�����ƽ��

The easiest method is to obtain a biopsy sample at the time of diagnosis. Alternatively, if a tumor is surgically removed, it can also be used as source material for creating organoids. For intestinal tissue, refer to our protocol to isolate human colonic crypts from intestinal biopsies for downstream intestinal organoid culture, as well as the product information sheet for ���Գٱ���پ���ܱ���™ Organoid Growth Medium (Human). To establish a hepatic organoid culture from liver tissue samples, refer to the technical manual for HepatiCult™ Organoid Kit (Human). If you do not have access to biopsy or tumor samples, biobanks, such as ATCC, NCI, and HUB Organoids, can provide you with relevant patient samples for your research.

As part of �����ƽ�� Technologies’ agreement with HUB Organoids, we have access to the HUB's biobank and can select both healthy and disease-derived tissue from this resource.

The viability of organoids in 3D Matrigel® cultures can be measured using the CellTiter-Glo® 3D Viability Assay (Promega Catalog #G9683), while monolayer culture viability can be assessed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega Catalog #G7570). For more information on how to measure cell viability as part of toxicity testing for drug development using intestinal organoids, please refer to our protocol "Toxicity Testing for Drug Development Using Human Intestinal Organoids and ���Գٱ���پ���ܱ���™".

For monolayer studies, dissociating the 3D organoids into single-cell suspensions (as much as possible) at the time of seeding is critical for good monolayer differentiation. We utilize this procedure that combines enzymatic dissociation (0.05% Trypsin ETDA) and mechanical disruption to achieve a cell suspension composed of single cells, or small fragments.

To establish large numbers of organoid monolayers, it is recommended to passage organoids from Matrigel® domes into a suspension culture 7 days prior to establishing monolayer cultures, as sufficient cell numbers are required to seed monolayers. This suspension method allows for the generation of a higher number of organoids in a smaller number of wells to avoid having to combine a large number of separate matrigel domes to seed large numbers of monolayer cultures. Once the organoids are harvested and seeded as monolayers using ���Գٱ���پ���ܱ���™ Monolayer Growth Medium (���Գٱ���پ���ܱ���™ Organoid Differentiation Medium [Human] + RHO/ROCK Pathway Inhibitor [Y-27632]), the organoids will begin to differentiate and become less proliferative. For more information, refer to section VI of How to Generate Human Intestinal Organoid-Derived Monolayers Using ���Գٱ���پ���ܱ���™ and Passaging Intestinal Organoids into Suspension Culture Using ���Գٱ���پ���ܱ���™.

When grown in a monolayer, the cells are confluent, polarized, and contain tight junctions. Many diverse cell types are present, including stem cells, enterocytes, and functional goblet cells. In my experience, more proliferative cells and stem cells tend to cluster, as do more differentiated cells. For more information, refer to our research poster on "".

Both are possible, but our internal characterization suggests that cytochrome P450 (CYP450) expression (a key marker for studying metabolism) is higher in organoids cultured in a differentiation medium (���Գٱ���پ���ܱ���™ Organoid Differentiation Medium [Human]) than those cultured in a growth medium because the CYP activity is found in the enterocytes, which are only present under differentiation conditions. Furthermore, air-liquid interface (ALI)-maintained monolayers, which require differentiation medium, have higher CYP450 activity than those maintained in the submerged state and therefore could be a beneficial model to study intestinal organoid metabolism.

In 3D culture, differentiated cultures can be maintained for up to 2 weeks. In the monolayer format, especially with ALI, cultures can be maintained for up to a few months.

To make any adjustments to the ���Գٱ���پ���ܱ���™ formulation itself, customers can work with our product and scientific support services to either help plan their experiments to work with our existing products, or to arrange for the manufacture of a custom batch of the medium that is modified to accommodate their experimental plan.

Organoid counting is conducted before seeding when the organoids are still in fragments. This process involves using an appropriate cell counting instrument to standardize seeding densities. Accurate seeding is crucial for proper dome and monolayer formation and differentiation. During the dissociation of organoids for seeding, creating a suspension of single cells and small clusters enhances the uniform formation of domes or monolayers. Typically, achieving a confluent monolayer within two days of seeding is necessary to ensure proper differentiation.

