For the STEMdiff™ Intestinal Organoid Kit, we do not recommend the use of antibiotics in the early stages of culture (Stages 1 - 2). While not advisable, antibiotics could be added to spheroids if desired after the first passage post-embedding. Once organoids are fully formed (> P2), it should be safe to add any standard antibiotic to the medium (e.g. gentamicin at a final concentration of 50 µg/ml).

Clump passaging results in the formation of larger mid-/hindgut spheroids, which ultimately lead to a higher differentiation efficiency, and is more robust. It is recommended that customers optimize their seeding densities to achieve optimal differentiation results if starting with single cells.

Mid-/hindgut spheroids released between Day 5 - 9 are capable of giving rise to human small intestinal organoids. Note that the duration that the cells are exposed to mid-/hindgut medium will steer the regional identity of the developing small intestinal organoids to an identity similar to duodenum (shorter exposure) or ileum (longer exposure). For reproducible results, it is recommended to consistently harvest and initiate human intestinal organoid cultures using mid-/hindgut spheroids generated on the same day of differentiation. Day 8 is often when the most budding is observed, but this depends on the cell line.

Spheroids are budded from the monolayer during Stage 2 and embedded into Matrigel® at P0. After 7 - 10 days of culture, spheroids differentiate and are thereby referred to as human intestinal organoids (HIOs). Mid-/hindgut spheroids are composed of polarized epithelium expressing CDX2, E-Cadherin, and EpCam. In addition, they contain mesoderm-derived cell types that express vimentin and CDX2, but not EpCam. The mesenchyme can be seen through the microscope and often envelops the spheroids (Figure 4 on the STEMdiff™ Intestinal Organoid Kit Product Landing Page). HIOs express intestinal, goblet, and enteroendocrine cell-specific markers, such as CDX2, MUC2, EpCam, CHGA, SOX9, and KRT20. They also express mesenchymal markers desmin and vimentin. The presence of Ki67 confirms cell proliferation and the presence of putative intestinal stem cells, which enable long-term organoid culture (Figure 5 on the STEMdiff™ Intestinal Organoid Kit Product Landing Page).

No, we advise against this. We tested longer and shorter durations of Stage 1, which both resulted in lower efficiencies of spheroid formation during Stage 2.

Yes, as long as the cell monolayer is still giving rise to spheroids. However, it has been observed with 5 different PSC cell lines that after 9 days in differentiation, the Stage 2 monolayer no longer produces spheroids.

No, but earlier days generally produce smaller spheroids.

It may be possible but we haven’t tested it. It may be more challenging to initiate organoid cultures from smaller spheroids.

This is not a good idea as you may carry over unwanted cells from the monolayer.

Yes, the Technical Manual (Generation of Human Intestinal Organoids Using STEMdiff™ Intestinal Organoid Kit) suggests seeding a range of clump densities (e.g. 4000, 5000, and 6000 clumps per well in a 24-well plate). Optimizing the initial seeding density is critical for reproducible and robust spheroid formation at Stage 2 of differentiation. Customers can try greater than 6000 clumps per well if needed.

There is intrinsic variability between cell lines in the efficiency of differentiation into mid-/hindgut progenitors. We have tested multiple embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) lines and observed similar effects (Figure 3 in STEMdiff™ Intestinal Organoid Kit Brochure).

Both dome formation and morphology are better preserved in a pure Matrigel® dome (100% Matrigel®). We don't recommend a 1:1 dilution for hPSC-derived organoids, especially at early passages, due to the presence of outgrowing mesenchyme, which digests and re-models its environment. Diluted Matrigel® accelerates this remodeling process, resulting in the intestinal organoids sinking to the bottom of the well and attaching to the plastic, which is not desirable.

It is better to establish the organoids first in a 24-well plate and then scale down the format. Customers can use the 24-well seeding densities as a starting point and scale appropriately, however, we haven't done any of this optimization in-house.

For the generation of mid-/hindgut spheroid cultures, they can be scaled down into 96-well plates. However, for hPSC-derived organoids, we would advise starting with 24-well plates to generate enough mid-/hindgut spheroids from multiple samples/donors, which will allow them to embed into multiple replicate Matrigel® domes in a smaller format, e.g. 96-well.

