TITLE: Duke - Isolation, Culture, and Maintenance of Patient-Derived Tumor Biopsy
AUTHORS: Xiling Shen, Marcos Negrete, Kun Xiang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is split into 3 sections: collecting tumor cells, passaging, and cryopreserving organoids. </div><div class = "text-block">Phase 1: </div><div class = "text-block"><span style = "font-weight:bold;">Aim:</span><span>Collect viable cells from biopsy samples </span></div><div class = "text-block"><span style = "font-weight:bold;">Tumor organoid: </span><span>Colorectal Cancer</span></div><div class = "text-block"><span style = "font-weight:bold;">Source: </span><span>Human primary tissue </span></div><div class = "text-block">Phase 2:</div><div class = "text-block"><span style = "font-weight:bold;">Aim: </span><span>Passage and expand organoid samples</span></div><div class = "text-block">Phase 3:</div><div class = "text-block"><span style = "font-weight:bold;">Aim: </span><span>Organoid cryopreservation</span></div><div class = "text-block"><span style = "font-weight:bold;">Tumor organoid: </span><span>Colorectal Cancer</span></div><div class = "text-block"><span style = "font-weight:bold;">Source: </span><span>Human primary tissue</span></div></div>

[STEPS]
?. [Disassociation of Tumor Cells]
Store tissue samples in cold transport media (). Keep samples on ice at all time and process within
10 mL
?. [Disassociation of Tumor Cells]
Transfer the tumor biopsy sample (1-2 cm3) and transport media into a petri dish and remove remnant non-tumor tissue with sterile tweezers.
The size of the tissue sample will affect the cells yield and the end of the protocol. Make sure to get enough tissue sample (~ 1-2cm3)
?. [Disassociation of Tumor Cells]
Transfer the tumor tissue to a new petri dish and cut the sample into small pieces with a sterile razor blade (2).
?. [Disassociation of Tumor Cells]
Add  of cold sterile PBS to the dish and transfer the tumor fragments and PBS to a 15 ml centrifuge tube, pipetting up and down for three times.
5 mL
?. [Disassociation of Tumor Cells]
Allow the tissue fragments to settle by gravity for , remove and discard the supernatant
on ice
?. [Disassociation of Tumor Cells]
Resuspend the tissue fragments in  HBSS buffer plus Collagenase and transfer to a fresh 15 ml tube.
5 mL
?. [Disassociation of Tumor Cells]
Place the tube in  and incubate the enzymatic digestion at for .
37 °C
The time of cell digestion will affect the cells yield and the end of the protocol. Make sure to incubate for enough time
?. [Disassociation of Tumor Cells]
Quench the enzymatic digestion by adding of cold, sterile FBS to the reaction and proceed immediately to plating for organoid culture.
1 mL
?. [Plating Cancer Cells for 3D Organoid Culture]
Warm up a 24-well flat bottom plate in the incubator and cool the 10% FBS solution on ice.
?. [Plating Cancer Cells for 3D Organoid Culture]
Strain the quenched enzyme solution through a 70 um cell strainer into a 15 ml tube using a reducing adaptors assembly. Use one cell strainer, reducing adaptor and tube per sample.
?. [Plating Cancer Cells for 3D Organoid Culture]
Wash the cells twice in  of 10% FBS by pipet up and down at least 10 times then centrifuging the sample for 3 minutes at 2000g to pellet the cells.
5 mL
?. [Plating Cancer Cells for 3D Organoid Culture]
Resuspend the cell pellet in 1 ml basal media and determine the cellular yield using a hemocytometer. For instructions on using hemocytometer:
?. [Plating Cancer Cells for 3D Organoid Culture]
Transfer the desired cell sample to a fresh tube and  to pellet the cells. Discard the supernatant. Do not remove the pellet.
Centrifuge: 2000 34, 7 min
The following steps are for plating 4 x   culture domes. If fewer or additional culture domes are required based on the counts in step 5, adjust the volume of Matrigel and PBS.
?. [Plating Cancer Cells for 3D Organoid Culture]
Thoroughly resuspend the cells in  of cold PBS by pipetting up and down 10 times. Avoid introducing bubbles.
40 µl
?. [Plating Cancer Cells for 3D Organoid Culture]
Add  of Matrigel to the cell solution and mix by pipetting up and down 10 times. Avoid introducing bubbles.
160 µl
Keep the Matrigel   all the time, at room temperature will start to polymerization
?. [Plating Cancer Cells for 3D Organoid Culture]
Gently plate  domes of the Matrigel-cell suspension in the center of the 4 central wells in the pre-warm 24 well flat bottom plate.
50 µl
?. [Plating Cancer Cells for 3D Organoid Culture]
Carefully transfer the plate to  incubator and incubate for at least  to allow domes to solidify (polymerize).
37 °C
?. [Plating Cancer Cells for 3D Organoid Culture]
Gently add  of basal media + small molecules to each well by pipetting the media gently down the wall of the well.
1 mL
?. [Plating Cancer Cells for 3D Organoid Culture]
Optional: Add sterile PBS to the unused wells of the 24 well plate to limit evaporation.
