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We have tested several storage solutions for transportation, such as advanced DMEM, Wisconsin solution, and HypoThermosol® FRS Preservation Medium. For short-term storage/shipment of tissue biopsies, we recommend immersing the sample in pre-cooled (2 - 8°C) HypoThermosol® FRS so that the tissue may be completely bathed in solution.

We have initiated and established human hepatic organoid cultures from 16 biopsy samples.

Pre-wetting (during the initial sample processing) and passaging is good practice, therefore, you may choose to include this as part of your processing. Pre-wetting can be done either using AdvDMEM/DMEM+1% BSA or Anti-Adherence Rinsing Solution (recommended for the mouse HepatiCult™ workflow).

We have received biopsies in the range of 20 g - 700 mg, therefore, there can be some variation in the size of the biopsy. If the tissue weighs less than 1 g, there is no need to scale down. If it is more than 1 g, the volume of the tissue digestion cocktail can be adjusted to ensure optimal digestion. For example, if starting with 2 g, double the volume of the enzymatic solution used.

Although this varies from sample to sample, as well as by user, we expect 2 - 3 g of liver tissue to be enough to initiate 70 wells of hepatic organoids (in our experience, more tissue does not necessarily mean more wells). It really depends on the efficiency of the digestion. We find that smaller biopsies are more manageable to get the maximal surface area for digestion.

Organoid yield is not correlated to tissue sample size. We have used tissue weighing < 0.5 g and were able to successfully establish human hepatic organoids.

Polypropylene tubes (e.g. Falcon® Conical Tubes, 15 mL and Falcon® Conical Tubes, 50 mL) are recommended to minimize cell adhesion to plasticware.

Skipping filtration with a 100 µm reversible cell strainer may result in dirtier or denser cultures. Filtration helps clean up the prep and remove any larger ductal material from the tissue preparation. You have the option of working with the flow-through and therefore smaller fragments/single cells, working with the ductal material (flip over the strainer and wash it out), or alternatively seeding both fractions (the flow-through and the larger ductal material caught on the strainer). Ultimately, it will be up to you to decide what material you want to work with. Depending on whether you are starting with single cells or ducts, you will notice differences in growth.

RBC contamination can affect the integrity of the domes depending on how significant it is. The contamination can be seen as "red cells" in the domes. Larger tissue pieces often yield very red pellets and can result in the seeding of very crowded or dense domes without RBC lysis. Therefore, we suggest including the ACK lysis step, if the pellet looks red.

We have not tested or validated any other dissociation reagents. If using a different dissociation reagent, some optimization in terms of the incubation time/reagent strength might be required.

There are two reasons for seeding at higher densities:

• Not every single cell or fragment will develop into an organoid; seeding at higher densities gives a better chance of establishing organoids.
• Liver organoids are much happier when they have "neighboring" or "surrounding cells". There is a lot of paracrine signaling that is advantageous for these organoids.

It is also important to not overseed as this can negatively affect the quality and viability of the cultures. Therefore, it is important to find an optimal seeding density.

We recommend optimizing differentiation protocol parameters for each donor and assay of interest. Please refer to the HepatiCult™ Technical Manual for recommended ranges to test.

In general, for differentiation experiments, seeding ~2000 fragments between 30 - 100 µm in size and expanding these in HepatiCult™ Organoid Growth Medium (OGM) for 5 days before switching to HepatiCult™ Organoid Differentiation Medium (ODM) works well.

Yes, human hepatic organoid cultures can be passaged and maintained long-term. Figure 4 on the Product Landing Page shows the expansion of hepatic organoids in HepatiCult™ Organoid Growth Medium (OGM) across multiple donors and passages with potential for indefinite culture. We typically maintain our cultures up to passage 14 - 15.

Differentiated cultures cannot be passaged or cryopreserved.

Working with the Costar® 24-Well Flat-Bottom Plate, Tissue Culture-Treated (Catalog# 38017) is recommended (see the Materials section in the HepatiCult™ Technical Manual). We have found that this plate type is ideal for plating domes that don't spread or flatten out, which is important for optimal organoid culture.

We do not recommend using an incubator to thaw organoids. The rate at which the sample is warmed and therefore thawed will be much slower in an incubator than if using a water bath, and prolonged thawing times can decrease cell viability and recovery efficiency.

When thawing organoids in a water bath, it will also be important to keep an eye on your sample and to transfer it to the biosafety cabinet for subsequent steps when there is a small piece of ice remaining in the vial—this typically thaws as soon as thaw or wash buffer is added to the vial.

Heterogeneity in organoid size is normal, especially if the fragments generated for passaging are not uniform in size. We aim to generate fragments between 30 - 100 µm in size and this helps standardize organoid size. If this has proven to be challenging, using reversible strainers can help generate more uniformly sized fragment suspensions.

Ductal marker expression is observed in ODM organoids but at lower levels than in OGM organoids. ODM is designed to mature organoids to a hepatocyte fate. Further characterization needs to be done to comment on hepatocyte vs cholangiocyte fate on a per-organoid or per-cell level.

CYP3A4 activity has been captured with the P450-Glo™ CYP3A4 Assay from Promega (Catalog #V9001) and we follow the manufacturer's instructions to generate the RLU values. We normalize these values to ng of RNA.

Albumin was analyzed in the supernatant of our organoid dome cultures using the Abcam albumin ELISA kit (Catalog #ab108788) by following the manufacturer’s instructions. We use “ng of RNA” as a way to normalize the albumin measurements to the number of cells used in the assay, hence the data is represented as (pg Albumin) / (ng RNA). In our assays, we normally use the same culture wells to do our ELISAs and then isolate RNA, so this is the most convenient normalization approach for us.

We have looked at albumin and A1AT secretion, total bile acid production, urea synthesis, and CYP3A4 activity, and confirmed that these functions are present in differentiated hepatic organoids (Figure 9 of the Product Landing Page).

Section 7.0 of our human HepatiCult™ describes a protocol for culturing the hepatic organoids in 96-well plates. We have used this 96-well protocol to perform toxicity screens in-house and found that differentiated hepatic organoids exhibited increased sensitivity to ketoconazole-, troglitazone-, and rifampicin-induced hepatotoxicity when compared to undifferentiated organoids, HepG2 cells and primary human hepatocytes (PHH).

The following protocol describes a hepatotoxicity screening assay using human liver organoids grown with the HepatiCult™ Organoid Kit (Human): How to Perform Cytotoxicity Screening Using Hepatic Organoids Cultured in HepatiCult™ (Human).

Please refer to this (Figures 5 - 8) for data that compares hepatotoxic responses of human liver organoids to HepG2 across different pharmaceutical compounds. For some compounds, comparison to PHHs was included.

We have not looked for the presence of non-parenchymal cells in human hepatic organoids.