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There are a few major advantages. First, IntestiCult™ Plus is a brand new, proprietary formulation that improves on existing literature. Second, it's consistent. There’s no conditioned medium involved, which eliminates a major source of variability, and it’s quality controlled, so there’s no lot-to-lot variation to worry about. That kind of reliability makes a big difference when you're trying to get reproducible data.

When using IntestiCult™ Plus as recommended, achieving a balanced cell population is straightforward and occurs naturally. Media components can be adjusted to shift the balance between stem and differentiated cells. For example, increasing Wnt levels favors stem cells, while its removal promotes differentiation.

Yes, we have. For example, goblet cells can be enriched using a gamma-secretase inhibitor like DAPT. Just be cautious with dosing, since overdoing it can affect viability. We’ve also successfully enriched enteroendocrine cells using a combination of MEK and gamma-secretase.

We have, and it does work. That said, ESC-derived systems using protocols like those from our STEMdiff™ kits or those from labs like Jim Wells’ or Michael Helmrath’s often run into issues like fibroblast overgrowth. Fibroblasts love this media too. So while IntestiCult™ Plus can support those organoids, you might get better results with the STEMdiff™ Intestinal Organoid Kit, which is designed specifically for ESC-derived systems.

We’ve confirmed compatibility with human, mouse, and rat organoids. It actually works really well with mouse-derived cells, though we also offer a mouse-specific formulation. As for other species—we haven’t tested every model, but given how the core components are designed, we suspect it will support a wide range of mammalian cells. We’d love to hear from researchers working with less common species.

M cells differentiate quite fast when using RANKL (Receptor Activator of Nuclear Factor κB Ligand) or TNF-alpha, sometimes almost too fast. You can quickly get a large population, and if you're not careful, they can overtake the culture and reduce viability. Tuft cells are slower to develop, but you can enrich them over several days to a couple of weeks without seeing the same viability issues we’ve seen with M cells.

Either approach works. We often prefer to seed crypts or intestinal fragments, which naturally contain LGR5+ stem cells. But IntestiCult™ Plus also supports single-cell seeding and passaging. Regardless of the starting material, whether that’s single LGR5+ stem cells or intestinal crypts or fragments, you’ll end up with all the relevant intestinal cell types when cultured with IntestiCult™ Plus.

In general, IntestiCult™ Plus works well with tumor-derived organoids, including those from subcutaneous PDX models. Depending on the tumor’s genetic makeup, you might see differences in how well they grow, but overall, we’ve had good success.

We usually use Promega’s CellTiter-Glo® 3D to measure ATP levels, which correlates with cell viability. We’ve also used live/dead staining and the LDH-Glo® assay, which detects cell death. Each method gives you slightly different information, so sometimes we use a combination to get a fuller picture of what's going on in the culture.

If you’re working with primary, patient-derived organoids, IntestiCult™ Plus supports both small intestine and colon cultures. For human pluripotent stem cell (hPSC)-derived systems, we do offer a publicly available protocol that adapts the STEMdiff™ Intestinal Organoid Kit for colonic differentiation.

Great question. Most of our toxicity testing is done using 3D organoids, where compounds are applied to the basolateral side. For many drugs, that works well. But depending on the drug’s mechanism of action, the apical side may be more relevant. In those cases, a monolayer format can be more appropriate.

That said, monolayers grow more slowly and tend to have lower proliferation, which can influence how drugs behave, especially those targeting dividing cells. So it adds another variable to consider.

We do use monolayers frequently for barrier function assays, like transepithelial electrical resistance (TEER) or FITC-dextran permeability. In those cases, the apical side is exposed and we can track both permeability and viability. So while we don't typically use monolayers for routine toxicity testing, they are definitely a valid and informative model when used for the right application.

We use RANKL because it's a well-established way to induce M cell differentiation. That’s the main context we’ve tested. We can’t speak to its effects in other tissues.

Yes, it can have a significant impact. Larger organoids tend to contain a higher population of differentiated cells, while smaller ones tend to be more proliferative. This difference can influence how the organoids respond to drugs, especially if the compound targets a specific cell type or relies on proliferation status.

To get consistent results across experiments, it’s important to standardize both organoid size and density. To support this, �����ƽ�� Technologies has developed Organoid Culture Plates, designed to help with this standardization. These tools can be especially valuable when you’re conducting drug testing and need reproducible, reliable outcomes.

In many ways, it’s quite different. First-generation differentiation media (e.g. IntestiCult™ Organoid Differentiation Medium) tends to eliminate the stem cell population, which limits proliferation and restricts some assay types. IntestiCult™ Plus keeps that balance—you retain stem cells while also achieving differentiation. This makes it more versatile and relevant for a broader range of experiments, especially those involving toxicity or rare cell types.

We do not recommend IntestiCult™ Plus for organoid-monolayer establishment or for hPSC-derived organoids at this time. We recommend following our protocol on “How to Generate Monolayers from hPSC-Derived Organoids Using IntestiCult™” which uses IntestiCult™ Organoid Differentiation Medium with hPSC-derived intestinal organoids previously expanded with the STEMdiff™ Intestinal Organoid Kit.

IntestiCult™ Plus does not currently support gastric organoids or cancerous tissue from other GI regions such as pancreas. Gastric organoids need something a bit different than what is currently in IntestiCult™ Plus. We’re actively exploring these areas for future development, so stay tuned.

We recommend dissociating a representative sample to single cells using 0.05% Trypsin-EDTA or a similar reagent, then counting using a hemocytometer or automated counter. Organoids can take longer to break apart than 2D cultures, so give it some time.

If you’re growing mouse colonic organoids for a monolayer, you typically use the first-generation human expansion medium (i.e. IntestiCult™ Organoid Growth Medium Human) to expand the organoids, followed by the same expansion medium plus 10 µM Y-27632 for the monolayer itself. The human IntestiCult™ Organoid Differentiation Medium is ideal for human organoid-monolayers, but not for mouse cultures. For mouse monolayer applications, we recommend sticking with the first-generation human expansion medium. It contains serum, which helps mouse cells adhere, a known challenge when working with this model. IntestiCult™ Plus isn’t optimal for this step due to its serum-free formulation.

Yes, �����ƽ�� Technologies offers a wide range of organoid media products for various organ types including, lung, hepatic, pancreatic, kidney, neural, and reproductive. Explore our organoids products here.

Please see the following publication as one of several published methods: Wood MB, Rios D, Williams IR. TNF-α augments RANKL-dependent intestinal M cell differentiation in enteroid cultures. Am J Physiol Cell Physiol. 2016 Sep 1;311(3):C498-507. .

In vitro systems don’t allow for constant replenishment like in vivo, so we use slightly elevated concentrations of factors like insulin and glucose. We’ve been careful not to overshoot, and the formulation aims to maintain a practical balance between physiological realism and in vitro viability.

Not extensively. Although, we recommend using the first-generation expansion and differentiation media for these platforms as it supports better cell adhesion in those systems. We currently have collaborations with both MIMETAS and Emulate that leverage our first-generation media to support their MPS platforms.

We recommend following our published protocol, “Toxicity Testing for Drug Development Using Human Intestinal Organoids and IntestiCult™” for evaluating drug toxicity, which uses Promega CellTitler-Glo® for cytotoxicity following the treatment phase.

Yes, while P-gp has been the transporter we have evaluated in the most depth, we have also done some work with BCRP, MRP2, OAT, and SLC family transporters as well.