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.

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.

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.

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.

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.

Preperitoneal Pelvic Packing versus Angioembolization for Patients with Hemodynamically Unstable Pelvic Fractures with Pelvic Bleeding: A Single-Centered Retrospective Study.

The aim of this study was to compare the outcomes of preperitoneal pelvic packing (PPP) and angioembolization (AE) for patients with equivocal vital signs after initial resuscitation. This single-centered retrospective study included information from the database of a regional trauma center from April 2014 to December 2022 for patients with pelvic fractures with a systolic blood pressure of 80-100 mmHg after initial fluid resuscitation. The patients' characteristics, outcomes, and details of AE after resuscitative endovascular balloon occlusion of the aorta (REBOA) placed in zone III were collected. The follow-up duration was from hospital admission to discharge. A total of 65 patients were enrolled in this study. Their mean age was 59.2 ± 18.1 years, and 40 were males. We divided the enrolled patients into PPP (n = 43) and AE (n = 22) groups. The median time from emergency department (ED) to procedure and the median duration of ED stay were significantly longer in the AE group than in the PPP group (p ≤ 0.001 for both). The median mechanical ventilation (MV) duration was significantly shorter (p = 0.046) in the AE group. The number of patients with complications, overall mortality, and mortality due to hemorrhage did not differ between the two groups. Three patients (13.6%) were successfully treated with AE after REBOA. AE may be beneficial for patients with hemodynamically unstable pelvic fractures who show equivocal vital signs after initial fluid resuscitation in terms of reducing the MV duration and incidence of infectious complications.

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.

Outcomes improvement despite continuous visits of severely injured patients during the COVID-19 outbreak: experience at a regional trauma centre in South Korea.

BACKGROUND: Understanding the changes in characteristics of patients who visited trauma centres during the coronavirus disease 2019 (COVID-19) pandemic is important to facilitate aneffective response. This retrospective study was conducted to analyse differences in the characteristics and outcomes of patients who visited our trauma centre between pre-COVID-19 and COVID-19 eras. METHODS: Medical data of trauma patients enrolled in the Korean trauma database from 1 January 2018 to 31 August 2021 were collected. The number of trauma centre visits, patient characteristics, factors associated with in-hospital intervention, and outcomes werecompared between patients in the two time periods. Propensity score matching was performed to analyse the outcomes in patients with similar characteristics and severitybetween patients in the two time periods. RESULTS: The number of emergency department (ED) trauma service visits reduced in the COVID-19 era. Based on the mean age, the patients were older in the COVID-19 era. Abbreviated injury scale (AIS) 1, AIS3, AIS5, and injury severity score (ISS) were higher in the COVID-19 era. The proportion of motor vehicle collisions decreased, whereas falls increased during the COVID-19 era. Ambulance transportation, admission to the general ward, and time from injury to ED visit significantly increased. Patient outcomes, such as hospital length of stay (LOS), intensive care unit (ICU) LOS, and duration of mechanical ventilation improved, while injury severity worsened during the COVID-19 era. After adjusting for patient characteristics and severity, similar findings were observed. CONCLUSION: The small reduction in the number of trauma patients and visits by patients who hadhigher ISS during the COVID-19 pandemic highlights the importance of maintaining trauma service capacity and capability during the pandemic. A nationwide or nationalmulticentre study will be more meaningful to examine the impact of the COVID-19 outbreak on the changes in trauma patterns, volume, and patient outcomes.

SH005S7 Overcomes Primary and Acquired Resistance of Non-Small Cell Lung Cancer by Combined MET/EGFR/HER3 Inhibition.

In this study, we have examined the anticancer effects of SH005S7 on MET-amplified and (HCC827GR) NSCLC cells and their primary HCC827 cells. In vitro, first of all, cell viability and colony formation assay confirmed the growth inhibitory effects of SH005S7 on both cells. Second, SH005S7 inactivated EGFR-related multiple cell signaling, which was associated with a marked decrease in the constitutive phosphorylation of EGFR, HER3, MET, AKT, and ERK. Third, SH005S7 attenuated the anchorage-independent cell growth. Fourth, SH005S7 blocked invasive and metastatic capability by downregulation of mesenchymal markers-vimentin, snail, and MMP-9. Fifth, BrdU assay confirmed the cell cycle arrest of SH005S7 on these cells. When administered orally to nude mice xenografically transplanted human NSCLC, SH005S7 inhibited the growth of tumor and did not cause hepatotoxicity and nephrotoxicity in animals. Immunohistochemical and Western blot analyses of tissue showed that the suppression of growth correlated with inhibition of proliferation (Ki-67, PCNA), invasiveness (vimentin, snail), and angiogenesis (CD31) marker and decrement in the constitutive and phosphorylation of EGFR, HER3, MET, AKT, and ERK. Additionally, SH005S7 had immune stimulatory effects by TNF-α cytokine release on macrophage, without cell cytotoxicity. Overall, our results suggest that SH005S7 can inhibit the growth of MET-amplified and gefitinib-resistant NSCLC cells through the suppression of EGFR-related multiple targets linked to overcome gefitinib resistance.

