פרוטוקול פשוט ומהיר לללא אנזימי רקמות אנושיות טריות לנתק לניתוח להחדרה לימפוציטים
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
היכולת של תאים ממאירים ללהתחמק ממערכת החיסון, המתאפיינת בבריחת גידול משני תגובות חיסון מולדים ובעלי כושר הסתגלות, מקובל כיום כסימן היכר חשוב של הסרטן. המחקר שלנו על סרטן השד מתמקד בתפקיד הפעיל שלימפוציטים גידול הסתננות לשחק בהתקדמות גידול ותוצאת מטופל. לקראת מטרה זו, פיתחנו מתודולוגיה לבידוד המהיר של תאי הלימפה שלמים מרקמות נורמליות ולא נורמליות במאמץ כדי להעריך אותם בסמוך למדינה האם שלהם. Homogenates הוכנה באמצעות תכנית Dissociator מכאנית שתי התאוששות כדאיות ותא גדלה תוך שמירת רקמות לעומת ביטוי קולט משטח לאנזים-מעוכלות. יתר על כן, עיכול אנזימטי של החומר מסיס שנותר לא התאושש CD45 תאים + נוספים מצביעים על כך שמדידות כמותיות ואיכותיות בhomogenate העיקרי העשויים באמת משקפות תת-אוכלוסיות שחדרו בfragm הרקמהאף אוזן גרון. תאי הלימפה בhomogenates אלה יכולים להיות בקלות מאופיינת באמצעות חיסוני (פנוטיפ, הפצה, וכו ') או מולקולרי (DNA, RNA ו / או חלבון) מתקרב. גם תאי CD45 + יכולים לשמש לטיהור תת-אוכלוסייה, התרחבות במבחנה או הקפאה. יתרון נוסף של גישה זו הוא שsupernatant הרקמה העיקרי מhomogenates יכול לשמש כדי לאפיין ולהשוות ציטוקינים, כמוקינים, נוגדנים ואנטיגנים הנמצאים ברקמות נורמליות וממאירים. פונקציות זה פרוטוקול טוב מאוד לרקמות שד אנושיות וצריכה להיות ישימות למגוון רחב של רקמות נורמליות ולא נורמליות.
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
מחקר זה מתאר שיטה מותאמת להכנה המהירה של homogenates רקמת שד הנורמלית וממאירים ללא עיכול אנזימטי למיון לאחר תא, חילוץ, הקפאה ו / או ניתוח פנוטיפי של CD45 + תת-אוכלוסיות. המטרה של הגישה הניסויית הזה היא לייצר תמונות של TIL המשקף באופן הדוק את מדינתם in vivo ולהשוות אותם לר…
Disclosures
The authors have nothing to disclose.
Acknowledgements
עבודה זו נתמכה על ידי מענקי fromthe קרן בלגית למחקר מדעי (FNRS), Les Amis de l'Institut Bordet, FNRS הפעולה Télévie, התכנית למלחמה בסרטן בבלגיה, Fonds לאמבו-Marteaux, Fonds JC Heuson וFonds 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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