Comprehensive analysis of resilience of human airway epithelial barrier against short-term PM2.5 inorganic dust exposure using in vitro microfluidic chip and ex vivo human airway models.

BACKGROUND AND OBJECTIVE: The updated World Health Organization (WHO) air quality guideline recommends an annual mean concentration of fine particulate matter (PM2.5) not exceeding 5 or 15 µg/m3 in the short-term (24 h) for no more than 3-4 days annually. However, more than 90% of the global population is currently exposed to daily concentrations surpassing these limits, especially during extreme weather conditions and due to transboundary dust transport influenced by climate change. Herein, the effect of respirable

Humanized brain organoids-on-chip integrated with sensors for screening neuronal activity and neurotoxicity.

The complex structure and function of the human central nervous system that develops from the neural tube made in vitro modeling quite challenging until the discovery of brain organoids. Human-induced pluripotent stem cells-derived brain organoids offer recapitulation of the features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation into mature neurons and micro-macroglial cells, as well as the complex interactions among these diverse cell types of the developing brain. Recent advancements in brain organoids, microfluidic systems, real-time sensing technologies, and their cutting-edge integrated use provide excellent models and tools for emulation of fundamental neurodevelopmental processes, the pathology of neurological disorders, personalized transplantation therapy, and high-throughput neurotoxicity testing by bridging the gap between two-dimensional models and the complex three-dimensional environment in vivo. In this review, we summarize how bioengineering approaches are applied to mitigate the limitations of brain organoids for biomedical and clinical research. We further provide an extensive overview and future perspectives of the humanized brain organoids-on-chip platforms with integrated sensors toward brain organoid intelligence and biocomputing studies. Such approaches might pave the way for increasing approvable clinical applications by solving their current limitations.

Aligned with sustainable development goals: microwave extraction of astaxanthin from wet algae and selective cytotoxic effect of the extract on lung cancer cells.

Astaxanthin is one of the most attractive carotenoid in the cosmetic, food, pharmaceutical, and aquaculture industries due to its strong bioactive properties. Among the various sources, several algae species are considered as rich sources of astaxanthin. Downstream processing of algae involves the majority of the total processing costs. Thus, elimination of high energy involved steps is imperative to achieve cost-effective scale in industry. This study aimed to determine operation conditions for astaxanthin extraction from wet Haematococcus pluvialis using microwave-assisted extraction. The isolated astaxanthin extract was evaluated for cytotoxicity on human lung cancer cells. The microwave-assisted extraction process at 75 °C under the power of 700 Watt for 7 min gave the highest astaxanthin yield (12.24 ± 0.54 mg astaxanthin/g wet cell weight). Based on MTT cell viability and Hoechst 33342 nuclear staining assays on A549 lung cancer cells, astaxanthin inhibited cell growth in dose- and time-dependent manners, where IC50 value was determined as 111.8 ± 14.8 µg/mL and apoptotic bodies were observed along with positive control group at 72 hr. These results showed that the treatment with astaxanthin extracted from wet H. pluvialis by microwave-assisted extraction exhibited anti-cancer activity on lung cancer cells indicating a newly potential to be utilized in industry.

Decellularised extracellular matrix-based biomaterials for repair and regeneration of central nervous system.

The central nervous system (CNS), consisting of the brain and spinal cord, regulates the mind and functions of the organs. CNS diseases, leading to changes in neurological functions in corresponding sites and causing long-term disability, represent one of the major public health issues with significant clinical and economic burdens worldwide. In particular, the abnormal changes in the extracellular matrix under various disease conditions have been demonstrated as one of the main factors that can alter normal cell function and reduce the neuroregeneration potential in damaged tissue. Decellularised extracellular matrix (dECM)-based biomaterials have been recently utilised for CNS applications, closely mimicking the native tissue. dECM retains tissue-specific components, including proteoglycan as well as structural and functional proteins. Due to their unique composition, these biomaterials can stimulate sensitive repair mechanisms associated with CNS damages. Herein, we discuss the decellularisation of the brain and spinal cord as well as recellularisation of acellular matrix and the recent progress in the utilisation of brain and spinal cord dECM.

