Jul 07, 2020
Human Kidney Tumor Dissociation for single-cell genomics
- Evrard Bertrand1,
- Saout Judikael1,
- Lardenois Aurelie1,
- Kammerer-Jacquet Solène-Florence1,
- Rioux-Leclercq Nathalie1,
- Chalmel Frederic1
- 1Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S 1085, F-35000 Rennes, France.
Protocol Citation: Evrard Bertrand, Saout Judikael, Lardenois Aurelie, Kammerer-Jacquet Solène-Florence, Rioux-Leclercq Nathalie, Chalmel Frederic 2020. Human Kidney Tumor Dissociation for single-cell genomics. protocols.io https://dx.doi.org/10.17504/protocols.io.2n2gdge
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 07, 2019
Last Modified: July 07, 2020
Protocol Integer ID: 22970
Keywords: Clear cell renal cell carcinoma; single-cell genomics; tissue dissociation; cell viability,
Abstract
This protocol was developed to dissociate (clear cell) renal cell carcinoma tumors in order to study single cell genomics using the Chromium Single Cell Gene Expression technology (10X Genomics). Starting from fresh biological material, the protocol was optimized to ensure fast and reproducible processing, both of which are very important criteria in single-cell are analyzes. This protocol allowed us to obtain biologically relevant high-quality data. It can easily be adapted according to the following optional steps: i) multiple sampling in several macroscopic areas of the tumor; ii) depletion of CD45+ cell populations; iii) and, progressive freezing of the dissociated tumor cells for biobanking.
Materials
MATERIALS
C TubeMiltenyi BiotecCatalog #130-096-334
gentleMACS DissociatorMiltenyi BiotecCatalog #130-093-235
Tumor Dissociation Kit humanMiltenyi BiotecCatalog #130-095-929
Tumor Preparation
Tumor Preparation
Note
This step should be designed according to the biological question. As this protocol was developed to catch the tumor heterogeneity, each tumor was sampled in multiple areas.
Material
| Material | Supplier Info | |
| Petri Dish, 100 mm | Corning (430591) | |
| Biopsy Punch 8mm | Dutscher (030740) | |
| Ice |
Transport the fresh tumor resection on ice and collect evenly distributed punch biopsies in multiple macroscopic areas.
Note
- Avoid all fibrous and necrotic areas. If these areas are of interest, please consider using Fluorescence Activated Cell Sorting (FACS) after dissociation to sort viable cells (not tested).
- Up to 9 biopsies per tumor were routinely collected (in the case of radical nephrectomies).
Place each biopsy onto 100mm Petri Dish, on ice
Note
Depending on the amount of tissue, put aside parts of each biopsy for further complementary analyses.
- Number and identify each punch biopsy as well as its following match samples.
- Cut each biopsy in three parts (¼, ¼, ½) consisting of approximately ¼ for formaldehyde treatment and immunohistochemistry staining; ¼ for snap freezing, -80°C storage and further DNA/RNA extraction; and ½ for tissue dissociation of tumor punches.
Tumor Dissociation
Tumor Dissociation
Note
This core step consists of a combined mechanical and enzymatic disaggregation of the tumor tissue. It requires GentleMACS Dissociator devices.
Material
| Material | Supplier Info | |
| Tumor Dissociation Kit, Human | Miltenyi Biotec (130-095-929) | |
| Gentle MACS Octo Dissociator | Miltenyi Biotec (130-096-427) | |
| GentleMACS C tubes | Miltenyi Biotec (130-096-334) | |
| MACS SmartStrainers 70 µm | Miltenyi Biotec (130-098-462) | |
| RPMI 1640 | Thermofisher (11835030) | |
| Surgical Blade | Dutscher (132522) | |
| Forceps | Dutscher (711200) | |
| 50ml Falcon Tubes / 15ml Falcon Tubes | Dutsher (352098 / 352097) | |
| 1.5 ml Eppendorf tube | Eppendorf (0030 108.035) | |
| Trypan Blue | SIGMA Aldrich (T8154) | |
| Malassez Chamber | Dutscher (140501) | |
| Petri Dish, 100 mm | Corning (430591) |
In the petri dish, pool the punch biopsies dedicated to further dissociation.
Note
Do not exceed 1g of total tumor tissue.
Prepare Digest Mix ON ICE by adding into a gentleMACS C Tube 4.7ml RPMI 1640, 200µl Enzyme H, 100µl Enzyme R and 25µl Enzyme A.
In the petri dish, pour 500ml Digest Mix onto the punch biopsies.
Gently mince the punch biopsies into small pieces of about 2 mm3 using a scalpel.
Using forceps, gently transfer tissue fragments into the gentle MACS C tube containing the Digest Mix (be careful not to crush the fragments). Add the remaining digest mix solution from the petri dish and tightly close C Tube.
Run the gentle MACS Octo Dissociator Program “37_h_TDK_1”RUN 01:00:00
Note
In case you own the gentleMACSTMDissociator, please successively run programs h_tumor_01, h_tumuor_02 and h_tumor_03 with 2x 30 minutes of incubation at 37°C under continuous rotation between programs 01-02 and programs 02-03.
Humidify the MACS SmartStrainer 70 µm with 5ml RPMI 1640 above a 50ml Falcon Tube.
Apply cell suspension through the strainer.
Rinse the remaining cells in the MACS C tube and reapply it through the strainer.
Wash the strainer and complete the suspension to a final volume of 40ml RPMI 1640.
