Summary

Un protocolo simple y rápida para no enzimáticamente disociarse Tejidos Humanos frescas para el Análisis de los linfocitos infiltrantes

Published: December 06, 2014
doi:

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

La capacidad de las células malignas para evadir el sistema inmune, caracterizado por el escape del tumor de ambos respuestas inmunitarias innatas y adaptativas, se acepta ahora como un sello importante de cáncer. Nuestra investigación sobre el cáncer de mama se centra en el papel activo que desempeñan linfocitos infiltrantes de tumores en la progresión tumoral y la evolución del paciente. Hacia este objetivo, hemos desarrollado una metodología para el aislamiento rápido de células linfoides intactos de tejidos normales y anormales en un esfuerzo para evaluar los próximo a su estado nativo. Los homogeneizados preparados usando un espectáculo disociador mecánica tanto una mayor recuperación de la viabilidad celular y preservando al mismo tiempo la expresión receptor de superficie en comparación tejidos digeridos con enzima para. Por otra parte, la digestión enzimática del material insoluble restante no se recuperó células CD45 + adicionales que indican que las mediciones cuantitativas y cualitativas en el homogeneizado primaria probablemente reflejen realmente la infiltración de las subpoblaciones en la Fragm tejidoent. Las células linfoides en estos homogeneizados pueden ser fácilmente caracterizado usando inmunológica (fenotipo, la proliferación, etc.) o molecular (ADN, ARN y / o proteína) enfoques. Células CD45 + también pueden ser utilizados para la purificación subpoblación, la expansión in vitro o crioconservación. Un beneficio adicional de este enfoque es que el sobrenadante tejido primario a partir de los homogeneizados se puede utilizar para caracterizar y comparar citocinas, quimiocinas, inmunoglobulinas y antígenos presentes en tejidos normales y malignos. Este funciones de protocolo extremadamente bien para los tejidos de mama humanos y deberían ser aplicables a una amplia variedad de tejidos normales y anormales.

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

NOTA: Todos los especímenes fueron adquiridos utilizando un protocolo aprobado por el Comité de Ética Médica del Instituto Jules Bordet con el consentimiento informado por escrito obtenido de cada paciente. 1. Preparación del homogeneizado de tejido Diseccionar tejidos resecados (tejido maligno y normal resecado de la sala de operaciones) están en el laboratorio de patología por personal capacitado para su recogida inmediata. Tumor, NANT (tomada la distancia más alejada de…

Representative Results

La digestión enzimática de fragmentos de tejido, ya sea con soluciones disponibles en el mercado de disociación de tejido o diversas mezclas de laboratorio de colagenasa, DNasa y / o inhibidores de hialuronidasa, escinden una amplia variedad de receptores en la superficie de las células. Nuestros estudios, se centró inicialmente en las células T CD4 + que infiltran los tumores de mama, se presentaron rápidamente con un problema técnico importante debido a la escisión de los receptores de CD4 de la su…

Discussion

Este estudio describe un método optimizado para la rápida preparación de los homogeneizados de tejidos normales y malignos de mama sin digestión enzimática para la clasificación posterior de células, la extracción, la criopreservación y / o análisis fenotípico de CD45 + subpoblaciones. El objetivo de este enfoque experimental es para producir imágenes de la TIL que refleja de cerca su estado in vivo y compararlos con los tejidos normales con mínima manipulación de los tejidos frescos de…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Este trabajo fue apoyado por becas fromthe Fondo Belga para la Investigación Científica (FNRS), Les Amis de l'Institut Bordet, FNRS-Opération Télévie, Cáncer del Plan de Bélgica, Fonds Lambeau-Marteaux, Fonds JC Heuson y 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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Cite This Article
Garaud, S., Gu-Trantien, C., Lodewyckx, J., Boisson, A., De Silva, P., Buisseret, L., Migliori, E., Libin, M., Naveaux, C., Duvillier, H., Willard-Gallo, K. A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes. J. Vis. Exp. (94), e52392, doi:10.3791/52392 (2014).

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