We have not tested flasks such as T-75 or T-175. Further optimization of the cell seeding density for the scale-up would be required. The recommended seeding density for 24-well plates is between 4000 - 6000 clumps per well (= 1.9 cm²), which corresponds to a T-75 flask of a range between approximately 160,000 - 240,000 clumps per flask. Customers should initiate differentiation (Stage 1 medium) when the density of hPSCs reaches 85 - 90%.

We have not optimized the suspension culture format. Matrigel® domes are the standard culture format in the field, and we expect to see the best morphologies of intestinal organoids in domes rather than suspension.

At earlier time points (Days 4 - 5), fewer spheroids are shed. Later on, it is common to get at least 50 spheroids per well, with sometimes as many as 300 - 500 per well toward Day 8.

Organoids derived from primary human biopsies using IntestiCult™ Organoid Growth Medium (Human) are less differentiated, as demonstrated by the positive staining of Ki67 (proliferation marker), EpCAM (epithelial marker), and Vilin (luminal marker) (Figure 2 on the IntestiCult™ Organoid Growth Medium (Human) Product Landing Page). Organoids derived using STEMdiff™ Intestinal Organoid Kit contain differentiated cell types, as well as stem and progenitor types (Figure 5 in the STEMdiff™ Intestinal Organoid Kit Brochure, Figure 7 in the Scientific Poster). These organoids also contain mesenchyme which contributes to the niche factors.

Yes, because the organoids contain goblet cells, as evidenced by the fact that we can detect mucin 2 (MUC2) protein within the cytoplasm and at the apical surface of goblet cells (Figure 5B in the STEMdiff™ Intestinal Organoid Kit Brochure).

It is more difficult to establish organoids from smaller spheres (under 75 μm); larger spheroids have a higher efficiency in forming organoids.

Different cell lines display different organoid morphologies (mixed morphologies of cystic and budding organoids, or either cystic or budding). Over time and multiple passages in STEMdiff™ Intestinal OGM, the cystic organoids may become more prevalent than budding organoids.

Many more mesenchymal cells can be observed at earlier passages (Figure 2B in the STEMdiff™ Intestinal Organoid Kit Brochure). The characterization of brachyury, vimentin, ACTA2, and desmin markers in the DE stage versus other stages (organoids) was presented in STEMdiff™ Intestinal Organoid Kit Brochure (Figures 4A and 5A).

There is a decrease of mesenchyme due to fragmentation of organoids at each passage. After 40+ passages, some mesenchyme is still present.

The highest number of in-house passages tested is 40 passages with one freeze/thaw cycle. We have successfully maintained hPSC-derived HIOs for more than one year in culture (n = 3) by passaging them every 7 - 10 days.

To scale up organoid cultures, it is advised to generate multiple Matrigel® domes per well (e.g. in a 6-well plate), as well as to increase the number of organoids per Matrigel® dome (up to 100 organoids per dome). In this scenario, we recommend performing medium changes three times a week, instead of only twice a week.

Organoids generated using STEMdiff™ Intestinal Organoid Kit are considered to be fetal in nature and not as matured as in fully developed primary intestinal tissue. However, these organoids can be further differentiated to express markers of differentiated intestinal cells, such as goblet cells and enteroendocrine cells, as an end-point assay. Organoids must first be passaged at least 2 - 3 times or cultured for at least 30 days during Stage 3. Cultures are then maintained for up to 2 weeks with regular media changes using the STEMdiff™ Intestinal Organoid Growth Medium to allow further differentiation of organoids. Disruption will reactivate the stem cells; by leaving the cultures without passaging, we can allow the organoids to further differentiate. We do not have a recommendation for differentiation media.

Human PSC-derived intestinal organoids can be frozen as early as passage 3 once they have fully established and display mature, budding, or cystic morphologies. Please refer to Section 8.1.1 of the Technical Manual (Generation of Human Intestinal Organoids Using STEMdiff™ Intestinal Organoid Kit) for a detailed protocol.

Although it has not been extensively tested or validated, we expect that the mid-/hindgut spheroids can be cryopreserved. One reason this may not succeed could be due to a low number of spheroids, which is usually the case with the daily harvest of spheroids. Instead of daily spheroid harvests, our recommendation is to increase the number of spheroids on one single day by harvesting released spheroids and subsequently adding them back to the monolayer cultures with the fresh medium change. This way, you can cryopreserve the spheroids on a single day, rather than cryopreserving just a few spheroids daily and running into the challenges associated with low spheroid quantities in the early days.