Human Hepatic Organoids

Transcription

1 Human Hepatic Organoids

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3 Product Information Catalog. No. Product Name Format ax41163 Human Hepatic Organoids Stock Conc. Storage on Arrival 1 cryopreserved vial N/A Liquid Nitrogen Thawing Instructions Follow protocol Storage Once Thawed N/A Additional Reagents Item Supplier SKU Culture Plate Greiner Bio-one Centrifuge tube Greiner Bio-one ROCK inhibitor Y ax68168 TrypLE Express 1X Thermo Fisher (w/o Phenol Red)

4 Recommendations Strict adherence to this protocol will allow optimal Human Hepatic Organoid expansion. Human Hepatic Organoids are cryo-preserved using an optimal organoid cryoprotectant in a controlled freezing environment. All vials are standardized to 1,000 hepatic organoid fragments per vial. A maximum number of 5 passages is recommended. ECM recommendations are available upon request. The recommended organoid fragment seeding density is 500 organoid fragments per well. The recommended maximum culture period for Human Hepatic Organoids is days. Human Hepatic Organoids can be stored at -150 C for 3 months. The recommended differentiation time period for Human Hepatic Organoids is 10 days. Stem cell marker LGR5, ductal marker SOX9, and hepatocyte marker HNF4α gene expression markers are used to characterize the Human Hepatic Organoids. The hepatic function of differentiated hepatic organoids has been tested. Albumin, CYP3A4 expression and function have been confirmed in differentiated hepatic organoids. Human Hepatic Organoids Adult liver stem cells cultured within an extracellular matrix self-assemble to form 3D organoids, and when exposed to appropriate culture conditions, differentiate into functional hepatocytes. These organoids are available cryopreserved, ready for revival into 3D cell culture conditions. Content provided is suitable for re-embedding into 3 wells of a 24 well plate. These features make the organoids a novel tool for use in in vitro hepatotoxicity analysis. Organoids display ductal marker SOX9 and hepatocyte marker HNFα and show increased expression of functional hepatocyte markers ALB and CYP3A4 following differentiation. Derived from stem cells, the organoids are self-renewing and proliferative, allowing further expansion to meet the needs of your study. Their physiological responses are retained from their tissue of origin which provides relative toxicological data. Applicable in multiple bioscience applications these organoids are a valuable alternative to hepatic cell lines and primary hepatocytes.

5 Preparation for Thawing & Plating Thaw the selected extracellular matrix (ECM) on ice until liquid and pre-chill sterile pipette tips for ECM handling at 2-8 C. Pre-warm a 24-well plate at 37 C for at least 30 minutes. Prepare and pre-warm a 13 ml volume of hepatic wash medium at 37 C in a sterile 15 ml conical centrifuge tube. Thaw Prepare a sufficient volume Thawing Remove the vial of human hepatic organoids from liquid nitrogen storage and quickly place the cryovial in a sterile cell culture water bath set at 37 C. The cryovial of human hepatic organoids is ready for processing when liquid and a small ice crystal can be observed. NOTE: Extended thawing of frozen organoids can affect the survival of the thawed organoids in culture. To prepare the hepatic organoids for transfer, wipe the outside of the cryovial with 70% ethanol or isopropanol. Transfer the thawed organoids to the pre-warmed hepatic wash medium. Rinse the inside of the cryovial with 1 ml of hepatic wash solution and add to the tube containing thawed organoids. Centrifuge the tube at 200 x g for 5 minutes. Carefully aspirate all the supernatant and discard. Place the tube containing the cell pellet of thawed organoids on ice. Using pre-chilled pipette tips, gently resuspend the cell organoids in 120 µl of ECM. Immediately dispense 35 µl of organoid/ecm as a droplet in the center of each well of the pre-warmed 24-well plate. NOTE: This dispensing step must be performed in a proficient manner to prevent solidifying of the ECM/organoid mixture in the tube. Proficient dispensing will also ensure avoidance of air bubble generation within the ECM of the 3D culture. Transfer the plate to an incubator for 15 minutes at 37 C until the ECM has completely solidified. Remove the plate from the incubator and place it in a Class II Biosafety cabinet. Carefully add 500µl f Hepatic Organoid Expansion Media (components listed at the end of this protocol) Add 500 µl of sterile PBS to any unused cells. Incubate the 24-well plate containing the 3D organoid/ecm cultures at 37 C and 5% CO2. NOTE: Monitoring organoid growth is recommended via bright field microscopy imaging every 2-3 days. Perform a full medium change every 2 days until organoid culture is at the required density.

6 Culturing Pre-warm a 24-well plate at 37 C for 30 minutes. Prepare a sufficient volume of Hepatic Organoid Expansion Media Thaw the ECM on ice until it becomes liquid and pre-chill sterile pipette tips for ECM handling at 2-8 C. Pre-wet a 15 ml conical centrifuge tube with 2 ml of hepatic wash solution at 2-8 C, keeping the tube on ice. NOTE: Pre-chill a centrifuge to 8 C before proceeding with passaging of organoid cultures. It is recommended that all passaging work is performed at 2-8 C. To prevent hepatic organoids from adhering to the pipette tip and decreasing the organoid yield, pre-wet pipette tips with a sterile 1% BSA solution. Carefully aspirate and discard the spent medium from each well to be passaged. Add 1 ml of chilled hepatic wash medium to each well and disrupt the ECM droplet containing hepatic organoids by vigorously pipetting 5-10 times. NOTE: Care must be taken to avoid generation of air bubbles within the ECM/organoid suspension. If pooling multiple organoid/suspension(s), no more than 3 wells (24 well plate) should be combined, to maintain efficient washing of the hepatic organoids. Transfer the 1 ml organoid/ecm suspension to the pre-wetted 15 ml conical centrifuge tube and add 13 ml of chilled hepatic wash medium. Gently mix by pipetting the solution three to five times to disperse the ECM. Centrifuge the tube at 200 x g for 5 minutes at 8 C. Carefully aspirate the supernatant so no liquid remains, and the pellet is undisturbed. Using pre-chilled pipette tips, resuspend the cell aggregates in 1 ml of TrypLE Express 1X (w/o Phenol Red) supplemented with 10 µm RHO/ROCK pathway inhibitor. Incubate the suspension for 2 minutes at 2-8 C. To de-activate the TrypLE Express, add 1 ml of hepatic wash medium and vigorously pipette. NOTE: It is recommended that the pipetting be carried out using a 200 µl pipette tip. To calculate an organoid fragment seeding density, dispense 3 x 10 µl of fragment suspension into an empty well of a 6-well plate to create 3 separate droplets. Using a light microscope, count the number of hepatic organoid fragments in each 10 µl droplet and determine the average number of organoid fragments across all organoid suspension(s), i.e. total number of organoid fragments / 3 = average number of organoid fragments per 10 µl. NOTE: Single cells are not included in the organoid fragment counting. Determine the total number of fragments in 2 ml volume i.e. average number of organoid fragments per 10 µl x 200. Add a further 7 ml of hepatic wash medium to the 15 ml conical tube and centrifuge the tube at 200 x g for 5 minutes at 8 C. Carefully aspirate the supernatant without disturbing the resultant pellet. NOTE: it is recommended that when removing this supernatant, a 5-10 µl volume of supernatant should remain on the pellet to prevent damage to the organoid/ecm suspension. Using pre-chilled pipette tips, gently resuspend the cell organoids in 120 µl of

7 ECM. Immediately dispense 35 µl of organoid/ecm as a droplet in the center of each well of the pre-warmed 24-well plate. NOTE: This dispensing step must be performed in a proficient manner to prevent solidifying of the ECM/organoid mixture in the tube. Proficient dispensing will also ensure avoidance of air bubble generation within the ECM of the 3D culture. Transfer the plate to an incubator for 15 minutes at 37 C until the ECM has completely solidified. Remove the plate from the incubator and place it in a Class II Biosafety cabinet. Carefully add 500 µl of Hepatic Organoid Expansion Media. Add 500 µl of sterile PBS to any unused cells. Incubate the 24-well plate containing the 3D organoid/ecm cultures at 37 C and 5% CO2. NOTE: Monitoring organoid growth is recommended via bright field microscopy imaging every 2-3 days. Perform a full medium change every 2 days until organoid culture is at the required density. Seed new hepatic organoid cultures at 500 organoid fragments per 35 µl of organoid/ecm suspension. Using a split ratio of 1:2-1:3, human hepatic organoid cultures are typically ready to be passaged every 4-6 days after seeding (user discretion advised). NOTE: adjust the ECM and Hepatic Organoid Expansion Media according to the required number of wells. Pathogen Testing Samples from each donor are tested via PCR and found non-reactive to viral DNA from HIV and Hepatitis B, and viral RNA from Hepatitis C. However, since no known test can offer complete assurance, please treat the culture as a potentially infectious agent. Product Warranty Ltd. warrants the performance of cells only if the recommended media/reagents are used and the recommended protocols are followed. Cryopreserved cells are assured to be viable when thawed according to the recommended protocol on recommended culture ware. Usage Statement: Our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans.

8 Recommended Medium Formulations Hepatic Organoid Expansion Media. 50ml is sufficient for initiation and expansion up to second passage. Component Supplier Product Code Final Conc. Human Recombinant R-Spondin-1 ax µg/ml Y (hydrochloride) ROCK inhibitor ax µm Normocin Invivogen Ant-nr µg/ml N-Acetylcysteine AKT Labs A mm Nicotinamide mm [Leu]15-Gastrin1-stock G nm Human Recombinant EGF ax ng/ml Human Recombinant FGF-10 ax ng/ml Human Recombinant HGF ax ng/ml Forskolin Tocris Bio µm A83-01 SML µm B27 without Vitamin A X N2 Supplement X Advanced DMEM/F N/A HEPES Fisher Bioreagents BP mm Glutamax X Pen/Strep P U/ml Penicillin, 100 µg/ml Streptomycin Human Hepatic Organoids Differentiation Medium Component Supplier Product Code Final Conc. N-Acetylcysteine AKT Labs A mm [Leu]15-Gastrin1-stock G nm Human Recombinant EGF ax ng/ml Human Recombinant HGF ax ng/ml A83-01 SML µm DAPT D µm Dexamethasone D µm BMP-7 ax ng/ml Human Recombinant FGF-19 R&D Systems 969-FG ng/ml B27 without Vitamin A X N2 Supplement X Advanced DMEM/F N/A HEPES Fisher Bioreagents BP mm Glutamax X Normocin Invivogen Ant-nr µg/ml Pen/Strep P U/ml Penicillin, 100 µg/ml Streptomycin

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10 Got any questions? Need help with the protocol? Contact Axol Technical Support at Or call +44 (0)