En enkel og hurtig protokol til ikke-enzymatisk dissociere frisk humant væv til analyse af lymfocytter
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
Evnen af maligne celler til at unddrage sig immunsystemet, kendetegnet ved, tumorundvigelse fra både medfødte og adaptive immunrespons, er nu accepteret som et vigtigt kendetegn for cancer. Vores forskning i brystkræft fokuserer på den aktive rolle, tumorinfiltrerende lymfocytter spille i tumor progression og patient resultat. Mod dette mål, udviklede vi en metode til hurtig isolering af intakte lymfoide celler fra normale og unormale væv i et forsøg på at vurdere dem nærmest til deres oprindelige tilstand. Homogenater fremstillet ved hjælp af en mekanisk dissociator show både øget rentabilitet og celle opsving mens overfladen receptorekspression forhold til enzym-fordøjede væv bevares. Desuden har enzymatisk nedbrydning af det resterende uopløselige materiale ikke gendanne yderligere CD45 + celler indikerer, at kvantitative og kvalitative målinger i det primære homogenat sandsynligvis reelt afspejler infiltrerende delpopulationer i vævet Fragment. De lymfoide celler i disse homogenater kan let karakteriseres ved immunologiske (fænotype, proliferation, etc.) eller molekylær (DNA, RNA og / eller protein) nærmer sig. CD45 + celler kan også anvendes til subpopulation oprensning in vitro ekspansion eller kryopræservering. En yderligere fordel ved denne fremgangsmåde er, at den primære væv supernatanten fra homogenaterne kan anvendes til at karakterisere og sammenligne cytokiner, kemokiner, immunoglobuliner og antigener til stede i normale og maligne væv. Denne protokol fungerer særdeles godt for menneskerettighederne brystvæv og bør gælde for en lang række af normale og unormale væv.
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
Denne undersøgelse beskriver en optimeret fremgangsmåde til hurtig fremstilling af normale og maligne bryst vævshomogenater uden enzymatisk fordøjelse til efterfølgende cellesortering, ekstraktion, kryopræservering og / eller fænotypisk analyse af CD45 + subpopulationer. Målet med denne eksperimentelle fremgangsmåde er at producere billeder af TIL, der nøje afspejler deres in vivo tilstand og sammenligne dem med normale væv med minimal manipulation af væv frisk fra operationsstuen. Hidtil…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af tilskud fromthe belgiske fond for videnskabelig forskning (FNRS), Les Amis de l'Institut Bordet, FNRS bejde Télévie, Plan Cancer Belgien, Fonds Lambeau-Marteaux, Fonds JC Heuson og Fonds 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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