目的: 通过创新的酶消化配方及机械处理方式,快速有效解离皮肤组织,满足单细胞研究的需求。方法: 酶消化方法: 酶消化方法分为两组,A组为Ⅰ型胶原酶+DNase I,B组为Ⅱ型胶原酶+0.25%胰酶+DNase I,两组均使用Hanks缓冲液配制。机械方法为恒温震荡和恒温拍打。将酶消化方法与机械处理方法进行两两组合,处理皮肤组织。结果: 最佳解离方法为酶消化A组(Ⅰ型胶原酶2 mg/mL,DNase I 10 U/mL)+恒温拍打(180 次/min,37 ℃),该组合在15 min内实现了皮肤组织的有效消化。随后通过透明质酸酶消化、红细胞裂解液处理,结合30 μm滤膜过滤及多次离心纯化,最终获得高纯度且保持高活性的单细胞悬液。结论: 通过对酶种类、浓度以及机械处理方式的优化组合,在15 min内实现了皮肤组织的高效消化,获得了高纯度、高活性的单细胞悬液,极大地提升了研究的效率和准确性,为后续流式细胞分析、细胞分选及高通量测序等研究提供了可靠的技术支持。
Objective: With the development of high-throughput sequencing technology, the demand for research at the single-cell level is constantly increasing. Especially for single-cell sequencing studies in skin tissues, the demand for dissociation into single-cell suspensions is growing day by day. However, the existing technology is difficult to dissociate skin tissue into single-cell suspensions in a short period of time. The dense structure of skin tissue leads to the fact that the existing dissociation methods are time-consuming and difficult to meet the timeliness requirements of downstream omics research. This experiment rapidly and effectively dissociates skin tissue through an innovative enzymatic digestion formula and mechanical processing method, meeting the requirements of single-cell research. Methods: Enzyme digestion method: Enzyme digestion method was divided into two groups, group A: type I collagenase+DNase I, group B: type II collagenase+0.25% trypsin+DNase I, both groups were prepared with Hanks buffer. The mechanical method was constant temperature shock and constant temperature beat. The enzyme digestion method and the mechanical treatment method were combined in pairs to treat the skin tissue. Results: The optimal dissociation method was enzyme digestion group A (type I collagenase 2 mg/mL, DNase I 10 U/mL)+constant temperature beating (180 times/min, 37 ℃). This combination achieved effective digestion of skin tissue within 15 min. The single cell suspension with high purity and high activity was successfully prepared by further treatment of hyaluronidase and red blood cell lysate, filtration (30 μm) and centrifugation. Conclusion: By optimizing the combination of enzyme types, concentrations and mechanical treatment methods, efficient digestion of skin tissue was achieved within 15 minutes, obtaining high-purity and high-activity single-cell suspensions. This greatly enhanced the efficiency and accuracy of the research, providing reliable technical support for subsequent flow cytometry analysis, cell sorting and high-throughput sequencing studies..
[1] Wang H, Van Blitterswijk CA, Bertrand-De Haas M, et al. Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes[J]. In Vitro Cell Dev Biol Anim, 2004, 40(8-9): 268-277.
[2] Clister T, Fey RM, Garrison ZR, et al. Optimization of tissue digestion methods for characterization of photoaged skin by single cell RNA sequencing reveals preferential enrichment of T cell subsets[J]. Cells, 2024, 13(3): 266.
[3] Novelli M, Savoia P, Cambieri I, et al. Collagenase digestion and mechanical disaggregation as a method to extract and immunophenotype tumour lymphocytes in cutaneous T-cell lymphomas[J]. Clin Exp Dermatol, 2000, 25(5): 423-431.
[4] Ikeuchi T, Akhi R, Cardona Rodriguez B, et al. Dissociation of murine oral mucosal tissues for single cell applications[J]. Immunol Methods, 2024, 525: 113605.
[5] Nayar S, Campos J, Steinthal N, et al. Tissue digestion for stromal cell and leukocyte isolation[J]. Methods Mol Biol, 2017, 1591: 225-234.
