Intestinal Organoid from Human iPSC, NGL-SC001

Catalog Number: NGL-COr003

Ask For A Price or Quote –

Product Overview

NeyroblastGX generated highly characterized and fully functional mature intestinal organoids from NGL-SC001 HiPSC-derived 3D gut spheroids, a patent-pending new and novel approach for intestinal development. This novel intestinal organoid (NGL-GIOr004)  tissue complexity of the human intestinal epithelial structures surrounded by stratified mesenchyme, which contains muscle cells and sub-epithelial fibroblasts. NeyroblastGX intestinal system can produce mature intestinal phenotypes within 18 days after the expression from gut spheroids expressing CDX2 markers, further expressing restricted crypts (Ki67+), goblet cells (MUC2+), Paneth cells (lysozyme+), endocrine cells (PDX1+), and enteroendocrine cells (chromogranin A+). These specialized intestinal cells can transport protein and have anti-microbial action, which usually takes a long time, more than 12 weeks, to generate. However, NGL-GIOr004 only requires 28 days to produce robust, complex, and highly functional intestinal organoids up to 3mm via maintaining with NGL-hGIOM004 media. NeyroblastGX system is more rapid and cost-effective and can be provided in large quantities if needed for scaling up the rapid drug, toxicity, and pathogen screening.

NGL-GIOr004 was tested for mycoplasma or other pathogens and found no mycoplasma or any other contamination. One vial of Intestinal organoids (~50 Mature Intestinal Organoids = ³100 million viable cells/vial) contains enough to grow a full 24-well plate and can be further passaged for a long time with obtaining a large number of intestinal organoids in the culture from the same batch to use or study downstream analysis, intestinal disease mechanism and/or drug, toxicity, and pathogen screening. NGL-GIOr004  intestinal organoids are optimized to grow in treated 24-well plates. NGL-GIOr004 can be cultured on a robust scale, e.g.,  one vial of frozen viable NGL-GIOr004 can be passaged for a long time and produce large quantities of intestinal organoids following our User Guideline.

Product Benefits

  • NeyroblastGX healthy control NGL-SC001 HiPSC line-derived NGL-GIOr004 intestinal organoids were derived from NeyroblastGX patent pending gut spheroids, which provides tissue complexity like a human and recapitulate human in vivo system to the screening drug, toxicity, pathogens of your research interest.
  • Naturally grown NGL-GIOr004 are immune stable and generated without viral integration, producing a highly efficient epithelium with tight junction protein and epithelium structures surrounded by mesenchyme for gut study.
  • Ready-to-use highly characterized NGL-GIOr004 intestinal organoid contains foregut, midgut, and hindgut markers in robust amounts, which is intermediate for your immediate downstream analysis of your project.
  • NGL-GIOr004 is highly functional and can secrete inflammatory cytokines with the induction of bacterial lipopolysaccharides or pathogens to study underlying disease mechanisms such as intestinal inflammation, bowel syndrome, Crohn’s disease, or intestinal degeneration.
  • NGL-GIOr004 intestinal organoids consist of highly convoluted tissue such as epithelium surrounded by non-polarized mesenchymal CDX2+ cells, and E-Cadherin expression demonstrates that the inner layer of polarized cells provides an ideal avenue to study disease complexity like humans to develop personalized medicine.
  • NGL-GIOr004 consists of a mature intestine containing crypts, villi, goblet, enteroendocrine, and Paneth cells, which can easily maintain and obtain many intestinal organoids for your study needs.

 

Storage, Stability, and Biosafety: NGL-GIOr004s are frozen in an Intestinal cryopreservation medium (NGL-hGIOFM004), preformulated with 10% DMSO, providing a safe, protective environment during the freezing, storage, and thawing process for intestinal organoid and storing. NGL-GIOr004 intestinal organoids are highly stable in liquid nitrogen (LN2) -130°C or colder and 12 months of date receipt. We strongly recommended storing the NGL-GIOr004 in LN2 to avoid undesirable cell damage or death. Thawed samples must be used immediately.

Caution: Please ensure the sample keeps storing LN2 (-130°C) upon receiving until further use. NeyroblastGX is not responsible if NGL-GIOr004 does not work due to your LN2 temperature being compromised. NGL-GIOr004 intestinal organoids are free from viral or harmful pathogen infections. However, this product should treated as potentially hazardous materials and only used in appropriate biosafety level handling procedures such as those described in Biological Safety Level 2.

 

LIMITED USE LICENSE

NGL-GIOr004 AND ITS DERIVED PRODUCT ARE FOR RESEARCH USE ONLY AND ARE NOT INTENDED USE FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC PURPOSES OR COMMERCIALLY RESALED UNLESS OTHERWISE STATED, or you can purchase a license for commercial usage and contact NeyroblastGX for this purchase. Please review the terms and conditions for the usage of NGL-GIOr004.

Characterization

A time course of NGL-GIOr004 mature intestinal organoids generation from healthy control NGL-SC001 HiPSC, as shown in Figure 1

Figure 1. A time course of NGL-GIOr004 mature intestinal organoids generation. (A) Our method first generates 3D gut spheroid from HiPSCs at 50-150 µm and maintains it until day 10. On Day (D) 10, mature gut spheroids started to become big with bright edges cells with darker cores (>200 µm) with high potential to generate high functional intestinal organoids (Intestinal OrG). This gut spheroid is our patent pending 3D spheroid able to develop a big intestine up to 3 mm. (B) After that, gut spheroids seed with matrigel-matrix domes and (C) maintained with NGL-hGIOM004 intestinal growth medium started to become >1mm intestinal OrG at about D11-D12. Gut spheroids started attaching and appearing paracellular barrier with intestinal OrG architecture, as shown in the D 12-13 images (scale bar 550 µm). (D) On day 18, GIO confluent >90% of domes, and this GIO is ready to passage. This GIO can be passage >10 times and expanded in culture or cryopreserved (Cryo) using NGL-hGIOFM004 crypreserve media. (E) Further, GIO started to become larger with microscopic visualized epithelial, lumen, and others (scale bar 270 µm) with the expression of (F) E-Cadherin (red) and (G) MUC2 (green) with size ~3 mm. Nuclei represent DAPI and scale bar 130 µm. D; Day.

Formation of fully functional NGL-GIOr004 mature intestinal organoids with the expression of prominent human intestinal cell types, as shown in Figure 2.  

Figure 2. Formation of HiPSC-derived fully functional NGL-GIOr004 intestinal organoids. NGL spheroid, when plated with in-house gel matrix domes, enables the generation of fully functional GIO by 18 days with the expression of (A) crypt-type proliferating cells (Ki67, red), (B) polarized inner endodermal and mesenchyme (CDX2+, green). (C) We also quantified crypt-type proliferating cells via Ki67 marker cells, which were determined with the comparison of nuclei (DAPI) in the same proliferating zone. We found that ~80% of our cells represent crypt-type cells. (D-F) On day 28, robust expression of endocrine cells (PDX1+, red) and highly proliferative SOX9+ (green), goblet cells (MUC2, red), and highly differentiated intestinal crypts proliferating cells (Ki67+, red). (G-I) ɑ-smooth muscle actin also expressed in our intestine including enteroendocrine (ChromgA, chromogranin A+, green) and highly convoluted epithelial E-cadherin (E-cad+, red) and Paneth cell-derived lysozyme (red). All these expressions indicate our intestinal organoids are fully functional and capable of studying pathogens and chemically-induced intestinal toxicity, inflammation, bowel syndrome, or other intestinal disorders: scale bar 130 µm or higher, Nuclei, DAPI. The arrow indicates a specific marker expression.

NGL-GIOr004 intestinal organoids showed robust expression of ZO1 representing tight junction protein 1 (TJP1) to model disease of intestinal inflammation or related disorders Figure 3.

Figure 3. Effect of chronic bacterial lipopolysaccharide (LPS) induction on TJP1 (tight junction protein 1) of NGL-GIOr004 intestinal organoids. (A) Tight junctions are protein complexes that create intercellular boundaries between the plasma membrane domains of epithelial and endothelial cells. One critical TJP is the ZO1 protein located on a cytoplasmic membrane surface of tight intercellular junctions that can be used as the marker of intestinal damage or dysfunction and modeling disease of the intestine. NGL-GIOr004 intestinal organoid study with the LPS (50-100 µg/ml) injection affects GIO TJP1 at 96h. We also performed ZO1 immunostaining at an earlier point (24-48h) but could not see any effect on ZO1 (green). Ki67 (red), an intestinal crypt cell type, decreased by LPS 100 µg/ml. (B-C) ZO1 and Ki67 quantification of fluorescence intensity (FI) showed 50-100 µg/ml LPS effect ZO1 and Ki67 significantly at 96h time. Scale bar 70 µm. We have performed one-way ANOVA with Tukey’s multiple comparisons using GraphPad Prism. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 denotes significance. Star (*) compared untreated (UT) control (Ctrl)  vs. LPS, DAPI staining represents Nuclei.

N NGL-GIOr004 intestinal organoids actively secrete proinflammatory cytokines using bacterial lipopolysaccharide (LPS) to study host-pathogen interaction, as shown in Figure 4.

