Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; Beijing Clinical Research Institute, Beijing 100050, China; Beijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing 100050, China; National Clinical Research Center for Digestive Diseases, Beijing 100050, China.
Department of Liver Transplantation Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China.
Int Immunopharmacol. 2020 Aug;85:106632. doi: 10.1016/j.intimp.2020.106632. Epub 2020 May 27.
Isolating and purifying liver immune cells are crucial for observing the changes in intrahepatic immune responses during the development of liver diseases and exploring the potential immunological mechanisms. Therefore, the aim of this study was to provide an optimal protocol for isolating immune cells with a high yield and less damage. We compared mechanical dissection and collagenase digestion, and the results were represented by the proportion of lymphocytes, Kupffer cells and neutrophils. The apoptosis rates of liver immune cells resulted by different isolation protocols were compared by Annexin V-staining using flow cytometric analysis. Our data indicated that the enzymatic digestion in vitro was more efficient than the mechanical dissection in vitro with a suitable collagenase IV concentration of 0.01%, and the purification of liver immune cells by a one-step density gradient centrifugation in 33% Percoll had the definite advantage of a higher proportion of the target cells. We also provided evidence that enzymatic digestion in vitro method was superior to collagenase digestion in situ for liver T lymphocytes, NK cells and NKT cells isolation and purification. This protocol was also validated in human liver samples. In conclusion, we developed an optimal protocol for isolating and purifying immune cells from mouse and human liver samples in vitro by 0.01% collagenase IV and 33% Percoll density gradient centrifugation with the advantages of higher cell yields and viability. This method provides a basis for further studying liver immune cells and liver immunity with a wide range of applications.
分离和纯化肝脏免疫细胞对于观察肝脏疾病发展过程中肝内免疫应答的变化以及探索潜在的免疫学机制至关重要。因此，本研究旨在提供一种优化的方案，用于分离具有高产量和低损伤的免疫细胞。我们比较了机械分离和胶原酶消化，并用淋巴细胞、枯否细胞和中性粒细胞的比例来表示结果。通过流式细胞术分析 Annexin V 染色比较不同分离方案对肝脏免疫细胞凋亡率的影响。我们的数据表明，在合适的胶原酶 IV 浓度为 0.01%的情况下，体外酶消化比体外机械分离更有效，用 33%Percoll 一步密度梯度离心法纯化肝脏免疫细胞具有更高比例靶细胞的明显优势。我们还提供了证据表明，体外酶消化法在分离和纯化肝 T 淋巴细胞、NK 细胞和 NKT 细胞方面优于原位胶原酶消化法。该方案在人肝组织样本中也得到了验证。总之，我们通过 0.01%胶原酶 IV 和 33%Percoll 密度梯度离心法开发了一种从鼠和人肝组织样本中体外分离和纯化免疫细胞的最佳方案，该方案具有更高的细胞产量和活力优势。该方法为进一步研究肝脏免疫细胞和肝脏免疫提供了基础，具有广泛的应用前景。