[6] 黄昕, 徐祥文, 高雅姗, 等. 改良联合酶消化法制备人瘢痕疙瘩组织单细胞悬液的实验研究[J]. 组织工程与重建外科杂志, 2019, 15(6): 398-402.
[7] 张远圆, 张杉杉, 常旭, 等. 机械法与机械-酶消化法制备大鼠膈肌组织单细胞悬液的比较[J]. 中国组织工程研究, 2020, 24(8): 1213-1217.
[8] Amosu M, Gregory AJ, Murtagh JD, et al. Mechanical dissociation of tissues for single cell analysis using a motorized device[J]. J Vis Exp, 2023, (201): 10.3791/65866.
[9] 张育飞, 夏育民. 单细胞RNA测序技术在皮肤病研究中的应用[J]. 中国皮肤性病学杂志, 2021, 35(11): 1314-1318.
[10] Trujillo M, McElroy T, Brown T, et al. Combined mechanical and enzymatic dissociation of mouse brain hippocampal tissue[J]. J Vis Exp, 2021, (176): 10.3791/63007.
[11] Benck CJ, Martinov T, Fife BT, et al. Isolation of infiltrating leukocytes from mouse skin using enzymatic digest and gradient separation[J]. J Vis Exp, 2016, (107): e53638.
[12] Fernandez J, Torchia EC. Isolation and staining of mouse skin keratinocytes for cell cycle specific analysis of cellular protein expression by mass cytometry[J]. Vis Exp, 2019, (147): 10.3791/59353.
[13] DaMata JP, Zelkoski AE, Nhan PB, et al. Dissociation protocols influence the phenotypes of lymphocyte and myeloid cell populations isolated from the neonatal lymph node[J]. Front Immunol, 2024, 15:1368118.
[14] Zhou X, Meng M, Wu Y, et al. Protocol to dissociate and isolate wide-diversity single cells by density gradient centrifugation from human hepatoblastoma tissue[J]. STAR Protoc, 2023, 4(3): 102449.
[15] He X, de Oliveira VL, Keijsers R, et al. Lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture[J]. J Vis Exp, 2016, (110): e52564.
[16] Ascensión AM, Araúzo-Bravo MJ, Izeta A. The need to reassess single-cell RNA sequencing datasets: the importance of biological sample processing[J].F1000Res, 2021, 10:767.
[17] Januszyk M, Gurtner GC. High-throughput single-cell analysis for wound healing applications[J]. Adv Wound Care (New Rochelle), 2013, 2(9): 457-469.
[18] Broggi A, Cigni C, Zanoni I, et al. Preparation of single-cell suspensions for cytofluorimetric analysis from different mouse skin regions[J]. J Vis Exp, 2016, (110): e52589.
[19] Zhou J, Wei A, Bertsch A, et al. High precision,high throughput generation of droplets containing single cells[J]. Lab Chip, 2022, 22(24): 4841-4848.
[20] Sierra-Sánchez Á, Barbier MA, Magne B, et al. Comparison of two human skin cell isolation protocols and their influence on keratinocyte and fibroblast culture[J]. Int J Mol Sci, 2023, 24(19): 14712.
[21] Fu S, Panayi A, Fan J, et al. Mechanotransduction in wound healing: from the cellular and molecular level to the clinic[J]. Adv Skin Wound Care, 2021, 34(2): 67-74.
[22] Zhang P, Han X, Yao J, et al. High-throughput isolation of cell protrusions with single-cell precision for profiling subcellular gene expression[J]. Angew Chem Int Ed Engl, 2019, 58(39): 13700-13705.
[23] 何杰, 王宁, 张杉杉, 等. 机械—酶消化法制备大鼠膈肌单细胞悬液的最佳消化酶浓度组合筛选[J]. 山东医药, 2022, 62(26): 45-48.
[24] Mainali BB, Yoo JJ, Ladd MR. Tissue engineering and regenerative medicine approaches in colorectal surgery[J]. Ann Coloproctol, 2024, 40(4): 336-349.
