Dec 19, 2022

Public workspaceUPitt TriState SenNet TMC Human lung single cell suspension (test run)

DOI
dx.doi.org/10.17504/protocols.io.eq2ly768plx9/v1
  • 1Division of Pulmonary and Critical Care Medicine. Department of Medicine. School of Medicine
  • TriState SenNet
  • Cellular Senescence Network (SenNet) Method Development Community
Marta Bueno
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly768plx9/v1
Protocol Citation: Marta Bueno, Lanping Guo, Melanie Königshoff, Oliver Eickelberg 2022. UPitt TriState SenNet TMC Human lung single cell suspension (test run). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly768plx9/v1
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: December 14, 2022
Last Modified: December 19, 2022
Protocol Integer ID: 73971
Funders Acknowledgements:
TriState SenNET (Lung and Heart) Tissue Map and Atlas consortium – NIA
Grant ID: U54AG075931
Abstract
Human lung tissue dissociation protocol to produce single cell suspension that can be used for different purposes. Optimal incubation times to obtain cell suspension with great cell numbers and high cell viability from non-disease lungs.
Protocol materials
ReagentDMEM high glucose HEPESThermo ScientificCatalog #12-430-062
ReagentRPMI-1640Thermo Fisher ScientificCatalog #22400089
ReagentFBSInvitrogen - Thermo Fisher
Reagent1X PBS (Phosphate-buffered saline )
ReagentRed Blood Cell Lysis Buffer (Roche)Merck MilliporeSigma (Sigma-Aldrich)Catalog #11814389001
ReagentLiberase TLRocheCatalog #05 401 020 001
ReagentElastase (3U/mg) 100mgWorthington Biochemical CorporationCatalog #LS002292
ReagentCollagenase IV (160U/mg) 1gWorthington Biochemical CorporationCatalog #LS004188
ReagentDNase I from bovine pancreas (Roche)Merck MilliporeSigma (Sigma-Aldrich)Catalog #11284932001
ReagentAntibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
Before you start
Before you start
Stock solutions
Can be prepared beforehand & stored Temperature-20 °C :

ReagentAntibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062 :
Aliquot in Amount5 mL in sterile conditions.
Store at Temperature-20 °C

ReagentDNase I from bovine pancreas (Roche)Sigma AldrichCatalog #11284932001 Amount0 mg :
Stock of Amount100 mg in Amount10 mL of sterile ddH2O
Aliquot in Amount300 µL in sterile conditions (Amount3 mg per aliquot)
Store at Temperature-20 °C

ReagentElastase (3U/mg) 100mgWorthington Biochemical CorporationCatalog #LS002292 :
Stock of Amount100 mg in Amount1 mL of sterile ddH2O
The enzyme should be 0.22 microns filtered after reconstitution and prior to use
Aliquot in Amount500 µL in sterile conditions (150U per aliquot)
Store at Temperature-20 °C

ReagentCollagenase IV (160U/mg) 1gWorthington Biochemical CorporationCatalog #LS004188 :
Stock of Amount1 g in Amount10 mL of sterile ddH2O
Aliquot in Amount500 µL in sterile conditions (8000U per aliquot)
Store at Temperature-20 °C

ReagentLiberase TLRocheCatalog #05 401 020 001 :
Stock of Amount5 mg in Amount2 mL of sterile ddH2O
Aliquot in Amount500 µL in sterile conditions (1.25mg per aliquot)
Store at Temperature-20 °C

Same-day preparation:
  • ReagentDMEM high glucose HEPESThermo ScientificCatalog #12-430-062
  • ReagentRPMI-1640Thermo Fisher ScientificCatalog #22400089
  • ReagentFBSInvitrogen - Thermo Fisher
  • Reagent1X PBS (Phosphate-buffered saline )
  • ReagentRed Blood Cell Lysis Buffer (Roche)Sigma AldrichCatalog #11814389001


