bims-orenst 2021-08-01 papers

bims-orenst

Biomed News

on Organs-on-chips and engineered stem cell models

Issue of 2021–08–01
thirteen papers selected by
Joram Mooiweer, University of Groningen

Multiplex recreation of human intestinal morphogenesis on a multi-well insert platform by basolateral convective flow.
An engineered microfluidic blood-brain barrier model to evaluate the anti-metastatic activity of β-boswellic acid.
A fully defined matrix to support a pluripotent stem cell derived multi-cell-liver steatohepatitis and fibrosis model.
[Development of a novel high throughput brain-on-chip with 3D structure and its application in evaluation of pesticide-induced-neurotoxicity].
Mesenchymal Stromal Cell-Derived Extracellular Vesicles Pass through the Filtration Barrier and Protect Podocytes in a 3D Glomerular Model under Continuous Perfusion.
RNA-Sequencing and Transcriptome Analysis after Fluid Shear Stress Stimulation in Lymphatic Endothelial Cells.
A robust vasculogenic microfluidic model using human immortalized endothelial cells and Thy1 positive fibroblasts.
Bioengineering a humanized 3D tri-culture osteosarcoma model to assess tumor invasiveness and therapy response.
Three dimensionally printed nitrocellulose-based microfluidic platform for investigating the effect of oxygen gradient on cells.
High shear stress enhances endothelial permeability in the presence of the risk haplotype at 9p21.3.
Osteogenic and Angiogenic Synergy of Human Adipose Stem Cells and Human Umbilical Vein Endothelial Cells Cocultured in a Modified Perfusion Bioreactor.
Human Serum Enhances Biomimicry of Engineered Tissue Models of Bone and Cancer.
Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations in vitro.

Lab Chip. 2021 Jul 29.

Multiplex recreation of human intestinal morphogenesis on a multi-well insert platform by basolateral convective flow.

Hyeon Beom Chong, Jaeseung Youn, Woojung Shin, Hyun Jung Kim, Dong Sung Kim.

Here, we report a multiplex culture system that enables simultaneous recreation of multiple replications of the three-dimensional (3D) microarchitecture of the human intestinal epithelium in vitro. The "basolateral convective flow-generating multi-well insert platform (BASIN)" contains 24 nano-porous inserts and an open basolateral chamber applying controllable convective flow in the basolateral compartment that recreates a biomimetic morphogen gradient using a conventional orbital shaker. The mechanistic approach by which the removal of morphogen inhibitors in the basolateral medium can induce intestinal morphogenesis was applied to manipulate the basolateral convective flow in space and time. In a multiplex BASIN, we successfully regenerated a 3D villi-like intestinal microstructure using the Caco-2 human intestinal epithelium that presents high barrier function with minimal insert-to-insert variations. The enhanced cytodifferentiation and proliferation of the 3D epithelial layers formed in the BASIN were visualized with markers of absorptive (villin) and proliferative cells (Ki67). The paracellular transport and efflux profiles of the microengineered 3D epithelial layers in the BASIN confirmed its reproducibility, robustness, and scalability for multiplex biochemical or pharmaceutical studies. Finally, the BASIN was used to investigate the effects of dextran sodium sulfate on the intestinal epithelial barrier and morphology to validate its practical applicability for investigating the effects of external chemicals on the intestinal epithelium and constructing a leaky-gut model. We envision that the BASIN may provide an improved multiplex, scalable, and physiological intestinal epithelial model that is readily accessible to researchers in both basic and applied sciences.

DOI: https://doi.org/10.1039/d1lc00404b
Biotechnol J. 2021 Jul 27. e2100044

An engineered microfluidic blood-brain barrier model to evaluate the anti-metastatic activity of β-boswellic acid.

Saeid Vakilian, Khurshid Alam, Juhaina Al-Kindi, Fatemeh Jamshidi-Adegani, Najeeb Ur Rehman, Rezvan Tavakoli, Khamis Al-Riyami, Anwarul Hasan, Fahad Zadjali, Rene Csuk, Ahmed Al-Harrasi, Sulaiman Al-Hashmi.

