Dec 19, 2022

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

DOI
https://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: https://dx.doi.org/10.17504/protocols.io.eq2ly768plx9/v1
Protocol Citation: Marta Bueno, Lanping Guo, koenigshoffm , 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
Keywords: single cell suspension, human lung tissue dissociation protocol, cell suspension, upitt tristate sennet tmc, cell suspension with great cell number, human lung, high cell viability, lung, cell, incubation, sennet, tissue
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
DMEM high glucose HEPESThermo ScientificCatalog #12-430-062
RPMI-1640Thermo Fisher ScientificCatalog #22400089
FBSInvitrogen - Thermo Fisher
1X PBS (Phosphate-buffered saline )
Red Blood Cell Lysis Buffer (Roche)Merck MilliporeSigma (Sigma-Aldrich)Catalog #11814389001
Liberase TLRocheCatalog #05 401 020 001
Elastase (3U/mg) 100mgWorthington Biochemical CorporationCatalog #LS002292
Collagenase IV (160U/mg) 1gWorthington Biochemical CorporationCatalog #LS004188
DNase I from bovine pancreas (Roche)Merck MilliporeSigma (Sigma-Aldrich)Catalog #11284932001
Antibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
Before you start
Stock solutions
Can be prepared beforehand & stored -20 °C :

Antibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062 :
Aliquot in 5 mL in sterile conditions.
Store at -20 °C

DNase I from bovine pancreas (Roche)Merck MilliporeSigma (Sigma-Aldrich)Catalog #11284932001 0 mg :
Stock of 100 mg in 10 mL of sterile ddH2O
Aliquot in 300 µL in sterile conditions (3 mg per aliquot)
Store at -20 °C

Elastase (3U/mg) 100mgWorthington Biochemical CorporationCatalog #LS002292 :
Stock of 100 mg in 1 mL of sterile ddH2O
The enzyme should be 0.22 microns filtered after reconstitution and prior to use
Aliquot in 500 µL in sterile conditions (150U per aliquot)
Store at -20 °C

Collagenase IV (160U/mg) 1gWorthington Biochemical CorporationCatalog #LS004188 :
Stock of 1 g in 10 mL of sterile ddH2O
Aliquot in 500 µL in sterile conditions (8000U per aliquot)
Store at -20 °C

Liberase TLRocheCatalog #05 401 020 001 :
Stock of 5 mg in 2 mL of sterile ddH2O
Aliquot in 500 µL in sterile conditions (1.25mg per aliquot)
Store at -20 °C

Same-day preparation:
  • DMEM high glucose HEPESThermo ScientificCatalog #12-430-062
  • RPMI-1640Thermo Fisher ScientificCatalog #22400089
  • FBSInvitrogen - Thermo Fisher
  • 1X PBS (Phosphate-buffered saline )
  • Red Blood Cell Lysis Buffer (Roche)Merck MilliporeSigma (Sigma-Aldrich)Catalog #11814389001


Base media
500 mL DMEM + 5 mL anti/anti (100x)

Digestion media (10 mL per digestion -> scale accordingly)

I. Modified base media with Final DNAseI 100 µL per 10 mL (final 0.1mg/ml)

II. Then add corresponding proteases:
II.a. Elastase / Collagenase (EC tubes)
Final elastase 250 µL per 10 mL (final 7.5U/ml) +
Final collagenase IV 250 µL per 10 mL (final 400U/ml)

II.b Liberase TL (TL tubes)
Final Liberase TL 1 mL per 10 mL (final 0.25mg/ml)

Tissue dissociation
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 with10 mL added digestion media. Use one tube per piece of tissue.

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

30m
Add 5 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 5 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 5 mL of plain RPMI.

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

10m
Remove and discard the supernatant.
Resuspend the cell pellets with 10 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.
300 x g, 15°C, 00:10:00

10m
Remove supernatant and resuspend the cell pellets with 5 mL of Red Blood Cell Lysis Buffer. Mix tubes by inversion and incubate for 00:01:00 at Room temperature .
(Note: It can be done by one after another)
1m
Fill up the tubes with 9 mL of plain RPMI.

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

10m
Remove and discard supernatant. Resuspend cell pellets with 10 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.
300 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