Tubular human brain organoids to model microglia-mediated neuroinflammation
Open accessAbstract
We report tubular organoid-on-a-chip devices to generate better human brain organoids and model neuroinflammation.
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Written from the paper’s full text.
TL;DR
The paper presents a 3D-printed tubular organoids-on-a-chip device that uses rocking flow to perfuse human brain organoids and incorporate isogenic microglia, reducing hypoxia/necrosis and heterogeneity while increasing neural progenitor proliferation. As proof of concept, microglia in tubular forebrain organoids respond to LPS/ATP and DAMGO with activation, NLRP3 inflammasome assembly, and cytokine secretion, and are partially rescued by CB2 agonist LY2828360.
Key findings
- The tubular device uses 250 μm perfusable gaps, limits organoid thickness to <500 μm, holds 60 μL medium per side, and at 1 rpm rocking generates a constant flow of 2 μL/second; tubular N2a cultures showed no detectable hypoxia and tubular cerebral organoids had significantly lower HIF1a expression (p<0.005).
- Compared with conventional organoids, tubular human forebrain organoids showed more uniform VZ/SVZ zones, higher EdU-labeled newly divided cells, and higher expression of PAX6, SOX2, and TBR1 (p<0.05, n=5) with less variation; on-chip tubular organoid formation success was ~60%, compared with ~70% intrinsic success for the cerebral organoid protocol.
- Isogenic WA01-derived microglia migrated into tubular hFOs over 3-day co-culture and showed homeostatic ramified morphology; after LPS/ATP or DAMGO (100 nM, 24 h), they became amoeboid with lower fractal dimension, lower lacunarity, and higher convex-hull circularity, and this activation was partially reversed by LY2828360.
- NLRP3/ASC colocalization increased with LPS/ATP and DAMGO and was partially rescued by LY2828360; IL-1β and IL-18 secretion increased after LPS/ATP, and TNF-α was not detected at high levels in 2D iMG cultures but increased significantly when iMG were incorporated into tubular brain organoids.
Methods
- Device design/fabrication: AutoCAD design of 20 × 4 × 10 mm device with 25 μm tubular layer, printed by stereolithography (Form 3B, Formlabs) using Clear Resin V4; device fits multiwell plates and can be cultured on a rocking platform.
- Organoid generation: WA01 hESCs were aggregated into 300 μm EBs (9,000 cells), loaded into the device on day 3, induced with dual-SMAD inhibitors dorsomorphin and A83, then CHIR-99021/SB-431542 for forebrain identity, embedded in Matrigel, and matured with rocking flow; comparisons included tubular vs conventional forebrain organoids and cerebral organoids, with qRT-PCR at days 16 and 35 (n=5 organoids/EBs, triplicates).
- Microglia and assays: WA01-derived induced microglia (iMG) were loaded into the medium reservoir and co-cultured for 3 days; treatments were LPS 100 ng/mL for 24 h plus ATP 2 mM for 30 min, DAMGO 100 nM for 24 h, and LY2828360 100 nM for 24 h after DAMGO. Analyses included live/dead and hypoxia staining, cryosectioning, immunofluorescence, EdU, qPCR, FracLac morphology, NLRP3/ASC proximity ligation assay, and ELISA for IL-1β, IL-18, and TNF-α; statistics used Student’s t-test in GraphPad Prism 7.
Limitations
- Stated: the current model has limited throughput with a 2 mm × 2 mm inner basket for EB loading, and the authors plan higher-resolution 3D printing to scale to 96- or 384-well formats; the study used only hESC-derived culture, and future work is planned with iPSC-derived culture for patient-relevant genetics.
- Stated/evident: microglial morphological and inflammasome rescue by LY2828360 was partial, not complete.
- Evident from the text: hypoxia/viability characterization in the tubular device used mouse N2a neuroblast spheroids as a control, and the on-chip tubular organoid formation success rate (~60%) was lower than the cited intrinsic cerebral organoid protocol success rate (~70%).
Model-written summary. Check it against the paper before relying on it.
Integrity signals
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Cites
Statistics
Claims the paper makes
21 of 30 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured
- Premisecitation
Colony-stimulating factors have opposite effects on MHCII, and subsequently on neuroinflammation, in mouse and human microglia.
“Moreover, colony-stimulating factors have been shown to have opposite effects on MHCII, and subsequently, on neuroinflammation, in mouse and human microglia.20, 21”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [20] Lambert C, Desbarats J, Arbour N, Hall JA, Olivier A, Bar-Or A and Antel JP, The Journal of Immunology, 2008, 181, 8288– 8297. 19050245 source
- cites [21] Re F, Belyanskaya SL, Riese RJ, Cipriani B, Fischer FR, Granucci F, Ricciardi-Castagnoli P, Brosnan C, Stern LJ, Strominger JL and Santambrogio L, The Journal of Immunology, 2002, 169, 2264– 2273. 12193691 source
The Ag presentation section reports that GM-CSF increased class II MHC protein in neonatal mouse microglia, while M-CSF did not; the Discussion links GM-CSF to proinflammatory activity. The paper does not establish opposite MHCII effects in mouse versus human microglia or show that such a difference subsequently changes neuroinflammation. (LLM, conf=0.87)
Experimental — accuracy not yet measured
- Findingown data
Upon LPS/ATP treatment, iMG infused into tubular organoids displayed lower complexity in patterns and a spherical shape, exemplified by lower fractal dimensions, lower lacunarity, and higher convex-hull circularity.
“We found that, upon LPS/ATP treatment, iMG infused into our tubular organoids displayed a lower complexity in patterns as well as a spherical shape, exemplified by lower fractal dimensions, lower lacunarity, and higher convex-hull circularity (Figure 6c).56”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [56] Fernández-Arjona M. d. M., Grondona JM, Granados-Durán P, Fernández-Llebrez P and López-Ávalos MD, Frontiers in Cellular Neuroscience, 2017, 11. PMC5550745 28848398 source
- Findingnone given
By employing 3D printed hollow mesh scaffolds, the device can be easily incorporated into multiwell-plates for reliable, scalable, and reproducible generation of tubular organoids.
“By employing 3D printed hollow mesh scaffolds, our device can be easily incorporated into multiwell-plates for reliable, scalable, and reproducible generation of tubular organoids.”
- Also stated in: § Introduction · plain
“We integrated 3D printed hollow mesh scaffolds into multiwell-plates for the reliable, scalable, and reproducible generation of tubular organoids.”
Quote in the paperSubstantial claim - Also stated in: § Introduction · plain
- Findingown data
Microglia incorporated into tubular organoid culture can respond to external stimulation such as LPS and DAMGO.
“Moreover, the microglia incorporated into our tubular organoid culture can respond to external stimulation such as LPS and DAMGO, recapitulating the in vivo phenotype and functions during neuroinflammation.”
Quote in the paperSubstantial claim - Findingown data
The thFO culture system allows the growth of more uniformly distributed, size-consistent VZ/SVZ zones compared with the more random and heterogeneous VZ/SVZ zones inside conventional hFO.
“We found that our thFO culture system allows the growth of more uniformly distributed, size-consistent VZ/SVZ zones as compared with the more random and heterogeneous VZ/SVZ zones inside the conventional hFO (Figure 4b).”
Quote in the paperSubstantial claim
- Findingown data
Elevated levels of proinflammatory cytokines post-opioid exposure were dampened by LY2828360 treatment.
“Finally, the elevated levels of proinflammatory cytokines post-opioid exposure were dampened by LY2828360 treatment (Figure 7d).”
Quote in the paperSubstantial claim - Findingown data
Post-DAMGO treatment, iMG in iMG-infused thFO displayed an ameboid morphology similar to that following LPS/ATP treatment.
“We discovered that indeed, post-DAMGO treatment, iMG (identified by Iba1 staining) displayed an ameboid morphology similar to that following LPS/ATP treatment (Figure 6b,c).”
Quote in the paperSubstantial claim- Rests on an assumption: DAMGO at 100 nM for 24 hours specifically activates μ-opioid receptors to activate microglia.
- Findingown data
iMG derived from WA01 hPSC were confirmed by double staining with Iba1 and CD68.
“We confirmed iMG derived from WA01 by double staining with Iba1 and CD68 (Supplementary Figure S1).”
Quote in the paperSubstantial claim - Findingown data
The activated morphology of iMG after DAMGO treatment can be partially reversed by treatment with CB2 agonist LY2828360.
“Interestingly, this activated morphology can be partially reversed by treatment of cannabinoid receptor 2 (CB2) agonist LY2828360 (Figure 6b,c).”