Figure 4. NGL-GIOr004 intestinal organoids to study host-pathogen interaction. GIO was treated with bacterial endotoxin lipopolysaccharide (LPS) with different concentrations to understand our GIO can recapitulate host-microbe interactions similar to humans in vivo. We found 6 out of 10 different proinflammatory cytokines (ProC) and chemokines such as interleukin (IL)-2, IL-6, IL-8, tumor necrosis factor (TNF)-ɑ, macrophage inflammatory protein (MIP)-1ɑ, and MIP-1β were upregulated using LPS (10, 50 and100 µg/ml) in a dose and time-dependent fashion [24h (blue bar) and 48h (purple bar) time point] using 10-plex cytokine array, but IL-10, IL-1β, and IFNγ were upregulated only in 48 hours (h) time points with 100 µg/mL LPS dose. IL-13 was unable to detect with this time point or any concentration of LPS. GraphPad Prism was used for comparing significant differences between treatments using an unpaired t-test* p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 denotes significance. Star (*) compared untreated (UT) control (Ctrl) vs. LPS.

 NGL-GIOr004 intestinal organoids can distinguish pathogen versus non-pathogenic bacterial response and provide a platform for chronic intestinal infection models, as shown in Figure 5.

Figure 5. Pathogen versus non-pathogenic bacterial response with 10-plex cytokine array. To study the pathogen versus non-pathogenic effect on NGL-GIOr004 intestinal organoids, we have used three strains of pathogenic bacteria (Escherichia coli strains: TY-2482O104:H4 and O103:H11GFP and Salmonella Enterica serovar Typhi) and two strains of non-pathogenic bacteria (Lactobacillus Brevis; Bb14Bifidobacterium adolescentis) from ATCC. These bacteria were cultured using TSB (Tryptic Soy Agar/Broth), NB (Nutrient Agar/Broth), and LB (Lactobacilli MRS Agar/Broth). We maintained a pure and stable bacterial culture, producing over 1×10bacterial cells / mL within 24h. We have found some important proinflammatory cytokines (ProC) such as (A) IL (interleukin)-1β, (B) tumor necrosis factor (TNF-ɑ),  (C) macrophage inflammatory protein (MIP)-1β, (D) IL-6, and (E) IL-8 were upregulated with pathogens with 8 hours (h) treatment significantly. Some of the ProCs were also undetected or lower, such as IL-10, IFNγ, and MIP-1ɑ. We have observed a pathogen versus non-pathogen effect significantly different on the ProC production. We have found that most of the beneficial non-pathogenic bacteria could not produce ProC except IL-6. We suspect IL-6 has dual functions in signaling immune cells via proinflammatory and anti-inflammatory action, which might be part of the beneficial factor that raises IL-6 levels in NGL-GIOr004 intestinal organoids. We have performed two-way ANOVA with Tukey’s multiple comparisons using GraphPad Prism. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 denotes significance. Star (*) compared control vs. pathogens. N (normal control), UT (untreated intestinal organoids), GP1 (E. coli-GFP), P2 (E. coli second pathogenic strain), P3 (S.Typhi), NP1(Lac, L. brevis), NP2 (Bf, B. adolescents).

NGL-GIOr004 viability and stability by comparing freshly (Fresh) versus cryopreserved (Cryo) thawed cultured intestinal organoids, as shown in Figure 6.

Figure 6. NGL-GIOr004 viability and stability by comparing Fresh versus Cryopreserved intestinal organoid culture (A) The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)] assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. Viable cells of intestinal organoids with an active metabolism converted MTT (yellow color) into a purple-colored formazan product and were measured with a microplate reader with an absorbance near 570 nm. (B) To perform MTT assay, we grew GIO in 24 well plates and then treated intestinal organoids with or without bacterial lipopolysaccharide (LPS) for 96h to observe intestinal organoids’ viability and stability. LPS lost cell viability <15-20%, a minimal and non-significant difference to perform downstream analysis of intestinal disease and immune response kinetics analysis. (C-E) NGL-GIOr004 cryopreserve intestinal organoids (Cryo-GIO) are highly viable after the freeze-thaw cycle and can be used extensively for clinical or research purposes. Scale bar 550 µmUT: Untreated control; LPS: Lipopolysaccharide; Cryo: cryopreserve; Inst. OrG: Intestinal organoids.

User Guideline

Frozen NGL-GIOr004 should be cultured and maintained with the procedure indicated in the User Guidelines. We will be sent a QR code for “User Guideline” upon ordering the product. QR code is for downloading the digital copy of a detailed procedure for culturing and maintaining the NGL-GIOr004 intestinal organoids. If you need a paper copy of the User Guidelines, please request it during ordering, and then we can ship it with NGL-GIOr004 frozen vials. NeyroblastGX scientists always guide you if you need consultation for your culture or face any challenges.

Additional Information

Required Additional Materials

NGL-GIOr004 intestinal organoid culture maintenance and characterization will need the following associated materials but not included with our product: