May 06, 2020
Single-Cell Dissociation of Human Trabecular Meshwork
- Alexi Mcadams1,2,
- Tavé van Zyl1,2
- 1Department of Ophthalmology, Harvard Medical School;
- 2Glaucoma Service, Mass. Eye and Ear, Boston, MA
- Human Cell Atlas Method Development Community
Protocol Citation: Alexi Mcadams, Tavé van Zyl 2020. Single-Cell Dissociation of Human Trabecular Meshwork . protocols.io https://dx.doi.org/10.17504/protocols.io.bfdyji7w
Manuscript citation:
van Zyl T, Yan W, McAdams A, Peng YR, Shekhar K, Regev A, Juric D, Sanes JR. Cell atlas of aqueous humor outflow pathways in eyes of humans and four model species provides insight into glaucoma pathogenesis. Proceedings of the National Academy of Sciences. 2020 Apr 27.
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: April 21, 2020
Last Modified: May 06, 2020
Protocol Integer ID: 35992
Keywords: trabecular meshwork, scRNAseq, single cell
Abstract
Fresh trabecular meshwork tissue is dissected from post-mortem human eyes and dissociated into a single cell suspension.
Guidelines
The stock solutions can be prepared up to 6 months in advance and aliquots can be stored at -20˚C.
Materials
MATERIALS
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
RNase ZapMerck MilliporeSigma (Sigma-Aldrich)Catalog #R2020-250ML
Razor blade
PapainWorthington Biochemical CorporationCatalog #LS003126
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
Bovine Serum Albumin (Non-acetylated)Merck MilliporeSigma (Sigma-Aldrich)Catalog #B6917
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9418
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
Trypsin Inhibitor OvomucoidWorthington Biochemical CorporationCatalog #LS003087
L-Cysteine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C1276
40 um Falcon® Cell Strainers Sterile CorningVWR International (Avantor)Catalog #21008-949
Millex-GP Syringe Filter Unit 0.22 µm polyethersulfone 33 mm gamma sterilizedMerck Millipore (EMD Millipore)Catalog #SLGP033RS
70% EtOH
Disposable Petri Dishes
Blunt curved Westcott scissors
Vannas Microscissors
Jewellers Forceps
50 mL Falcon Tubes
15 mL Falcon Tubes
1.7 mL Eppendorf Tubes
STEP MATERIALS
L-Cysteine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C1276
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
Bovine Serum Albumin (Non-acetylated)Merck MilliporeSigma (Sigma-Aldrich)Catalog #B6917
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9418
Trypsin Inhibitor OvomucoidWorthington Biochemical CorporationCatalog #LS003087
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
PapainWorthington Biochemical CorporationCatalog #LS003126
RNaseZap™ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
Protocol materials
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Trypsin Inhibitor OvomucoidWorthington Biochemical CorporationCatalog #LS003087
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
Disposable Petri Dishes
Jewellers Forceps
15 mL Falcon Tubes
Razor blade
RNase ZapMerck MilliporeSigma (Sigma-Aldrich)Catalog #R2020-250ML
Millex-GP Syringe Filter Unit 0.22 µm polyethersulfone 33 mm gamma sterilizedMerck Millipore (EMD Millipore)Catalog #SLGP033RS
PapainWorthington Biochemical CorporationCatalog #LS003126
L-Cysteine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C1276
50 mL Falcon Tubes
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9418
RNaseZap™ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
40 um Falcon® Cell Strainers Sterile CorningVWR International (Avantor)Catalog #21008-949
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
PapainWorthington Biochemical CorporationCatalog #LS003126
Bovine Serum Albumin (Non-acetylated)Merck MilliporeSigma (Sigma-Aldrich)Catalog #B6917
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9418
Trypsin Inhibitor OvomucoidWorthington Biochemical CorporationCatalog #LS003087
70% EtOH
Blunt curved Westcott scissors
Vannas Microscissors
1.7 mL Eppendorf Tubes
L-Cysteine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C1276
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
Bovine Serum Albumin (Non-acetylated)Merck MilliporeSigma (Sigma-Aldrich)Catalog #B6917
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
PapainWorthington Biochemical CorporationCatalog #LS003126
RNaseZap™ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
L-Cysteine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C1276
Bovine Serum Albumin (Non-acetylated)Merck MilliporeSigma (Sigma-Aldrich)Catalog #B6917
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9418
Trypsin Inhibitor OvomucoidWorthington Biochemical CorporationCatalog #LS003087
Safety warnings
Universal biosafety precautions should be observed with the handling of all human post-mortem tissue. Instruments should be cleaned and sterilized appropriately after each dissection.
Before start
Fresh Ames' medium should be prepared and oxygenated at the beginning of each dissection to allow for fresh working solutions.
Prepare Stock Solutions
Prepare Stock Solutions
2h
2h
Prepare Oxygenated Ames' Medium
- Add 1.9 g sodium bicarbonate to a 1L container (e.g. glass bottle or flask)
- Empty one container of Ames' Medium powder to 1L bottle
- Fill bottle with 1L of ddH2O, mix well
- Bubble oxygen gas (95% O2/5% CO2) through solution
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
DNase stock solution
- DNase I 40,000 Units
- oxygenated Ames' solution 3 mL
Filter solution into new 15 mL falcon tube with 0.22 um filter to sterilize
- Store 115 µL aliquots at -20˚C.
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
0.4% non-acetylated BSA stock solution
- NA BSA 25 mg
- oxygenated Ames' solution 6.25 mL
Filter solution into new 15 mL falcon tube with 0.22 um filter to sterilize
- Store 100 µL aliquots at -20˚C.
Bovine Serum Albumin (Non-acetylated)Merck MilliporeSigma (Sigma-Aldrich)Catalog #B6917
Prepare 10X Ovomucoid solution (LO)
- BSA 150 mg
- Ovomucoid 150 mg
- Oxygenated Ames' 10 mL
- dissolve and adjust pH to ~7.4 with a few µL of NaOH (2N)
Filter solution into new falcon tube with 0.22 um filter to sterilize
- Make 1 mL aliquots & store at -20˚C.
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9418
Trypsin Inhibitor OvomucoidWorthington Biochemical CorporationCatalog #LS003087
L-cysteine stock solution
- L-cysteine, anhydrous (sigma C1276): 24 mg
- ddH2O 1 mL
Filter into new 15 mL falcon tube with 0.22 um filter to sterilize
- Store 115 µL aliquots at -20˚C.
L-Cysteine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C1276
Prepare Working Solutions and Materials
Prepare Working Solutions and Materials
1h
1h
Prepare Fresh Oxygenated Ames' Medium
- Add 1.9 g sodium bicarbonate to a 1L container (e.g. glass bottle or flask)
- Empty one container of Ames' Medium powder to 1L container
- Fill container with 1L of ddH2O, shake to mix
- Bubble oxygen gas (95% O2/5% CO2) through solution
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Ames’ MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1420
Prepare papain solution
- Oxygenated Ames' 10 mL
- Papain 200 Units (ex: for a 44.8 mgP/mL, 26.6 u/mgP batch, use 168 uL)
- DNase I stock solution 100 uL
Filter into new 15 mL falcon tube with 0.22 um filter to sterilize
- L-cysteine aliquot (see above, stock solution) 100 uL
Activate the papain by adding the L-cysteine and incubating at 37 ˚C for 15 minutes before using for dissociation.
PapainWorthington Biochemical CorporationCatalog #LS003126
Prepare 1X Ovomucoid solution (LO)
- 10X LO (aliquot) 1 mL
- Oxygenated Ames' 9 mL
Prepare 0.04% non-acetylated BSA
- Oxygenated Ames' 900 uL
- 0.4% non-acetylated BSA stock 100 uL
Tissue collection & dissection
Tissue collection & dissection
45m
45m
Set up dissection area
- Clean all intruments and workspace with RNaseZap then 70% EtOH.
- Place all instruments on a Chux Pad or similar sheet to work on
Note: Keep the eye in immersed in Ames media or Optisol within a container on ice until dissection; and preferrably, the eye should be processed within 6 hours from death.
RNaseZap™ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
Start Dissection under microscope: Isolate the anterior segment
- Make a small incision at the pars plana (~4 mm posterior to the limbus) with a razor blade.
- Cut circumferentially with curved scissors to liberate the anterior portion from the rest of the eye.
- Place the anterior segment in oxygenated Ames' medium.
Expose the trabecular meshwork
- Free the lens from the ciliary body by carefully cutting the zonules with curved microscissors all the way around the lens
- Gently lift the lens from the rest of the structures with the scissor blades (holding the scissors parallel with the workbench)
- Take care and try to avoid violating or puncturing the lens capsule
- Gently peel the ciliary body and iris from the corneoscleral button with forceps.
- Trim any large chunks of ciliary muscle left behind from the trabecular meshwork with microscissors.
- This is an ideal time to begin activating the papain solution in a 37˚C waterbath
Isolate the trabecular meshwork
- Identify the TM and insert one prong of the Jewelers forceps into Schlemm’s canal
- Grab the TM with the forceps and gently pull the TM out in strips. Some of the ciliary muscle and possibly descemet membrane will come along, and this is okay for the purposes of this protocol
- The back wall of schlemm canal can also be gently scraped so that cells here can be transferred to the papain solution
Dissociate tissue into single cell solution
Dissociate tissue into single cell solution
1h
1h
Digest tissue
- Add TM strips to prewarmed, activated papain solution in an eppendorf tube
- Incubate for a total of 30 minutes at 37 ˚C
- Every 7-10 minutes, invert tube to agitate gently
Dissociate tissue into a single cell suspension
- When finished incubating, centrifuge digested tissue and papain solution for 30 seconds at 500 rcf
- Carefully remove supernatant to leave behind tissue
- Add 1 mL 1X LO to the tissue
- Triturate with a 1000 uL pipette
- Filter through a 40 um strainer into a 50 mL falcon tube
- If any large chunks of tissue are left behind, return to eppendorf and add another 1 mL of 1X LO and repeat.
- Rinse filter with 500 uL - 1000 uL of 1X LO and collect in the same 50 mL falcon tube
Prepare single cell solution for loading onto 10X
- Place the 50ml falcon tube containing the cell/1X LO suspension in centrifuge
- Centrifuge cells suspension at 500 rcf for 5 minutes (preferably at 4 ˚C)
- Carefully remove supernatant. Note where pellet should be given the falcon tubes' position in the centrifuge; sometimes the pellet is too small to see
- Resuspend the cells in 50 uL - 200 uL 0.04% non-acetylated BSA, depending on pellet size
- Transfer to a 1.7 mL eppendorf tube
- Count cells with hemocytometer and dilute with 0.04% non-acetylated BSA to desired concentration based on 10X protocol (we usually aim to achieve a concentration of 1000 cells per microliter and load the 10X for 6000 cells recovered)
- Cell viability assay can be performed at this stage as well prior to loading