TITLE: Isolation of cells from the epithelial layer of frozen human intestinal biopsies
AUTHORS: Oxford HCA, Nicholas Provine, Michael FitzPatrick, Sophie Irwin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for the isolation of the epithelial cells (and associated immune cells within the epithelial layer) from frozen human intestinal pinch biopsies, for use in single-cell sequencing or flow cytometry experiments. This protocol is adapted from Parikh, Antanaviciute, Fawkner-Corbett et al., (2019) Natuer (doi: 10.1038/s41586-019-0992-y). It has been modified specifically with the aim of increasing cell recovery from biopsies of the small intestine.</div></div>

[STEPS]
?. [Warming media]
Per sample: pre-warm 10ml of Transport medium in the waterbath.
?. [Warming media]
Per sample: pre-warm 20ml of chelation medium in the waterbath.
?. [Collection/Wash]
Partially thaw frozen biopsies in 37°C waterbath until only a little ice remains.
?. [Collection/Wash]
Dropwise add 1ml of pre-warmed transport media to the sample to thaw completely.
?. [Collection/Wash]
Use a pastette to transfer the biopsies into the conical of pre-warmed transport media.
?. [Collection/Wash]
Place a 70 micron filter onto a 50ml conical.
?. [Collection/Wash]
Transfer biopsies onto the filter.
?. [Collection/Wash]
Wash with 20ml of cold wash medium.
?. [Crypt isolation]
Transfer biopsies to 5ml warm chelation medium (in 50ml conical).
?. [Crypt isolation]
Incubate at 37°C for 10 mins (in incubator).
?. [Crypt isolation]
Vortex 2 x 5-10s.
?. [Crypt isolation]
Allow biopsies to settle. Remove biopsies from the chelation medium (containing potential isolated crypts) with FCS-coated pastette and transfer to fresh Falcon with 5ml warm chelation medium. Return the biopsies in fresh medium to 37°C incubator and repeat steps 10-12.
?. [Crypt isolation]
Store chelation medium with the isolated crypts at 4°C while proceeding with subsequent isolation steps.
?. [Crypt isolation]
Repeat steps 9-13 for a total of 4 times, and pool crypt containing fractions.
?. [Single cell suspention from isolated crypts]
Spin down the pooled crypt suspension (300 g, 5 mins).
?. [Single cell suspention from isolated crypts]
Resuspend in 3 ml warm TrypLE Express with 50 µg/ml DNAse at 37°C (75 µl of 2 mg/ml stock).
?. [Single cell suspention from isolated crypts]
Incubate 60 mins at 37°C in incubator shaking incubator (220 RPM).
?. [Single cell suspention from isolated crypts]
Filter with 70 µm cell strainer (prepped with FCS) into 50 ml Falcon, wash through with 5 ml of T5 media.
?. [Single cell suspention from isolated crypts]
Spin filtrate 1500 RPM 5mins.
?. [Single cell suspention from isolated crypts]
Wash pellet with 30ml T5 media.
?. [Single cell suspention from isolated crypts]
Spin 1500 RPM 5mins.
?. [Single cell suspention from isolated crypts]
Very carefully remove supernatant until less than 1ml remains. Resuspend pellet in this.
?. [Single cell suspention from isolated crypts]
Run through 30um cell strainer (prepped with FCS) and wash through with 5ml of PBS.
?. [Single cell suspention from isolated crypts]
Spin 1500 RPM 5 mins, re-suspend in approximately 1ml of remaining PBS, transfer to Eppendorf.
?. [Single cell suspention from isolated crypts]
Make up to 1ml with PBS and take 10 uL for count (1:1 with Trypan blue).
?. [Single cell suspention from isolated crypts]
For 10X single-cell sequencing, count each sample twice using countess to obtain average count and viability:
?. [Single cell suspention from isolated crypts]
Spin down in micro centrifuge (1500 RPM, 5 mins) and re-suspend using live count to concentration of 2000 cells/µl (2x10^6 cells/ml).
?. [Single cell suspention from isolated crypts]
re-check count and make volume to 1500 cells/µl (1.5x10^6 cells/ml) with PBS.
?. [Single cell suspention from isolated crypts]
Transfer to single cell facility on ice.
?. [Flow cytometry to confirm cell recovery]
Transfer epithelial cells to a round bottom 96 well plate.
?. [Flow cytometry to confirm cell recovery]
Spin plate at 2200RPM for 2 mins, and dispose of supernatant.
?. [Flow cytometry to confirm cell recovery]
Add 200 µl wash buffer, and spin at 2200 RPM for 1min x2.
?. [Flow cytometry to confirm cell recovery]
Remove supernatant and add 100 µl of FACS buffer.
?. [Flow cytometry to confirm cell recovery]
Add 1:100 EpCAM-PE (1 µl), 1:50 CD24-PE-CF594 (2 µl) and 1:100 CD45-AF700 (1 µl).
?. [Flow cytometry to confirm cell recovery]
Place in fridge for 20 mins.
?. [Flow cytometry to confirm cell recovery]
Spin plate at 2200 RPM for 1 min, and dispose of supernatant.
?. [Flow cytometry to confirm cell recovery]
Add 200µl wash buffer, and spin at 2200 RPM for 1min x2.
?. [Flow cytometry to confirm cell recovery]
Resuspend in 300µl of FACS buffer and add 1:100 (3 µl) of diluted Sytox Green.
?. [Flow cytometry to confirm cell recovery]
a.1:60 (5 µl Sytox Green + 295 µl FACS buffer) – final dilution 1:6000.
?. [Flow cytometry to confirm cell recovery]
10.Transfer to tubes and run through FACS.