Feb 04, 2020

Public workspaceEnzymatic disaggregation of human myometrium for 10X Single Cell RNA-seq

DOI
dx.doi.org/10.17504/protocols.io.bb5miq46
  • 1Igenomix Foundation, INCLIVA, Valencia, Spain;
  • 2Valencia University, Valencia, Spain;
  • 3Harvard University, Boston, USA
Aymara Mas
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bb5miq46
Protocol Citation: Aymara Mas, Alba Machado-Lopez, Patricia Escorcia, Carlos Simón 2020. Enzymatic disaggregation of human myometrium for 10X Single Cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bb5miq46
Manuscript citation:
Mas A, et al. Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells. Fertil Steril. 2012 Sep;98(3):741-751.
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
Working. We and other researchers are using and published the results using this protocol (Mas et al., 2015; Prusinski et al., 2018; Corachán et al., 2019).
Created: February 04, 2020
Last Modified: February 04, 2020
Protocol Integer ID: 32653
Abstract
This protocol describes the procedure for dissociating myometrial samples, which is based in an enzymatic disaggregation using Collagenase IV and DNAse I. This protocol is adapted from Mas et al. 2012 Fertil Steril with minor modifications.
Guidelines
Storage conditions of reagents
ReagentStorage condition
Hypothermosol4ºC
HBSS4ºC
HEPES 1M4ºC
DNAse type IResuspend in PBS and store 100 µl aliquots at -20ºC
Collagenase type IV4ºC
DMEM low glucose4ºC
FBSStore 30 ml aliquots at -20ºC
Penicillin-Streptomycin (10000 U/ml)Store 5 ml aliquots at -20ºC


Required equipment
EquipmentSupplierCatalog nº
Heracell 150i CO2 incubatorThermo Scientific51026280
New Brunswick Incubator shaker Innova 40EppendorfM1299-0092
Centrifuge Mega Star 600VWR collection521-1893
Microscope Nikon eclipse TE200Nikon-
Class II microbiological safety cabinet MSC advantageThermo Scientific51025411


The protocol workflow is as follows:
  1. Tissue collection and cold transportation until processing
  2. Dissociation: mechanic and enzymatic
  3. Remove red blood cells if necessary
  4. Prepare cells for Chromium
Materials
MATERIALS
ReagentBRAND® counting chamber BLAUBRAND® Neubauer improved New without clips, double ruledMerck MilliporeSigma (Sigma-Aldrich)Catalog #BR717805
ReagentPenicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
ReagentCollagenase Type 4Worthington Biochemical CorporationCatalog #LS004188
Reagent100 µm Cell StrainerFalconCatalog #352360
ReagentHypoThermosol® FRS Preservation SolutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #H4416
ReagentTrypLE™ Select Enzyme (1X), no phenol redThermo FisherCatalog #12563029
ReagentHEPES (1 M)Thermo FisherCatalog #15630080
ReagentNalgene™ Rapid-Flow™ Sterile Disposable Filter Units with PES Membrane, 250mL, 0.2μm pore, 50mm membraneThermo FisherCatalog #568-0020
ReagentACK Lysing BufferThermo FisherCatalog #A1049201
ReagentHBSS (1x)Gibco - Thermo Fisher ScientificCatalog #14175-095
ReagentDeoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4513
ReagentDulbecco’s Modified Eagle’s Medium - low glucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5921
ReagentFetal bovine serum (FBS)BioWestCatalog #S181B-500
ReagentFalcon 40 µm Cell StrainerCorningCatalog #352340
ReagentSterile surgical bladesAesculapCatalog #7806607

Safety warnings
Human samples including tissue, blood and bodily fluids have the potential to harbour HG2 and Hazard Group 3 (HG3) organisms, specifically Blood Borne Viruses (BBVs). As security warning, please follow the procedures related to Biological Safety at Containment Level 2.
Before start
Prepare Wash buffer:
Make stock of wash buffer and store at 4 °C. Aliquot 49.5 mL of HBSS in a 50 ml conical Falcon tube and add 500 µl of Pen/Strep (100 U/ml).

Prepare Enzyme buffer:
To make 100 ml of enzyme buffer, combine 95.6 ml of HBSS with 1 ml of Pen/Strep (100 U/ml); 1 ml of HEPES (1M); 4.6 ml of wash buffer; 95 µl of DNAse and 135 mg of collagenase IV and filter in a 0.2 µm bottle-top sterile filter unit.

Prepare resuspension buffer:
Add 12 ml of inactivated FBS and 1 ml of Pen/Strep (10000 U/ml) to 87 ml of DMEM medium (low glucose) to prepare 100 ml of resuspension buffer.
Tissue collection and transportation
Tissue collection and transportation
Uterine tissues should be transported in preservation solution (HypoThermosol® FRS) at Temperature4 °C from the surgery room to the lab.

Dissociation
Dissociation
Rinse the sample with wash buffer, removing blood or mucus.
Place wet tissue under a petri dish. Isolate with a sterile scalpel the myometrial layer by removing the endometrium, serosa and laser-burnt zones.
Mechanical disaggregation by chopping and thoroughly mincing up the tissue into small pieces (<1 mm3).
Transfer contents to 50 ml falcon tubes containing Amount30 mL of enzyme buffer. Tighten lid, seal with parafilm and incubate at Temperature37 °C overnight horizontally.

Afterwards, cell suspensions should be filtered through 100 to 50-μm polyethylene filters to remove cellular clumps and undigested tissue.
Centrifuge the filtered medium Centrifigation400 x g, 00:05:00 . Remove supernatant and add at least Amount1 mL of resuspension buffer (depending of the pellet content).
Remove red blood cells
Remove red blood cells
[Optional] If after centrifugation there is a ring of red blood cells, add ACKL lysis buffer, consisting in hypotonic shock, and incubate at Temperature37 °C for Duration00:05:00 . Then, follow step 7 again.

Dissociaton
Dissociaton
Add Amount400 µL Tryple Select Enzyme to resuspended media containing cells and mix thoroughly by pipetting. Incubate for Duration00:10:00 - Duration00:15:00 at Temperature37 °C and centrifuge at Centrifigation300 rpm .

Add Amount100 µL DNAse I to digest the extracellular genomic DNA and mix thoroughly by pipetting. Incubate forDuration00:05:00 atTemperatureRoom temperature .

Add at least Amount1 mL of resuspension buffer. Filter through a 40 μm cell strainer and centrifuge at Centrifigation400 x g, 00:05:00 .

Remove supernatant and resuspend the content in at least Amount1 mL of resuspension buffer (depending of the pellet content).

Prepare cells for chromium
Prepare cells for chromium
Count the total number of cells (at least twice on two different cell counters).
Proceed to 10x experiment.
[Optional] Resuspend cells in freezing medium to a concentration of 1 x 106 cells for storage in cryogenic vials.