Cerebral Organoid form Human iPSC, NGL-SC001

Catalog Number: NGL-COr003

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Product Overview

NeyroblastGX generated highly characterized and mature Healthy control NGL-COr003 cerebral organoids (OrGs) from NGL-SC001 HiPSC-derived 3D OrGspheroids, a patent-pending new and novel method for high functional cerebral development. NGL high-quality cerebral OrG (NGL-COr003)  tissue complexity of the human cerebral structures consisting of all the cortical layering neurons, astrocytes, and glutamatergic synaptic neurons to study neurodevelopmental and neurodegenerative disorders. NGL cerebral OrG maintenance with complete neural growth media (NGL-hNGM005) in culture, capable of generating neuroepithelium, and cortical patterning, as shown in NGL-COr003 culture, such as PAX6+, TBR1+, TBR2+, Reelin+, SATB2+, β III Tubulin+, and MAP2+ by using immunocytochemistry (ICC). Once we re-seeded them in the gel matrix and maintained them in the floating conditions, we obtained various layers of cortical neurons, including glutamatergic excitatory synaptic neurons, by day 28. However, NGL-COr003 only requires 28 days to produce robust, complex, and highly functional cerebral OrG up to 3 mm sizes via maintaining with NGL-hNGM005 neural complete growth media (see related product). NeyroblastGX system is more rapid and cost-effective and can be provided in large quantities if needed for scaling up the rapid drug, toxicity, disease pathology specifically affected by excitatory cortical neurons such as Autism spectrum disorder, Fragile X syndrome, Frontotemporal dementia, and Alzheimer’s disease.

NGL-COr003 was tested for mycoplasma or other pathogens and found no mycoplasma or any other contamination. One vial of cerebral OrGs (~50 Mature cerebral Organoids = ³100 million viable cells/vial) contains enough to grow an entire 24-well plate and can be further passaged for a long time with obtaining a large number of cerebral OrGs in the culture from the same batch to use or study downstream analysis, understanding neurodevelopmental or neurodegenerative disease mechanism and/or screening drug, toxicity, and pathogens. NGL-COr003  cerebral OrGs are optimized to grow in treated 24-well plates. NGL-COr003  can be cultured on a robust scale, e.g.,  one vial of frozen viable NGL-COr003 can be passaged for a long time and produce large quantities of cerebral OrGs following our User Guideline.

 

Product Benefits

  • Easy culture routine only using NGL-hNGM005 neural complete growth media, no need for extra steps like an additional kit or costly growth factor for this culture.
  • Naturally grown healthy control human NGL-COr003 generated from healthy control HiPSC-derived patent pending OrGspheroid led to having high quality various cortical layers neurons.
  • NGL-COr003  expresses robust glutamatergic excitatory neurons, and cortical neurons are defined, immune stable, and generated without viral integration.
  • NGL-COr003 produces a human CNS cell-enriched cerebral cortex that can recapitulate the human postnatal cerebral cortex.
  • NGL-COr003 when we integrated ready-to-use microglia, which integrated well and expressed a higher amount of IBA1 and GFAP markers cells.
  • Ready-to-use highly characterized NGL-COr003 is easy to use for your downstream analysis.
  • Cost-effective and time-saving for screening drug, toxicity, or underlying disease mechanisms using high-quality NGL-COr003 human cerebral.
  • Compatible with the same genetic background of the NGL-SC001 HiPSC line, NGL-NPC005, NGL-NeuR006, NPCs, microglia, and cortical neurons to use for studying various disease mechanisms and compare. See related products from similar backgrounds to your needs.

Storage, Stability, and Biosafety: NGL-COr003 is frozen in neural cryopreservation medium (NGL-hNFM005), preformulated with 10% DMSO, providing a safe, protective environment during the freezing, storage, and thawing process for stem-derived NPCs and neurons. NGL-COr003 is highly stable in liquid nitrogen (LN2) -130°C or colder and 12 months from the date of receipt. We strongly recommended storing the NGL-COr003 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-COr003 does not work due to your LN2 temperature being compromised. NGL-COr003 is 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-COr003 AND ITS DERIVED CELL 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 purchasing a license for commercial usage. please get in touch with NeyroblastGX for this purchase. Please review the terms and conditions for the usage of NGL-COr003.

