Um protocolo simples e rápido para não enzimática Separar frescos Tecidos Humanos para a Análise de infiltrar Linfócitos
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
A capacidade das células malignas para evadir o sistema imune, caracterizado por escape do tumor a partir de ambas as respostas imunes inatas e adaptativas, é agora aceite como uma característica importante do cancro. Nossa pesquisa sobre câncer de mama centra-se no papel ativo que os linfócitos tumor infiltrando desempenhar na progressão do tumor e evolução de pacientes. Com esse objetivo, foi desenvolvida uma metodologia para o rápido isolamento de células linfóides intactas de tecidos normais e anormais em um esforço para avaliá-los próximo ao seu estado natal. Homogeneizados preparados utilizando um show Dissociator mecânica tanto maior recuperação e viabilidade celular, preservando a expressão do receptor de superfície em comparação tecidos para enzima-digerida. Além disso, a digestão enzimática do material insolúvel restante não recuperou células adicionais CD45 +, indicando que as medidas quantitativas e qualitativas no homogeneizado primária provável genuinamente refletem infiltrando as subpopulações no fragm tecidoent. As células linfóides em homogenatos estes podem ser facilmente caracterizadas utilizando imunológica (fenótipo, proliferação, etc.) ou molecular (DNA, RNA e / ou proteína) se aproxima. As células CD45 + pode também ser utilizado para a purificação subpopulação, expansão in vitro ou de criopreservação. Um benefício adicional desta abordagem é que o sobrenadante de tecido primário a partir dos homogeneizados podem ser utilizados para caracterizar e comparar as citocinas, quimiocinas e imunoglobulinas, antigénios presentes em tecidos normais e malignos. Este funções de protocolo extremamente bem para tecidos mamários humanos e deve ser aplicável a uma grande variedade de tecidos normais e anormais.
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
Este estudo descreve um método otimizado para o preparo rápido de homogeneizados normais e malignas do tecido mamário, sem digestão enzimática para classificação subseqüente celular, extração, criopreservação e / ou análise fenotípica de CD45 + subpopulações. O objetivo dessa abordagem experimental é produzir imagens da TIL que refletem de perto seu estado in vivo e compará-los aos tecidos normais com mínima manipulação dos tecidos frescos da sala de cirurgia. Até o momento, o n…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Este trabalho foi financiado por doações fromthe Fundo Belga de Pesquisa Científica (FNRS), Les Amis de l'Institut Bordet, FNRS-operação Télévie, Câncer da Bélgica Plan, Fonds Lambeau-Marteaux, Fonds JC Heuson e 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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