Jul 14, 2020
Version 2
Duke - Isolation, Culture, and Maintenance of Patient-Derived Tumor Biopsy V.2
- Xiling Shen1,
- Marcos Negrete1,
- Kun Xiang1
- 1Duke University, Department of Biomedical Engineering
- NCI PDMC consortium
- Organoid and Assembloid
Protocol Citation: Xiling Shen, Marcos Negrete, Kun Xiang 2020. Duke - Isolation, Culture, and Maintenance of Patient-Derived Tumor Biopsy. protocols.io https://dx.doi.org/10.17504/protocols.io.bijikcke
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 14, 2020
Last Modified: December 14, 2020
Protocol Integer ID: 39242
Abstract
This protocol is split into 3 sections: collecting tumor cells, passaging, and cryopreserving organoids.
Phase 1:
Aim:Collect viable cells from biopsy samples
Tumor organoid: Colorectal Cancer
Source: Human primary tissue
Phase 2:
Aim: Passage and expand organoid samples
Phase 3:
Aim: Organoid cryopreservation
Tumor organoid: Colorectal Cancer
Source: Human primary tissue
Materials
MATERIALS
PBS
HEPESFisher ScientificCatalog #BP310-500
FBSInvitrogen - Thermo Fisher
MACS 15 mL Tube RackMiltenyi BiotecCatalog #130-091-052
CollagenaseSigmaCatalog #C5138
Cell strainer 70um filterFalconCatalog #352350
HBSSGibco - Thermo FischerCatalog #14060040
B-27™ Supplement (50X), minus antioxidantsThermo FisherCatalog #10889038
Externally and Internally Threaded Cryogenic Storage VialsThermo FisherCatalog #12567501
Advanced DMEM/F-12Thermo FisherCatalog #12634010
Recovery™ Cell Culture Freezing MediumThermo FisherCatalog #12648010
Antibiotic-Antimycotic (100X)Thermo FisherCatalog #15240062
L-GlutamineThermo FisherCatalog #21051040
Cryogenic Box Divider, vertical, 2mL with 100-cellsThermo FisherCatalog #4000014
N-Acetyl-L-cysteineSigma AldrichCatalog #A9165
Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane MatrixCorningCatalog #356231
Rock Inhibitor Y-27632 Dihydrochloride TocrisCatalog #1254
N2 supplement (100x supplement)Gibco, ThermoFisherCatalog #17502048
Razor blades (brand new)
Ice
Petri Dish (100X15 mm)
15 ml tube
Ethanol 70%
Tweezers
Ice
Ethanol 70%
Biosafety cabinet
Basal media (L-glutamine, HEPES, B27, N2, AA, NACE)
Basal media (L-glutamine, HEPES, B27, N2, AA, NACE) human (1mM), A-83-01 (500 uM), Rock inhibitor Y-27631 (10 mM), SB 202190 (30 mM))
Basal Media Formulation
Formulation of Basal Media + Small Molecules
Small Molecule Preparation
Transport Media Formulation
Cell Digestion Solution
Disassociation of Tumor Cells
Disassociation of Tumor Cells
Store tissue samples in cold transport media (10 mL ). Keep samples on ice at all time and process within 00:45:00
Transfer the tumor biopsy sample (1-2 cm3) and transport media into a petri dish and remove remnant non-tumor tissue with sterile tweezers.
Note
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)
Transfer the tumor tissue to a new petri dish and cut the sample into small pieces with a sterile razor blade (<2 mm2).
Add 5 mL 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.
Allow the tissue fragments to settle by gravity for 00:01:00 On ice , remove and discard the supernatant
Resuspend the tissue fragments in 5 mL HBSS buffer plus Collagenase and transfer to a fresh 15 ml tube.
Place the tube in
Equipment
MACSmix™ Tube Rotator
NAME
Miltenyi Biotec
BRAND
130-090-753
SKU
LINK
and incubate the enzymatic digestion at37 °C for 01:30:00 .
Note
The time of cell digestion will affect the cells yield and the end of the protocol. Make sure to incubate for enough time
Quench the enzymatic digestion by adding 1 mL of cold, sterile FBS to the reaction and proceed immediately to plating for organoid culture.