Once the organoids are differentiated using ���Գٱ���پ���ܱ���™ Organoid Differentiation Medium in either 3D or 2D ALI, we see a mix of intestinal stem cells, transit-amplifying cells, enterocytes, goblet cells, and enteroendocrine cells in roughly physiological proportions. Paneth cells may appear, as well as other rare cell types, such as tuft or microfold cells, but may require external stimuli to promote differentiation and maturation.

Yes they can, however, these differences are only those that might be intrinsic to the epithelial cells themselves.

When using marker molecules to assess barrier integrity, versus a cell viability readout (i.e. CellTiter-Glo®) to assess cytotoxicity, the effect on marker molecule permeability is typically observed at concentrations lower than those that affect viability. This is true for both the Caco-2 and organoid lines, even though the relative sensitivity of Caco-2 and organoids can be quite different and is compound-dependent.

Consistent well-to-well variability can be a challenge especially when working with HTS formats and small Matrigel® domes. We have found that achieving a homogeneous suspension of organoid fragments that is periodically resuspended facilitates consistency. Consistent placement and volumes during Matrigel® spotting will also reduce variability (and may be assisted by automation).

The IC50 of control compounds in our assays depends on the type of readout and mechanism of action of the compound in question, as discussed in our webinar, and thus estimations regarding the therapeutic window are subject to the method used to derive them. This may also be compounded by the clinical route of administration of the control compound(s). For example, is the maximum concentration (C_max) of an IV-administered drug in the blood comparable to the localized gastrointestinal (GI) exposure of an orally delivered drug concerning drug-induced GI toxicity? The data shown in our presentation indicates that the Caco-2 cell line does not indicate toxicity to reference compounds like Colchicine (IC50 > 100 uM), and thus would not be a predictive model for this compound with a known high clinical incidence of diarrhea. The therapeutic index is estimated to be quite large, and ultimately a false negative result for Caco-2.

It is true that FITC and Lucifer Yellow have similar emission spectrums and can interfere even though the excitation wavelengths are different. We recommend incorporating a reconditioning/washout step in between the use of each marker, which reduces potential cross-talk.

We have used organoids from a variety of donors and we have seen some donor-specific responses. We observed more notable donor-specific differences in colorectal cancer-derived tumor organoids.

The organoid imaging analysis algorithm that we use in CAS was developed internally at �����ƽ�� Technologies and is optimized for mouse organoid morphology. Since human organoids can have a large cystic morphology, individual organoids can appear to overlap when viewed as a 2D image. It is challenging to analyze each organoid object with image-based tools when it is not identifiable as a discrete object. We are in the process of developing new tools and methods to apply image-based analysis to human organoids.

�����ƽ�� Technologies has a non-exclusive license with HUB Organoids to provide these services to our customers. Customers who use our services do not need any additional licensing to use the data we generate on their behalf. Customers who choose to perform their own experiments in-house using our organoid media products should note these products were developed under a license to intellectual property owned by HUB Organoids. These products are sold for research use only. Purchase of these products does not include the right to use them for drug screening aiming for commercial gain, equipment validation, biobanking, or other commercial purposes. Purchasers wishing to use the HUB Organoid technology for purposes other than basic research use should contact HUB Organoids at to obtain a further license. Purchasers may apply for a license from HUB Organoids, which will not be unreasonably withheld by HUB Organoids.

Our Z-planes are evenly spaced, though it's important to validate appropriate Z-planes that will ensure that every organoid is captured in focus in at least one plane on your instrument. One function of our internally developed algorithm is that the final composited image maintains the focus of the organoid object even if some Z-planes are out of focus, which is critical for accurate object identification.

We have not done these experiments, but publications comparing healthy to inflammatory bowel disease-derived organoids' responses to stimuli suggest that pathogenic phenotypes are maintained in the organoid cultures.