Synthesis of Resveratrol Loaded Hybrid Silica-PAMAM Dendrimer Nanoparticles With Emphases on Inducible Nitric Oxide Synthase and Cytotoxicity.

Resveratrol is a naturally occurring polyphenolic compound exhibiting therapeutic activities. However, the stability can be altered by UV light, pH and changes in temperature. Encapsulation would be an ideal strategy to improve the stability and bioavailability. Thus, trans-resveratrol (Res) was encapsulated within hybrid nanoparticles consisted with silica and G4 polyamidoamine dendrimer (PAMAM) by sol-gel method. The diameters of synthesized nanoparticles (NPs) were at a range of 212-574 nm and the encapsulation efficiency was 86 %. RAW 264.7 murine macrophage cell line induced with endotoxin/lipopolysaccharide was treated with free resveratrol and Res-loaded NPs for assessing inhibition of inducible nitric oxide synthase (iNOS), where IC50 values of free resveratrol and Res-loaded NPs were 122.68 µM and 249.74 µM. As for cytotoxicity, IC50 values of free resveratrol were found as 176.57 µM and 201.54 µM for MCF-7 and MDA-MB-231 cells, whereas 197.16 µM and 219.07 µM for Res-loaded NPs for the respective cell lines. Overall, sol-gel technique proved to be an ideal technology as can be carried out under mild conditions and Res-loaded NPs have potential to be utilized in the industry.

Quantitative determination of H2O2 for detection of alanine aminotransferase using thin film electrodes.

The abnormal concentrations or absence of biomolecules (e.g., proteins) in blood can further be used in diagnosis of a particular pathology at an early stage. Current studies are intensely focusing on the analysis of interaction and detection of biomolecules via point-of-care systems (POCs), allowing miniaturized and parallelized reactions, simultaneously. Recent developments have shown that the collaboration of electrochemical sensing techniques and POCs to overcome challenging problems in health-care settings provides new approaches in diagnosis and treatment of diseases. The aim of this study was to adapt the alanine aminotransferase (ALT) enzyme to the platinum (Pt) thin film electrode system and quantitatively determine the enzyme levels via enzymatically generated H2O2 with differential pulse voltammetry (DPV). A simple potentiostat architecture with expanded sweep range utilizing dual LMP91000 devices was developed and adapted to the needs of the biosensor. In order to calibrate the system, known concentrations of H2O2 were also tested. Moreover, signals associated with the other electroactive species coming from the ALT reaction were eliminated. Resulted potential range has been achieved between +500 mV and +900 mV and the linear range was found to be 0.05 M-0.5 M for H2O2, whereas 5 UL-1 to 120 UL-1 for ALT enzyme.

Cytotoxic, antimicrobial and nitric oxide inhibitory activities of supercritical carbon dioxide extracted Prunus persica leaves.

Different parts of Prunus persica as fruits, flowers, leaves and kernels have been consumed with dietary and therapeutic purposes traditionally. During fruit production, remarkable amount of leaves which can hold important bioactive groups as phenolics, have been left unutilized. The aim of this study was to investigate cytotoxic, antimicrobial and nitric oxide inhibitory activities of supercritical carbondioxide extracts of Prunus persica leaves. Among studied cell lines, supercritical carbon dioxide extract which was processed at 150 bar, 60 °C, and 6% co-solvent ethanol, exhibited remarkable cytotoxic activity against HeLa, MPanc-96 and MCF-7 cell lines with IC50 values of 12.22 µg/ml, 28.17 µg/ml and 35.51 µg/ml respectively, whereas IC50 value of conventional solvent extract was above 50 µg/ml. Minimum inhibitory concentration values determined for antibacterial and antifungal activities against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium and Candida albicans were found as 62.50 µg/ml. Strong nitric oxide inhibition was achieved with IC50 of 9.30 µg/ml. The promising results revealed that Prunus persica leaves may have remarkable potential as supplement both for drug and food industries. This study is the first report revealing cytotoxic, antimicrobial and nitric oxide inhibitory activity of supercritical carbon dioxide extract of Prunus persica leaves.

Nano-vesicular formulation of propolis and cytotoxic effects in a 3D spheroid model of lung cancer.

