Dissociation of fresh colorectal biopsies

Hydrate, aliquot, and freeze 2x DNAse

Hydrate to 5mg/ml in DPBS; keep cold; aliquot 1ml each into 2ml tubes and freeze immediately.

Store at -20˚c and use within 6 months.

Hydrate, aliquot, and freeze 20x Cold protease

Hydrate to 100mg/ml in molecular grade H2O; keep cold; aliquot 100ul each into 2ml tubes and freeze immediately.

Store at -20˚c and use within 6 months.

Dilute to 0.1M, aliquot, and freeze stock DTT

Dilute the 1M stock to 0.1M by adding 1 part 1M DTT to 9 parts Molecular bio grade H2O. Mix well and aliquot at 1-2ml each. Freeze and store at -20˚c for up to 6months. Use 0.1M aliquots within 1 week of thawing.

On the day of tissue reciept, prepare chelation buffer:

A	B
DPBS	49.35ml
0.5M EDTA	400µl
0.1M DTT (diluted from 1M stock)	250µl

Upon reciept, inspect tissue and record information such as:

Accession \# and other relevant collection metadata

Size - this can be as simple as drawing a spot the size of the tissue with description (flat, round, etc.)

Appearance

Presence of blood

Weight - in the interest of moving forward and not drying out a sample, we do not weigh tissue. Our samples are typically 20 - 40mg or less 

If tissue is solid/pedunculated, it may need to be bisected or trisected to increase surface area, do not cut smaller than 2mm^2.

Wash tissue gently in DPBS to remove any debris or excess transport media.

Move tissue to a 2ml tube containing ~1.8ml of chelation buffer and place on a tube rotator with continuous rotation.

Equipment

Value	Label
Labquake Rotisserie	NAME
Tube Rotator	TYPE
Thermo Scientific	BRAND
TS-4002110Q	SKU
https://www.fishersci.com/us/en/home.html	LINK

Gently replace chelation buffer on sample at 30 minutes and 1 hour into chelation, ensuring that enough air remains in tube to displace (agitate) tissue during rotation

Prepare DNAse for use before chelation is over by adding 1ml of DPBS to a frozen 1ml aliqout of 2x DNAse and leaving on bench to thaw. Move DNAse to Ice after it has thawed.

Gently remove chelation buffer and re-suspend tissue in DPBS.

Quickly prepare Dissociation solution by adding 1.9ml of 1x DNAse to a frozen 100µl aliquot of 20x cold Protease. Mix by pipetting.

Gently remove DPBS from tissue and resuspend in 1.8ml of Dissociation solution. Make sure there is enough air in the tube to displace the tissue on inversion.

Place on a tube rotator with continuous rotation.

Equipment

Value	Label
Labquake Rotisserie	NAME
Tube Rotator	TYPE
Thermo Scientific	BRAND
TS-4002110Q	SKU
https://www.fishersci.com/us/en/home.html	LINK

At 25 minutes (end of dissociation) triturate using a normal p-1000 tip 5-10 times, such that tissue is deformed as it enters the tip to yield material into the supernatant, and clusters are broken apart. The supernatant should become somewhat cloudy with cells. Inspect supernatant on a hemocytometer. Cells shoud be mostly single or in clusters of 10 or less. There should be at least 100K cells total. If tissue does not adequately yield cells, continue incubation for another 5 minutes before triturating again.

Pipette cells to mix and re-suspend before transfering onto a 70µm filter on a clean tube in a cold rack. Allow cells to filter through by gravity and check filtrate for cells before proceeding.

Spin cells at 700xg for 5 minutes to pellet.

700rcf,4-6°C

Ensure that cells have visibly pelleted before removing supernatant. Resuspend in DPBS using a wide-bore p-1000 tip. If cells do not pellet, you may need to increase speed by 100xg. As cells continue to die and secrete material, you may observe stringy aggregates in the supernatant or pellet. Ideally this material remains in the supernatant and is removed during washes, however if persists and becomes too large it may prevent cells from pelleting.

Repeat the previous 2 steps (wash) twice, checking cell number and quality upon resuspension.

On the 3rd resuspension into DPBS (or single cell running buffer), gently resuspend cells at roughly 2x the desired final concentration or greater and pass through a 70µm flowmi filter into a clean tube.

Check cells, dilute as needed and proceed to encapsulation.