Online Learning versus Hands-On Learning of Basic Ocular Ultrasound Skills: A Randomized Controlled Non-Inferiority Trial.

Background and objectives: Ocular ultrasound is a core application of point-of-care ultrasound (POCUS) to assist physicians in promptly identifying various ocular diseases at the bedside; however, hands-on POCUS training is challenging during a pandemic. Materials and Methods: A randomized controlled non-inferiority trial was conducted in an academic emergency department from October 2020 to April 2021. Thirty-two participants were randomly assigned to one of two groups. Group H (hands-on learning group) participated individually in a hands-on session with a standardized patient for 30 min, whereas Group O (online learning group) learned training materials and video clips for 20 min. They scanned four eyeballs of two standardized patients sequentially following the ocular POCUS scan protocol. Repeated POCUS scans were performed 2 weeks later to assess skill maintenance. Both groups completed the pre- and post-surveys and knowledge tests. Two emergency medicine faculty members blindly evaluated the data and assigned a score of 0−25. The primary endpoint was the initial total score of scan quality evaluated using non-inferiority analysis (generalized estimating equation). The secondary endpoints were total scores for scan quality after 2 weeks, scan time, and knowledge test scores. Results: The least squares means of the total scores were 21.7 (0.35) for Group O and 21.3 (0.25) for Group H, and the lower bound of the 95% confidence interval (CI) was greater than the non-inferiority margin of minus 2 (95% CI: −0.48−1.17). The second scan scores were not significantly different from those of the first scan. The groups did not differ in scanning time or knowledge test results; however, Group H showed higher subjective satisfaction with the training method (p < 0.001). Conclusion: This study showed that basic online ocular ultrasound education was not inferior to hands-on education, suggesting that it could be a useful educational approach in the pandemic era.

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.

Impact of Insurance Benefits and Education on Point-of-Care Ultrasound Use in a Single Emergency Department: An Interrupted Time Series Analysis.

Background and Objectives: Point-of-care ultrasound (POCUS) is a useful tool that helps clinicians properly treat patients in emergency department (ED). This study aimed to evaluate the impact of specific interventions on the use of POCUS in the ED. Materials and Methods: This retrospective study used an interrupted time series analysis to assess how interventions changed the use of POCUS in the emergency department of a tertiary medical institute in South Korea from October 2016 to February 2021. We chose two main interventions-expansion of benefit coverage of the National Health Insurance (NHI) for emergency ultrasound (EUS) and annual ultrasound educational workshops. The primary variable was the EUS rate, defined as the number of EUS scans per 1000 eligible patients per month. We compared the level and slope of EUS rates before and after interventions. Results: A total of 5188 scanned records were included. Before interventions, the EUS rate had increased gradually. After interventions, except for the first workshop, the EUS rate immediately increased significantly (p < 0.05). The difference in the EUS rate according to the expansion of the NHI was estimated to be the largest (p < 0.001). However, the change in slope significantly decreased after the third workshop during the coronavirus disease 2019 pandemic (p = 0.004). The EUS rate increased significantly in the presence of physicians participating in intensive POCUS training (p < 0.001). Conclusion: This study found that expansion of insurance coverage for EUS and ultrasound education led to a significant and immediate increase in the use of POCUS, suggesting that POCUS use can be increased by improving education and insurance benefits.

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.

High-content imaging of 3D-cultured neural stem cells on a 384-pillar plate for the assessment of cytotoxicity.

The assessment of neurotoxicity has been performed traditionally with animals. However, in vivo studies are highly expensive and time-consuming, and often do not correlate to human outcomes. Thus, there is a need for cost-effective, high-throughput, highly predictive alternative in vitro test methods based on early markers of mechanisms of toxicity. High-content imaging (HCI) assays performed on three-dimensionally (3D) cultured cells could provide better understanding of the mechanism of toxicity needed to predict neurotoxicity in humans. However, current 3D cell culture systems lack the throughput required for screening neurotoxicity against a large number of chemicals. Therefore, we have developed miniature 3D neural stem cell (NSC) culture on a unique 384-pillar plate, which is complementary to conventional 384-well plates. Mitochondrial membrane impairment, intracellular glutathione level, cell membrane integrity, DNA damage, and apoptosis have been tested against 3D-cultured ReNcell VM on the 384-pillar plate with four model compounds rotenone, 4-aminopyridine, digoxin, and topotecan. The HCI assays performed in 3D-cultured ReNcell VM on the 384-pillar plates were highly robust and reproducible as indicated by the average Z' factor of 0.6 and CV values around 12%. From concentration-response curves and IC50 values, mitochondrial membrane impairment appears to be the early stage marker of cell death by the compounds.

