Feb 13, 2022

Public workspaceHuman Fallopian Tube and Ovary Dissociation for Single Cell RNA-Seq

DOI
dx.doi.org/10.17504/protocols.io.bfudjns6
  • 1University of Chicago
Melissa Javellana
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DOI: dx.doi.org/10.17504/protocols.io.bfudjns6
Protocol Citation: Melissa Javellana, Mark Eckert, Ernst Lengyel 2022. Human Fallopian Tube and Ovary Dissociation for Single Cell RNA-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bfudjns6
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 30, 2020
Last Modified: February 13, 2022
Protocol Integer ID: 36453
Keywords: Single Cell, Ovary, Fallopian Tube,
Abstract
This protocol provides a procedure for human fallopian tube and ovary dissociation into single cell suspension prior to single cell RNA-sequencing. It involves removing the epithelial fraction and then further digesting the remaining solid tissue fraction to gain maximum yield from all tissue compartments.

Materials
MATERIALS
ReagentFBSInvitrogen - Thermo Fisher
ReagentPronase from Streptomyces griseusRocheCatalog #10165921001
ReagentOpti-MEMInvitrogenCatalog #31985-070
ReagentHBSSMerck MilliporeSigma (Sigma-Aldrich)Catalog #H4891
ReagentBSAFisher ScientificCatalog #BP1600
ReagentDMEM with L-glutamineCorningCatalog #10-013-CV
ReagentCollagenase IVMerck MilliporeSigma (Sigma-Aldrich)Catalog #C5138
ReagentHyaluronidaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3884
ReagentDNaseIMerck MilliporeSigma (Sigma-Aldrich)Catalog #4536282001
ReagentDNA LoBind 50mL Conical EppendorfCatalog #0030122232
ReagentEDTACatalog #BP2482100
ReagentEasySep™ RBC Depletion ReagentSTEMCELL Technologies Inc.Catalog #18170
Before start
Prepare DMEM/10%FBS stock
Prepare 0.5M EDTA stock
Prepare 1mL PBS/0.05% BSA per sample


Note: Our samples are retrieved from the OR immediately upon removal from patient and transported to pathology. The pathologist grossly examines the sample and provides 100-200mg transverse tissue segments from the isthmus, ampulla and fimbriae of the fallopian tube and a longitudinal segment from the center of the ovary. Tissue segments are transported to the lab in DMEM/10% FBS.
Solution Preparation
Solution Preparation
5m
5m
Prepare Amount10 mL of Pronase solution per sample in 50ml LoBind conical :



ReagentQuantity
Opti-MEM10 ml
Pronase (~7 U/mg)18 mg

CITATION
Karst AM, Drapkin R (2012). Primary culture and immortalization of human fallopian tube secretory epithelial cells.. Nature protocols.
LINK
https://doi.org/10.1038/nprot.2012.097

Prepare Amount10 mL of digestion buffer per sample in a 50 mL LoBind conical:

ReagentQuantity
HBSS10 ml
Collagenase IV (>120 U/mg)15 mg
Hyaluronidase (750-3000 U/mg)10 mg
DNaseI (20,000 U/ml)10 μl

Note: Keep all solutions at Temperature37 °C throughout protocol.

Dissociation
Dissociation
1h 30m
1h 30m
Rinse gross blood off samples with DMEM/10% FBS.


Fallopian tube isthmus: bivalve
Fallopian tube ampulla: bivalve
Fallopian tube fimbriae: leave whole
Ovary: mince Amount100 mg tissue into 1-2 mm pieces.
Place each sample into Amount10 mL of Pronase solution. Place in Temperature37 °C orbital shaker at 200 rpm for Duration00:30:00 .

30m
Using a 10 ml pipette, triturate tissue to remove loose epithelial fraction. Pass all of the pronase solution through 70 μm filter into new 50 ml LoBind conical and rinse filter with Amount10 mL DMEM/10%FBS. Recover solid fraction from original conical and/or off of filter.

NOTE: You now have an epithelial fraction and a solid fraction. You will continue to process the epithelial fraction and solid fraction separately in the next steps. Both contain potentially unique cell types.
Critical
Place remaining solid fraction in digestion buffer. Place on Temperature37 °C orbital shaker at 200 rpm for Duration00:30:00 to Duration00:45:00

45m
Spin down epithelial fraction at 400 rcf for Duration00:04:00 . Aspirate and discard supernatant. Re-suspend in Amount3 mL DMEM/FBS and wait for solid tissue fraction to complete digestion.

4m
Pass solid fraction solution through a 70 μm filter into conical containing epithelial fraction and rinse filter with Amount10 mL DMEM/FBS.

5m
Spin down at 400 rcf for Duration00:04:00 . Aspirate and discard supernatant. Re-suspend in Amount1 mL PBS/0.05% BSA

4m
RBC Clean-up
RBC Clean-up
10m
10m
Move solution to Eppendorf tube and add Amount12 µL Concentration0.5 Molarity (M) EDTA

Vortex the EasySep RBC depletion reagent bottle for Duration00:00:30 to re-suspend

Add Amount25 µL reagent and gentle vortex for Duration00:00:03 until solution appears homogeneous

Place on magnet and incubate Duration00:05:00

5m
Leaving on magnet, pipet off clear solution
Re-suspend in pre-warmed Temperature37 °C DMEM/10%FBS.

Assess viabilty with trypan blue if proceeding immediately to single cell library creation or method of choice if optimizing protocol.
Citations
Step 1
Karst AM, Drapkin R. Primary culture and immortalization of human fallopian tube secretory epithelial cells.
https://doi.org/10.1038/nprot.2012.097