   BACKGROUND: The development of anti-cancer drugs with the ability to inhibit brain metastasis through the blood-brain barrier (BBB) is substantially limited due to the lack of reliable in vitro models.
MAIN METHODS: In this study, the Geltrex™-based Transwell® and microfluidic BBB models were applied to screen the effect of β-boswellic acid (β-BA) on the metastasis of MDA-MB-231 cells through the BBB in static and dynamic conditions, respectively.
MAJOR RESULTS: The toxicity assay revealed that β-BA deteriorates MDA-MB-231 cells, while β-BA had no detectable toxic effects on human umbilical vein endothelial cells (HUVECs) and astrocytes. Trans-endothelial electrical resistance evaluation showed sustainable barrier integrity upon treatment with β-BA. Vimentin expression in HUVECs, evaluated using western blot, confirmed superior barrier integrity in the presence of β-BA. The obtained results were confirmed using an invasion study with a cell tracker and a scanning electron microscope. β-BA significantly inhibited metastasis by 85%, while cisplatin (Cis), a positive control, inhibited cancer cell migration by 12% under static conditions. Upon applying a dynamic BBB model, it was revealed that β-BA-mediated metastasis inhibition was significantly higher than that mediated by Cis.
CONCLUSIONS AND IMPLICATIONS: In summary, the current study proved the anti-metastatic potential of β-BA in both static and dynamic BBB models. This article is protected by copyright. All rights reserved.

Keywords:  Transwell®; blood-brain barrier; boswellic acid ; metastasis; microfluidics

DOI:  https://doi.org/10.1002/biot.202100044
Biomaterials. 2021 Jul 09. pii: S0142-9612(21)00362-8. [Epub ahead of print]276 121006

A fully defined matrix to support a pluripotent stem cell derived multi-cell-liver steatohepatitis and fibrosis model.

Manoj Kumar, Burak Toprakhisar, Matthias Van Haele, Asier Antoranz, Ruben Boon, Francois Chesnais, Jonathan De Smedt, Tine Tricot, Teresa Izuel Idoype, Marco Canella, Pierre Tilliole, Jolan De Boeck, Manmohan Bajaj, Adrian Ranga, Francesca Maria Bosisio, Tania Roskams, Leo A van Grunsven, Catherine M Verfaillie.

  Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells, that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC) derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGFβ-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures of its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects.

Keywords:  Fibrosis; Hydrogels; Multi-cell-liver model; Pluripotent stem cells; Steatohepatitis; Synthetic matrices

DOI:  https://doi.org/10.1016/j.biomaterials.2021.121006
Sheng Wu Gong Cheng Xue Bao. 2021 Jul 25. 37(7): 2543-2553

[Development of a novel high throughput brain-on-chip with 3D structure and its application in evaluation of pesticide-induced-neurotoxicity].

Chenyu Zhao, Haidi Li, Xiaoping Chen.

  We designed and fabricated a novel high throughput brain-on-chip with three dimensional structure with the aim to simulate the in vivo three-dimensional growth environment for brain tissues. The chip consists of a porous filter and 3D brain cell particles, and is loaded into a conventional 96-well plate for use. The filter and the particle molds were fabricated by using computer modeling, 3D printing of positive mold and agarose-PDMS double reversal mold. The 3D cell particles were made by pouring and solidifying a suspension of mouse embryonic brain cells with sodium alginate into a cell particle mold, and then cutting the resulting hydrogel into pieces. The loaded brain-on-chip was used to determine the neurotoxicity of pesticides. The cell particles were exposed to 0, 10, 30, 50, 100 and 200 µmol/L of chlorpyrifos or imidacloprid, separated conveniently from the medium by removing the porous filter after cultivation. Subsequently, cell proliferation, acetylcholinesterase activity and lactate dehydrogenase release were determined for toxicity evaluation. The embryonic brain cells were able to grow and proliferate normally in the hydrogel particles loaded into the filter in a 96-well plate. Pesticide neurotoxicity test showed that both chlorpyrifos and imidacloprid presented dose-dependent inhibition on cell growth and proliferation. Moreover, the pesticides showed inhibition on acetylcholinesterase activity and increase release of lactate dehydrogenase. However, the effect of imidacloprid was significantly weaker than that of chlorpyrifos. In conclusion, a novel brain-on-chip was developed in this study, which can be used to efficiently assess the drug neurotoxicity, pharmacodynamics, and disease mechanism by combining with a microtiterplate reader.

Keywords:  3D cell culture; brain-on-chip; high-throughput detection; neurotoxicity; pesticides

DOI:  https://doi.org/10.13345/j.cjb.200538
Tissue Eng Regen Med. 2021 Jul 27.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Pass through the Filtration Barrier and Protect Podocytes in a 3D Glomerular Model under Continuous Perfusion.

Linda Bellucci, Giovanni Montini, Federica Collino, Benedetta Bussolati.

   BACKGROUND: Dynamic cultures, characterized by continuous fluid reperfusion, elicit physiological responses from cultured cells. Mesenchymal stem cell-derived EVs (MSC-EVs) has been proposed as a novel approach in treating several renal diseases, including acute glomerular damage, by using traditional two-dimensional cell cultures and in vivo models. We here aimed to use a fluidic three-dimensional (3D) glomerular model to study the EV dynamics within the glomerular structure under perfusion.
METHODS: To this end, we set up a 3D glomerular model culturing human glomerular endothelial cells and podocytes inside a bioreactor on the opposite sides of a porous membrane coated with type IV collagen. The bioreactor was connected to a circuit that allowed fluid passage at the rate of 80 µl/min. To mimic glomerular damage, the system was subjected to doxorubicin administration in the presence of therapeutic MSC-EVs.
RESULTS: The integrity of the glomerular basal membrane in the 3D glomerulus was assessed by a permeability assay, demonstrating that the co-culture could limit the passage of albumin through the filtration barrier. In dynamic conditions, serum EVs engineered with cel-miR-39 passed through the glomerular barrier and transferred the exogenous microRNA to podocyte cell lines. Doxorubicin treatment increased podocyte apoptosis, whereas MSC-EV within the endothelial circuit protected podocytes from damage, decreasing cell death and albumin permeability.
CONCLUSION: Using an innovative millifluidic model, able to mimic the human glomerular barrier, we were able to trace the EV passage and therapeutic effect in dynamic conditions.

Keywords:  Exosomes; Glomerular permeability; Mesenchymal stromal cell; Podocytes

DOI:  https://doi.org/10.1007/s13770-021-00374-9
Methods Mol Biol. 2021 ;2319 25-30

RNA-Sequencing and Transcriptome Analysis after Fluid Shear Stress Stimulation in Lymphatic Endothelial Cells.

Hongjiang Si, Xu Peng.

  Fluid shear stress (FSS) is an important mechanical force that regulates endothelial and vascular functions in the blood and lymphatic vasculature. RNA-sequencing (RNA-Seq), an emerging next-generation sequencing (NGS) technique, facilitates us to quantify the global gene expression levels and, thereby, provides us with a powerful tool to study the transcriptional reprograming in the cells. Here, we describe the RNA-Seq procedures and the subsequent bioinformatic analysis to understand the transcriptome regulation by FSS in lymphatic endothelial cells (LECs).

Keywords:  Fluid shear stress; Lymphatic endothelial cells; RNA-sequencing; Transcriptome

DOI:  https://doi.org/10.1007/978-1-0716-1480-8_3
Biomaterials. 2021 Jul 16. pii: S0142-9612(21)00388-4. [Epub ahead of print]276 121032

A robust vasculogenic microfluidic model using human immortalized endothelial cells and Thy1 positive fibroblasts.

Zhengpeng Wan, Shun Zhang, Amy X Zhong, Sarah E Shelton, Marco Campisi, Shriram K Sundararaman, Giovanni S Offeddu, Eunkyung Ko, Lina Ibrahim, Mark F Coughlin, Tiankun Liu, Jing Bai, David A Barbie, Roger D Kamm.

