A Pillar/Perfusion Plate Enhances Cell Growth, Reproducibility, Throughput, and User Friendliness in Dynamic 3D Cell Culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from a necrotic core due to limited nutrient and oxygen diffusion and waste removal and have a limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids were loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow velocity was maintained within perfusion wells and the pillar plate was separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in a dynamic 3D cell culture.

Dynamic culture of cerebral organoids using a pillar/perfusion plate for the assessment of developmental neurotoxicity.

Despite the potential toxicity of commercial chemicals to the development of the nervous system (known as developmental neurotoxicity or DNT), conventional in vitro cell models have primarily been employed for the assessment of acute neuronal toxicity. On the other hand, animal models used for the assessment of DNT are not physiologically relevant due to the heterogenic difference between humans and animals. In addition, animal models are low-throughput, time-consuming, expensive, and ethically questionable. Recently, human brain organoids have emerged as a promising alternative to assess the detrimental effects of chemicals on the developing brain. However, conventional organoid culture systems have several technical limitations including low throughput, lack of reproducibility, insufficient maturity of organoids, and the formation of the necrotic core due to limited diffusion of nutrients and oxygen. To address these issues and establish predictive DNT models, cerebral organoids were differentiated in a dynamic condition in a unique pillar/perfusion plate, which were exposed to test compounds to evaluate DNT potential. The pillar/perfusion plate facilitated uniform, dynamic culture of cerebral organoids with improved proliferation and maturity by rapid, bidirectional flow generated on a digital rocker. Day 9 cerebral organoids in the pillar/perfusion plate were exposed to ascorbic acid (DNT negative) and methylmercury (DNT positive) in a dynamic condition for 1 and 3 weeks, and changes in organoid morphology and neural gene expression were measured to determine DNT potential. As expected, ascorbic acid didn't induce any changes in organoid morphology and neural gene expression. However, exposure of day 9 cerebral organoids to methylmercury resulted in significant changes in organoid morphology and neural gene expression. Interestingly, methylmercury did not induce adverse changes in cerebral organoids in a static condition, thus highlighting the importance of dynamic organoid culture in DNT assessment.

Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting.

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15 - 18%, without any detrimental effect on cell viability. Despite utilizing 10 - 50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 ± 1.6 µM and 25.4 ± 8.3 µM, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting.

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15-18%, without any detrimental effect on cell viability. Despite utilizing 10-50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 ± 1.6 µM and 25.4 ± 8.3 µM, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays.

Human liver organoids (HLOs) differentiated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can recapitulate structure and function of human fetal liver tissues, thus, considered as a promising tissue model for liver diseases and predictive compound screening. Nonetheless, there are still several technical challenges to adopt HLOs in the drug discovery process, which include relatively long-term cell differentiation with multiple culture media (3 - 4 weeks) leading to batch-to-batch variation, short-term hepatic function after maturation (3 - 5 days), low assay throughput due to Matrigel dissociation and HLO transfer to a microtiter well plate, and insufficient maturity as compared to primary hepatocytes. To address these issues, expandable HLOs (Exp-HLOs) derived from human iPSCs were generated by optimizing differentiation protocols, which were rapidly printed on a 144-pillar plate with sidewalls and slits (144PillarPlate) and dynamically cultured for up to 20 days into differentiated HLOs (Diff-HLOs) in a 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for in situ organoid culture and analysis. Dynamically cultured Diff-HLOs were generated robustly and reproducibly in the pillar/perfusion plate with higher maturity as compared to those in statically cultured HLOs by differentiating Exp-HLOs for 10 days. In addition, Diff-HLOs in the pillar/perfusion plate were tested with acetaminophen and troglitazone for 3 days to assess drug-induced liver injury (DILI) and then incubated in an expansion medium for 10 days to evaluate the recovery of the liver from DILI. The assessment of liver regeneration post injury is critical to understand the mechanism of recovery and determine the threshold drug concentration beyond which there will be a sharp decrease in the liver's regenerative capacity. We envision that bioprinted Diff-HLOs in the pillar/perfusion plate could be used for high-throughput screening (HTS) of hepatotoxic compounds due to short-term differentiation of passage-able Exp-HLOs necessary, stable hepatic function after maturation, high reproducibility, and high throughput with capability of in situ organoid culture, testing, staining, imaging, and analysis.

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate.

Human induced pluripotent stem cell (iPSC)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effectivein vitromodel for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies in an ultra-low attachment 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95%-100%. Using this approach, we robustly generated cerebral organoids on the pillar plate and demonstrated an intra-batch coefficient of variation below 9%-19% based on ATP-based cell viability and compound treatment. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowingin situorganoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

Brain organoids: A revolutionary tool for modeling neurological disorders and development of therapeutics.

Brain organoids are self-organized, three-dimensional (3D) aggregates derived from pluripotent stem cells that have cell types and cellular architectures resembling those of the developing human brain. The current understanding of human brain developmental processes and neurological disorders has advanced significantly with the introduction of this in vitro model. Brain organoids serve as a translational link between two-dimensional (2D) cultures and in vivo models which imitate the neural tube formation at the early and late stages and the differentiation of neuroepithelium with whole-brain regionalization. In addition, the generation of region-specific brain organoids made it possible to investigate the pathogenic and etiological aspects of acquired and inherited brain disease along with drug discovery and drug toxicity testing. In this review article, we first summarize an overview of the existing methods and platforms used for generating brain organoids and their limitations and then discuss the recent advancement in brain organoid technology. In addition, we discuss how brain organoids have been used to model aspects of neurodevelopmental and neurodegenerative diseases, including autism spectrum disorder (ASD), Rett syndrome, Zika virus-related microcephaly, Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).

SOX7: Novel Autistic Gene Identified by Analysis of Multi-Omics Data.

Background: Despite thousands of variants identified by genome-wide association studies (GWAS) to be associated with autism spectrum disorder (ASD), it is unclear which mutations are causal because most are noncoding. Consequently, reliable diagnostic biomarkers are lacking. RNA-seq analysis captures biomolecular complexity that GWAS cannot by considering transcriptomic patterns. Therefore, integrating DNA and RNA testing may reveal causal genes and useful biomarkers for ASD. Methods: We performed gene-based association studies using an adaptive test method with GWAS summary statistics from two large Psychiatric Genomics Consortium (PGC) datasets (ASD2019: 18,382 cases and 27,969 controls; ASD2017: 6,197 cases and 7,377 controls). We also investigated differential expression for genes identified with the adaptive test using an RNA-seq dataset (GSE30573: 3 cases and 3 controls) and DESeq2. Results: We identified 5 genes significantly associated with ASD in ASD2019 (KIZ-AS1, p = 8.67×10- 10; KIZ, p = 1.16×10- 9; XRN2, p = 7.73×10- 9; SOX7, p = 2.22×10- 7; LOC101929229 (also known as PINX1-DT), p = 2.14×10- 6). Two of the five genes were replicated in ASD2017: SOX7 (p = 0.00087) and LOC101929229 (p = 0.009), and KIZ was close to the replication boundary of replication (p = 0.06). We identified significant expression differences for SOX7 (p = 0.0017, adjusted p = 0.0085), LOC101929229 (p = 5.83×10- 7, adjusted p = 1.18×10- 5), and KIZ (p = 0.00099, adjusted p = 0.0055). SOX7 encodes a transcription factor that regulates developmental pathways, alterations in which may contribute to ASD. Limitations: The limitation of the gene-based analysis is the reliance on a reference population for estimating linkage disequilibrium between variants. The similarity of this reference population to the population of study is crucial to the accuracy of many gene-based analyses, including those performed in this study. As a result, the extent of our findings is limited to European populations, as this was our reference of choice. Future work includes a tighter integration of DNA and RNA information as well as extensions to non-European populations that have been under-researched. Conclusions: These findings suggest that SOX7 and its related SOX family genes encode transcription factors that are critical to the downregulation of the canonical Wnt/ß-catenin signaling pathway, an important developmental signaling pathway, providing credence to the biologic plausibility of the association between gene SOX7 and autism spectrum disorder.

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate.

Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. By differentiating the EBs on the pillar plate, we achieved robust generation of cerebral organoids with a coefficient of variation (CV) below 19%. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

High-throughput assessment of metabolism-mediated neurotoxicity by combining 3D-cultured neural stem cells and liver cell spheroids.

Despite the fact that biotransformation in the liver plays an important role in the augmented toxicity and detoxification of chemicals, relatively little efforts have been made to incorporate biotransformation into in vitro neurotoxicity testing. Conventional in vitro systems for neurotoxicity tests lack the capability of investigating the qualitative and quantitative differences between parent chemicals and their metabolites in the human body. Therefore, there is a need for an in vitro toxicity screening system that can incorporate hepatic biotransformation of chemicals and predict the susceptibility of their metabolites to induce neurotoxicity. To address this need, we adopted 3D cultures of metabolically competent HepaRG cell line with ReNcell VM and established a high-throughput, metabolism-mediated neurotoxicity testing system. Briefly, spheroids of HepaRG cells were generated in an ultralow attachment (ULA) 384-well plate while 3D-cultured ReNcell VM was established on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds were added in the ULA 384-well plate with HepaRG spheroids and coupled with 3D-cultured ReNcell VM on the 384PillarPlate, which allowed us to generate metabolites in situ by HepaRG cells and test them against neural stem cells. We envision that this approach could be potentially adopted in pharmaceutical and chemical industries when high-throughput screening (HTS) is necessary to assess neurotoxicity of compounds and their metabolites.

Suppression of the antitumoral activity of natural killer cells under indirect coculture with cancer-associated fibroblasts in a pancreatic TIME-on-chip model.

BACKGROUND: Recently, natural killer (NK) cells emerged as a treatment option for various solid tumors. However, the immunosuppressive tumor immune microenvironment (TIME) can reduce the cytotoxic ability of NK cells in pancreatic ductal adenocarcinoma. Cancer-associated fibroblasts within the tumor stroma can suppress immune surveillance by dysregulating factors involved in the cellular activity of NK cells. Herein, the effect of activated pancreatic stellate cells (aPSCs) on NK cell-mediated anticancer efficacy under three-dimensional (3D) coculture conditions was investigated. METHODS: 3D cocultures of PANC-1 tumor spheroids (TSs) with aPSCs and NK-92 cells in a collagen matrix were optimized to identify the occurring cellular interactions and differential cytokine profiles in conditioned media using microchannel chips. PANC-1 TSs and aPSCs were indirectly cocultured, whereas NK-92 cells were allowed to infiltrate the TS channel using convective medium flow. RESULTS: Coculture with aPSCs promoted PANC-1 TSs growth and suppressed the antitumor cytotoxic effects of NK-92 cells. Mutual inhibition of cellular activity without compromising migration ability was observed between aPSCs and NK-92 cells. Moreover, the reduced killing activity of NK-92 cells was found to be related with reduced granzyme B expression in NK cells. CONCLUSIONS: Herein, a novel TIME-on-chip model based on the coculture of PANC-1 TSs, aPSCs, and NK-92 cells was described. This model may be useful for studying the detailed mechanisms underlying NK cells dysregulation and for exploring future therapeutic interventions to restore NK cell activity in the tumor microenvironment.

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis.

Human organoids have the potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by the lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, these challenges are overcome by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing, and encapsulation techniques are demonstrated on a pillar plate, which is coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels are differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

SOX7: Novel Autistic Gene Identified by Analysis of Multi-Omics Data.

Background: Genome-wide association studies and next generation sequencing data analyses based on DNA information have identified thousands of mutations associated with autism spectrum disorder (ASD). However, more than 99% of identified mutations are non-coding. Thus, it is unclear which of these mutations might be functional and thus potentially causal variants. Transcriptomic profiling using total RNA-sequencing has been one of the most utilized approaches to link protein levels to genetic information at the molecular level. The transcriptome captures molecular genomic complexity that the DNA sequence solely does not. Some mutations alter a gene's DNA sequence but do not necessarily change expression and/or protein function. To date, few common variants reliably associated with the diagnosis status of ASD despite consistently high estimates of heritability. In addition, reliable biomarkers used to diagnose ASD or molecular mechanisms to define the severity of ASD do not exist. Objectives: It is necessary to integrate DNA and RNA testing together to identify true causal genes and propose useful biomarkers for ASD. Methods: We performed gene-based association studies with adaptive test using genome-wide association studies (GWAS) summary statistics with two large GWAS datasets (ASD 2019 data: 18,382 ASD cases and 27,969 controls [discovery data]; ASD 2017 data: 6,197 ASD cases and 7,377 controls [replication data]) which were obtained from the Psychiatric Genomics Consortium (PGC). In addition, we investigated differential expression for genes identified in gene-based GWAS with a RNA-seq dataset (GSE30573: 3 cases and 3 controls) using the DESeq2 package. Results: We identified 5 genes significantly associated with ASD in ASD 2019 data (KIZ-AS1, p=8.67×10-10; KIZ, p=1.16×10-9; XRN2, p=7.73×10-9; SOX7, p=2.22×10-7; PINX1-DT, p=2.14×10-6). Among these 5 genes, gene SOX7 (p=0.00087), LOC101929229 (p=0.009), and KIZ-AS1 (p=0.059) were replicated in ASD 2017 data. KIZ (p=0.06) was close to the boundary of replication in ASD 2017 data. Genes SOX7 (p=0.0017, adjusted p=0.0085), LOC101929229 (also known as PINX1-DT, p=5.83×10-7, adjusted p=1.18×10-5), and KIZ (p=0.00099, adjusted p=0.0055) indicated significant expression differences between cases and controls in the RNA-seq data. SOX7 encodes a member of the SOX (SRY-related HMG-box) family of transcription factors pivotally contributing to determining of the cell fate and identity in many lineages. The encoded protein may act as a transcriptional regulator after forming a protein complex with other proteins leading to autism. Conclusion: Gene SOX7 in the transcription factor family could be associated with ASD. This finding may provide new diagnostic and therapeutic strategies for ASD.

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis.

Human organoids have potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo . This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, we overcome these challenges by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

A pillar/perfusion plate enhances cell growth, reproducibility, throughput, and user friendliness in dynamic 3D cell culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from the necrotic core due to limited nutrient and oxygen diffusion and waste removal and have limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids have been loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow rates were maintained within perfusion wells, and the pillar plate could be separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in dynamic 3D cell culture.

Effect of paclitaxel priming on doxorubicin penetration in a multicellular layer model of human colorectal cancer cells.

Tumor priming is considered a promising strategy for improving drug distribution in malignant tissues. Multicellular layers (MCLs) of human cancer cells are potentially useful models for evaluating tumor-priming agents. We evaluated the priming effects of paclitaxel (PTX) on doxorubicin (DOX) penetration using MCLs of the human colorectal cancer cell lines including DLD-1, HCT-116, and HT-29. The penetration of DOX treated at 50 µM for 3 h was highly limited in all three MCLs. The penetration of the priming agent PTX into MCLs was determined using rhodamine-labeled PTX and appeared to be cell line-dependent: full penetration was observed in HCT-116 and HT-29 MCLs, whereas only limited penetration occurred in DLD-1 MCLs. PTX pretreatment at 20 µM for 24 or 48 h induced a tumor-priming effect in DOX distribution, with a 3 to 5.6-fold-increase in HCT-116 and HT-29 MCLs but a less than two-fold increase in DLD-1 MCLs. PTX treatment decreased fibronectin expression in HCT-116 and HT-29 MCLs but not in DLD-1, suggesting that the prominent priming effect of PTX in HCT-116 and HT-29 MCLs may be associated with the downregulation of fibronectin expression. Our study demonstrated that MCLs of human cancer cells are a useful model not only for the study of drug penetration into tumor tissues but also for screening and evaluating tumor-priming agents.

Current methods for fabricating 3D cardiac engineered constructs.

3D cardiac engineered constructs have yielded not only the next generation of cardiac regenerative medicine but also have allowed for more accurate modeling of both healthy and diseased cardiac tissues. This is critical as current cardiac treatments are rudimentary and often default to eventual heart transplants. This review serves to highlight the various cell types found in cardiac tissues and how they correspond with current advanced fabrication methods for creating cardiac engineered constructs capable of shedding light on various pathologies and providing the therapeutic potential for damaged myocardium. In addition, insight is given toward the future direction of the field with an emphasis on the creation of specialized and personalized constructs that model the region-specific microtopography and function of native cardiac tissues.

Establishment of ion channel and ABC transporter assays in 3D-cultured ReNcell VM on a 384-pillar plate for neurotoxicity potential.

Neurotoxicity potential of compounds by inhibition of ion channels and efflux transporters has been studied traditionally using two-dimensionally (2D) cultured cell lines such as CHO and HEK-293 overexpressing the protein of interest. However, these approaches are time consuming and do not recapitulate the activity of ion channels and efflux transporters indigenously expressed in neural stem cells (NSCs) in vivo. To overcome these issues, we established ion channel and transporter assays on a 384-pillar plate with three-dimensionally (3D) cultured ReNcell VM and demonstrated high-throughput measurement of ion channel and transporter activity. RNA sequencing analysis identified major ion channels and efflux transporters expressed in ReNcell VM, followed by validating 3D ReNcell-based ion channel and transporter assays with model compounds. Major ion channel activities were measured by specifically inhibiting potassium channels Kv 7.2 with XE-991 and Kv 4.3 with fluoxetine, and a calcium channel with 2-APB. Activities of major efflux transporters, MDR1, MRP1, and BCRP, were assessed using their respective blockers, verapamil, probenecid, and novobiocin. From this study, we demonstrated that 3D-cultured ReNcell VM on the 384-pillar plate could be a good alternative to rapidly identify environmental chemicals and therapeutic compounds for their role in modulating the activity of ion channels and efflux transporters, potentially leading to neurotoxicity.

Recent advances in microarray 3D bioprinting for high-throughput spheroid and tissue culture and analysis.

Three-dimensional (3D) cell culture in vitro has proven to be more physiologically relevant than two-dimensional (2D) culture of cell monolayers, thus more predictive in assessing efficacy and toxicity of compounds. There have been several 3D cell culture techniques developed, which include spheroid and multicellular tissue cultures. Cell spheroids have been generated from single or multiple cell types cultured in ultralow attachment (ULA) well plates and hanging droplet plates. In general, cell spheroids are formed in a relatively short period of culture, in the absence of extracellular matrices (ECMs), via gravity-driven self-aggregation, thus having limited ability to self-organization in layered structure. On the other hand, multicellular tissue cultures including miniature tissues derived from pluripotent stem cells and adult stem cells (a.k.a. 'organoids') and 3D bioprinted tissue constructs require biomimetic hydrogels or ECMs and show highly ordered structure due to spontaneous self-organization of cells during differentiation and maturation processes. In this short review article, we summarize traditional methods of spheroid and multicellular tissue cultures as well as their technical challenges, and introduce how droplet-based, miniature 3D bioprinting ('microarray 3D bioprinting') can be used to improve assay throughput and reproducibility for high-throughput, predictive screening of compounds. Several platforms including a micropillar chip and a 384-pillar plate developed to facilitate miniature spheroid and tissue cultures via microarray 3D bioprinting are introduced. We excluded microphysiological systems (MPSs) in this article although they are important tissue models to simulate multiorgan interactions.

High-Throughput Screening of Compound Neurotoxicity Using 3D-Cultured Neural Stem Cells on a 384-Pillar Plate.

Assessing the neurotoxicity of test chemicals has typically been performed using two-dimensionally (2D)-cultured neuronal cell monolayers and animal models. The in vitro 2D cell models are simple and straightforward compared to animal models, which have the disadvantage of being relatively low throughput, expensive, and time consuming. Despite their extensive use in this area of neurotoxicology research, both models often do not accurately recapitulate human outcomes. To bridge this gap and attempt to better replicate what happens in vivo, three-dimensionally (3D) cultured neural stem cells (NSCs) encapsulated in hydrogels on a 384-pillar plate have been developed via miniature 3D bioprinting. This technology allows users to print NSCs on a pillar plate for rapid 3D cell culture as well as high-throughput compound screening. For this, the 384-pillar plate with bioprinted NSCs is sandwiched with a standard 384-well plate with growth medium for 3D culture, allowing researchers to expose the cells to test compounds and stain them with various fluorescent dyes for a suite of high-content imaging assays, including assays for DNA damage, mitochondrial impairment, cell membrane integrity, intracellular glutathione levels, and apoptosis. After acquiring cell images from an automated fluorescence microscope and extracting fluorescence intensities, researchers can obtain the IC50 value of each compound to evaluate critical parameters in neurotoxicity. Here, we provide a detailed description of protocols for cell printing on a 384-pillar plate, 3D NSC culture, compound testing, 3D cell staining, and image acquisition and analysis, which altogether will allow researchers to investigate mechanisms of compound neurotoxicity with 3D-cultured NSCs in a high-throughput manner. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Three-dimensional neural stem cell culture on a 384-pillar plate Basic Protocol 2: Compound treatment and cell staining Basic Protocol 3: Image acquisition, processing, and data analysis.