?. [Plating Cancer Cells for 3D Organoid Culture]
Place the lid on the culture plate and return it to the tissue culture incubator.
?. [Plating Cancer Cells for 3D Organoid Culture]
Perform a full media change every  to expand organoids. Observe cancer organoids daily and replace the media every 2 days after cancer cell isolation. Proceed to the next phases (drug screening, passage organoids and cryopreservation) after 4 or 5 days.
?. [Passaging Cancer Organoids]
Carefully remove media from each well.
Passage must be performed between 7 or 10 days depending on organoid size and density, passaging helps to avoid the organoid overgrowth and keeps the culture healthy and expanding.
?. [Passaging Cancer Organoids]
Gently add  cold PBS to each well by pipetting the PBS gently down the wall of the well.
1 mL
?. [Passaging Cancer Organoids]
Carefully remove and discard PBS from each well.
?. [Passaging Cancer Organoids]
Add  of accumax on the top of the dome in each well and perform mechanical detach from the bottom (gently scrape).
1 mL
?. [Passaging Cancer Organoids]
Carefully transfer the plate to 37°C incubator. Incubate at  for
37 °C
?. [Passaging Cancer Organoids]
Collect and transfer the accumax-cell suspension to a 15 ml tube.
?. [Passaging Cancer Organoids]
Add of FBS to get 10% FBS and pipette up and down the accumax-cell suspension at least 10 times.
2000 µl
?. [Passaging Cancer Organoids]
Centrifuge the sample for  to pellet the cells. Do not remove the pellet.
Centrifuge: 200 34, 5 min
?. [Passaging Cancer Organoids]
Remove the 24 well flat bottom plate from the incubator.
37 °C
?. [Passaging Cancer Organoids]
Add of PBS to the sample tube. Pipette up and down 10 times to thoroughly resuspend the pellet. Avoid introducing bubbles.
40 µl
?. [Passaging Cancer Organoids]
Add of Matrigel to the sample tube. Pipette up and down 10 times to thoroughly resuspend the pellet. Avoid introducing bubbles.
160 µl
?. [Passaging Cancer Organoids]
Gently add  of Matrigel-cell suspension in 4 central wells of a pre-warm 24 well plate. This protocol uses a 1:2 split ratio on passage organoid between 7 to 10 days after plating, or when the density reaches 150 organoids per well.
50 µl
?. [Passaging Cancer Organoids]
Carefully transfer the plate to 37°C incubator. Incubate at for at least  to allow domes to solidify (polymerization).
37 °C
?. [Passaging Cancer Organoids]
Gently add  of conditioned media to each well by pipetting the media gently down the wall of the well.
1 mL
?. [Passaging Cancer Organoids]
Place the lid on the culture plate and incubate at  and 5% CO2.
37 °C
?. [Passaging Cancer Organoids]
Every 2 days perform a full media change.
?. [Cryopreserving Cancer Organoids]
Carefully remove media from each well and add 1 ml of recovery cell culture freezing medium per well.
?. [Cryopreserving Cancer Organoids]
Scrape the Matrigel off the bottom of the wells with a 1,000 ul pipette and transfer the cancer organoids into a one cryovial. Label with date and tissue source or any other specifications.
?. [Cryopreserving Cancer Organoids]
Place each tube in a freezing container and incubate the tubes at for at least 1 day.
-80 °C
?. [Cryopreserving Cancer Organoids]
Transfer the frozen cryovials to a liquid nitrogen storage tank. Cancer organoids can be kept in liquid nitrogen storage for at least 3 years.
?. [Recovery of Frozen Organoids]
Remove the cryovials from storage and thaw them quickly in a  water bath.
37 °C
?. [Recovery of Frozen Organoids]
Collect the organoids with a 1,000 µl pipette into a 15-ml centrifuge tube.
?. [Recovery of Frozen Organoids]
Add  of basal media and spin the organoids down at
10 mL
Centrifuge: 200 34, 5 min, 4 10
?. [Recovery of Frozen Organoids]
Remove and discard the supernatant and suspend the organoids with Matrigel.
?. [Recovery of Frozen Organoids]
The appropriate volume of Matrigel depends on the number of the cancer organoids. In most cases splitting one vial (1000 µl) of preserved organoids to 4-6 wells of a 24 well flat bottom plate is enough.
?. [Recovery of Frozen Organoids]
Gently add  of Matrigel-cell suspension in 4 central wells of a pre-warm 24 well plate.
50 µl
?. [Recovery of Frozen Organoids]
Carefully transfer the plate to 37°C incubator. Incubate at  for at least  to allow domes to solidify (polymerization).
37 °C
?. [Recovery of Frozen Organoids]
Gently add  of conditioned media to each well by pipetting the media gently down the wall of the well.
1 mL
?. [Recovery of Frozen Organoids]
Place the lid on the culture plate and incubate at 37°C and 5% CO2.
?. [Recovery of Frozen Organoids]
Every 2 days perform a full media change.