Quote in the paperSubstantial claim- Rests on an assumption: LY2828360 acts as a CB2 agonist to reverse microglial activation.
- Findingown data
Upon LPS/ATP treatment, activated iMG secreted higher IL-1β and IL-18.
“We found that, upon LPS/ATP treatment, activated iMG also secreted higher IL-1β and IL-18, which indicates a functional output of inflammasome activation (Figure 7d).”
Quote in the paperSubstantial claim - Findingown data
The tubular 3D cultures of N2a cells exhibited good viability over a 7-day culture.
“We found that these tubular 3D cultures exhibited good viability over a 7-day culture (Figure 2d).”
Quote in the paperSubstantial claim - Findingown data
The activated microglia transformed back to the homeostatic status with further treatment by LY2828360.
“and transformed back to the homeostatic status with further treatment by a cannabinoid receptor 2 agonist (LY2828360).”
Quote in the paperSubstantial claim - Findingown data
When iMG were incorporated into tubular brain organoids and treated with LPS/ATP, TNF-α secretion significantly increased.
“However, when we incorporated iMG into tubular brain organoids and then treated the culture with LPS/ATP, we observed a significant increase in TNF-α secretion.”
Quote in the paperSubstantial claim - Findingown data
The tubular 3D cultures of N2a cells showed no detectable signs of hypoxia.
“We found that the tubular 3D cultures of N2a cells showed no detectable signs of hypoxia.”
Quote in the paperSubstantial claim - Findingown data
The more guided human forebrain differentiation protocol limited organoid differentiation fate toward dorsal forebrain in both conventional protocols as well as the tubular culture protocol.
“Additionally, as expected, the more guided human forebrain differentiation protocol limited organoid differentiation fate toward dorsal forebrain in both conventional protocols as well as the tubular culture protocol.”
Quote in the paperNot a substantial claim - Findingown data
Isogenic microglia were activated after exposure to DAMGO.
“We found isogenic microglia were activated after exposure to an opioid receptor agonist (DAMGO)”
Quote in the paperSubstantial claim - Findingnone given
Tubular organoid culture eliminated hypoxic core formation by scaffold guided growth and constant passive flow perfusion using a rocking-platform system.
“Our tubular organoid culture eliminated hypoxic core formation by scaffold guided growth and constant passive flow perfusion using a rocking-platform system.”
Quote in the paperSubstantial claim - Findingown data
Such elevated inflammasome activation was partially rescued by LY2828360 treatment.
“Such elevated inflammasome activation was partially rescued by LY2828360 treatment (Figure 7b,c).”
Quote in the paperSubstantial claim - Findingown data
The activated microglia in tubular organoids had a stronger cytokine response compared to those in 2D microglial cultures.
“Importantly, the activated microglia in tubular organoids had a stronger cytokine response compared to those in 2D microglial cultures.”
Quote in the paperSubstantial claim - Findingown data
Post LPS/ATP treatment, ramified microglia in iMG-infused thFO became activated and transformed into an amoeboid-like morphology.
“Post LPS/ATP treatment, we observed the ramified microglia became activated and transformed into an amoeboid-like morphology (Figure 6b).”
Quote in the paperSubstantial claim - Findingown data
TNF-α was not detected in high levels in 2D iMG cultures post LPS/ATP treatment.
“Interestingly, TNF-α, a key mediator in neuroinflammation was not detected in high levels in 2D iMG cultures post LPS/ATP treatment.”
Quote in the paperSubstantial claim - Findingown data
NLRP3/ASC colocalization is significantly increased in LPS/ATP and DAMGO treatment conditions in iMG infused into tubular organoids.
“As we expected, NLRP3/ASC colocalization is significantly increased in LPS/ATP and DAMGO treatment conditions.”
Quote in the paperSubstantial claim - Findingown data
When placed onto a rocking platform with a rocking speed of 1 rpm, the device induces a constant flow of 2 μL/second.
“When placed onto a rocking platform with a rocking speed of 1 rpm, this induces a constant flow of 2 μL/second. We validated this flow with fluorescent beads (Supplementary Video S1).”
Quote in the paperNot a substantial claim - Premisecitation
Microglia orchestrate brain inflammatory responses as the resident myeloid cells of the brain.
“As the resident myeloid cells of the brain, microglia orchestrate brain inflammatory responses.2”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [2] Salter Michael W. and Beggs S, Cell, 2014, 158, 15– 24. 24995975 source
- Interpretationreasoning
The above methods are still not sufficient to fully support microglia-organoid interaction studies, due to reasons such as low throughput, poor reproducibility, labor intensiveness, complicated fabrication, or difficulty in immune cell introduction.
“However, the above methods are still not sufficient to fully support microglia-organoid interaction studies, due to reasons such as low throughput, poor reproducibility, labor intensiveness, complicated fabrication, or difficulty in immune cell introduction.”
Quote in the paperSubstantial claim - Interpretationown data
The neuroinflammation model can be utilized for screening of therapeutics as evidenced by its clear responses to LY2828360 treatment.
“Our neuroinflammation model can also be utilized for the screening of therapeutics as evidenced by its clear responses to LY2828360 treatment.”
Quote in the paperSubstantial claim - Interpretationown data
Perfusion from both the inner lumen and outside skirt gaps provides sufficient oxygen in the tubular 3D culture.
“Suggesting that the perfusion from both the inner lumen and outside skirt gaps provides sufficient oxygen (Figure 2f).”
Quote in the paperSubstantial claim - Interpretationown data
The response of microglia in tubular organoid culture to external stimulation recapitulates the in vivo phenotype and functions during neuroinflammation.
“Moreover, the microglia incorporated into our tubular organoid culture can respond to external stimulation such as LPS and DAMGO, recapitulating the in vivo phenotype and functions during neuroinflammation.”
Quote in the paperSubstantial claim - Interpretationown data
CB2 agonist can partially rescue the activated microglia morphology by DAMGO in the 3D co-culture system.
“This result demonstrated that CB2 agonist can partially rescue the activated microglia morphology by DAMGO in our 3D co-culture system.”
Quote in the paperSubstantial claim
- Classified as probably not a substantial claim. 11
- May combine two claims. 27
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationActivated microglia can secrete pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β, IL-18) or anti-inflammatory cytokines (e.g., IL-4, IL-10, TGF-β) depending on the dose, frequency, and context of stimulation.
“Activated microglia can either secrete pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-18 or anti-inflammatory cytokines such as IL-4, IL-10, and transforming growth factor (TGF)-β depending on the dose, frequency, and context of the stimulation.4–7”
Quote in the paperNot a substantial claim2 of 4 sources checked- cites [4] Renno T, Krakowski M, Piccirillo C, Lin JY and Owens T, The Journal of Immunology, 1995, 154, 944– 953. 7814894 source
- cites [5] Qin Y, Garrison BS, Ma W, Wang R, Jiang A, Li J, Mistry M, Bronson RT, Santoro D, Franco C, Robinton DA, Stevens B, Rossi DJ, Lu C and Springer TA, Cell, 2018, 174, 156– 171.e116. 29909984 PMC6089614 source
The Introduction states that TGF-β1 is constitutively expressed in microglia, and the Discussion describes localized activation of microglia-associated TGF-β1. The paper does not establish the claimed pro- and anti-inflammatory cytokine secretion patterns or their dependence on stimulation dose, frequency, and context. No page numbers are available in our copy. (LLM, conf=0.83)
Experimental — accuracy not yet measured - cites [6] Hickman S, Izzy S, Sen P, Morsett L and El Khoury J, Nature Neuroscience, 2018, 21, 1359– 1369. 30258234 PMC6817969 source
The “Protection against injurious self and non-self stimuli” section says that stimulated microglia produce cytokines including TNF and IL-1; the “Huntington’s disease” section also reports increased IL6 and TNF expression. Our copy does not substantiate the listed anti-inflammatory cytokines or the claim that cytokine secretion depends on stimulation dose and frequency. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [7] Hanisch U-K and Kettenmann H, Nature Neuroscience, 2007, 10, 1387– 1394. 17965659 source
May combine two claims.
PremisecitationThese microglia/organoid models still suffer from variation of organoid size and composition, confounds stemming from organoid hypoxia/necrosis, and physical damage from microglia injection.
