بروتوكول بسيطة وسريعة لغير إنزيمي فصل الطازجة الأنسجة البشرية لتحليل التسلل اللمفاويات
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
يتم قبول قدرة الخلايا الخبيثة للتهرب من الجهاز المناعي، ويتميز ورم الهروب من كل من الاستجابات المناعية الفطرية والتكيفية، والآن باعتبارها السمة المميزة مهمة من السرطان. أبحاثنا حول سرطان الثدي يركز على الدور الفعال الذي تلعبه الورم تسلل الخلايا الليمفاوية في تطور الورم ونتائج المرضى. لتحقيق هذا الهدف، وضعنا منهجية لعزل السريع للخلايا اللمفاوية سليمة من الأنسجة العادية وغير العادية في محاولة لتقييمها الداني إلى حالتها الأصلية. الخليط أعدت باستخدام عرض dissociator الميكانيكية على حد سواء زيادة سلامة وخلية الانتعاش مع الحفاظ على مستقبلات سطح التعبير مقارنة الأنسجة هضمها انزيم ل. وعلاوة على ذلك، لم الهضم الأنزيمي من المواد غير القابلة للذوبان المتبقية لا يتعافى CD45 + خلايا إضافية تشير إلى أن القياسات الكمية والنوعية في جناسة الأساسي من المرجح أن تعكس بصدق تسلل مجموعات سكانية فرعية في fragm الأنسجةوالأنف والحنجرة. الخلايا اللمفاوية في هذه الخليط يمكن أن يكون بسهولة تتميز باستخدام المناعية (النمط الظاهري، والانتشار، وغيرها) أو الجزيئية (DNA، RNA و / أو البروتين) النهج. ويمكن أيضا خلايا CD45 + أن تستخدم لتنقية حيوانية، والتوسع في المختبر أو الحفظ بالتبريد. فائدة إضافية لهذا النهج هو أن طاف النسيج الأساسي من الخليط يمكن استخدامها لتوصيف ومقارنة السيتوكينات، كيموكينات، والمناعية المستضدات الموجودة في الأنسجة الطبيعية والخبيثة. وينبغي لهذه الوظائف البروتوكول بشكل جيد للغاية لأنسجة الثدي الإنسان وتكون قابلة للتطبيق لمجموعة واسعة من أنسجة طبيعية وغير طبيعية.
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
توضح هذه الدراسة طريقة الأمثل لإعداد السريع من الخليط أنسجة الثدي العادية والخبيثة دون الهضم الأنزيمي للفرز لاحق الخلية، والاستخراج، والحفظ بالتبريد و / أو تحليل المظهري من CD45 + القطعان. والهدف من هذا النهج التجريبي لإنتاج صور للسمسم التي تعكس بشكل وثيق دولتهم…
Disclosures
The authors have nothing to disclose.
Acknowledgements
كان مدعوما من المنح هذا العمل fromthe صندوق البلجيكي للبحوث العلمية (FNRS)، LES AMIS DE L'معهد بورديه، FNRS في العملية Télévie، سرطان خطة بلجيكا، فون مبو]-Marteaux، فون JC Heuson وفون برسي.
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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