  Human umbilical vein endothelial cells (HUVECs) and stromal cells, such as human lung fibroblasts (FBs), have been widely used to generate functional microvascular networks (μVNs) in vitro. However, primary cells derived from different donors have batch-to-batch variations and limited lifespans when cultured in vitro, which hampers the reproducibility of μVN formation. Here, we immortalize HUVECs and FBs by exogenously expressing human telomerase reverse transcriptase (hTERT) to obtain stable endothelial cell and FB sources for μVN formation in vitro. Interestingly, we find that immortalized HUVECs can only form functional μVNs with immortalized FBs from earlier passages but not from later passages. Mechanistically, we show that Thy1 expression decreases in FBs from later passages. Compared to Thy1 negative FBs, Thy1 positive FBs express higher IGFBP2, IGFBP7, and SPARC, which are important for angiogenesis and lumen formation during vasculogenesis in 3D. Moreover, Thy1 negative FBs physically block microvessel openings, reducing the perfusability of μVNs. Finally, by culturing immortalized FBs on gelatin-coated surfaces in serum-free medium, we are able to maintain the majority of Thy1 positive immortalized FBs to support perfusable μVN formation. Overall, we establish stable cell sources for μVN formation and characterize the functions of Thy1 positive and negative FBs in vasculogenesis in vitro.

Keywords:  Fibroblasts; HUVECs; Immortalization; Microfluidic; Thy1; Vasculogenesis

DOI:  https://doi.org/10.1016/j.biomaterials.2021.121032
Acta Biomater. 2021 Jul 21. pii: S1742-7061(21)00472-4. [Epub ahead of print]

Bioengineering a humanized 3D tri-culture osteosarcoma model to assess tumor invasiveness and therapy response.

Cátia F Monteiro, Catarina A Custódio, João F Mano.

  To date, anticancer therapies with evidenced efficacy in preclinical models fail during clinical trials. The shortage of robust drug screening platforms that accurately predict patient's response underlie these misleading results. To provide a reliable platform for tumor drug discovery, we herein propose a relevant humanized 3D osteosarcoma (OS) model exploring the potential of methacryloyl platelet lysates (PLMA)-based hydrogels to sustain spheroid growth and invasion. The architecture and synergistic cell-microenvironment interaction of an invading tumor was recapitulated encapsulating spheroids in PLMA hydrogels, alone or co-cultured with osteoblasts and mesenchymal stem cells. The stem cells alignment toward OS spheroid suggested that tumor cells chemotactically attracted the surrounding stromal cells, which supported tumor growth and invasion into the hydrogels. The exposure of established models to doxorubicin revealed an improved drug resistance of PLMA-based models, comparing with scaffold-free spheroids. The proposed OS models highlighted the feasibility of PLMA hydrogels to support tumor invasion and recapitulate tumor-stromal cell crosstalk, demonstrating the potential this 3D platform for complex tumor modelling. STATEMENT OF SIGNIFICANCE: Cell invasion mechanisms involved in tumor progression have been recapitulated in the field of 3D in vitro modeling, leveraging the great advance in biomimetic materials. In line with the growing interest in human-derived biomaterials, the aim of this study is to explore for the first time the potential of methacryloyl platelet lysates (PLMA)-based hydrogels to develop a humanized 3D osteosarcoma model to assess tumor invasiveness and drug sensitivity. By co-culturing tumor spheroids with human osteoblasts and human mesenchymal stem cells, this study demonstrated the importance of the synergistic tumor cell-microenvironment interaction in tumor growth, invasion and drug resistance. The established 3D osteosarcoma model highlighted the feasibility of PLMA hydrogels as a relevant 3D platform for complex tumor modelling.

Keywords:  3D in vitro tumor model; Co-culture; Drug screening; Human platelet lysates; Osteosarcoma

DOI:  https://doi.org/10.1016/j.actbio.2021.07.034
Analyst. 2021 Jul 29.

Three dimensionally printed nitrocellulose-based microfluidic platform for investigating the effect of oxygen gradient on cells.

Ping Liu, Longwen Fu, Zhihua Song, Mingsan Man, Huamao Yuan, Xiaoli Zheng, Qi Kang, Dazhong Shen, Jinming Song, Bowei Li, Lingxin Chen.

