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.
Muligheten av ondartede celler for å unngå immunforsvaret, preget av svulst flukt fra både medfødte og ervervede immunresponser, er nå akseptert som et viktig kjennetegn på kreft. Vår forskning på brystkreft fokuserer på den aktive rollen som tumor infiltrerer lymfocytter spiller i tumorprogresjon og pasient utfall. Mot dette målet, har vi utviklet en metodikk for rask isolering av intakte lymfoide celler fra normale og unormale vev i et forsøk på å vurdere dem andreplassen til sin opprinnelige tilstand. Homogenates utarbeidet ved hjelp av en mekanisk dissociator showet både økt levedyktighet og celle utvinning samtidig bevare overflaten reseptor uttrykk sammenlignet med enzym-fordøyd vev. Videre gjorde enzymatisk fordøyelse av de resterende uløselige materialet ikke gjenopprette flere CD45 + celler som indikerer at kvantitative og kvalitative målinger i primær homogenate sannsynlig genuint reflektere infiltrere subpopulasjoner i vevet fragment. Den lymfoide celler i disse homogenater lett kan karakteriseres ved hjelp av immunologiske (fenotype, proliferasjon, etc.) eller molekyl (DNA, RNA og / eller protein) nærmer seg. CD45 + celler kan også brukes til rensing subpopulasjon, in vitro ekspansjon eller nedfrysing. En ytterligere fordel ved denne tilnærmingen er at den primære vev supernatanten fra homogenater kan brukes til å karakterisere og sammenligne cytokiner, kjemokiner, immunoglobuliner og antigener til stede i normale og maligne vev. Denne protokollen fungerer svært godt for menneske brystvev og bør være knyttet til et bredt utvalg av normale og unormale vev.
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.
Denne studien beskriver en optimalisert metode for rask tilberedning av normale og maligne bryst vevshomogenater uten enzymatisk fordøyelse for påfølgende celle sortering, utvinning, nedfrysing og / eller fenotypiske analyse av CD45 + subpopulasjoner. Målet med denne eksperimentelle tilnærming er å produsere bilder av TIL som tett reflekterer deres in vivo staten og sammenligne dem med normalt vev med minimal manipulering av vev frisk fra operasjonsstuen. Til dags dato har vårt laboratorium br…
The authors have nothing to disclose.
Dette arbeidet ble støttet med tilskudd Bergem belgiske Fund for Scientific Research (FNRS), Les Amis de l'Institut Bordet, FNRS-drift Télévie, Plan Kreft i Belgia, Fonds Lambeau-marteaux, Fonds JC Heuson og Fonds Barsy.
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 |