge, Navajas Daniel, Farré Ramon, Gavara Núria, Almendros Isaac
Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, Barcelona, Spain.
The Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Barcelona, Spain.
Front Bioeng Biotechnol. 2022 Mar 9;10:832178. doi: 10.3389/fbioe.2022.832178. eCollection 2022.
Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue's vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices-with an emphasis on lung-while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies.
去细胞化程序已针对整个器官或组织块进行了开发和优化，方法是通过组织血管灌注去细胞化剂，或将大的组织切片浸入去细胞化溶液中。然而，一些研究目标需要对天然组织切片和去细胞化组织切片进行并排分析，但尚未建立最佳方案来满足这一需求。因此，这项工作的主要目标是开发一种快速有效的组织切片去细胞化方法，重点是附着在载玻片上的肺组织切片。为此，比较了不同去细胞化剂在去除细胞同时保留细胞外基质方面的有效性。DNA染色强度作为剩余细胞的指标，并与未处理的切片进行比较。使用免疫染色和信号定量对胶原蛋白、弹性蛋白和层粘连蛋白的存在进行定量。使用原子力显微镜对优化方案得到的支架进行力学表征。用间充质基质细胞对肺支架进行再细胞化，以评估其生物相容性。一些去细胞化剂（CHAPS、曲拉通和氢氧化铵）未能实现充分的细胞去除。十二烷基硫酸钠（SDS）在细胞去除方面有效（剩余DNA信号为1%），但其弹性蛋白信号急剧降低（仅剩余6%）以及较低的附着率（32%），使得脱氧胆酸钠（SD）成为该应用的最佳处理方法（剩余DNA信号为6.5%），因为其弹性蛋白保留率更高（34%）且附着率更高（60%）。层粘连蛋白和胶原蛋白在所有处理中均得到充分保留。SD去细胞化方案对猪和鼠（小鼠和大鼠）的肺以及心脏、肾脏和膀胱等其他组织也同样成功。样品去细胞化前后未发现明显的力学差异。所得的无细胞肺支架显示具有生物相容性（培养72小时后细胞存活率为98%）。这种使组织切片去细胞化的新方法为更好地理解细胞外基质在多种疾病背景下的作用以及组织工程研究开辟了新的方法可能性，并且可以很容易地应用于临床活检等稀缺样本。