We have evaluated both P-gp and BCRP transport function in organoid monolayers using substrates and inhibitors, following a protocol based on the —, also summarized in . These efflux studies indicate that organoid-derived monolayers have much greater functional transporter activity compared to Caco-2, though there are also regional differences in activity. For CYP3A4, we have also demonstrated much higher metabolic activity in organoid-derived monolayers compared to Caco-2 monolayers. For UGT and other Phase II metabolic processes, we have not yet characterized those properties in organoid monolayers but that is part of our future planned activities.

We currently have functional transporter data for the efflux transporter substrates methotrexate and quinidine which suggest expression of the BCRP and P-gp transporters, though some non-specificity of the substrates is possible. Confirmation of expression localized to the apical border via immunostaining is in progress.

We are continuously validating our systems using physiologically relevant substrates per FDA guidance and focusing our current efforts on the most clinically relevant mediators of transport and metabolism. If you would like more information on this topic, please reach out to contractassay@stemcell.com.

Increased differentiation and decreased proliferation is likely the reason for the increase in TransEpithelial Electrical Resistance (TEER) between ���Գٱ���پ���ܱ���™ Organoid Growth Medium vs. ���Գٱ���پ���ܱ���™ Organoid Differentiation Medium. The changes we see in TEER from submerged versus ALI cultures are due to an increase in the secretory cell types present along the crypt-villus axis. The greater the diversity of cell types between secretory and absorptive cells, the weaker the tight junctions, and the more physiologically relevant the system becomes.

We have not specifically assessed NSB molecules in the barrier integrity assay. Our characterizations in the monolayer model have been limited to molecules with known low passive permeability as a marker to identify loss of barrier integrity, as well as transporter substrates and CYP450 substrates.

We are currently developing our high-throughput immunofluorescence processes in a monolayer format and that in combination with our burgeoning robotics protocols will vastly increase the scope and scale in which our assays will be run. We don't currently have an organoid immunofluorescence assay that is amenable to high throughput. Our best staining protocols are available as part of our online resources.

The intestinal organoids are comparable to in vivo conditions with their heterogeneous cell populations. Our reference compounds, such as Colchicine and Afatinib, demonstrate toxicity in our organoid assays and these compounds have a high GI toxicity clinical incidence. For more information on the clinical relevance of organoid models, please watch our webinar "Optimizing Organoid Models to Better Predict Clinical Outcomes".

We have gone through several optimization processes for our intestinal organoid assays in which we have found ideal seeding densities and technical replicates to maximize consistency and decrease variability. Additionally, using �����ƽ�� Technologies' high-quality, standardized media products, like our ���Գٱ���پ���ܱ���™ Organoid Growth Medium, significantly contributes to the reliability of our results. Our specialized media are designed to provide uniform performance, reducing variability and ensuring reproducibility across your studies.

Integrating high-content screening techniques with intestinal organoid assays can significantly improve the predictability and robustness of drug toxicity and efficacy assessments by closely mimicking human intestinal biology. Challenges in this integration include managing the inherent heterogeneity of organoids, standardizing culture conditions, and effectively quantifying complex cellular responses. To mitigate these challenges, using organoid biobanks, such as the HUB, will help reduce variability and manage the inherent heterogeneity of organoids, while our high-quality media products and established protocols ensure our culture conditions are standardized. Additionally, the intestinal organoid viability assay can be adapted to automation to further optimize standardization and we are currently working on the expansion of automation into new capabilities and readout formats.

In addition to the standard readouts, we have begun to characterize cytochrome P450 metabolism and transport function. We have data showing greatly increased CYP450 and transporter activity in organoid-derived monolayers compared to Caco-2 monolayers. Additional characterization, such as gene expression through qPCR and protein expression through immunostaining, may also be performed or requested as an add-on service.

Each study can be customized according to the client's needs. Clients don't have to meet a minimum format with CAS for their services, there's no baseline you need to hit with regards to sample numbers, though of course there can be great economy of scale benefits. Assay parameters, such as the number of test articles, concentrations, replicates, treatment regimen, and duration, are flexible and generally can be accommodated in our workflow.

For study sizes, there is certainly a wide range of requested formats. 6 - 12 compounds tested at 3 - 8 concentrations in triplicate (i.e. 200 - 300 data points) is common, though we can accommodate well over 1,000 data points in a single study with our workflow if needed.