In this article, we present a novel nitrocellulose-based microfluidic chip with 3-dimensional (3D) printing technology to study the effect of oxygen gradient on cells. Compared with conventional polydimethylsiloxane (PDMS) chips of oxygen gradient for cell cultures that can only rely on fluorescence microscope analysis, this hybrid nitrocellulose-based microfluidic platform can provide a variety of analysis methods for cells, including flow cytometry, western blot and RT-PCR, because the nitrocellulose-based chips with cells can be taken out from the growth chambers of 3D printed microfluidic chip and then used for cell collection or lysis. These advantages allow researchers to acquire more information and data on the basic biochemical and physiological processes of cell life. The effect of oxygen gradient on the zebrafish cells (ZF4) was used as a model to show the performance and application of our platform. Hypoxia caused the increase of intercellular reactive oxygen species (ROS) and accumulation of hypoxia-inducible factor 1α (HIF-1α). Hypoxia stimulated the transcription of hypoxia-responsive genes vascular endothelial growth factor (VEGF) and induced cell cycle arrest of ZF4 cells. The established platform is able to obtain more information from cells in response to different oxygen concentration, which has potential for analyzing the cells under a variety of pathological conditions.

DOI: https://doi.org/10.1039/d1an00927c
APL Bioeng. 2021 Sep;5(3): 036102

High shear stress enhances endothelial permeability in the presence of the risk haplotype at 9p21.3.

Evan L Teng, Evan M Masutani, Benjamin Yeoman, Jessica Fung, Rachel Lian, Brenda Ngo, Aditya Kumar, Jesse K Placone, Valentina Lo Sardo, Adam J Engler.

Single nucleotide polymorphisms (SNPs) are exceedingly common in non-coding loci, and while they are significantly associated with a myriad of diseases, their specific impact on cellular dysfunction remains unclear. Here, we show that when exposed to external stressors, the presence of risk SNPs in the 9p21.3 coronary artery disease (CAD) risk locus increases endothelial monolayer and microvessel dysfunction. Endothelial cells (ECs) derived from induced pluripotent stem cells of patients carrying the risk haplotype (R/R WT) differentiated similarly to their non-risk and isogenic knockout (R/R KO) counterparts. Monolayers exhibited greater permeability and reactive oxygen species signaling when the risk haplotype was present. Addition of the inflammatory cytokine TNFα further enhanced EC monolayer permeability but independent of risk haplotype; TNFα also did not substantially alter haplotype transcriptomes. Conversely, when wall shear stress was applied to ECs in a microfluidic vessel, R/R WT vessels were more permeable at lower shear stresses than R/R KO vessels. Transcriptomes of sheared cells clustered more by risk haplotype than by patient or clone, resulting in significant differential regulation of EC adhesion and extracellular matrix genes vs static conditions. A subset of previously identified CAD risk genes invert expression patterns in the presence of high shear concomitant with altered cell adhesion genes, vessel permeability, and endothelial erosion in the presence of the risk haplotype, suggesting that shear stress could be a regulator of non-coding loci with a key impact on CAD.

DOI: https://doi.org/10.1063/5.0054639
Organogenesis. 2021 Jul 29. 1-16

Osteogenic and Angiogenic Synergy of Human Adipose Stem Cells and Human Umbilical Vein Endothelial Cells Cocultured in a Modified Perfusion Bioreactor.

Fatemeh Mokhtari-Jafari, Ghasem Amoabediny, Mohammad Mehdi Dehghan, Sonia Abbasi Ravasjani, Massoumeh Jabbari Fakhr, Yasaman Zamani.

  Synergistic promotion of angiogenesis and osteogenesis in bone tissue-engineered constructs remains a crucial clinical challenge, which might be overcome by simultaneous employment of superior techniques including coculture systems, differentiation-stimulated factors, combinatorial scaffolds and bioreactors.Current study investigated the effect of flow perfusion along with coculture of human adipose stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) on osteogenic and angiogenic differentiation.Pre-treated hASCs with 1,25-dihydroxyvitamin D3 were seeded onto poly(lactic-co-glycolic acid)/β-tricalcium phosphate/polycaprolactone (PLGA/β-TCP/PCL) scaffold with/without HUVECs, and cultured for 14 days within a flask or modified perfusion bioreactor. Analysis of osteogenic and angiogenic gene expression, alkaline phosphatase (ALP) activity and ALP staining indicates a synergistic effect of perfusion flow and coculture system on osteogenic and angiogenic differentiation. The advantage of modified perfusion bioreactor is its five-branch flow distributor which directly connect to the porous PCL hollow fibers embedded in the 3D scaffold to improve flow and flow-induced shear stress uniformity.Dynamic coculture increased VEGF165 by 6-fold, VEGF189 by 2-fold, and Endothelin-1 by 4-fold, relative to dynamic monoculture. Static coculture enhanced osteogenic and angiogenic differentiation, compared with static monoculture. Although dynamic coculture is in preference to static coculture due to significant increase in ALP activity and promoted angiogenic marker expression. Our finding is the first to indicate that the modified perfusion bioreactor combined with the beneficial cell-cell crosstalk in pre-treated hASC/HUVEC cocultures provides a synergy between osteogenic and angiogenic differentiation of the accumulation of cells, suggesting that it represents a promising approach for regeneration of critical-sized bone defects.

