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首页> 外文期刊>Materials science & engineering >Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion
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Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion

机译：使用温和的去污剂和压力控制的灌注，可快速，稳固和有效地使整个人类肝脏脱细胞

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Human whole-liver perfusion-decellularization is an emerging technique for producing bio-scaffolds for tissue engineering purposes. The native liver extracellular matrix (ECM) provides a superior microenvironment for hepatic cells in terms of adhesion, survival and function. However, current decellularization protocols show a high degree of variation in duration. More robust and effective protocols are required, before human decellularized liver ECM can be considered for tissue engineering applications. The aim of this study is to apply pressure-controlled perfusion and test the efficacy of two different detergents in porcine and human livers.To test this, porcine livers were decellularized using two different protocols; a triton-x-100 (Tx100)-only protocol (N = 3) and a protocol in which Tx100 was combined with SDS (N = 3) while maintaining constant pressure of 120 mm Hg. Human livers (N = 3) with different characteristics (age, weight and fat content) discarded for transplantation were decellularized using an adapted version of the Tx-100-only protocol. Decellularization efficacy was determined by histology and analysis of DNA and RNA content. Furthermore, the preservation of ECM components was assessed.After completing the perfusion cycles with detergents the porcine livers from both protocols were completely white and transparent in color. After additional washing steps with water and DNase, the livers were completely decellularized, as no DNA or cell remnants could be detected. The Tx100-only protocol retained 1.5 times more collagen and 2.5 times more sGAG than the livers decellularized with Tx100 + SDS. The Tx100-only protocol was subsequently adapted for decellularizing whole-organ human livers. The human livers decellularized with pressure-controlled perfusion became off-white in color and semi-transparent within 20 h. Livers decellularized without pressure-controlled perfusion took 64-96 h to completely decellularize, but did not become white or transparent. The addition of pressure-controlled flow did remove all cells and double stranded DNA, but did not damage the ultra-structure of the ECM as was analyzed by histology and scanning electron microscopy. In addition, collagens and sGAG were maintained with the decellularized ECM.In conclusion, we established effective, robust and fast decellularization protocols for both porcine and human livers. With this protocol the duration of decellularization for whole-organ human livers has been shortened considerably. The increased pressure and flow did not damage the ECM, as major ECM components remained intact.
机译：人全肝灌注脱细胞是一种用于组织工程目的生产生物支架的新兴技术。就粘附，存活和功能而言，天然肝细胞外基质（ECM）为肝细胞提供了优越的微环境。然而，当前的脱细胞方案显示持续时间的高度变化。在考虑将人脱细胞的肝ECM用于组织工程应用之前，需要更强大有效的方案。本研究的目的是应用压力控制的灌注并测试两种不同去污剂在猪和人肝脏中的功效。仅限Triton-x-100（Tx100）方案（N = 3）和将Tx100与SDS结合的方案（N = 3），同时保持120 mm Hg的恒定压力。使用仅Tx-100协议的改编版，将废弃的具有不同特征（年龄，体重和脂肪含量）的人类肝脏（N = 3）进行脱细胞处理。通过组织学和DNA和RNA含量的分析来确定脱细胞效力。此外，评估了ECM成分的保存性。用洗涤剂完成灌注周期后，两种方案的猪肝脏均为完全白色且颜色透明。用水和DNase进行额外的洗涤步骤后，肝脏完全脱细胞，因为无法检测到DNA或细胞残留物。仅Tx100的方案保留的胶原蛋白和sGAG的含量是用Tx100 + SDS脱细胞的肝脏的1.5倍。随后将仅Tx100的协议修改为使全器官人类肝脏脱细胞。在压力控制的灌注下脱细胞的人类肝脏在20小时内颜色变为灰白色并半透明。没有压力控制的灌注而脱细胞的肝花了64-96 h才能完全脱细胞，但是没有变白或透明。如通过组织学和扫描电子显微镜分析的，添加压力控制的流动确实去除了所有细胞和双链DNA，但是没有破坏ECM的超微结构。此外，脱细胞ECM可以维持胶原蛋白和sGAG。总之，我们为猪和人的肝脏建立了有效，强大和快速的脱细胞方案。通过该协议，全器官人类肝脏的脱细胞持续时间已大大缩短。压力和流量的增加并没有损坏ECM，因为主要的ECM组件仍然完好无损。

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