Oct 21, 2022
Version 2
Adult human lung cell dissociation (on ice) V.2
- 1CCHMC
Protocol Citation: Andrew Potter 2022. Adult human lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr13zgmkn/v2Version created by Andrew Potter
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: October 20, 2022
Last Modified: October 21, 2022
Protocol Integer ID: 71589
Keywords: lung, single cell, dissociation, CAP
Abstract
This protocol was used to generate a single cell suspension from transbronchial biopsies from human lung. The procedure is carried out on ice, reducing artifact gene expression changes. Cell yield / mg is >5000 cells/mg with 80-90% viability (trypan blue dye exclusion). 10X scRNA-seq data is high quality (3'v3.1 and 5' v1.1) with >74% of cells retained after initial QC filtering (<20% mitochondrial gene reads, >500 genes/cell and >800 RNA molecules/cell). Expression of IER genes FOS and JUN is low in integrated, QC filtered dataset (normalized avg gene expression <3). Cell populations identified in single cell data include:
-CD8+ T
-CD4+ T
-γδ+ T
-NK
-Mast
-mDC/pDC
-B / plasma
-Monocyte
-Macrophage
-AT1 / AT2
-Ciliated
-Goblet
-Pericyte
-SMC
-Endothelial (several populations)
-Fibroblast
-Basal
If you have any questions regarding the single cell analysis thus far, please contact Andrew Potter.
Guidelines
Enzyme Mix (2 x 1 mL for 13 mg tissue made up in 1.5 mL tubes)
(If digesting more than 13 mg tissue, prepare more enzyme mix as necessary)
Coll. A/Elastase/Dispase Enzyme Mix (make two tubes, each 1 mL for 13 mg starting tissue)
60 µL Collagenase A 100 mg/mL – 6 mg/mL final (Sigma, 10103578001)
100 µL Elastase 43 u/mL - 4.3 U/mL final (Worthington, LS002292)
100 µL Dispase 90 u/mL – 9 U/mL final (Worthington, LS02100)
100 µL soybean trypsin inhibitor 1 mg/mL - 100 µg/mL final (Roche, 10109886001)
5 µL 1 M CaCl2 – 5 mM final (MLS)
5 µL DNAse - 125 U/mL final (StemCell, 07469)
630 µL DPBS (no Ca, no Mg - MLS)
+13 mg tissue per 1 mL enzyme mix
Safety warnings
Dissociation of human tissue must be performed in biosafety cabinet. Use caution when handling tissue, and pipetting as aerosols may be generated.
Please review health safety warnings for enzymes and trypan blue, handle with caution.
Before start
Just prior to starting
- Prepare enzyme mixes and leave on ice.
- Cool centrifuges to 4 °C.
Enzyme Mixtures
- Collagenase, elastase and dispase enzyme stock mixes are prepared ahead of time by diluting in PBS, and frozen in 500 µL aliquots at -80 C.
- StemCell DNAse (#07469) has an activity of 2,000 U / mg. We weigh out the powder, dilute in PBS to 125 U per 5 µL, make single-use aliquots to 20-40 µL (depending on samples run at a time), and freeze at -80 C. For 10 mg of weighed powder (20,000 U of activity total), we add 800 µL PBS, mix gently, make aliquots and freeze.
10% FBS/PBS is made up with heat-inactivated, sterile filtered FBS (MLS) with Ca/Mg-free PBS.
Equipment
- Centrifuge for 1.5 mL, 15 mL conicals (preferably swinging bucket)
- Pipettes and pipet tips.
- 15 mL conicals (MLS)
- 1.5 mL tubes (MLS)
- 30 µM filters (Miltenyi, 130-098-458)
- Petri dish (MLS)
- Razor blades (MLS)
- Ice bucket w/ice
- Hemocytometer - InCyto Neubauer Improved (DHC-N01-5)
Isolate and mince tissue
Isolate and mince tissue
Transport tissue in ice-cold PBS.
Transfer 13 mg tissue to petri dish on ice with 100 µL enzyme mix; using razor blade mince thoroughly for 2 min until large chunks are broken up finely.
00:02:00 mince on ice
Layer 1
Layer 1
Add 13 mg minced tissue to 900 µL enzyme mix (1 mL total) in 1.5 mL tube using cut p200 pipet to transfer.
13 mg minced human lung tissue
Incubate on ice. Triturate 10x using 1 mL pipet set to 700 µL every 3 min (w/tip cut). Shake 3-5X to re-suspend every 2 min.
00:03:00 triturate 10X
00:02:00 shake 3-5X
2d 2h 2m
After 30 minutes of incubation let settle on ice 1 min.
00:30:00 incubate on ice
00:01:00 settle one min
2d 2h 1m
Pipet and save 80% of the supernatant (consisting of released cells), leaving undigested tissue chunks on the bottom of the tube. Add released cells (supernatant) to sterile 30 µM filter on 15 mL conical. Rinse filter w/12 mL ice-cold 10% FBS/PBS. Tap filter to ensure passage of cells.
12 mL ice-cold 10% FBS/PBS
Remove filter from 15 mL conical. Spin the 15 mL conical with released cells 300 g for 5 min at 4 °C.
00:05:00 spin at 300 g
4 °C
5m
Remove supernatant for the 15 mL conical with pelleted supernatant. Re-suspend the pellet in 14 mL ice-cold 10% FBS/PBS and leave on ice while performing next steps.
14 mL ice-cold 10% FBS / PBS
Layer 2
Layer 2
Add additional 1 mL enzyme mix to residual tissue chunks from Layer 1.
1 mL enzyme mix
Continue incubating on ice for 40 additional minutes (1 hr. 10 min. total). Triturate 10x using 1 mL pipet set to 700 µL every 3 min (w/tip cut if necessary). Shake 3-5X to re-suspend every 2 min.
00:40:00 incubate on ice
00:03:00 triturate 10x
00:02:00 shake 3-5X
45m
After 1 hr. 10 min total incubation time triturate digest mix 10X and add total digest mix (including any chunks) to a new sterile 30 µM filter on 15 mL conical.
Rinse filter w/12 mL ice-cold 10% FBS/PBS.
12 mL ice-cold 10% FBS/PBS
Remove filter. Spin the 15 mL conicals from the first and second layer, 300 g for 5 min at 4 ºC.
00:05:00 spin 300 g
4 °C
5m
RBC Lysis
RBC Lysis
Remove supernatant for all tubes. Re-suspend combined volume in 5 mL RBC lysis buffer. Pipet 20x to mix. Incubate on ice 5 min.
5 mL RBC lysis buffer
00:05:00 incubate on ice
Add 9 mL ice-cold 10% FBS/PBS to 5 mL RBC lysis buffer. Triturate and apply to sterile 30 µM filter on 15 mL conical.
9 mL ice-cold 10% FBS/PBS
Separate flow-through to two 15 mL conicals. Bring volume for each to 14 mL with ice-cold 10% FBS/PBS.
14 mL 10% FBS/PBS
Re-suspend and prepare for scSeq
Re-suspend and prepare for scSeq
5m
5m
Spin 300 g for 5 min at 4 °C. Remove supernatant.
00:05:00 spin 300 g
4 °C
5m
Re-suspend cells in 100 µL ice-cold 10% FBS/PBS. Analyze viability and cell yield using a hemocytometer with trypan blue.
100 µL 10% FBS/PBS
Adjust cell concentration to 700-1200 cells / µL for 10x chromium.