Jun 04, 2024
Version 5
Preparation of single cell suspensions from human intestinal biopsies for single cell genomics applications V.5
- 1University of Chicago
Protocol Citation: Ran RZ Zhou, Oni Basu 2024. Preparation of single cell suspensions from human intestinal biopsies for single cell genomics applications. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7bxqklwz/v5Version created by Ran RZ Zhou
Manuscript citation:
1. Fujii M, Matano M, Toshimitsu K, Takano A, Mikami Y, Nishikori S, Sugimoto S, Sato T. Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition. Cell Stem Cell. 2018 Dec 6;23(6):787-793.e6. doi: 10.1016/j.stem.2018.11.016. 2.Smillie CS, Biton M, Ordovas-Montanes J, Sullivan KM, Burgin G, Graham DB, Herbst RH, Rogel N, Slyper M, Waldman J, Sud M, Andrews E, Velonias G, Haber AL, Jagadeesh K, Vickovic S, Yao J, Stevens C, Dionne D, Nguyen LT, Villani AC, Hofree M, Creasey EA, Huang H, Rozenblatt-Rosen O, Garber JJ, Khalili H, Desch AN, Daly MJ, Ananthakrishnan AN, Shalek AK, Xavier RJ, Regev A. Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis.Cell. 2019 Jul 25;178(3):714-730.e22. doi: 10.1016/j.cell.2019.06.029.
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: September 09, 2022
Last Modified: June 04, 2024
Protocol Integer ID: 101193
Keywords: single cell, RNA-seq, human intestines,
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Abstract
The protocol is adapted from Fujii's and Smillies's reports for single cell transcriptome analysis from human intestines. It provides details on acquirement of single cell suspension from epithelium and lamina propria. This methods is modified to generate appropriate meterials from patient's intestinal biopsies for sinlge-cell transcriptome and genomic applications.
Guidelines
The human intestinal tissue are obtained after patients' consents and approval from Institutional Review Board at the University of Chicago (IRB Number: 15573A). All the samples are processed for research use only.
Materials
MATERIALS
BSASigma AldrichCatalog #A7906
Liberase TMSigma AldrichCatalog #000000005401119001
FBSInvitrogen - Thermo Fisher
Corning® 40µm Cell StrainerCorningCatalog #431750
TrypLE™ Express EnzymeThermo Fisher ScientificCatalog #12604013
Trypan Blue Solution 0.4% (w/v) in PBS pH 7.5 ± 0.5CorningCatalog #25-900-CI
Eppendorf tubes 1.5 mL uncoloredEppendorf CentrifugeCatalog #022363204
NxGen® RNAse InhibitorLucigenCatalog #30281-2
Penicillin-Streptomycin (10,000 U/mL)Thermo FisherCatalog #15140163
DNase I, RNase-free, HC (50 U/µL)Thermo FisherCatalog #EN0523
HEPES 1MThermo Fisher ScientificCatalog #15630080
15 ml corniacal tubeCorningCatalog #352095
0.5M EDTAFisher ScientificCatalog #2482-500
HBSS no calsium no magnesiumThermo ScientificCatalog #14175-095
10 x PBS no calsium no magnesiusmFisher ScientificCatalog #BP399500
UltraPure™ DNase/RNase-Free Distilled WaterThermofisherCatalog #10977023
50 ml Conical tubeGenesee ScientificCatalog #28106
0.2 micron syring filterCatalog #7232520
Filter top vaccum unit 1000 mlThermo ScientificCatalog #5670020
RBC lysis bufferSigma AldrichCatalog #11814389001
1x PBS 1000 ml
10x PBS 100 ml
Water 900 ml
Filtered through 1000 ml Filter-top vaccum unit and stored at room temperature.
Wash media 20 ml
HBSS. 18 ml
FBS 2 ml
Keep the reagents sterilized and prepare freshly
Dissociation media 10 ml
HBSS 8.8 ml
EDTA 0.5M 0.1 ml (final concentration 5 mM)
FBS 1 ml
HEPE 1M 0.1 ml (final concentration 10 mM)
Digestion media 10 ml
Wash media (recipe above) 7.8 ml
Liberase TM (1 mg/ml) 2 ml (final concentration 200 ug/mL)
DNase (5 mg/ml) 0.2 ml (final concentration 100 ug/mL)
Cell suspension buffer 10 ml
PBS 10 ml
2%BSA 50 ul
Filter through 0.2 micron syringe filter.
Safety warnings
Sharp-end forceps and Iris scissors are used in the protocol. Primary tissues from patients are treated with cautions for unknown infectious status.
Before start
Generally two samples are obtained from each patient. The sample information from tissue containers (1.7 ml Eppendorf tube in our facility) are recorded. Each sample contains 2-3 pinches. Samples from the same patients but from different biopsy location (ileum and ascending colon) are processed separately. Wet weight of the tissues from each sample are measured by a micro-scale and recorded.
Pre-Dissociation
Pre-Dissociation
Chill 1x PBS, wash media and dissociation media on ice. Samples are transfered with Advanced DMEM/F12 based media in 1.7 ml eppendorf tubes on ice. Once received in lab, samples are transfered to 35 mm dish using sharp-end forceps. Alternatively, tissues can be transferred to a 5 ml conical tube using a P200 pipette.
