Nov 09, 2023
Preparation of Single Cell Suspensions of the Intra-Epithelial Layer and Lamina Propria from Human Intestinal Tissue
- Steven B. Wells1,
- Pranay Dogra2,
- Peter A. Szabo2,
- Joshua I. Gray2,
- Daniel P. Caron2,
- Yoonseung Lee2,
- Rory Morrison-Colvin2
- 1Department of Systems Biology, Columbia University Irving Medical Center, New York, NY 10032, USA;
- 2Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA
- Columbia
Protocol Citation: Steven B. Wells, Pranay Dogra, Peter A. Szabo, Joshua I. Gray, Daniel P. Caron, Yoonseung Lee, Rory Morrison-Colvin 2023. Preparation of Single Cell Suspensions of the Intra-Epithelial Layer and Lamina Propria from Human Intestinal Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bwq7pdzn
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: July 20, 2021
Last Modified: November 09, 2023
Protocol Integer ID: 51711
Keywords: Gut, Intestine, Jejunum, Ileum, Colon, GI, CD45, Lymphocytes, Myeloid, Isolation, Density Gradient, Ficoll, Immune, 10x, scRNAseq, Flow cytometry, Leukocyte, Single cell suspension, T cell, Epithelium, Lamina propria,
Abstract
This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from the epithelial layer and the lamina propria of human gut sections of about one gram of tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples. This protocol can be used for any section of the intestinal tract from duodenum to distal colon.
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Materials
Materials:
Fisherbrand™ Sterile Syringes for Single UseFisher ScientificCatalog #14955459
Benzonase nucleaseSigma AldrichCatalog #E1014-5KU
Dulbeccos phosphate-buffered saline (DPBS)Gibco - Thermo FischerCatalog #14190144
Penicillin-Streptomycin-Glutamine (100X)Thermo FisherCatalog #10378016
Thermo Scientific™ Nunc™ 50mL Conical Sterile Polypropylene Centrifuge TubesFisher ScientificCatalog #12-565-271
Thermo Scientific™ 250mL Conical Centrifuge TubeFisher ScientificCatalog #12566441
DTT (Dithiothreitol)Cell Signaling TechnologyCatalog #7016
Gibco™ IMDM (Iscoves Modified Dulbeccos Medium)Fisher ScientificCatalog #12-440-053
Gibco™ Fetal Bovine Serum qualified AustraliaFisher ScientificCatalog #10-099-141
UltraPure 0.5M EDTA pH 8.0Invitrogen - Thermo FisherCatalog #15575020
100μM cell strainer (Fisher Scientific, Cat. No.: 50-146-1428)
Ficoll-Paque™ PLUS MediaFisher ScientificCatalog #45-001-749
Collagenase DSigma AldrichCatalog #11088882001
Mr. Frosty™ Freezing ContainerFisher ScientificCatalog #5100-0001
DNASE 1 100MGFisher ScientificCatalog #NC9709009
CryoStor CS10 100MLFisher ScientificCatalog #NC9930384
Corning™ Externally Threaded Cryogenic VialsFisher ScientificCatalog #09-761-71
5mL Falcon™ Round-Bottom Polypropylene Test TubesFisher ScientificCatalog #14-959-11A
Solution 13 AO – DAPIChemometecCatalog #910-3013
NC-Slide A8™ box with 25 SlidesChemometecCatalog #942-0003
Equipment:
- Multi-Axle-Rotating Mixer
- Centrifuge
- Cell Counter - NC-3000
- Surgical scissors
- Scale
Preparing Mediums and Buffers
Preparing Mediums and Buffers
Create the following IMDM-FBS-PSQ Media in a 500mL bottle of IMDM by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc. | |
| IMDM | 500 | - | - | |
| Penicillin-Streptomycin-Glutamine | 5 | 100X | 1X | |
| FBS | 50 | 100% | 10% |
Table 1.
Create the following DPBS-FBS Solution in a bottle of DPBS by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc. | |
| DPBS | 500 | - | - | |
| FBS | 25 | 100% | 5% |
Table 2.
Create the following IMDM-FBS-PSQ-EDTA-DTT Media in a 500mL bottle of IMDM by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc. | |
| IMDM | 500 | - | - | |
| FBS | 50 | 100% | 10% | |
| Penicillin-Streptomycin-Glutamine | 5 | 100X | 100X | |
| EDTA | 10 | 0.5M | 10mM | |
| DTT | 1 | 1M | 2mM |
Table 2.
Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc. | |
| DPBS | 500 | - | - | |
| FBS | 25 | 100% | 5% | |
| EDTA | 1 | 0.5M | 10mM |
Table 2.
Tissue Preparation
Tissue Preparation
Use a surgical scissors to remove about 7 cm -8 cm of intestinal tissue section from the mysentary. Remove any remaining mysentary from the intestinal tissue.
Gently massage the chyme or fecal matter out of the tissue over a bucket.
Cut open the tissue on a tray containing cold DPBS-FBS Solution and add the tissue to a 250 mL conical with 100 mL of cold DPBS-FBS Solution and using a forceps gently agitate the tissue to remove yellow/brown chyme, fecal matter and/or mucus.
Discard the DPBS-FBS solution into a bucket, replace with 100 mL of cold DPBS-FBS Solution and continue to wash the tissue until the DPBS-FBS Solution is no longer brown – when it has been successfully cleaned the DPBS-FBS Solution it should appear cloudy yellow/white. Depending upon how clean the tissue is this may take numerous washes (anywhere between 3-10, perhaps more).