Keywords:  angiogenic differentiation; bone; human adipose stem cells; human umbilical vein endothelial cells; modified perfusion bioreactor; osteogenic differentiation

DOI:  https://doi.org/10.1080/15476278.2021.1954769
Front Bioeng Biotechnol. 2021 ;9 658472

Human Serum Enhances Biomimicry of Engineered Tissue Models of Bone and Cancer.

Aranzazu Villasante, Samuel T Robinson, Andrew R Cohen, Roberta Lock, X Edward Guo, Gordana Vunjak-Novakovic.

  For decades, fetal bovine serum (FBS) has been used routinely for culturing many cell types, based on its empirically demonstrated effects on cell growth, and the lack of suitable non-xenogeneic alternatives. The FBS-based culture media do not represent the human physiological conditions, and can compromise biomimicry of preclinical models. To recapitulate in vitro the features of human bone and bone cancer, we investigated the effects of human serum and human platelet lysate on modeling osteogenesis, osteoclastogenesis, and bone cancer in two-dimensional (2D) and three-dimensional (3D) settings. For monitoring tumor growth within tissue-engineered bone in a non-destructive fashion, we generated cancer cell lines expressing and secreting luciferase. Culture media containing human serum enhanced osteogenesis and osteoclasts differentiation, and provided a more realistic in vitro mimic of human cancer cell proliferation. When human serum was used for building 3D engineered bone, the tissue recapitulated bone homeostasis and response to bisphosphonates observed in native bone. We found disparities in cell behavior and drug responses between the metastatic and primary cancer cells cultured in the bone niche, with the effectiveness of bisphosphonates observed only in metastatic models. Overall, these data support the utility of human serum for bioengineering of bone and bone cancers.

Keywords:  3D cancer models; 3Rs; Ewing’s sarcoma; cypridina luciferase; human serum

DOI:  https://doi.org/10.3389/fbioe.2021.658472
Stem Cell Reports. 2021 Jul 13. pii: S2213-6711(21)00326-X. [Epub ahead of print]

Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations in vitro.

Britt Mossink, Anouk H A Verboven, Eline J H van Hugte, Teun M Klein Gunnewiek, Giulia Parodi, Katrin Linda, Chantal Schoenmaker, Tjitske Kleefstra, Tamas Kozicz, Hans van Bokhoven, Dirk Schubert, Nael Nadif Kasri, Monica Frega.

  Micro-electrode arrays (MEAs) are increasingly used to characterize neuronal network activity of human induced pluripotent stem cell (hiPSC)-derived neurons. Despite their gain in popularity, MEA recordings from hiPSC-derived neuronal networks are not always used to their full potential in respect to experimental design, execution, and data analysis. Therefore, we benchmarked the robustness of MEA-derived neuronal activity patterns from ten healthy individual control lines, and uncover comparable network phenotypes. To achieve standardization, we provide recommendations on experimental design and analysis. With such standardization, MEAs can be used as a reliable platform to distinguish (disease-specific) network phenotypes. In conclusion, we show that MEAs are a powerful and robust tool to uncover functional neuronal network phenotypes from hiPSC-derived neuronal networks, and provide an important resource to advance the hiPSC field toward the use of MEAs for disease phenotyping and drug discovery.

Keywords:  human induced pluripotent stem cells; micro-electrode arrays; neuronal differentiation; neuronal network activity

DOI:  https://doi.org/10.1016/j.stemcr.2021.07.001