“However, these microglia/organoid models still suffer from (1) variation of organoid size and composition; (2) confounds stemming from organoid hypoxia/necrosis; and (3) physical damage from microglia injection.29–31”
Quote in the paperSubstantial claim3 of 3 sources checked- cites [29] Bhaduri A, Andrews MG, Mancia Leon W, Jung D, Shin D, Allen D, Jung D, Schmunk G, Haeussler M, Salma J, Pollen AA, Nowakowski TJ and Kriegstein AR, Nature, 2020, 578, 142– 148. 31996853 PMC7433012 source
The sections “Cellular Stress Increases in Organoids” and “Organoid Environment Activates Stress” report stress in organoid cells, supporting a general concern about culture-related confounds. They do not establish hypoxia or necrosis as the cause. “Impaired Cell Type Fidelity in Organoids” says composition was similar across organoids of the same age, and the paper does not report organoid-size variation or damage from injecting microglia; its injection methods concern transplanting organoid cells into mice. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [30] Boisvert EM, Means RE, Michaud M, Madri JA and Katz SG, Cell Death & Disease, 2019, 10, 325. 30975982 PMC6459920 source
The Discussion (page unavailable) reports substantial batch-to-batch variability in several brain organoid systems and identifies necrosis as a potential concern in existing models. It does not specify variation in size and composition or discuss physical damage from microglia injection. The authors also report no apparent necrosis in their own organoids for up to 5–6 months. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [31] Pașca AM, Park J-Y, Shin H-W, Qi Q, Revah O, Krasnoff R, O’Hara R, Willsey AJ, Palmer TD and Pașca SP, Nature Medicine, 2019, 25, 784– 791. PMC7020938 31061540 source
The study-limitations passage (section labeled Abstract; page unavailable) says this model does not include microglia and proposes incorporating them in future models. Our copy discusses experimentally induced hypoxia, but does not identify organoid hypoxia or necrosis as a confound of microglia/organoid models. Searches for size and composition variation and damage from microglia injection likewise found no supporting passage. (LLM, conf=0.78)
Experimental — accuracy not yet measured
May combine two claims.
PremisecitationThe IFNγ-induced MHCII/HLA response can be blocked by TGFβ1 in mice but not in human microglia.
“This IFNγ-induced response can be blocked by TGFβ1 in mice,18 but not in human microglia.19”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [18] O’Keefe GM, Nguyen VT and Benveniste EN, European Journal of Immunology, 1999, 29, 1275– 1285. 10229095
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [19] Smith AM, Graham ES, Feng SX, Oldfield RL, Bergin PM, Mee EW, Faull RLM, Curtis MA and Dragunow M, PloS one, 2013, 8, e80463. 24339874 PMC3855168 source
May combine two claims.
PremisecitationAnimal microglia are often inaccessible to real-time imaging and cannot fully reflect human biology.
“Animal microglia are often inaccessible to real-time imaging, and cannot fully reflect human biology.15–17”
Quote in the paperSubstantial claim3 of 3 sources checked- cites [15] Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian JL, Fernhoff NB, Mulinyawe SB, Bohlen CJ, Adil A, Tucker A, Weissman IL, Chang EF, Li G, Grant GA, Hayden Gephart MG and Barres BA, Proceedings of the National Academy of Sciences, 2016, 113, E1738– E1746. PMC4812770 26884166 source
The introduction describes difficulty distinguishing microglia from other immune cells, not a lack of access to real-time imaging. A later RNAseq discussion mentions transcranial two-photon imaging of mouse microglia, but does not establish how often imaging is possible. The passages examined also do not say that animal microglia cannot fully reflect human biology (searched: real-time imaging, live imaging, mouse–human differences). (LLM, conf=0.72)
Experimental — accuracy not yet measured - cites [16] Hoogland ICM, Houbolt C, van Westerloo DJ, van Gool WA and van de Beek D, J Neuroinflammation, 2015, 12, 114. 26048578 PMC4470063 source
The Discussion questions the clinical relevance of LPS-based animal models because their microglial responses differ from those produced by live bacteria. That supports a narrower caution about applying some animal-model findings, but the paper does not say animal microglia cannot fully reflect human biology or that they are often inaccessible to real-time imaging. (LLM, conf=0.70)
Experimental — accuracy not yet measured - cites [17] Galatro TF, Holtman IR, Lerario AM, Vainchtein ID, Brouwer N, Sola PR, Veras MM, Pereira TF, Leite REP, Möller T, Wes PD, Sogayar MC, Laman JD, den Dunnen W, Pasqualucci CA, Oba-Shinjo SM, Boddeke E, Marie SKN and Eggen BJL, Nat Neurosci, 2017, 20, 1162– 1171. 28671693 source
The Discussion (p. 10) reports critical differences between human and mouse microglia, especially with aging, supporting the point that animal microglia cannot fully reflect human biology. The paper does not say that animal microglia are often inaccessible to real-time imaging; a search for real-time and in vivo imaging found no support for that part. (LLM, conf=0.88)
Experimental — accuracy not yet measured
May combine two claims.
PremisecitationMouse microglia respond to IFNγ by increasing MHCII expression whereas human microglia increase HLA expression.
“For example, mouse microglia respond to interferon-γ (IFNγ) by increasing the expression levels of class II major histocompatibility complex proteins (MHCII) whereas human microglia increase human leukocyte antigen (HLA).”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [18] O’Keefe GM, Nguyen VT and Benveniste EN, European Journal of Immunology, 1999, 29, 1275– 1285. 10229095
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [19] Smith AM, Graham ES, Feng SX, Oldfield RL, Bergin PM, Mee EW, Faull RLM, Curtis MA and Dragunow M, PloS one, 2013, 8, e80463. 24339874 PMC3855168 source
The Results section reports that IFNγ increased HLA expression in adult human microglia. The Discussion reports IFNγ-mediated MHC-II induction in rodent tumor microglia/macrophages, but does not specifically establish the stated response in mouse microglia alone; that part of the claim is more specific than the evidence in our copy. (LLM, conf=0.83)
Experimental — accuracy not yet measured
May combine two claims.
PremisecitationDuring substance abuse, microglia-mediated neuroinflammation can exert direct neural toxicity and potentially exacerbate addiction via modulation of synapse plasticity.
“Particularly, during substance abuse, microglia-mediated neuroinflammation can exert direct neural toxicity and potentially exacerbate addiction via modulation of synapse plasticity.13, 14”
Quote in the paperSubstantial claim2 of 2 sources checked- cites [13] Lacagnina MJ, Rivera PD and Bilbo SD, Neuropsychopharmacology, 2017, 42, 156– 177. 27402494 PMC5143481 source
- cites [14] Loftis JM and Huckans M, Pharmacol Ther, 2013, 139, 289– 300. 23631821 PMC3725980 source
Section 2.2 (Stimulants; page unavailable) links activated microglia during stimulant exposure to neuroinflammation, neurodegeneration and neuronal damage. Section 2.3 (Opiates; page unavailable) links microglial signaling to morphine-induced reward. Neither passage identifies modulation of synaptic plasticity as the mechanism by which inflammation might exacerbate addiction. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim. May combine two claims.
PremisecitationHuman brain organoids are 3D cultures derived from human pluripotent stem cells and recapitulate native brain cellular components, cytoarchitecture, and exhibit neural activity.
“Human brain organoids are 3D cultures derived from human pluripotent stem cells and recapitulate native brain cellular components, cytoarchitecture, and exhibit neural activity.22–24”
Quote in the paperSubstantial claim3 of 3 sources checked- cites [22] Lancaster MA, Renner M, Martin C-A, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP and Knoblich JA, Nature, 2013, 501, 373– 379. 23995685 PMC3817409 source
- cites [23] Amin ND and Paşca SP, Neuron, 2018, 100, 389– 405. 30359604 source
The accessible copy yields only the title and a garbled list of section headings, with no readable passage addressing the claim. Searches for pluripotent stem cells, neural activity, and organoid structure did not recover evidence about their cellular components, cytoarchitecture, or activity. (LLM, conf=0.72)
Experimental — accuracy not yet measured - cites [24] Trujillo CA, Gao R, Negraes PD, Gu J, Buchanan J, Preissl S, Wang A, Wu W, Haddad GG, Chaim IA, Domissy A, Vandenberghe M, Devor A, Yeo GW, Voytek B and Muotri AR, Cell stem cell, 2019, 25, 558– 569.e557. 31474560 PMC6778040 source
Classified as probably not a substantial claim.
PremisecitationUpon stimulation by factors such as TLR ligands, Aβ aggregates, complement factors, or opioids, microglia can shift from a homeostatic state to an activated state.
“Upon stimulation by factors such as toll-like receptor (TLR) ligands, β-amyloid (Aβ) aggregates, complement factors, or opioids, microglia can shift from homeostatic state to an activated state.”