BACKGROUND: Propolis exhibits therapeutic properties due to the presence of phenolic acids, esters, and flavonoids. The scope of this study was to develop a nano-vesicular formulation and establish a three-dimensional (3D) spheroid model in which lung cancer is recapitulated. RESULTS: Niosome vesicles doped with galangin-rich propolis extract were synthesized by the ether injection method using a cholesterol : surfactant mass ratio of 1 : 3 at 40 °C for 1 h. Formulated niosomes were administered to 3D lung cancer spheroid model and the cytotoxicity was compared with that of a two-dimensional (2D) setting. The galangin content was determined as 86 µg mg-1 propolis extract by ultra-performance liquid chromatography (UPLC). The particle size of loaded niosome was 151 ± 2.84 nm with a polydispersity index (PDI) of about 0.232, and an encapsulation efficiency of 70% was achieved. CONCLUSION: The decrease in cell viability and the scattering in the 3D spheroids of A549 lung cancer cells treated with propolis-loaded niosomes were notable, indicating a profound cytotoxic effect and suggesting that they can be utilized as an effective nano-vesicle. © 2020 Society of Chemical Industry.

Cytotoxicity screening of supercritical fluid extracted seaweeds and phenylpropanoids.

Detached leaves of Posidonia oceanica and Zostera marina creating nuisance at the shores were extracted by means of supercritical CO2 enriched with a co-solvent, compared with that of soxhlet extraction. The extracts and their active compounds which are phenylpropanoids (chicoric, p-coumaric, rosmarinic, benzoic, ferulic and caffeic acids) were screened for cytotoxicity in cancer cell lines including human breast adenocarcinoma (MCF-7, MDA-MB-231, SK-BR-3), human colon adenocarcinoma (HT-29), human cervix adenocarcinoma (HeLa), human prostate adenocarcinoma (PC-3), Mus musculus neuroblastoma (Neuro 2A) cell lines and African green monkey kidney (VERO) as healthy cell line. Supercritical CO2 extracts proved to be more active than soxhlet counterparts. Particularly, Zostera marina extract obtained by supercritical CO2 at 250 bar, 80 °C, 20% co-solvent and a total flow rate of 15 g/min revealed the best IC50 values of 25, 20, 8 µg/ml in neuroblastoma, colon and cervix cancer cell lines. Among the major compounds tested, p-coumaric acid exhibited the highest cytotoxic against colon and cervix cell lines by with IC50 values of 25, 11 µg/ml. As for the effects on healthy cells, the extract was not cytotoxic indicating a selective cytotoxicity. Obtained supercritical CO2 extracts can be utilized as a supplement for preventive purposes.

Application of ß-glucuronidase-immobilised silica gel formulation to microfluidic platform for biotransformation of ß-glucuronides.

OBJECTIVE: To improve the efficiency of reactions of ß-glucuronidase (GUS)-assisted glucuronic acid (GluA) removal within a microfluidic system. RESULTS: ß-glucuronidase from Helix pomatia was immobilised and characterised in silica-based sol-gel monoliths. Efficiency of the GUS-doped silica monoliths was tested for hydrolysis of p-Nitrophenyl-ß-D-glucuronide (pNP-GluA) in both ml-scaled medium via batch reactions and microfluidic environment via continuous-flow reactions. In the microfluidic platform, within a duration of 150 min of continuous operation (flow rate: 1 µL/min), the obtained highest pNP yield was almost 50% higher than that of the corresponding batchwise reaction. However, increased flow rates (3, 5, and 10 µL/min) resulted in lower conversion yields compared to 1 µL/min. The microfluidic platform demonstrated continuous hydrolytic activity for 7 days with considerable reaction yields while using a small amount of the enzyme. CONCLUSION: These results revealed that usage of the microreactors has considerable potential to efficiently obtain bioactive GluA-free aglycons from various plant-derived ß-glucuronides for pharmaceutical applications.

Synthesis of silica-PAMAM dendrimer nanoparticles as promising carriers in Neuro blastoma cells.