Characterization

Timelines of NGL patent pending human cerebral organoids (NGL-COr003) generation from NGL-SC001 healthy control HiPSC, as shown in Figure 1.  

Figure 1. A time course of mature CNS cerebral organoids (OrGs) development. (A) Our method first generates OrGspheroids from HiPSCs at 100-200 µm within the day (D)3. On D3, OrGspheroids were treated with dual inhibition of SMAD signaling small molecules SB431542 and dorsomorphin. On D10, OrGspheroids became big with bright translucent edges and darker colors in the middle (400-500 µm) by treating both small molecules and producing a cortical progenitor-rich corticosphere (Csphere). Scale bar 150 µm. (B) After that, Csphere was seeded in the gel matrix in 24 well-treated plates.  (C) On Day 12, Csphere started to produce buds in the gel-matrix domes as early as two days. D15 Csphere started to produce large numbers of buds, D18 buds matured, and cortical patterning was visualized. In this stage, cerebral OrG needs to be re-seed or cryopreserved. Scale bar 300 µm. (D) To re-seed and generate central nervous system (CNS) mimic cerebral OrGs, we culture them on the ultra-low attachment 24-well plates with gel-matrix and maintained in floating condition with an orbital shaker with continuous rotation using complete neural growth media (NGL-hNGM005). On D28, the Cerebral OrG becomes large with a dark core up to 3 mm for downstream analysis, passage, or cryopreservation. This cerebral OrG can maintain and passage for a long time. Our unique and complex CNS cerebral OrG is ready to use for screening drugs, toxicity, chemicals, disease pathological proteins, or underlying disease mechanisms of brain disorders using microelectrode plates or any other culture plate for downstream analysis.  Scale bar 500 µm, D; Day.

 

NGL-COr004 cerebral organoids (OrGs) efficiently generated early cortical patterning buds, further influencing the outcome of robust cortical neuronal marker expression by Day 18, as shown in Figure 2.

Figure 2. Morphogenesis of neuroepithelium and early cortical patterning NGL-COr003 produced a large number of buds in the gel-matrix domes of Csphere; the outcome of the neuroepithelium further maintained the neuroepithelium and generation of early cortical layering cells with the expression of  (A) ZO1+ (green) and PAX6(red), (B) β III Tubulin(green) and SOX2+(red), (C)  SATB2+ (green) and MAP2+(red),  (D)  β III Tubulin+ (green) and TBR1+(red), (E) TBR2+ (green) and MAP2+(red), and (F) Reelin+ (green) and MAP2+(red). We have observed distinct neuroepithelium (ZO1) and SOX2 during the formation of neuroepithelium into radial glial cells and further differentiation of ventricular (VZ) and subventricular zone (SVZ) with expression of early cortical neurons using complete neural growth media (NGL-hNGM005). In early cortical layering, usually appeared PAX6, TBR1, TBR2, Reelin, β III Tubulin, and MAP2 in the apical surface in the ventricular zone (VZ), as shown in our culture. SATB2 usually appeared late, but we still found some expression in our early cortical layering culture. Scale bar 150 µm for all the images except images B and F (80 µm). This stage of cortical layering is recapitulated in the developing human brain, which can be studied and screened for neurodevelopmental disorders and their complex nature and developed into new therapeutics.

 

NGL-Cor003 cerebral organoids (OrGs) represent distinct cortical projecting neurons expressing various layers of cortical neurons and glial marker expression, as shown in Figure 3.