On ice
Note
Pinched tissues range from 6 to 20 mm3 in size.
Sample Wash before dissociation
Sample Wash before dissociation
Place 3 ml ice cold PBS in each dish with each sample (containing 2-3 pinches). Shake the dish on ice to rinse off mucus. Alternatively, add 4 ml ice cold PBS to the 5 ml conical tube and invert the tube 5 times with the lid closed.
Repeat step 2 three more times or until the PBS becomes clear.
Transfer tissues from the 35 mm dish to a clean 5 ml conical tube (tissues are remained in the same tube if washed in the conical tube). Add 0.2 ml Dissociation media and mince the tissues with Iris scissors for 1 minute on ice.
Note
The minced tissues range from 1 to 3 mm3 in size, can easily pass a P1000 tip.
00:01:00 On ice
Dissociate Epithelial layers from Lamina propria
Dissociate Epithelial layers from Lamina propria
Add 3 ml ice-cold Dissociation media to each 5 ml tube.
place the tubes on ice on a linear rocker and rock the tubes (end to end) at 100/min for 60 mins. Alternatively rotate the tubes in a 37 Celsius oven for 15 mins.
37 °C 00:15:00
15m
Pipet the tissue in dissociation media in a 5 ml serological pipet 5 times and let the tissue settle in the tube on ice for 5 mins. Collect the supernatant and continue with Epithelium Digestion. Add 10 ml dissociation media to the tissue and repeat step 6.
Collected supernantant will be processed in the following section. Sedimented tissue represents the lamina propria and will be processed in the "Lamina propria Digestion" section.
Note
The epithelial sheets isolated from lamina propria remain in the Dissociation media supernatant. The lamina propria will be processed separately from epithelium.
Epithelium Digestion
Epithelium Digestion
Centrifuge the epithelium in the supernatant in 5 ml conical tubes at 300 g x 5 mins at 4 celsius.
300 x g, 4°C, 00:05:00
Remove supernatant and add 2 ml warmed TrypLE express to the pellet. Incubate the mixture at 37 celsius for 5 mins with rotation at 20 rpm.
Note
A hybridation oven with a rotisserie is recommended.
Neutralize TrypLE express by adding 2 ml wash media, agitate the tissue by pipetting up and down and filter through a 40 micron cell strainer.
Centrifuge at 300 g x 5 mins at 4 celsius.Remove supernatant. Resuspend cells in 1 ml wash media and place on ice.
300 x g, 4°C, 00:05:00
Take 20 ul cell suspension with 20 ul Tryplan blue and mix well. Check the cell viability on a light microscope.
If viability is higher than 85%, proceed with red blood cell removal.
Note
Either hemocytometer or Countess automated cell counter can be used for viability assessment. Live cells are excluded from staining blue and the numbers of live and dead cells are both counted for viablity rate = 100% x N of live cells/(N of live cells + N dead cells). At least 50 cells are counted in total.
Lamina propria Digestion
Lamina propria Digestion
Wash the tissue one times with 3 ml Wash media. Aspire the Wash media as much as possible.
Add 2 ml Digest media. Incubate the tissue at 37°C with end-over-end rotation 20 rpm for up to 30 mins.
Note
For samples less than 40 mg, a 20-mins digestion is sufficient.For larger samples, check the digestion after 20 mins incubation with Digest media. If cell clumps remains and cell viability > 95%, incubate for additional 5 minutes.
Neutralize Liberase TM by 1 ml Quench media. Leave tissue on ice for 5 mins. Agitate the tissue by pipetting up and down and filter through a 40 micron cell strainer.
Centrifuge at 300 g x 5 minus at 4 Celsius. Remove supernatant and suspend cells in 1 ml wash media.
300 x g, 4°C, 00:05:00
Take 20 ul cell suspension with 20 ul Trypan Blue. Mix well and check the viability on a light microscope. Proceed with red blood cell removal.
Red blood cell removal
Red blood cell removal
Dilute one volume of cell suspension (epithelium or lamina propria; keep tissue types separate) by two volumes of Red Blood Cell Lysis Solution.
Gently mix by inverting the tubes and incubate for 2 minutes at room temperature.
Centrifuge at 300 g for 5 minutes at 4 Celsius.
300 x g, 4°C, 00:05:00
Aspirate supernatant completely. Resuspend the cell pellet in 0.1 ml cell suspension buffer with RNase Inhibitor 0.1 U/ul (RNase Inhibitor is not needed if loading on 10x Genomics platform). Proceed with Cell suspension preparation.
Cell Suspension Preparation
Cell Suspension Preparation
Count the cells with Trypan Blue staining.
Adjust cell density by cell suspension buffer with RNase Inhibitor 0.1 U/ul (RNase Inhibitor is not needed if loading on 10x Genomics platform).
Note
Dead cell removal is optional after the viability is determined. Tested: EasySep‱ Dead Cell Removal (Annexin V) Kit from STEMCELL Technologies. Cell viability can be improved from 70% to 90%. Cell suspension with a viability lower than 60% is not recommended for single cell RNA-seq.