Plating Cancer Cells for 3D Organoid Culture
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.
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.
Wash the cells twice in 5 mL 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.
Resuspend the cell pellet in 1 ml basal media and determine the cellular yield using a hemocytometer.
For instructions on using hemocytometer:
Hemocytometer Protocol.docx Transfer the desired cell sample to a fresh tube and 2000 x g, Room temperature, 00:07:00 to pellet the cells. Discard the supernatant. Do not remove the pellet.
Note
The following steps are for plating 4 x 50 µL culture domes. If fewer or additional culture domes are required based on the counts in step 5, adjust the volume of Matrigel and PBS.
Thoroughly resuspend the cells in 40 µL of cold PBS by pipetting up and down 10 times. Avoid introducing bubbles.
Add 160 µL of Matrigel to the cell solution and mix by pipetting up and down 10 times. Avoid introducing bubbles.
Note
Keep the Matrigel On ice all the time, at room temperature will start to polymerization
Gently plate 50 µL domes of the Matrigel-cell suspension in the center of the 4 central wells in the pre-warm 24 well flat bottom plate.
Carefully transfer the plate to 37 °C incubator and incubate for at least 00:30:00 to allow domes to solidify (polymerize).
Gently add 1 mL of basal media + small molecules to each well by pipetting the media gently down the wall of the well.
Optional: Add sterile PBS to the unused wells of the 24 well plate to limit evaporation.
Place the lid on the culture plate and return it to the tissue culture incubator.
Perform a full media change every 48:00:00 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
Passaging Cancer Organoids
Note
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.
Carefully remove media from each well.
Gently add 1 mL cold PBS to each well by pipetting the PBS gently down the wall of the well.
Carefully remove and discard PBS from each well.
Add 1 mL of accumax on the top of the dome in each well and perform mechanical detach from the bottom (gently scrape).
Carefully transfer the plate to 37°C incubator. Incubate at 37 °C for 00:10:00
Collect and transfer the accumax-cell suspension to a 15 ml tube.
Add 2000 µL of FBS to get 10% FBS and pipette up and down the accumax-cell suspension at least 10 times.
Centrifuge the sample for 200 x g, Room temperature, 00:05:00 to pellet the cells. Do not remove the pellet.
Remove the 24 well flat bottom plate from the 37 °C incubator.
Add 40 µL of PBS to the sample tube. Pipette up and down 10 times to thoroughly resuspend the pellet. Avoid introducing bubbles.
Add 160 µL of Matrigel to the sample tube. Pipette up and down 10 times to thoroughly resuspend the pellet. Avoid introducing bubbles.
Gently add 50 µL 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.
Carefully transfer the plate to 37°C incubator. Incubate at 37 °C for at least 00:30:00 to allow domes to solidify (polymerization).
Gently add 1 mL of conditioned media to each well by pipetting the media gently down the wall of the well.
Place the lid on the culture plate and incubate at 37 °C and 5% CO2.
Every 2 days perform a full media change.
Cryopreserving Cancer Organoids
Cryopreserving Cancer Organoids
Carefully remove media from each well and add 1 ml of recovery cell culture freezing medium per well.
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.
Place each tube in a freezing container and incubate the tubes at -80 °C for at least 1 day.
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
Recovery of Frozen Organoids
Remove the cryovials from storage and thaw them quickly in a 37 °C water bath.
Collect the organoids with a 1,000 µl pipette into a 15-ml centrifuge tube.
Add 10 mL of basal media and spin the organoids down at 200 x g, 4°C, 00:05:00
Remove and discard the supernatant and suspend the organoids with Matrigel.
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.
Gently add 50 µL of Matrigel-cell suspension in 4 central wells of a pre-warm 24 well plate.
Carefully transfer the plate to 37°C incubator. Incubate at 37 °C for at least 00:30:00 to allow domes to solidify (polymerization).
Gently add 1 mL of conditioned media to each well by pipetting the media gently down the wall of the well.
Place the lid on the culture plate and incubate at 37°C and 5% CO2.
Every 2 days perform a full media change.