TITLE: Preparation of Single Cell Suspensions of the Intra-Epithelial Layer and Lamina Propria from Human Intestinal Tissue
AUTHORS: Steven B. Wells, Pranay Dogra, Peter A. Szabo, Joshua I. Gray, Daniel Caron, Yoonseung Lee, Rory Morrison-Colvin

[DESCRIPTION]
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.

[STEPS]
SECTION: Preparing Mediums and Buffers
1. Create the following IMDM-FBS-PSQ Media in a 500mL bottle of IMDM by using the table below:
 
            Component  Volume (mL)  Starting Conc.  Final Conc.*    IMDM  500  -  -    Penicillin-Streptomycin-Glutamine  5  100X  1X    FBS  50  100%  10%  
 

SECTION: Preparing Mediums and Buffers
2. Create the following DPBS-FBS Solution in a bottle of DPBS by using the table below:
 
            Component  Volume (mL)  Starting Conc.  Final Conc.*    DPBS  500  -  -    FBS  25  100%  5%  
 
SECTION: Preparing Mediums and Buffers
3. Create the following IMDM-FBS-PSQ-EDTA-DTT Media in a 500mL bottle of IMDM by using the table below: 
            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  
 
 
SECTION: Preparing Mediums and Buffers
4. Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS by using the table below: 
            Component  Volume (mL)  Starting Conc.  Final Conc.    DPBS  500  -  -    FBS  25  100%  5%    EDTA  1  0.5M  10mM  
 
SECTION: Tissue Preparation
5. 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.

SECTION: Tissue Preparation
6. Gently massage the chyme or fecal matter out of the tissue over a bucket.
SECTION: Tissue Preparation
7. 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.

SECTION: Tissue Preparation
8. 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).

SECTION: Tissue Dissociation – Epithelial Stripping (IE Fraction)
9. Add 1±0.2 grams of the cleaned intestinal tissue to a 50 mL centrifuge tube and record the weight below:
Total weight. __________g.
 

SECTION: Tissue Dissociation – Epithelial Stripping (IE Fraction)
10. Add 20 mL of Room temperature IMDM-FBS-PSQ-EDTA-DTT Media to the tissue-containing 50 mL tube. Incubate on a shaker for 30 min at 37 °C.

SECTION: Tissue Dissociation – Epithelial Stripping (IE Fraction)
11. 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.

SECTION: Tissue Dissociation – Epithelial Stripping (IE Fraction)
12. Re-add 20 mL IMDM-FBS-PSQ-EDTA-DTT Media to the tissue and incubate on a shaker for 30 min at 37 °C.

SECTION: Tissue Dissociation – Epithelial Stripping (IE Fraction)
13. 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.

SECTION: Tissue Dissociation – Lamina Propria Digestion (LP Fraction)
14. 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”.

SECTION: Tissue Dissociation – Lamina Propria Digestion (LP Fraction)
15. Add 40 mL of 37 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 30 min at 37 °C.

SECTION: Tissue Dissociation – Lamina Propria Digestion (LP Fraction)
16. After digestion, add 0.500 mL of EDTA 0.5 Molarity (M) pH 8.0 to the digested cell suspensions and incubate for 2 min at 20 °C.
  

SECTION: Tissue Dissociation – Lamina Propria Digestion (LP Fraction)
17. Distribute and filter the mash of tissue over 100 micromolar (µM) cell strainers above 50 mL tubes (about 4 filters/gram of tissue).
 

SECTION: Tissue Dissociation – Lamina Propria Digestion (LP Fraction)
18. 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.

SECTION: Ficoll-Paque
19. Centrifuge the cell suspensions (EL and LP fractions) for 10 min at 400 x g at 20 °C.

SECTION: Ficoll-Paque
20. 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 20 Room temperature IMDM (NO ADDITIVES).

SECTION: Ficoll-Paque
21. Add 10 µL of benzonase/1 gram of tissue to the EL and LP fractions and incubate at 37 °C for 30 min.

SECTION: Ficoll-Paque
22. Add 15 mL of IMDM (NO ADDITIVES) to the cell suspension, spike in 0.250 mL of EDTA 0.5 Molarity (M) pH 8.0 to all tubes.

SECTION: Ficoll-Paque
23. Filter both the EL and LP cell suspensions through a 100 micromolar (µM) cell strainer.

SECTION: Ficoll-Paque
24. Layer 25 mL of cell suspension (both IE and LP fractions) on top of 15 mL of Ficoll-Paque Media PLUS.

SECTION: Ficoll-Paque
25. Spin for 20 min, 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).

SECTION: Ficoll-Paque
26. 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 10 min at 400 x g, 4 °C.

SECTION: Ficoll-Paque
27. Remove the supernatant and re-suspend the cell pellet in 50 mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 10 min at 120 x g, 4 °C.

SECTION: Ficoll-Paque
28. Remove the supernatant and re-suspend the cell pellet in cold 10 mL IMDM-FBS-PSQ Media.

SECTION: Cell Count
29. 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: _______
SECTION: Cell Count
30. 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: ________
SECTION: Freeze-down
31. (Optional QC) Aliquot 2 x 106 cells to a 5 mL Falcon tube and place on ice for subsequent flow cytometric analysis.

SECTION: Freeze-down
32. 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: __________.