Mesoporous silica carriers are emerging as therapeutic drug delivery systems. The objective of this study was to develop a formulation for synthesizing silica-PAMAM dendrimer hybrid nanoparticles with sol-gel technique. Subsequently, black carrot anthocyanins were encapsulated and investigated for their capability in terms of inhibiting the proliferative effects of neuroblastoma (Neuro 2A). In this context, particle size distributions were ascertained followed by thermal analysis (DSC), scanning electron microscopy and encapsulation efficiency. Subsequently, in vitro release kinetics was determined along with cytotoxicity of empty and anthocyanin doped hybrid nanoparticles. The lowest particle size was 134.8 nm with a zeta potential of +19.78 mV which enhanced electrostatic interaction with the cell membrane in the cytotoxicity analyses. As the anthocyanin content was totally released at the end of 6 days, the cytotoxicity was observed for 134 h, reaching an inhibition of 87.9%. On the other hand, Neuro 2A cells incubated with empty nanoparticles exhibited a high proliferation indicating that hybrid nanoparticles were not toxic to the cells and the inhibitory effect was associated with the anthocyanins.

Cytotoxic and Nitric Oxide Inhibition Activities of Propolis Extract along with Microencapsulation by Complex Coacervation.

In this study, cytotoxicity of ethanol extract of propolis (EEP) originating from Sivas, Turkey was screened against several cancer cell lines, namely PC-3, U87MG, A-549, mPANC96, CaCo-2, MCF-7, HeLa, MDA-MB-231 and a non-tumor cell line HEK293 by MTT assay. The inhibition levels of inducible nitric oxide synthase (iNOS) were also determined by using RAW 264.7 macrophage cells following lipopolysaccharide (LPS) treatment. EEP exhibited significant cytotoxic nitric oxide inhibition activities with an IC50 value of 0.1 ± 0.1 µg/ml indicating a high potential as an anti-inflammatory agent. In spite of these promising results and the fact that propolis is a highly nutritive substance, its low solubility and bitter taste limit the applications as a natural supplement. Encapsulation might serve as a good strategy in order to overcome these problems. Complex coacervation was applied where the main focus was on surfactant type, polymer ratio (alginate:gelatin), stirring rate and concentration of core material. The mean particle size of unloaded microparticles were 22.62 µm obtained with gelatin:alginate ratio of 1:1 at a stirring rate of 1400 rpm with 2 ml of 1 % (w/v) sodium carboxymethyl cellulose (Na-CMC), whereas addition of EEP at a concentration of 100 mg/ml increased the mean particle size to 36.44 µm and yielded an encapsulation efficiency of 98.77 %. The cytotoxicities of EEP loaded microparticles were also assessed both on MCF-7 and MDA-MB-231 where similar results were achieved as free EEP which can enhance the possible use of propolis extract in the industry as a natural supplement.

Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones.

A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol-chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2min instead of 24h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8±0.2°C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40ng/µl and proved to be a superior alternative to the phenol-chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction.

Differentiation of Neurons, Astrocytes, Oligodendrocytes and Microglia From Human Induced Pluripotent Stem Cells to Form Neural Tissue-On-Chip: A Neuroinflammation Model to Evaluate the Therapeutic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells.

Advances in stem cell (SC) technology allow the generation of cellular models that recapitulate the histological, molecular and physiological properties of humanized in vitro three dimensional (3D) models, as well as production of cell-derived therapeutics such as extracellular vesicles (EVs). Improvements in organ-on-chip platforms and human induced pluripotent stem cells (hiPSCs) derived neural/glial cells provide unprecedented systems for studying 3D personalized neural tissue modeling with easy setup and fast output. Here, we highlight the key points in differentiation procedures for neurons, astrocytes, oligodendrocytes and microglia from single origin hiPSCs. Additionally, we present a well-defined humanized neural tissue-on-chip model composed of differentiated cells with the same genetic backgrounds, as well as the therapeutic potential of bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles to propose a novel treatment for neuroinflammation derived diseases. Around 100 nm CD9 + EVs promote a more anti-inflammatory and pro-remodeling of cell-cell interaction cytokine responses on tumor necrosis factor-α (TNF-α) induced neuroinflammation in neural tissue-on-chip model which is ideal for modeling authentic neural-glial patho-physiology.

Receptor mediated targeting of EGF-conjugated alginate-PAMAM nanoparticles to lung adenocarcinoma: 2D/3D in vitro and in vivo evaluation.

Carboplatin (cis-diamine (1,1-cyclobutandicarboxylaso)­platinum (II)) is a second-generation antineoplastic drug, which is widely used for chemotherapy of lung, colon, breast, cervix, testicular and digestive system cancers. Although preferred over cisplatin due to the lower incidence of nephrotoxicity and ototoxicity, efficient carboplatin delivery remains as a major challenge. In this study, carboplatin loaded alginate- poly(amidoamine) (PAMAM) hybrid nanoparticles (CAPs) with mean sizes of 192.13 ± 4.15 nm were synthesized using a microfluidic platform, then EGF was conjugated to the surface of CAPs (EGF-CAPs) for the receptor-targeted delivery. Hence, increased FITC+ cell counts were observed in A549 spheroids after EGF-CAP treatment compared to CAP in the 3D cellular uptake study. As such, the cytotoxicity of EGF-CAP was approximately 2-fold higher with an IC50 value of 35.89 ± 10.37 µg/mL compared to the CAPs in A549 spheroids. Based on in vivo experimental animal model, anti-tumor activities of the group treated with CAP decreased by 61 %, whereas the group treated with EGF-CAP completely recovered. Additionally, EGF-CAP application was shown to induce apoptotic cell death. Our study provided a new strategy for designing a hybrid nanoparticle for EGFR targeted carboplatin delivery with improved efficacy both in vitro and in vivo applications.

Organotypic lung tissue culture as a preclinical model to study host- influenza A viral infection: A case for repurposing of nafamostat mesylate.

Reliable and effective models for recapitulation of host-pathogen interactions are imperative for the discovery of potential therapeutics. Ex vivo models can fulfill these requirements as the multicellular native environment in the tissue is preserved and be utilized for toxicology, vaccine, infection and drug efficacy studies due to the presence of immune cells. Drug repurposing involves the identification of new applications for already approved drugs that are not related to the prime medical indication and emerged as a strategy to cope with slow pace of drug discovery due to high costs and necessary phases to reach the patients. Within the scope of the study, broad-spectrum serine protease inhibitor nafamostat mesylate was repurposed to inhibit influenza A infection and evaluated by a translational ex vivo organotypic model, in which human organ-level responses can be achieved in preclinical safety studies of potential antiviral agents, along with in in vitro lung airway culture. The safe doses were determined as 10 µM for in vitro, whereas 22 µM for ex vivo to be applied for evaluation of host-pathogen interactions, which reduced virus infectivity, increased cell/tissue viability, and protected total protein content by reducing cell death with the inflammatory response. When the gene expression levels of specific pro-inflammatory, anti-inflammatory and cell surface markers involved in antiviral responses were examined, the significant inflammatory response represented by highly elevated mRNA gene expression levels of cytokines and chemokines combined with CDH5 downregulated by 5.1-fold supported the antiviral efficacy of NM and usability of ex vivo model as a preclinical infection model.

Extracts from black carrot tissue culture as potent anticancer agents.

Black carrots contain anthocyanins possessing enhanced physiological activities. Explants of young black carrot shoots were cultured in Murashige and Skoog (MS) medium for callus initiation and were transferred to new MS medium supplemented with four different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Subsequently, the lyophilized calli and black carrot harvested from fields were subjected to ultrasound extraction with ethanol at a ratio of 1:15 (w:v). Obtained extracts were applied to various human cancer cell lines including MCF-7 SK-BR-3 and MDA-MB-231 (human breast adenocarcinomas), HT-29 (human colon adenocarcinoma), PC-3 (human prostate adenocarcinoma), Neuro 2A (Musmusculus neuroblastoma) cancer cell lines and VERO (African green monkey kidney) normal cell line by MTT assay. The highest cytotoxic activity was achieved against Neuro-2A cell lines exhibiting viability of 38-46% at 6.25 µg/ml concentration for all calli and natural extracts. However, a significantly high IC50 value of 170.13 µg/ml was attained in normal cell line VERO indicating that its natural counterpart is an ideal candidate for treatment of brain cancer without causing negative effects to normal healthy cells.

Comprehensive Transcriptomic Investigation of Rett Syndrome Reveals Increasing Complexity Trends from Induced Pluripotent Stem Cells to Neurons with Implications for Enriched Pathways.

Rett syndrome (RTT) is a rare genetic neurodevelopmental disorder that has no cure apart from symptomatic treatments. While intense research efforts are required to fulfill this unmet need, the fundamental challenge is to obtain sufficient patient data. In this study, we used human transcriptomic data of four different sample types from RTT patients including induced pluripotent stem cells, differentiated neural progenitor cells, differentiated neurons, and postmortem brain tissues with an increasing in vivo-like complexity to unveil specific trends in gene expressions across the samples. Based on DEG analysis, we identified F8A3, CNTN6, RPE65, and COL19A1 to have differential expression levels in three sample types and also observed previously reported genes such as MECP2, FOXG1, CACNA1G, SATB2, GABBR2, MEF2C, KCNJ10, and CUX2 in our study. Considering the significantly enriched pathways for each sample type, we observed a consistent increase in numbers from iPSCs to NEUs where MECP2 displayed profound effects. We also validated our GSEA results by using single-cell RNA-seq data. In WGCNA, we elicited a connection among MECP2, TNRC6A, and HOXA5. Our findings highlight the utility of transcriptomic analyses to determine genes that might lead to therapeutic strategies.

Biosensor integrated brain-on-a-chip platforms: Progress and prospects in clinical translation.

Because of the brain's complexity, developing effective treatments for neurological disorders is a formidable challenge. Research efforts to this end are advancing as in vitro systems have reached the point that they can imitate critical components of the brain's structure and function. Brain-on-a-chip (BoC) was first used for microfluidics-based systems with small synthetic tissues but has expanded recently to include in vitro simulation of the central nervous system (CNS). Defining the system's qualifying parameters may improve the BoC for the next generation of in vitro platforms. These parameters show how well a given platform solves the problems unique to in vitro CNS modeling (like recreating the brain's microenvironment and including essential parts like the blood-brain barrier (BBB)) and how much more value it offers than traditional cell culture systems. This review provides an overview of the practical concerns of creating and deploying BoC systems and elaborates on how these technologies might be used. Not only how advanced biosensing technologies could be integrated with BoC system but also how novel approaches will automate assays and improve point-of-care (PoC) diagnostics and accurate quantitative analyses are discussed. Key challenges providing opportunities for clinical translation of BoC in neurodegenerative disorders are also addressed.

Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids.

The bioengineerined and whole matured human brain organoids stand as highly valuable three-dimensional in vitro brain-mimetic models to recapitulate in vivo brain development, neurodevelopmental and neurodegenerative diseases. Various instructive signals affecting multiple biological processes including morphogenesis, developmental stages, cell fate transitions, cell migration, stem cell function and immune responses have been employed for generation of physiologically functional cerebral organoids. However, the current approaches for maturation require improvement for highly harvestable and functional cerebral organoids with reduced batch-to-batch variabilities. Here, we demonstrate two different engineering approaches, the rotating cell culture system (RCCS) microgravity bioreactor and a newly designed microfluidic platform (µ-platform) to improve harvestability, reproducibility and the survival of high-quality cerebral organoids and compare with those of traditional spinner and shaker systems. RCCS and µ-platform organoids have reached ideal sizes, approximately 95% harvestability, prolonged culture time with Ki-67 + /CD31 + /ß-catenin+ proliferative, adhesive and endothelial-like cells and exhibited enriched cellular diversity (abundant neural/glial/ endothelial cell population), structural brain morphogenesis, further functional neuronal identities (glutamate secreting glutamatergic, GABAergic and hippocampal neurons) and synaptogenesis (presynaptic-postsynaptic interaction) during whole human brain development. Both organoids expressed CD11b + /IBA1 + microglia and MBP + /OLIG2 + oligodendrocytes at high levels as of day 60. RCCS and µ-platform organoids showing high levels of physiological fidelity a high level of physiological fidelity can serve as functional preclinical models to test new therapeutic regimens for neurological diseases and benefit from multiplexing.