Base media
Amount500 mL DMEM + Amount5 mL anti/anti (100x)

Digestion media (Amount10 mL per digestion -> scale accordingly)

I. Modified base media with Final DNAseI Amount100 µL per Amount10 mL (final 0.1mg/ml)

II. Then add corresponding proteases:
II.a. Elastase / Collagenase (EC tubes)
Final elastase Amount250 µL per Amount10 mL (final 7.5U/ml) +
Final collagenase IV Amount250 µL per Amount10 mL (final 400U/ml)

II.b Liberase TL (TL tubes)
Final Liberase TL Amount1 mL per Amount10 mL (final 0.25mg/ml)

Tissue dissociation
Tissue dissociation
2h
2h
Cut the lung tissue with a scalpel into approximately 3 cm3 pieces (size of a thumb) upon receipt.
Place lung tissue into a 100mm petri dish. Place a ruler/control object next to it and take pictures for archives.
Transfer them to the lid of a fresh 100mm petri dish.
Each piece should be minced with scissors.
Locations: A-> Pleura; B-> bronchovascular bundle; C-> Parenchyma
(Label tubes accordingly)

AB
EC-ATL-A
EC-BTL-B
EC-CTL-C
Table 1. Samples generated.

Transfer to a 50 ml conical Falcon tube withAmount10 mL added digestion media. Use one tube per piece of tissue.

Repeat the procedure with the remaining pieces.
Incubate 30min at 37C, shaking 500rpm.
Shaker500 rpm, 37°C, 00:30:00

30m
Add Amount5 mL of FBS to the cell suspension to stop the digestion (final volume ~15ml)

Filter through 100 μm cell strainer using the syringe plunger to press and smash the tissue, washing the tube and rinse the strainer with Amount5 mL of plain RPMI.

Filter through 70 μm cell strainer using the syringe plunger to press and smash the tissue, washing tube and rinse the strainer with Amount5 mL of plain RPMI.

Centrifuge. 300g, 15°C for 10 minutes.
Centrifigation300 x g, 15°C, 00:10:00

10m
Remove and discard the supernatant.
Resuspend the cell pellets with Amount10 mL of plain RPMI and transfer to a fresh 15 ml Falcon conical tubes.

Count cells here. (This count can be skipped -not informative because of RBC numbers)
To count: Take a 20 µL aliquot to determine total cell number, and mix with 20μl of VitaStain. Run AO/PI program in Nexcelom cell counter.
Centrifuge. 300g, 15°C for 10 minutes.
Centrifigation300 x g, 15°C, 00:10:00

10m
Remove supernatant and resuspend the cell pellets with Amount5 mL of Red Blood Cell Lysis Buffer. Mix tubes by inversion and incubate for Duration00:01:00 at TemperatureRoom temperature .
(Note: It can be done by one after another)
1m
Fill up the tubes with Amount9 mL of plain RPMI.

Centrifuge. 300g, 15°C for 10 minutes.
Centrifigation300 x g, 15°C, 00:10:00

10m
Remove and discard supernatant. Resuspend cell pellets with Amount10 mL of RPMI buffer kept on ice at all times.

Count cells here: Take a 20 µL aliquot to determine total cell number, and mix with 20μl of VitaStain. Run AO/PI program in Nexcelom cell counter.
Centrifuge. 300g, 15°C for 10 minutes.
Centrifigation300 x g, 15°C, 00:10:00

10m
Resuspend in the corresponding media for the next step
For scRNA-seq:
  • Resuspend in 500μl of PBS+2%FBS in the 15ml tube and then take 200μl of that suspension to a new Eppendorf tube.
  • Pellet the cells by a centrifugation pulse, remove the supernatant and then resuspend to prepare ~4Million cells in 200μl of PBS+2%FBS (minimum 2M in 100μl)
For storage:
  • Resuspend in 500μl of PBS
  • Pellet the cells by a centrifugation pulse, remove the supernatant and stored at -80C