Figure 3. Formation of mature NGL-Cor003 cerebral organoids (OrGs) consisting of glial cells and neuroepithelium. NGL cerebral OrG was re-seeded within the gel-matrix domes on D18. In this step, cerebral OrG is maintained in a floating condition, leading to fully functional CNS cerebral OrG development within day 28. Our method required a time-dependent increase of our complete neural growth media (NGL-hNGM005) in culture, capable of generating neuroepithelium, ventricular and subventricular zones (VZ and SVZ), and mature cortical plates. For each stage of cerebral formation, such as neural induction to Csphere to cerebral OrGs, we have used the same neural media, unique to our system, for providing proper nutrition and integrating microglia without interrupting cortical patterning. Our cerebral OrG expresses (A-B) mature neuronal MAP2 (red) and astrocyte markers GFAP(green). Inserts showing visualize dendrites of neurons and spongiform morphology of astrocytes. (C) We have added cerebral OrG ready-to-use microglia, which efficiently integrated microglia with IBA1 (green) marker expression, (D) neuron-specific marker β III Tubulin (green), (E) late born superficial cortical layer marker (SATB2, green), (F) cortical deep layer marker FOXP2 (green), (G) cortical layers 2-3 marker cells BRN2 (green), and (H) cortical pre-plate to all the cortical layers marker TBR1 (red). (I) We have also observed epithelial tight junction protein ZO1 (green) marker expression. Our cerebral OrG is highly compatible with studying complex CNS diseases like Alzheimer’s, Parkinsons’, and many others—scale bar 130 µm, D; day.

 

NGL-Cor003 cerebral organoids (OrGs) represent robust marker expression of functional excitatory synaptic neuronal marker expression, as shown in Figure 4.

Figure 4. NGL-COr003 cerebral organoids (OrGs) showed robust expression of functional glutamatergic excitatory cortical neurons. NGL-Cor003 cerebral OrGs generate highly efficient glutamatergic excitatory neurons expressing (A) postsynaptic density marker postsynaptic density-95 (PSD-95, red) and presynaptic marker synaptophysin (green). Inserts with arrowheads indicate the interaction of presynaptic and postsynaptic proteins on the axons, prove synaptic transmission of cerebral neurons with an active action potential, (B) NGL-SC001 HiPSCs-derived corticosphere (Csphere) at day 18 produces large numbers of buds in the gel-matrix domes with neuron-specific marker expression β III Tubulin (green) and vesicular glutamate transporter 1 (Vglut1, red). During neurogenesis, Vglut1 started to express from as early as day 18. (C) In our cerebral OrGs, Vglut1 (red) and PSD-95 (green) were highly expressed. Inserts with arrowheads showed PSD-95 puncta associated with Vglut1, which provides insights into the neuronal glutamatergic synaptic signaling usually observed in the human postnatal developmental maturation stage. Our Cerebral OrG is functional and represents highly active synaptic interaction, providing a model for studying neurodevelopmental or neurodegenerative diseases or sensitive drug screening platforms. Scale bar 130 µm. The arrowheads indicate a specific marker expression. PSD-95 (green-SantaCruz, SC-32290, red-Invitrogen, 51-6900).

 

NGL-COr003 viability and stability compared with freshly (Fresh), prolonged, and cryopreserved (Cryo) thawed cultured cerebral organoids (OrGs), as shown in Figure 5.

Figure 5. NGL-COr003 viability and stability by comparing Freshly versus Cryopreserved and prolonged cerebral organoid (OrG) culture (A) The  MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. Viable cells of cerebral OrGs 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 the MTT assay, we grew cerebral OrGs in 24 well plates for 28 days and prolonged culture kept for 90 days. We also used cryopreserved (Cryo) cerebral OrG, which was thawed and immediately seeded using gel-matrix and maintained culture for ten days. All the freshly prolonged and Cryo cultures were maintained with complete neural growth media (NGL-hNGM005). MTT assay was performed on (i) freshly, (ii) prolonged, and (iii) Cryo Cerebral OrG cultures to determine cerebral OrG viability and stability. (B) MTT quantification of cerebral OrG results showed our Cryo cerebral OrG is highly viable (>80%) after the freeze-thaw cycle and comparable to freshly cultured OrG. We did not observe any necrotic core or the reduction of cerebral OrG viability over 90 days, and culture remains viable at > 80%, as shown with an arrow. Thus, NGL cerebral OrG culture is highly stable in freeze-thaw cycles or long cultures and can be used to HT screen via various agents for extended periods—scale bar 1375 µm.

User Guideline

Frozen human NGL-COr003 cerebral OrGs 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-COr003. If you need a paper copy of the User Guidelines, please request it during ordering, and then we can ship it with NGL-COr003 frozen vials.

Additional Information

Required Additional Materials

NGL-COr003 culture maintenance and characterization will need the following associated materials but not included with our product: