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.
Förmågan hos maligna celler att undvika immunsystemet, som kännetecknas av tumör flykt från både medfödda och adaptiva immunsvar, är nu accepterat som en viktig kännetecken för cancer. Vår forskning om bröstcancer fokuserar på den aktiva roll som tumörinfiltrerande lymfocyter spelar i tumörprogression och patient utfall. Mot detta mål har vi utvecklat en metod för snabb isolering av intakta lymfoida celler från normala och onormala vävnader i ett försök att utvärdera dem intill deras naturliga tillstånd. Homogen framställda med hjälp av en mekanisk dissociator show både ökad lönsamhet och cell återhämtning samtidigt som ytreceptor uttryck jämfört med enzym-spjälkade vävnader. Dessutom har enzymatisk nedbrytning av det återstående olösliga materialet inte återskapa ytterligare CD45 + celler indikerar att kvantitativa och kvalitativa mätningar i primär homogenatet sannolikt verkligen speglar infiltrerande subpopulationer i vävnaden fragment. De lymfoida celler i dessa homogenat kan lätt karakteriseras med användning av immunologiska (fenotyp, proliferation, etc.) eller molekylär (DNA, RNA och / eller protein) närmar. CD45 + celler kan också användas för subpopulationen rening in vitro expansion eller kryokonservering. En ytterligare fördel med detta tillvägagångssätt är att den primära vävnads supernatanten från homogenaten kan användas för att karakterisera och jämföra cytokiner, kemokiner, immunoglobuliner och antigener närvarande i normala och maligna vävnader. Detta protokoll fungerar extremt väl för mänskliga bröstvävnad och bör vara tillämplig på ett brett utbud av normala och onormala vävnader.
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.
Denna studie beskriver en optimerad metod för snabb beredning av normala och maligna bröstvävnadshomogen utan enzymatisk nedbrytning för efterföljande cellsortering, utvinning, frysförvaring och / eller fenotypisk analys av CD45 + subpopulationer. Målet med denna experimentella tillvägagångssätt är att producera bilder av TIL som nära återspeglar deras in vivo tillstånd och jämföra dem med normal vävnad med minimal manipulation av vävnader färska från operationssalen. Hittills har…
The authors have nothing to disclose.
Detta arbete har finansierats med bidrag fromthe belgiska fond för vetenskaplig forskning (FNRS), Les Amis de l'Institut Bordet, FNRS-Opération Télévie, Plan Cancer i Belgien, Fonds Lambeau-Marteaux, Fonds JC Heuson och 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 |