一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析
Summary
This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.
Abstract
恶性细胞逃避免疫系统，从先天性和适应性免疫应答特征为肿瘤逃逸的能力现在被接受为癌症的重要标志。我们对乳癌的研究主要集中在积极的作用，肿瘤浸润淋巴细胞肿瘤进展和患者预后发挥。为了实现这一目标，我们制定了正常和异常组织完整淋巴样细胞的快速分离的方法，努力以评估他们接近他们的原生状态。匀浆用机械离解秀既增加了活力和细胞的恢复，同时保持表面受体的表达相比，酶消化的组织准备。另外，剩余的不溶物的酶消化未恢复额外的CD45 +细胞表示在初级匀浆的定量和定性测量可能真正反映在组织fragm浸润亚耳鼻喉科。在淋巴样细胞中这些匀浆可以容易地使用免疫（表型，细胞增殖等 ）或分子（DNA，RNA和/或蛋白质），其特征接近。 CD45 +细胞也可以用于亚群纯化， 体外扩增或冷冻保存。这种方法的一个额外的好处是，从匀浆主组织上清液可用于表征和比较细胞因子，趋化因子，免疫球蛋白和抗原存在于正常和恶性组织。这个协议功能非常好为人类乳房组织和应适用于各种各样的正常和异常组织。
Introduction
The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.
Protocol
Representative Results
Discussion
该研究描述了在正常和恶性乳腺组织匀浆中迅速地制备无酶消化用于随后的细胞分选，提取，冷冻保存和/或CD45 +亚群的表型分析的优化方法。本实验方法的目标是产生紧密反映其在体内的状态，将它们与从手术室新鲜组织的最低限度的操作比较正常组织的TIL的图像。到今天为止，我们的实验室已使用此协议分析> 250新鲜BC组织（肿瘤和NANT），> 35正常乳腺组织，从乳腺减少。原?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了资金支持fromthe比利时科学研究基金（FNRS），莱斯阿美族DE L'研究所博尔德，FNRS操作Télévie，比利时计划癌症，全宗朗博-Marteaux，全宗JC Heuson和全宗Barsy。
Materials
| Equipment | Company | Catalog Number | Comments/Description |
| GentleMacs Dissociator | Miltenyi Biotec | 130-093-235 | BD Medimachine is somewhat equivalent |
| Centrifuge 5810 R | Eppendorf | N/A | or other standard table top centrifuge |
| Centrifuge 5417 R | Eppendorf | N/A | or other standard microcentrifuge |
| Esco Class II A2 Biosafety Cabinet | ESCO global | N/A | or other standard BSL2 hood |
| Inverted Microscope | Nikon eclipse TS100 | N/A | or other microscope compatible for a hemacytometer |
| Bürker Chamber | Marienfield | 640210 | or other standard hemacytometer |
| Navios Flow Cytometer | Beckman Coulter | N/A | or other flow cytometer (8-10 color recommended) |
| Materials | Company | Catalog Number | Comments/Description |
| GentleMacs C-Tube | Miltenyi Biotec | 130-096-344 | BD Medimachine uses Filcon |
| Cell Culture Dish | Sarstedt | 72,710 | or other non-pyrogenic plasticware |
| Disposable Scalpel | Swann-Morton | 0510 | or standard single use sterile scalpel |
| BD Cell Strainer 40µm | Becton Dickinson | 734-0002 | or other non-pyrogenic plasticware |
| BD Falcon Tube 50mL | Becton Dickinson | 352070 | or other non-pyrogenic plasticware |
| BD Falcon Tube 15mL | Becton Dickinson | 352097 | or other non-pyrogenic plasticware |
| BD FACS Tube 5mL | Becton Dickinson | 352008 | or other non-pyrogenic plasticware |
| Sterile Pasteur Pipette 5 mL | VWR | 612-1685 | or other non-pyrogenic plasticware |
| Microfuge Tube 1.5 mL | Eppendorf | 7805-00 | or other non-pyrogenic plasticware |
| Reagents | Company | Catalog Number | Comments/Description |
| X-Vivo 20 | Lonza | BE04-448Q | serum-free medium recommended |
| Phosphate buffered saline | Lonza | BE17-516F | standard physiological PBS |
| Trypan blue | VWR | 17942E | or other vital stain |
| VersaLyse | Beckman Coulter | A09777 | for flow cytometry experiments |
| Fixable viability Dye eFluor 780 | eBioscience | 65-0865-14 | for flow cytometry experiments |
| anti-CD3 FITC | BD Biosciences | 345763 | for flow cytometry experiments |
| anti-CD3 Vio Blue | Miltenyi Biotec | 130-094-363 | for flow cytometry experiments |
| anti-CD4 PE | BD Biosciences | 345769 | for flow cytometry experiments |
| anti-CD4 APC | Miltenyi Biotec | 130-091-232 | for flow cytometry experiments |
| anti-CD8 ECD | Beckman Coulter | 737659 | for flow cytometry experiments |
| anti-CD8 PerCP | BD Biosciences | 345774 | for flow cytometry experiments |
| anti-CD19 APC-Vio770 | Miltenyi Biotec | 130-096-643 | for flow cytometry experiments |
| anti-CD45 VioGreen | Miltenyi Biotec | 130-096-906 | for flow cytometry experiments |
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