Quote in the paperSubstantial claim2 of 4 sources checked- cites [4] Renno T, Krakowski M, Piccirillo C, Lin JY and Owens T, The Journal of Immunology, 1995, 154, 944– 953. 7814894 source
- cites [5] Qin Y, Garrison BS, Ma W, Wang R, Jiang A, Li J, Mistry M, Bronson RT, Santoro D, Franco C, Robinton DA, Stevens B, Rossi DJ, Lu C and Springer TA, Cell, 2018, 174, 156– 171.e116. 29909984 PMC6089614 source
The Results section (“Abnormalities in microglia, macrophages, and TGF-β1 in Lrrc33−/− mice”; page not provided) reports that loss of Lrrc33 gave microglia an activated, reactive-like phenotype compared with wild-type microglia. The paper does not identify TLR ligands, Aβ aggregates, complement factors, or opioids as stimuli for that shift; it discusses deficient TGF-β signaling instead. (LLM, conf=0.85)
Experimental — accuracy not yet measured - cites [6] Hickman S, Izzy S, Sen P, Morsett L and El Khoury J, Nature Neuroscience, 2018, 21, 1359– 1369. 30258234 PMC6817969 source
The sections “Protection against injurious self and non-self stimuli” and “Aβ-induced inflammation” (pages unavailable) describe microglial inflammatory responses to Aβ and interactions involving TLRs. They do not identify opioids or complement factors as stimuli that shift microglia into an activated state; the latter section mentions a complement receptor, which is not the same claim. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [7] Hanisch U-K and Kettenmann H, Nature Neuroscience, 2007, 10, 1387– 1394. 17965659 source
May combine two claims.
Findingown dataiMG have lower baseline activation compared with immortalized microglia like HMC3 and were significantly more activated upon LPS treatment followed by ATP.
“We discovered that iMG have lower baseline activation as compared with immortalized microglia like HMC3 and were significantly more activated upon LPS treatment followed by ATP, which facilitates NLRP3 inflammasome assembly (Supplementary Figure S2).55”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [55] Zha QB, Wei HX, Li CG, Liang YD, Xu LH, Bai WJ, Pan H, He XH and Ouyang DY, Front Immunol, 2016, 7, 597. 28018360 PMC5149551 source
May combine two claims.
Findingown dataThe success rate of on-chip tubular organoid formation is around 60%, comparable with the intrinsic success rate (70%) of the cerebral organoid protocol.
“Overall, we observe a success rate of on-chip tubular organoid formation around 60%, a rate comparable with the intrinsic success rate (70%) with the cerebral organoid protocol.52”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [52] Yakoub AM and Sadek M, Cell transplantation, 2018, 27, 393– 406. 29749250 PMC6038047 source
The Procedures section describes measuring successful embryoid-body formation, and the Characterization section describes successful cerebral organoids, but neither passage gives a 70% success rate. Searches for “70% success rate” and cerebral-organoid formation efficiency found no passage establishing that figure. The on-chip 60% rate is presented as the citing authors’ own observation, not a result attributed to this source. (LLM, conf=0.72)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S4 (based). May combine two claims.
Findingnone givenThe device provides perfusion of medium and oxygen from both the outside and an embedded hollow lumen in the center, limiting the distance between the medium and all cells within the organoid culture to < 500 μm.
“Our technology has the following advantages: (1) Excellent prefusion: The device provides perfusion of medium and oxygen from both the outside and an embedded hollow lumen in the center, limiting the distance between the medium and all cells within the organoid culture to < 500 μm.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataTubular culture for organoids derived from both hFO and hCO protocols have higher expression of dorsal forebrain neural progenitor cell markers PAX6 and SOX2 as well as the deep cortical neuron marker TBR1 (p<0.05), with the tubular culture showing less variation across organoids (n=5).
“We discovered that (1) tubular culture for organoids derived from both hFO and hCO protocols have higher expression of dorsal forebrain neural progenitor cell markers PAX6 and SOX2 as well as the deep cortical neuron marker TBR1 (p<0.05), with the tubular culture showing less variation across organoids (n=5) (Figure 5a).”
Quote in the paperSubstantial claimMay combine two claims.
Findingnone givenCompared with conventional protocols, the method increased neural progenitor proliferation and reduced heterogeneity of human brain organoids.
“Compared with conventional protocols, our method increased neural progenitor proliferation and reduced heterogeneity of human brain organoids.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataWith the tubular culture method, higher expression of PAX6, SOX2, and Tuj1 was detected in thFOs as compared with conventional hFOs, indicating an enhanced neural fate commitment as well as better neural differentiation.
“With the tubular culture method, higher expression of PAX6, SOX2, and Tuj1 was detected in thFOs as compared with conventional hFOs, indicating an enhanced neural fate commitment as well as better neural differentiation (Figure 5b).”
Quote in the paperSubstantial claimMay combine two claims.
Findingnone givenTubular organoids showed reduced heterogeneity and improved neural development.
“The tubular organoids showed reduced heterogeneity and improved neural development.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataOver a 3-day co-culture period, iMG migrated and integrated into tubular hFO and showed a homeostatic state ramified morphology.
“Over a 3-day co-culture period, iMG migrated and integrated into tubular hFO and showed a homeostatic state ramified morphology (Figure 6a).”
Quote in the paperSubstantial claim- Rests on an assumption: The ramified morphology of iMG in tubular hFO indicates a homeostatic state.
May combine two claims.
Findingown dataThe hCO has a more heterogeneous differentiation with expression of both dorsal and ventral forebrain differentiation markers as well as expression of some midbrain/hindbrain and even slight mesodermal and endodermal layer markers, with the diversity of differentiation increasing over time in culture (day 16 versus day 35).
“the hCO has a more heterogeneous differentiation with the expression of both dorsal and ventral forebrain differentiation markers as well as expression of some midbrain/hindbrain and even slight mesodermal and endodermal layer markers, with the diversity of differentiation increasing over time in culture (day 16 versus day 35).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThere is a higher number and more even distribution of newly divided cells inside thFOs compared with conventional hFOs.
“We found a higher number and more even distribution of newly divided cells inside thFOs as compared with conventional hFOs (Figure 4c).”
Quote in the paperSubstantial claimMay combine two claims.
Findingnone givenBy introducing rocking flows through the tubular device channel, the device can perfuse nutrients and oxygen to minimize organoid necrosis and hypoxia, and incorporate immune cells into organoids to model neuro-immune interactions.
“By introducing rocking flows through the tubular device channel, our device can perfuse nutrients and oxygen to minimize organoid necrosis and hypoxia, and incorporate immune cells into organoids to model neuro-immune interactions.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIsogenic microglia were incorporated with non-invasive flow, preserving their homeostatic morphology and function.
“Additionally, we demonstrated the incorporation of isogenic microglia introduced with the non-invasive flow, preserving their homeostatic morphology and function.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataTubular cerebral organoid culture showed significantly lower HIF1a expression than conventional cerebral organoid culture (p<0.005), indicating reduction of hypoxia conditions.
“Additionally, we analyzed HIF1a expression inside conventional and tubular cerebral organoid culture, the tubular culture showed significantly lower HIF1a expression (p<0.005), indicating reduction of hypoxia conditions (Figure 2g).”
Quote in the paperSubstantial claimClassified as probably not a substantial claim. May combine two claims.
Findingnone givenThe device can be inserted into multiwell-plates (24, 96, 384, or higher), followed by culturing in an incubator.
“(3) Compatibility with common laboratory settings: The device can be inserted into multiwell-plates (24, 96, 384, or higher), followed by culturing in an incubator.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S4 (based). May combine two claims.
FindingreasoningThe device allows simple and effective perfusion to introduce immunocytes such as microglia while avoiding invasive injection or manipulation and preserving the native, homeostatic status of microglia.
“(2) Noninvasive immune incorporation: The device allows simple and effective perfusion to introduce immunocytes such as microglia while avoiding invasive injection or manipulation and preserving the native, homeostatic status of microglia.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningThe results of these promising biotechnologies highlight the importance of nutrient/oxygen perfusion as well as the elimination of hypoxia/necrosis on organoid development and function.
“The results of these promising biotechnologies highlight the importance of nutrient/oxygen perfusion as well as the elimination of hypoxia/necrosis on organoid development and function.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningBecause increased MHCII and HLA levels are associated with neuroinflammatory response to disease and injury in mouse and human brains, mouse models can be limited in their usefulness in extrapolating the results of several therapeutic approaches into the clinic.
“Because increased MHCII and HLA levels are associated with neuroinflammatory response to disease and injury in mouse and human brains, mouse models can be limited in their usefulness in extrapolating the results of several therapeutic approaches into the clinic.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationNeuroinflammation plays a critical role in neurodegenerative diseases, autoimmune diseases, and substance abuse.
“Neuroinflammation plays a critical role in neurodegenerative diseases, autoimmune diseases as well as in substance abuse.1”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [1] Kempuraj D, Thangavel R, Natteru PA, Selvakumar GP, Saeed D, Zahoor H, Zaheer S, Iyer SS and Zaheer A, J Neurol Neurosurg Spine, 2016, 1. PMC5260818 28127589
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenEach qPCR reaction was repeated in triplicates and 5 organoids/EBs were analyzed for each group.
“Each qPCR reaction was repeated in triplicates and 5 organoids/EBs were analyzed for each group.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataBetter perfusion and constant passive flow stimulation increases cell division and guides the VZ/SVZ zone to form more uniformly.
“This suggests that better perfusion and constant passive flow stimulation increases cell division and guides the VZ/SVZ zone to form more uniformly.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenWA01 human embryonic stem cells were used following relevant laws and guidelines approved by Indiana University and the WiCell institute.
“Human embryonic stem cell WA01 was purchased from WiCell, and used following the relevant laws and guidelines approved by the Indiana University and the WiCell institute.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataEach ELISA sample was tested in triplicate.
“Each sample was tested in triplicate and read at 450 nm for absorbance.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningThe increase in TNF-α secretion upon LPS/ATP treatment of iMG in tubular brain organoids could be explained by iMG cross-talk with neurons and astrocytes inside the tubular organoids, inducing higher neuroinflammation and higher TNF-α production from iMG and potentially neurons and astrocytes.
“This could be explained by iMG cross-talk with neurons and astrocytes inside the tubular organoids, inducing a higher level of neuroinflammation and a significantly higher production of TNF-α, from not only iMG themselves but also potentially from neurons and astrocytes inside a tubular organoid culture.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataiMG infused to thFO could recapitulate key aspects of neuroinflammation including microglia morphology change, NLRP3 inflammasome activation as well as cytokine production.
“Thus, we demonstrated that iMG infused to thFO could recapitulate key aspects of neuroinflammation including microglia morphology change, NLRP3 inflammasome activation as well as cytokine production.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThis model could be used to study opioid-induced neural inflammation and the efficacy of therapeutics.
“We also demonstrated that this model could be used to study opioid-induced neural inflammation and the efficacy of therapeutics.”
Quote in the paperSubstantial claim
The paper relies on this without support.
Current in vitro neuroinflammation studies are largely based on 2D cultures, which lack a complex brain microenvironment to preserve the native state of microglia and the complex cross-talk between neurons and glial cells.
“Current in vitro neuroinflammation studies are largely based on 2D cultures, which lack a complex brain microenvironment to preserve the native state of microglia, as well as the complex cross-talk between neurons and glial cells.”
The paper relies on this without support.
Most current brain organoids lack mesoderm-derived microglia, prohibiting their application to model neuro-immune interactions.
“However, most current brain organoids lack mesoderm-derived microglia, prohibiting their application to model neuro-immune interactions.”
The paper relies on this without support.
The ramified morphology of iMG in tubular hFO indicates a homeostatic state.
“Over a 3-day co-culture period, iMG migrated and integrated into tubular hFO and showed a homeostatic state ramified morphology (Figure 6a).”
The paper relies on this without support.
DAMGO at 100 nM for 24 hours specifically activates μ-opioid receptors to activate microglia.
“Here, we treated iMG infused thFO with DAMGO, a synthetic μ-opioid receptor agonist, at a concentration of 100 nM for 24 hours.”
The paper relies on this without support.
LY2828360 acts as a CB2 agonist to reverse microglial activation.
“Interestingly, this activated morphology can be partially reversed by treatment of cannabinoid receptor 2 (CB2) agonist LY2828360 (Figure 6b,c).”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Current device compatibility conflicts with planned resizing
MinorThe paper says the device can already be inserted into 96- and 384-well plates, but later says it must be made smaller to fit those formats.
“The device can be inserted into multiwell-plates (24, 96, 384, or higher), followed by culturing in an incubator.”
“we plan to adopt higher resolution 3D printing to print finer and smaller tubular structure to scale our model to fit within a 96 or 384 well plate format.”
Microglial recovery is described as complete and partial
MinorThe abstract says LY2828360 returned activated microglia to a homeostatic state, whereas the results describe only a partial reversal of their activated morphology.
“transformed back to the homeostatic status with further treatment by a cannabinoid receptor 2 agonist (LY2828360).”
“Interestingly, this activated morphology can be partially reversed by treatment of cannabinoid receptor 2 (CB2) agonist LY2828360 (Figure 6b,c).”
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Microfluidic organoids-on-a-chip: The future of human models
Saorin, G., Caligiuri, I., & Rizzolio, F. (2022) · Seminars in Cell and Developmental Biology · doi:10.1016/j.semcdb.2022.10.001 · Cited by 206 · see citing works
Microglia-containing human brain organoids for the study of brain development and pathology
Zhang, W., Jiang, J., Xu, Z., Yan, H., Tang, B., Liu, C., Chen, C., & Meng, Q. (2022) · Molecular Psychiatry · doi:10.1038/s41380-022-01892-1 · Cited by 188 · see citing works
“[76] used a stereolithography 3D printer to develop a tubular device for brain organoid culture.”
MethodMicroglia-like Cells Promote Neuronal Functions in Cerebral Organoids
Fagerlund, I., Dougalis, A., Shakirzyanova, A., Gómez‐Budia, M., Pelkonen, A., Konttinen, H., Ohtonen, S., Fazaludeen, M. F., Koskuvi, M., Kuusisto, J., et al. (2021) · Cells · doi:10.3390/cells11010124 · Cited by 124 · see citing works
“Recently, the inflammatory functions of incorporated mature embryonic stem cell derived microglia were also assessed in tubular cortical organoids [55].”
BackgroundResultInfluentialAdvances in Spheroids and Organoids on a Chip
Fang, G., Chen, Y., Lu, H., & Jin, D. (2023) · Advanced Functional Materials · doi:10.1002/adfm.202215043 · Cited by 127 · see citing works
Self-citationPapamichail, L., Koch, L. S., Veerman, D., Broersen, K., & van der Meer, A. D. (2025) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2025.1515340 · Cited by 120 · see citing works
Neuropathogenesis-on-chips for neurodegenerative diseases
Amartumur, S., Nguyen, H. M., Huynh, T., Kim, T. S., Woo, R., Oh, E., Kim, K. K., Lee, L. P., & Heo, C. (2024) · Nature Communications · doi:10.1038/s41467-024-46554-8 · Cited by 120 · see citing works
“To enhance organoid viability, a dynamic medium fl ow can be achieved by integrating either a rocking platform or a syringe pump 49,543 .”
MethodUnderstanding Immune‐Driven Brain Aging by Human Brain Organoid Microphysiological Analysis Platform
Ao, Z., Song, S., Tian, C., Cai, H., Li, X., Miao, Y., Wu, Z., Krzesniak, J., Ning, B., Gu, M., et al. (2022) · Advanced Science · doi:10.1002/advs.202200475 · Cited by 77 · see citing works
Self-citationHuman iPSC-derived glia models for the study of neuroinflammation
Stöberl, N., Maguire, E., Salis, E., Shaw, B., & Hall-Roberts, H. (2023) · Journal of Neuroinflammation · doi:10.1186/s12974-023-02919-2 · Cited by 71 · see citing works
“Interestingly, the authors found that TNF levels were increased after LPS/ATP stimulation in tubular organoids but not in 2D iPSC-derived microglia monocultures, which could be explained by crosstalk of the different cell types (including astrocytes) present in the tubular organoids model [124].”
BackgroundPushing the boundaries of brain organoids to study Alzheimer’s disease
Cerneckis, J., Bu, G., & Shi, Y. (2023) · Trends in Molecular Medicine · doi:10.1016/j.molmed.2023.05.007 · Cited by 61 · see citing works
Global Literature Analysis of Organoid and Organ‐on‐Chip Research
Shoji, J., Davis, R. P., Mummery, C. L., & Krauss, S. J. K. (2023) · Advanced Healthcare Materials · doi:10.1002/adhm.202301067 · Cited by 48 · see citing works
Hu, Y., Zhu, T., Cui, H., & Cui, H. (2024) · Advanced Healthcare Materials · doi:10.1002/adhm.202403762 · Cited by 49 · see citing works
Brain organoids are new tool for drug screening of neurological diseases
Zhou, J.-Q., Zeng, L., Li, C.-T., He, D.-H., Zhao, H.-D., Xu, Y., Jin, Z.-T., & Gao, C. (2023) · Neural Regeneration Research · doi:10.4103/1673-5374.367983 · Cited by 47 · see citing works
“To minimize organoid necrosis and hypoxia, Ao et al. (2021) constructed a tubular device channel to control the perfusion of nutrients and oxygen and incorporate microglia into organoids.”
BackgroundVascular network-inspired diffusible scaffolds for engineering functional midbrain organoids
Cai, H., Tian, C., Chen, L., Yang, Y., Sun, A. X., McCracken, K. W., Tchieu, J. H., Gu, M., Mackie, K., & Guo, F. (2025) · Cell stem cell · doi:10.1016/j.stem.2025.02.010 · Cited by 55 · see citing works
Self-citationEngineering human spinal microphysiological systems to model opioid-induced tolerance
Cai, H., Ao, Z., Tian, C., Wu, Z., Kaurich, C., Chen, Z., Gu, M., Hohmann, A. G., Mackie, K., & Guo, F. (2022) · Bioactive Materials · doi:10.1016/j.bioactmat.2022.10.007 · Cited by 40 · see citing works
“Recently, we employed 3D printed scaffolds to generate tubular human brain organoids for reducing hypoxia/necrosis and improving the standardization of organoids.(43) In addition to growing better organoid cultures, several attempts have been made to enable monitoring of development as well as the response to drug tre…”
MethodSelf-citationShamul, J. G., Wang, Z., Gong, H., Ou, W., White, A. M., Garcia, D. P. M., Gu, S., Clyne, A. M., Quiñones‐Hinojosa, A., & He, X. (2024) · Nature Biomedical Engineering · doi:10.1038/s41551-024-01250-2 · Cited by 42 · see citing works
“Spheroids and organoids have also been used extensively in recapitulating CNS disease cellular state and biochemical milieu in diseases such as GBM 169–172 , AD 173–176 , PD 177,178 and ID 179–181 .”
D’Antoni, C., Mautone, L., Sanchini, C., Tondo, L., Grassmann, G., Cidonio, G., Bezzi, P., Cordella, F., & Angelantonio, S. D. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms241310762 · Cited by 39 · see citing works
“This finding is particularly intriguing considering that hypoxia is a recognized challenge commonly associated with organoid culture [65].”
BackgroundThe NLRP3 Inflammasome in Neurodegenerative Disorders: Insights from Epileptic Models
Palumbo, L., Carinci, M., Guarino, A., Asth, L., Zucchini, S., Missiroli, S., Rimessi, A., Pinton, P., & Giorgi, C. (2023) · Biomedicines · doi:10.3390/biomedicines11102825 · Cited by 38 · see citing works
Saglam‐Metiner, P., Yıldırım, E., Dincer, C., Başak, O., & Yeşil-Çeliktaş, Ö. (2024) · Microchimica Acta · doi:10.1007/s00604-023-06165-4 · Cited by 36 · see citing works
“…channels compatible with multi-well plates operated by dynamic rocking flow, for modeling of Alzheimer disease relevant pathology with microglia-mediated neuroinflammation after opioid exposure, after embryonic stem cell-derived microglia and tubular forebrain organoid co-culturing on device [30].”
Kofman, S., Mohan, N., Sun, X., Ibric, L., Piermarini, E., & Qiang, L. (2022) · Journal of Tissue Engineering · doi:10.1177/20417314221113391 · Cited by 30 · see citing works
Advances of 3D Cell Co-Culture Technology Based on Microfluidic Chips
Li, C., He, W., Song, Y., Zhang, X., Sun, J., & Zhou, Z. (2024) · Biosensors · doi:10.3390/bios14070336 · Cited by 33 · see citing works
“Research has indicated that the flow of mediators in liver cell secretions and the dynamic interaction with collagen play a crucial role in maintaining primary liver cell function [129–131].”
BackgroundInfluentialInduced pluripotent stem cell-derived macrophages as a platform for modelling human disease
Tiwari, S. K., Wong, W. J., Moreira, M., Pasqualini, C., & Ginhoux, F. (2024) · Nature reviews. Immunology · doi:10.1038/s41577-024-01081-x · Cited by 32 · see citing works
“Ao et al. 138 developed a device that allowed an ‘organoid on a chip’ model by providing 3D printing scaffolds.”
Functional Neural Networks in Human Brain Organoids
Gu, L., Cai, H., Chen, L., Gu, M., Tchieu, J. H., & Guo, F. (2024) · BME Frontiers · doi:10.34133/bmef.0065 · Cited by 27 · see citing works
Self-citationExploiting sound for emerging applications of extracellular vesicles
Wu, Z., Cai, H., Tian, C., Ao, Z., Jiang, L., & Guo, F. (2023) · Nano Research · doi:10.1007/s12274-023-5840-6 · Cited by 24 · see citing works
Self-citationTailoring biomaterials for biomimetic organs-on-chips
Sun, L., Bian, F., Xu, D., Luo, Y., Wang, Y., & Zhao, Y. (2023) · Materials Horizons · doi:10.1039/d3mh00755c · Cited by 24 · see citing works
Ma, C., Seong, H., Li, X., Yu, X., Xu, S., & Li, Y. (2022) · Stem Cells International · doi:10.1155/2022/2150680 · Cited by 23 · see citing works
Methodologies for Generating Brain Organoids to Model Viral Pathogenesis in the CNS
Hopkins, H. K., Traverse, E. M., & Barr, K. L. (2021) · Pathogens · doi:10.3390/pathogens10111510 · Cited by 20 · see citing works
iPSC-derived PSEN2 (N141I) astrocytes and microglia exhibit a primed inflammatory phenotype
Sullivan, M. A., Lane, S. D., McKenzie, A. D. J., Ball, S. R., Sunde, M., Neely, G. G., Moreno, C. L., Maximova, A., Werry, E. L., & Kassiou, M. (2024) · Journal of Neuroinflammation · doi:10.1186/s12974-023-02951-2 · Cited by 22 · see citing works
“To generate iPSC-derived microglia-like cells, we followed a method based on a previously published protocol which produced microglia-like cells with gene expression profiles and functional activity very similar to those of primary human fetal and adult microglia [22, 35].”
MethodEngineering human midbrain organoid microphysiological systems to model prenatal PFOS exposure
Tian, C., Cai, H., Ao, Z., Gu, L., Li, X., Niu, V. C., Bondesson, M., Gu, M., Mackie, K., & Guo, F. (2024) · The Science of The Total Environment · doi:10.1016/j.scitotenv.2024.174478 · Cited by 21 · see citing works
Self-citationGeneration of Neural Organoids and Their Application in Disease Modeling and Regenerative Medicine
Huang, R., Gao, F., Yu, L., Chen, H., & Zhu, R. (2025) · Advanced Science · doi:10.1002/advs.202501198 · Cited by 19 · see citing works
Wu, Z., Ao, Z., Cai, H., Li, X., Chen, B., Tu, H., Wang, Y., Lu, R. O., Gu, M., Liang Cheng, C., et al. (2023) · Journal of Nanobiotechnology · doi:10.1186/s12951-023-01786-6 · Cited by 18 · see citing works
Self-citationAlzheimer’s Disease: Current Perspectives and Advances in Physiological Modeling
Boder, E. J., & Banerjee, I. A. (2021) · Bioengineering · doi:10.3390/bioengineering8120211 · Cited by 18 · see citing works
“Ao and co-workers [119] recently developed a tubular organoid on-a-chip device to generate organoids and model neuroinflammation.”
BackgroundÖzçelik, A., Abas, B. İ., Erdoğan, Ö., Cevik, E., & Çevik, Ö. D. (2022) · Biosensors · doi:10.3390/bios12121177 · Cited by 15 · see citing works
“developed an acoustofluidic platform to study human brain organoids by implementing acoustic tweezers in microfluidic devices [27].”
BackgroundCerneckis, J., & Shi, Y. (2023) · Frontiers in Cellular Neuroscience · doi:10.3389/fncel.2023.1198715 · Cited by 14 · see citing works
“Exposing neuroimmune organoids to bacterial-derived lipopolysaccharide, amyloid β, physical injury, or virus infection leads to iMG activation, indicating a common response to an inflammatory stimulus (Abud et al., 2017; Ao et al., 2021; Xu et al., 2021; Cakir et al., 2022).”