Tissue Dissociation – Epithelial Stripping (IE Fraction)
Tissue Dissociation – Epithelial Stripping (IE Fraction)
1h
1h
Add 1±0.2 grams of the cleaned intestinal tissue to a 50 mL centrifuge tube and record the weight below:
Total weight. __________g.
Note
NOTE: Going beyond the 1±0.2 grams of tissue without concomitantly increasing the number of tubes reduces the efficacy of the eventual enzymatic digest and lowers yields.
Add 20 mL of Room temperature IMDM-FBS-PSQ-EDTA-DTT Media to the tissue-containing 50 mL tube. Incubate on a shaker for 00:30:00 at 37 °C .
30m
Filter the cell suspension through a 100 micromolar (µM) filter into a 50 mL conical, rinse the tissue and the filter with 20 mL of DPBS-FBS Solution. Set the cell suspension aside at 4 °C . Place the remaining tissue into a back into its original 50 mL conical.
Re-add 20 mL IMDM-FBS-PSQ-EDTA-DTT Media to the tissue and incubate on a shaker for 00:30:00 at 37 °C .
30m
Filter the cell suspension through a 100 micromolar (µM) filter into a 50 mL conical, rinse the tissue and the filter with 20 mL of DPBS-FBS Solution. Set the cell suspension aside at 4 °C . Place the remaining tissue into a back into its original 50 mL conical.
Tissue Dissociation – Lamina Propria Digestion (LP Fraction)
Tissue Dissociation – Lamina Propria Digestion (LP Fraction)
32m
32m
Add 5 mL of Room temperature IMDM (NO ADDITIVES! Just the base media formulation) to the tube and use a scissors to chop the tissue into a fine “mash”.
Add 40 mL of Room temperature IMDM (NO ADDITIVES) and spike in 0.400 mL of Collagenase D, and 0.400 mL of DNAse to the tube to begin the enzymatic digestion. Place on a shaker for 00:30:00 at 37 °C .
30m
After digestion, add 0.500 mL of EDTA 0.5 Molarity (M) 8.0 to the digested cell suspensions and incubate for 00:02:00 at 20 °C .
2m
Distribute and filter the mash of tissue over 100 micromolar (µM) cell strainers above 50 mL tubes (about 4 filters/gram of tissue).
Note
NOTE: Cell yields and ease of pushing through the filter are increased by using multiple filters/gram of tissue, default to using more filters to decrease processing time, and increase yields.
Apply pressure with the black rubber bottom or the plastic end of a 10 mL syringe plunger to any remaining, partially digested tissue on the cell strainers, and intermittently wash through with DPBS-FBS-EDTA Solution from a transfer pipet. When finished, combine the tubes of cell suspension and proceed to the next section.
Ficoll-Paque
Ficoll-Paque
1h 20m
1h 20m
Centrifuge the cell suspensions (EL and LP fractions) for 00:10:00 at 400 x g at 20 °C .
10m
Remove the EL and LP supernatants and combine the cell pellets down to a single 50 mL , 1 g /tube, keep the fractions distinct, add 10 mL with Room temperature IMDM (NO ADDITIVES).
Add 10 µL of benzonase/1 gram of tissue to the EL and LP fractions and incubate at 37 °C for 00:30:00 .
30m
Add 15 mL of IMDM (NO ADDITIVES) to the cell suspension, spike in 0.250 mL of EDTA 0.5 Molarity (M) 8.0 to all tubes.
Filter both the EL and LP cell suspensions through a 100 micromolar (µM) cell strainer.
Layer 25 mL of cell suspension (both IE and LP fractions) on top of 15 mL of Ficoll-Paque Media PLUS.
Spin for 00:20:00 , 1200 x g at 20 °C with 4 acceleration and 0 brake, evenly distribute the tubes across the entire rotor to prevent wobbling (use all four buckets if possible as opposed to just two).
20m
For both fractions, remove the mononuclear cell layer with a transfer pipet and transfer to a separate 50 mL tubes. Add cold DPBS-FBS-EDTA Solution to a final volume of 50 mL and centrifuge the cell suspensions for 00:10:00 at 400 x g , 4 °C .
10m
Remove the supernatant and re-suspend the cell pellet in 50 mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 00:10:00 at 120 x g , 4 °C .
10m
Remove the supernatant and re-suspend the cell pellet in cold 10 mL IMDM-FBS-PSQ Media.
Cell Count
Cell Count
IE Fraction - Count cells, and viability by using the NC-3000 cell counter. Calculate total viable cells and record below:
cell number: ________cells/mL, ________% viable
final volume: ________mL
𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 (𝑐𝑒𝑙𝑙𝑠/𝑚𝐿) ∗ 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦(%) ∗ 𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑚𝐿) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠
Total Viable Cells: _______
LP Fraction - Count cells, and viability by using the NC-3000 cell counter. Calculate total viable cells and record below:
cell number: ________cells/mL, ________% viable
final volume: ________mL
𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 (𝑐𝑒𝑙𝑙𝑠/𝑚𝐿) ∗ 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦(%) ∗ 𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒(𝑚𝐿) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠
Total Viable Cells: ________
Freeze-down
Freeze-down
(Optional QC) Aliquot 2 x 106 cells to a 5 mL Falcon tube and place on ice for subsequent flow cytometric analysis.
Aliquot cells for analysis or experimentation, and then freeze down cells in up to 5 x 106 aliquots using Cryostor CS10 Medium, a Mr. Frosty, and a-80 °C freezer (1 mL -1.5 mL aliquots, round down to the nearest 5 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.