High-Throughput Assessment of Metabolism-Induced Toxicity of Compounds on a 384-Pillar Plate.

A variety of oxidative and conjugative enzymes are involved in the metabolism of compounds including drugs, which can be converted into toxic metabolites by Phase I drug-metabolizing enzymes (DMEs), such as the cytochromes P450 (CYP450s), and/or detoxified by Phase II DMEs, such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), and glutathione S-transferases (GSTs). Traditionally, primary hepatocytes containing a complete set of DMEs have been widely used as a gold standard to assess metabolism-induced compound toxicity. However, primary hepatocytes are expensive, have high donor variability in expression levels of DMEs, and rapidly lose liver-specific functions when the cells are maintained under standard in vitro cell culture conditions over time. To address this issue and rapidly profile metabolism-induced drug toxicity, we have developed a 384-pillar plate, which is complementary to conventional 384-well plates. In this chapter, we provide step-by-step procedures for three-dimensional (3D) cell printing on the 384-pillar plate coupled with DMEs and compounds in the 384-well plate for high-throughput assessment of metabolism-induced toxicity.

Traditional Herbal Formula Taeeumjowi-Tang (TJ001) Inhibits p53-Mutant Prostate Cancer Cells Growth by Activating AMPK-Dependent Pathway.

Dysregulated lipid metabolism is a prominent feature of prostate cancers (PCas); several enzymes involved in lipid accumulation are highly expressed. Here, we elucidated efficacy of TJ001, a traditional herbal decoction, in inhibiting de novo lipogenesis. TJ001 had significant cytotoxicity against DU145 but not PC3 and LNCaP cells and, similarly, TJ001 markedly AMPK phosphorylation only in DU145 cells. This was accompanied by the downregulation of phosphorylated-acetyl coenzyme A carboxylase (ACC) expression and sterol regulatory element-binding protein 1 (SREBP1) proteolytic cleavage, thereby inhibiting its role as a transcription factor to induce lipid biosynthesis. When Oil Red O staining was performed, it is reflected in the reduction of lipid droplets (LDs). TJ001 also induced G1/S cell cycle arrest via a cell cycle inhibitor (CKI) p21WAF1/CIP1 upregulation. Although p53 proteins remained unchanged, both cyclin E and cyclin D1 were decreased. Moreover, TJ001 suppressed the mammalian target of rapamycin (mTOR) signaling pathway. Generally, the prolonged G1/S phase arrest accompanies apoptosis, but TJ001 failed to work as a trigger apoptosis in DU145 cells. We showed that mutant p53 proteins were required for the survival of DU145 cells. In presence of TJ001, inhibition of endogenous mutant p53 by RNAi led to cell viability reduction and induction of the p-AMPK/AMPK ratio. In addition, it induced apoptotic cell death in DU145 cells. At the cellular level, induction of PARP, caspase-3, and caspase-9 cleavages was observed, and caspase-3 activity was increased in the p53 knockdown cells treated with TJ001. Taken together, we demonstrated that TJ001 inhibited cell growth in DU145 prostate cancer cells as indicated by blocking lipogenesis and induction in G1/S cell cycle arrest. In addition, we may provide an evidence that mutant p53 protein has potential role as an oncogenic action in DU145 cells. Collectively, the combination of mutant p53 targeting and TJ001 treatment resulted in decreased cell growth in DU145 cells.

High-Throughput Assessment of Mechanistic Toxicity of Chemicals in Miniaturized 3D Cell Culture.

High-content imaging (HCI) assays on two-dimensional (2D) cell cultures often do not represent in vivo characteristics accurately, thus reducing the predictability of drug toxicity/efficacy in vivo. On the other hand, conventional 3D cell cultures are relatively low throughput and possess difficulty in cell imaging. To address these limitations, a miniaturized 3D cell culture has been developed on a micropillar/microwell chip platform with human cells encapsulated in biomimetic hydrogels. Model compounds are used to validate human cell microarrays for high-throughput assessment of mechanistic toxicity. Main mechanisms of toxicity of compounds can be investigated by analyzing multiple parameters such as DNA damage, mitochondrial impairment, intracellular glutathione level, and cell membrane integrity. IC50 values of these parameters can be determined and compared for the compounds to investigate the main mechanism of toxicity. This paper describes miniaturized HCI assays on 3D-cultured cell microarrays for high-throughput assessment of mechanistic profiles of compound-induced toxicity. © 2018 by John Wiley & Sons, Inc.