BackgroundMethodInfluentialRubio, A. D., Hamilton, L. L., Bausch, M., Jin, M., Papetti, A. V., Jiang, P., & Yelamanchili, S. V. (2025) · Journal of Neuroimmune Pharmacology · doi:10.1007/s11481-025-10181-x · Cited by 16 · see citing works
“Others have used an on-chip fabrication model to generate human brain organoids that do not have a core because they are shaped like a tube instead of a sphere (Ao et al. 2021).”
Experimental Models to Study Immune Dysfunction in the Pathogenesis of Parkinson’s Disease
Šaponjić, J., Mejı́as, R., Nikolovski, N., Dragić, M., Canak, A., Papoutsopoulou, S., Özdemir, Y., Fladmark, K. E., Ntavaroukas, P., Muluk, N. B., et al. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25084330 · Cited by 13 · see citing works
“Immune cells from PD animal models could be introduced into the organoids to simulate the immune response observed in PD [184,191].”
BackgroundCuní‐López, C., Stewart, R., White, A. R., & Quek, H. (2023) · Journal of Neuroimmunology · doi:10.1016/j.jneuroim.2023.578017 · Cited by 9 · see citing works
Advanced brain organoids for neuroinflammation disease modeling
Sabaté-Soler, S., Kurniawan, H., & Schwamborn, J. (2023) · Neural Regeneration Research · doi:10.4103/1673-5374.375321 · Cited by 10 · see citing works
“The brain organoids-microglia assembly could respond to inflammatory stimuli and were able to secrete various cytokines and chemokines (Ao et al., 2021).”
BackgroundHuman brain organoids for understanding substance use disorders
Li, K., Gu, L., Cai, H., Lu, H., Mackie, K., & Guo, F. (2024) · Drug Metabolism and Pharmacokinetics · doi:10.1016/j.dmpk.2024.101036 · Cited by 12 · see citing works
Self-citationGu, L., Mao, X., Tian, C., Yang, Y., Yang, K., Canfield, S. G., Zhu, D., Gu, M., & Guo, F. (2025) · Biomaterials Science · doi:10.1039/d5bm00204d · Cited by 12 · see citing works
Self-citation3D Printed Device for Separation of Cells and Particles by Tilted Bulk Acoustic Wave Actuation
Özçelik, A. (2022) · Actuators · doi:10.3390/act11090249 · Cited by 12 · see citing works
Modeling neurodegenerative diseases with brain organoids: from development to disease applications
Larriba-González, T., García-Martín, M., Ojeda-Hernández, D. D., Rincón-Cerrada, P., Martín-Blanco, L., Benito‐Martín, M. S., Selma-Calvo, B., de la Fuente-Martín, S., Matías‐Guiu, J. A., Matías‐Guiu, J., et al. (2025) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2025.1663286 · Cited by 12 · see citing works
Microfluidic Techniques for Next‐Generation Organoid Systems
Gong, J., Li, M., Kang, J., Yin, Z., Cha, Z., Yang, J., & Xu, H. (2022) · Advanced Materials Interfaces · doi:10.1002/admi.202200846 · Cited by 12 · see citing works
Neurogenesis and Neuroinflammation in Dialogue: Mapping Gaps, Modulating Microglia, Rewiring Aging
Tanaka, M. (2026) · Cells · doi:10.3390/cells15010078 · Cited by 11 · see citing works
“Rodent models remain indispensable for dissecting mechanisms and enabling invasive manipulations that cannot be performed in humans [335].”
Immunocompetent brain organoids—microglia enter the stage
Sabaté‐Soler, S., Bernini, M., & Schwamborn, J. C. (2022) · Progress in Biomedical Engineering · doi:10.1088/2516-1091/ac8dcf · Cited by 10 · see citing works
“For example, microglia co-cultured with tubular forebrain organoids, generated in a hollow mesh scaffold responded to pro-inflammatory stimuli in terms of inflammasome activation and cytokine production (Ao et al 2021). In that work, microglia activation was assessed, as well as their branching complexity, cytokine se…”
BackgroundInduced Pluripotent Stem Cell-Derived Organoids: Their Implication in COVID-19 Modeling
Csöbönyeiová, M., Klein, M., Kuniaková, M., Varga, I., & Danišovič, Ľ. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms24043459 · Cited by 10 · see citing works
“These iPSC-derived brain organoids have been used to model various neurological pathologies, such as the toxicity effects of the Zika virus [68,70], sporadic Alzheimer’s disease [71], neurodevelopmental disease [72–74], microglia-mediated neuroinflammation [75], and (most recently) the neurotoxic effects of SARS-CoV-2…”
MethodTian, C., Ao, Z., Cerneckis, J., Cai, H., Chen, L., Niu, H., Takayama, K., Kim, J., Shi, Y., Gu, M., et al. (2025) · Science Advances · doi:10.1126/sciadv.adu2708 · Cited by 9 · see citing works
“The device was designed using the AutoCAD software and then printed with a 3D printer (Form3B, Formlabs) using our well-developed protocols ( 77 – 81 ).”
Self-citationEmerging Models to Study Human Microglia In vitro
Jäntti, H., Kistemaker, L., Buonfiglioli, A., de Witte, L. D., Malm, T., & Hol, E. M. (2024) · Advances in neurobiology · doi:10.1007/978-3-031-55529-9_30 · Cited by 8 · see citing works
Human brain organoids: an innovative model for neurological disorder research and therapy
Li, H., Zhu, J., Li, J., Wu, Y., Luo, C., Huang, Y., Wu, J., Liu, W., Wang, H., & Mo, Z. (2025) · Frontiers in Cellular Neuroscience · doi:10.3389/fncel.2025.1658074 · Cited by 11 · see citing works
Functional bioengineered tissue models of neurodegenerative diseases
Mullis, A. S., & Kaplan, D. L. (2023) · Biomaterials · doi:10.1016/j.biomaterials.2023.122143 · Cited by 9 · see citing works
A review of regulated self-organizing approaches for tissue regeneration
Zhu, X., Wang, Z., & Teng, F. (2021) · Progress in Biophysics and Molecular Biology · doi:10.1016/j.pbiomolbio.2021.07.006 · Cited by 9 · see citing works
Saglam‐Metiner, P., Yanasik, S., Odabasi, Y. C., Modamio, J., Negwer, M., Avcı, Ç. B., Güler, A., Ertürk, A., Yıldırım, E., & Yeşil-Çeliktaş, Ö. (2024) · Communications Biology · doi:10.1038/s42003-024-07313-z · Cited by 8 · see citing works
Modeling neuro-immune interactions using human pluripotent stem cells
Garcia-Epelboim, A., & Christian, K. M. (2023) · Current Opinion in Neurobiology · doi:10.1016/j.conb.2022.102672 · Cited by 7 · see citing works
Dwivedi, I., & Haddad, G. G. (2024) · Frontiers in Cellular Neuroscience · doi:10.3389/fncel.2024.1403326 · Cited by 8 · see citing works
Pannexin1 Channel-Mediated Inflammation in Acute Ischemic Stroke
Huang, Y., Shi, Y., Wang, M., Liu, B., Chang, X., Xiao, X., Yu, H., Cui, X., & Bai, Y. (2023) · Aging and Disease · doi:10.14336/ad.2023.0303 · Cited by 7 · see citing works
“Specifically, the tubular human brain organoid chips proposed by Ao et al. in 2021 can be an alternative model for the preliminary screening of miRNAs that highly target PANX1 channels to lay a foundation for the clinical transformation of PANX1 channels [132].”
BackgroundMichalski, C., & Wen, Z. (2023) · Frontiers in Psychiatry · doi:10.3389/fpsyt.2023.1291115 · Cited by 7 · see citing works
A Battery-Powered Fluid Manipulation System Actuated by Mechanical Vibrations
Akkoyun, F., & Özçelik, A. (2022) · Actuators · doi:10.3390/act11050116 · Cited by 6 · see citing works
“Lab-on-a-chip systems that involve microfluidic channels require fluid manipulation systems t perform sample-preparation, bioanalysis, testi g, and diagnostics [40,41].”
BackgroundNeuroinflammatory response on a newly combinatorial cell–cell interaction chip
Zhao, Y., Lv, X., Chen, Y., Zhang, C., Zhou, D., & Deng, Y. (2024) · Biomaterials Science · doi:10.1039/d4bm00125g · Cited by 5 · see citing works
Asteroid impact: the potential of astrocytes to modulate human neural networks within organoids
Lavekar, S. S., Patel, M. D., Parra, M. D. M., & Krencik, R. (2023) · Frontiers in Neuroscience · doi:10.3389/fnins.2023.1305921 · Cited by 5 · see citing works
“In a different approach, a tubular organoid-on-a-chip device has been used that forces microglia to migrate into organoids to study neuroinflammation while ensuring oxygen perfusion, thereby minimizing necrosis and hypoxia (Ao et al., 2021b).”