Centrifuge cell suspension 300 x g 00:05:00
Red Blood Cell (RBC) Lysis Protocol
Red Blood Cell (RBC) Lysis Protocol
Note
In this step, red blood cells are lysed and washed away as they represent irrelevant cells for the study.
| Material | Supplier Info | |
| RBC Buffer (10x) | BioLegend (420301) | |
| Desionized water | Invitrogen (10977035) | |
| DPBS | Thermofisher (14190250) |
Take advantage of the centrifugation duration to prepare 1X RBC Lysis Buffer on ice: dilute 1ml 10X RBC Lysis Buffer to 1X working concentration with 9ml deionized water.
Discard the supernatant, gentlgy resuspend the cell pellet in 1ml RBC Lysis Buffer and quickly transfer it into a new 50ml Falcon.
Add the remaining RBC Lysis Buffer.
Incubate on Ice for 4 min with occasional shaking.
Stop the reaction by diluting the active RBC Lysis Buffer with 30ml 1X PBS.
Centrifuge cell suspension 300 x g 05:00:00
Discard the supernatant and resuspend the pellet in 1ml RPMI 1640
Note
Optional:
- Take 20µl from cell suspension for counting and microscopic quality control of the dissociation using trypan blue in a Malassez Chamber.
- 0.3 million cells were used for Flow Cytometry Assays to measure viability with 7‐amino‐actinomycin (7AAD), as well as CD45 (immune) and CD34 (endothelial) positive cells..
Low Centrifugation
Low Centrifugation
Note
This step aims at clearing the suspension from debris and dead cells while preserving tumor cells. When working on cancerous tissues, we recommend not using Annexin V-based dead cell removal kits as they also remove altered but yet viable cells and presumed pre-apoptotic tumor cells that often represent relevant populations in the framework of single-cell studies. Successive low centrifugations were chosen instead as they allow to recover a higher proportion of tumor cells. This solution was also preferred over FACS sorting because of its significant time savings.
| Material | Supplier Info | |
| 15ml Falcon Tubes | Dutsher (352097) | |
| 1.5 ml Eppendorf tube | Eppendorf (0030 108.035) | |
| RPMI 1640 | Thermofisher (11835030) |
Transfer the cells in a 15ml Falcon tube and complete the suspension to a final volume of 12ml RPMI 1640.
Centrifuge cell suspension at 800g 150 x g 00:06:00
Discard the supernatant and resuspend the pellet in 1 ml of RPMI 1640.
Transfer cells in a new 15ml Falcon Tube and repeat the low centrifugation step.
CD45 Positive Cells Depletion
CD45 Positive Cells Depletion
Note
- This step is optional and depends on the targeted cell population. It aims at enriching the cell suspension with tumor and stromal cells by depleting the population of leucocytes, mainly consisting of circulating lymphocytes, which may represent the vast majority of the cells after dissociation.
- The following volumes were adapted to a maximum population of 107 cells. In case you recover too much cells, please adapt the volumes according to the following ratios.
| Material | Supplier Info | |
| CD45(TIL) MicroBeads | Miltenyi Biotec (130-118-780) | |
| EDTA 0.5 M | Sigma Aldrich (03690) | |
| BSA | Sigma Aldrich (A7906) | |
| DPBS | Thermofisher (14190250) | |
| RPMI 1640 | Thermofisher (11835030) | |
| LS MACS Columns | Miltenyi Biotec (130-042-401) | |
| MACS Separator | Miltenyi Biotec (130-090-976) |
Prepare 10ml MACS Buffer solution containing PBS, 0.5% BSA and 2 mM EDTA.
Resuspend cell pellet (up to 107 cells) in 80 μl of MACS Buffer, and mix well by gently pipetting 10 to 15 times.
Add 20 μl CD45 micro beads, mix and incubate in fridge 4 °C 00:15:00
Add 400 µl of MACS Buffer, mix by pipetting
Place LS column in QuadroMACS Magnetic Cell Separator and prepare it by rinsing with 3ml MACS Buffer (discard effluent).
Apply cell suspension onto the column and immediately collect flow-through containing unlabeled cells in 1.5ml Falcon tube.
Rinse the column twice with 1ml of MACS Buffer, collect and pool the effluent.
Centrifuge cell suspension 300 x g 00:05:00
Discard the supernatant and resuspend the pellet in 1.5ml tubes with 250µl RPMI 1640.
Note
Optionnal:
- take 20µl from cell suspension for counting and microscopic quality control do the dissociation using trypan blue in a Malassez Chamber.
- 0.25 million cells were used for Flow Cytometry Assays to measure viability with 7‐amino‐actinomycin (7AAD), as well as CD45 (immune) and CD34 (endothelial) positive cells.
Expected Result
Expected Result
Cell dissociation control by Blue Trypan staining and flow cytometry. After GentleMacs dissociation (A, D), after Low Centrifugation (B, E) and after CD45+ depletion (C,F). CD34 expression reveals endothelial cells and CD45 expression reveals immune cells. Red squares indicate the proportions of CD45-/CD34- cells containing tumor cells and part of stromal cells.
Application
Application
Single Cell experiment
Note
If cell viability is higher than 70%, cells can then be processed for scRNA-seq on 10X genomics platform. Gently resuspend the cells in appropriate volume of PBS, 0.04% BSA and proceed to 10X protocol.
Freezing samples and biobanking
Note
- This step aims at storing the tumor cells after dissociation in order to allow their delayed study. This can be useful for the study of asynchronous samples such as matched primary and metastatic tumors.
- We tested this approach and were able to obtain high quality and comparable data in single-cell RNA-sequencing of fresh versus frozen samples (data not shown).
- Transfer enough cells (0.1 to 1 Million) in 1ml freezing solution consisting of 700µl RPMI 1640, 200µl Fetal Bovine Serum and 100µl DMSO). Immediately place the cells at -20°C for one hour before gradually freezing and storing them at -80°C overnight (~12h). Then, transfer cells to liquid nitrogen for long-term storage.