침투 림프구의 분석을위한 비 효소 해리 신선한 인간의 조직에 대한 간단하고 빠른 프로토콜
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
선천성 및 후천성 면역 반응 모두에서 종양 탈출 특징으로 면역 시스템을 무력화하는 악성 세포의 능력은 지금 암의 중요한 특징으로 받아 들여진다. 유방암에 대한 우리의 연구는 종양 침윤 림프구가 종양의 진행과 환자 결과에서 재생하는 적극적인 역할에 초점을 맞추고있다. 이 목표를 향해, 우리는 그들의 원래 상태로 그들을 근접 평가하기위한 노력의 일환으로 정상 및 비정상 조직에서 그대로 림프 세포의 신속한 분리를위한 방법론을 개발했다. 표면 수용체 발현 비교하는 효소 – 소화 된 조직을 유지하면서 기계적 dissociator 쇼 모두 증가 된 생존율 및 세포를 회수하여 제조 균질. 또한, 나머지 불용성 물질의 소화 효소는 기본 균질의 양적, 질적 측정 가능성이 진정으로 조직 fragm에 침투 부분 집단을 반영하는 것을 나타내는 추가 CD45 + 세포를 복구하지 않았다천만에. 이러한 균질 림프 세포는 쉽게 면역 (표현형, 확산 등) 또는 분자 (DNA, RNA 및 / 또는 단백질)을 이용하여 특성화 할 수 접근한다. CD45 + 세포는 모집단 정화 시험 관내 팽창 또는 냉동 보관을 위해 사용할 수있다. 이 접근법의 또 다른 장점은 균질 물에서 뜨는 기본 조직과 정상 조직에서 악성 사이토 카인, 케모카인, 면역 글로불린 및 본 항원을 특성화하고 비교하기 위해 사용될 수 있다는 점이다. 이 프로토콜 기능을 매우 잘 인간 유방 조직 및 정상 및 비정상 다양한 조직에 적용 할 수 있어야한다.
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
이 연구는 이후의 세포 분류, 추출, 동결 보존 및 / 또는 CD45 + 개체군의 표현형 분석을위한 소화 효소없이 정상 및 악성 유방 조직 균질의 빠른 준비를위한 최적화 된 방법을 설명합니다. 이 실험적인 접근의 목표는 밀접하게 자신의 생체 상태를 반영하고 수술실에서 신선한 조직의 최소한의 조작으로 정상 조직에 비교 TIL의 이미지를 생성하는 것입니다. 지금까지, 우리의 실험실>…
Divulgations
The authors have nothing to disclose.
Acknowledgements
보조금 지원이 작품은 과학 연구 (FNRS), 레 미스 드 난 연구소 보르, FNRS 조작 Télévie, 벨기에의 계획 암, 퐁 Lambeau-Marteaux, 퐁 JC Heuson과 퐁 Barsy에 대한 벨기에 기금 폴더 만.
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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