MethodBiofabrication of brain-like living tissue: structure to intelligence
Wang, L., Wang, S., Liu, Y., Zhang, B., Pan, Z., Bai, L., Yao, S., Zhang, C., Xie, H., He, J., et al. (2025) · International Journal of Extreme Manufacturing · doi:10.1088/2631-7990/ada7a8 · Cited by 6 · see citing works
Kanupriya, K., Verma, S., Sharma, V., Mishra, I., & Mishra, R. (2025) · Current Drug Research Reviews · doi:10.2174/0125899775369286250206050006 · Cited by 4 · see citing works
Tregub, P. P., Volegova, D. D., Berdnikov, A. K., Chekulaev, P. A., Bystrov, D. A., Komleva, Y. K., Kolotyeva, N. A., Illarioshkin, S. N., Salmina, A. B., & Yurchenko, S. O. (2025) · Reviews in the Neurosciences · doi:10.1515/revneuro-2025-0015 · Cited by 4 · see citing works
Sun, W., Zhang, S., Nie, B., et al. (2026) · Journal of Environmental Exposure Assessment · doi:10.20517/jeea.2025.75 · Cited by 3 · see citing works
Li, K., Gu, L., Cai, H., Lu, H., Mackie, K., & Guo, F. (2024) · Drug Metabolism and Pharmacokinetics · doi:10.1016/j.dmpk.2024.101031 · Cited by 3 · see citing works
Self-citationBrain organoids as models of extracellular vesicle–mediated human neural communication
Rosa, G. L., Pascale, E., Iazzetta, M. R., Sozzi, E., Fico, A., Parrotta, E. I., & Fiorenzano, A. (2026) · Neural Regeneration Research · doi:10.4103/nrr.nrr-d-25-01806 · Cited by 2 · see citing works
“Current state-of-the-art brain organoid systems strive to more accurately recapitulate the intricate cellular interactions that drive human brain development and sustain its complex functions (Oliveira et Ao et al., 2021; Hu et al., 2023; Liddell et al., 2024; Luongo et al., 2024).”
Organoid–microglia system for modeling the immune microenvironment of the brain and retina
Yu, J., Tang, B., Gu, Z., Wang, G., & Liu, A. (2026) · Frontiers in Immunology · doi:10.3389/fimmu.2026.1747589 · Cited by 2 · see citing works
“Zheng Ao and colleagues (80) developed a tubular brain-on-a-chip platform that directs the differentiation of neural progenitor cells along microchannels to form centrally organized structures with uniform polarity, enhancing tissue viability and perfusion ef fi ciency while optimizing conditions…”
Organoids: generation strategies, applications, and future challenges
Yin, H., Zhu, G., Hai-zhan, W., Yang, B., Wang, Z., & Xie, H. (2026) · Stem Cell Research & Therapy · doi:10.1186/s13287-026-05045-x · Cited by 3 · see citing works
“These genetically engineered cell lines are designed to evade the host’s immune response, enabling ready-to-use organoid therapies [155, 156].”
Farahani, N. I., Lin, L. D., Nazir, S., Naderi, A., Rokos, L., McIntosh, A. R., & Julian, L. M. (2025) · Frontiers in Cellular Neuroscience · doi:10.3389/fncel.2024.1478572 · Cited by 3 · see citing works
Understanding immune-driven brain aging by human brain organoid microphysiological analysis platformPreprint
Song, S., Ao, Z., Cai, H., Li, X., Miao, Y., Wu, Z., Krzesniak, J., Gu, M., Lee, L. P., & Guo, F. (2022) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2022.01.19.476989 · Cited by 2 · see citing works
Self-citationVascular network-inspired diffusible scaffolds for engineering functional neural organoidsPreprint
Cai, H., Tian, C., Chen, L., McCracken, K. W., Tchieu, J. H., Gu, M., Mackie, K., & Guo, F. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.08.31.610649 · Cited by 2 · see citing works
Self-citation“Armed in-vitro retina”-generating microglial retinal organoids, where are we now?
Liu, Y., Gao, L., Chen, W., Yan, Y., Ye, Z., & Li, Z. (2025) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2025.1574283 · Cited by 2 · see citing works
Alaeq, R. A., & Mohammedsaeed, W. M. (2025) · Microbial Pathogenesis · doi:10.1016/j.micpath.2025.107938 · Cited by 1 · see citing works
Engineering Human Retinal Organoids and Eye-on-a-Chip Models for Degenerative Eye Disease
Wang, J., Yang, Y., Hong, Z., Xing, Y., Gu, M., Sharma, T. P., Meyer, J. S., & Guo, F. (2026) · ACS Biomaterials Science & Engineering · doi:10.1021/acsbiomaterials.5c02202 · Cited by 1 · see citing works
Self-citationMechanics of morphogenesis in neural development: In vivo, in vitro, and in silico
Sutlive, J., Seyyedhosseinzadeh, H., Ao, Z., Xiu, H., Choudhury, S., Gou, K., Guo, F., & Chen, Z. (2023) · Brain Multiphysics · doi:10.1016/j.brain.2022.100062
“By 3D scaffolding, brain organoids could also be guided into a neural tube configuration with internal lumen flows [49].”
BackgroundSelf-citationApplications and prospect of human brain organoids in neurodegenerative diseases
Yan, H., Wang, H., Zhang, W., Jiang, J., Wan, J., Xu, Z., Yang, Z., Tang, B., & Meng, Q. (2023) · Scientia Sinica Vitae · doi:10.1360/ssv-2023-0116
Organoid Computing: Leveraging Organoid Neural Networks for Artificial Intelligence
Li, K., Gu, L., Cai, H., & Guo, F. (2024) · Collaborative bioethics · doi:10.1007/978-3-031-72371-1_12 · Cited by 1 · see citing works
Self-citationThe potential of brain organoids in addressing the heterogeneity of synucleinopathies
Diao, X.-J., Soto, C. A., Wang, F., Wang, Y., Wu, Y., & Mukherjee, A. (2025) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-025-05686-w
Beythien, G., Becker, S., Leitzen, E., Kohale, S. K., & Baumgärtner, W. (2025) · Veterinary Pathology · doi:10.1177/03009858251344819
Engineering immune microenvironments for organoid-on-a-chip systems
Li, L., Xu, Q., Zhao, J., Qu, Y., Luo, Y., & Zhang, X. (2025) · MedMat. · doi:10.1097/mm9.0000000000000030
Wu, Z., Ao, Z., Cai, H., Li, X., Chen, B., Tu, H., Wang, Y., Lu, R. O., Gu, M., Cheng, L., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26566340
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- Authors
- Ao, Z., Cai, H., Wu, Z., Song, S., Karahan, H., Kim, B., Lu, H.-C., Kim, J., Mackie, K., & Guo, F.
- Affiliations
- Indiana University Bloomington (US)
- Indiana University Indianapolis (US)
- Stark Neurosciences Research Institute, and Department of Medical and Molecular Genetics
- Corresponding author
- Guo, Feng
- ORCID
- Venue
- Lab on a Chip
- Publisher
- Royal Society of Chemistry (RSC)
- Published
- 2021
- Volume
- 21
- Issue
- 14
- Pages
- 2751-2762
- ISSN
- 1473-0189 (ISSN-L)
- 1473-0197
- PMC
- PMC8493632
- OpenAlex
- W3161619369
- Cited by
- 87
- References
- 64
- Citations by year
- 2026: 13 · 2025: 17 · 2024: 20 · 2023: 21 · 2022: 12 · 2021: 4
- FWCI
- 4.68
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Lab on a Chip (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (submittedVersion)
- PubMed Central (submittedVersion)
- IUScholarWorks (Indiana University) (submittedVersion)
- Funders
- Indiana University Bloomington (Departmental Start-Up Funds)
- National Institutes of Health (R03EB030331, DA047858, DP2AI160242)
- Topics
- Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neural Engineering
- Keywords
- human brain organoids, microglial activation, opioid agonists, DAMGO, cannabinoid receptor 2 agonist, cytokine response, neural progenitor proliferation, neuroimmune interactions, substance use disorders
- MeSH terms
- Brain, Humans, Organoids, Microglia, Pluripotent Stem Cells, Induced Pluripotent Stem Cells
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-17.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-17.
Cited sources are checked by gpt-6-sol.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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