3D cell subculturing pillar dish for pharmacogenetic analysis and high-throughput screening.

A pillar dishe for subculture of 3D cultured cells on hydrogel spots (Matrigel and alginate) have been developed. Cells cultured in 3D in an extracellular matrix (ECM) can retain their intrinsic properties, but cells cultured in 2D lose their intrinsic properties as the cells stick to the bottom of the well. Previously, cells and ECM spots were dispensed on a conventional culture dish for 3D cultivation. However, as the spot shape and location depended on user handling, pillars were added to the dish to realize uniform spot shape and stable subculture, supporting 3D cell culture-based high-throughput screening (HTS). Matrigel and alginate were used as ECMs during 6-passage subculture. The growth rate of lung cancer cell (A549) was higher on Matrigel than on alginate. Cancer cell was subcultured in three dimensions in the proposed pillar dish and used for drug screening and differential gene expression analysis. Interestingly, stemness markers, which are unique characteristics of lung cancer cells inducing drug resistance, were upregulated in 3D-subcultured cells compared with those in 2D-subcultured cells. Additionally, the PI3K/Akt/mTOR, VEGFR1/2, and Wnt pathways, which are promising therapeutic targets for lung cancer, were activated, showing high drug sensitivity under 3D-HTS using the 3D-subcultured cells.

In Vitro three-dimensional (3D) cell culture tools for spheroid and organoid models.

Three-dimensional (3D) cell culture technology has been steadily studied since the 1990's due to its superior biocompatibility compared to the conventional two-dimensional (2D) cell culture technology, and has recently developed into an organoid culture technology that further improved biocompatibility. Since the 3D culture of human cell lines in artificial scaffolds was demonstrated in the early 90's, 3D cell culture technology has been actively developed owing to various needs in the areas of disease research, precision medicine, new drug development, and some of these technologies have been commercialized. In particular, 3D cell culture technology is actively being applied and utilized in drug development and cancer-related precision medicine research. Drug development is a long and expensive process that involves multiple steps-from target identification to lead discovery and optimization, preclinical studies, and clinical trials for approval for clinical use. Cancer ranks first among life-threatening diseases owing to intra-tumoral heterogeneity associated with metastasis, recurrence, and treatment resistance, ultimately contributing to treatment failure and adverse prognoses. Therefore, there is an urgent need for the development of efficient drugs using 3D cell culture techniques that can closely mimic in vivo cellular environments and customized tumor models that faithfully represent the tumor heterogeneity of individual patients. This review discusses 3D cell culture technology focusing on research trends, commercialization status, and expected effects developed until recently. We aim to summarize the great potential of 3D cell culture technology and contribute to expanding the base of this technology.

Cytotoxicity of Human Hepatic Intrasinusoidal Gamma/Delta T Cells Depends on Phospho-antigen and NK Receptor Signaling.

BACKGROUND/AIM: We previously showed that human hepatic intrasinusoidal (HI) natural killer (NK) T cells selectively eliminate hepatocellular carcinoma (HCC) cell lines. In this study, we investigated the underlying mechanisms on how HI γδ T cells, expanded with zoledronate, exhibit a superior cytotoxic effect on HI NK-resistant Huh7 HCC cells. MATERIALS AND METHODS: γδ T cells were obtained from living liver transplant donors or from peripheral blood mononuclear cells (PBMC) of healthy volunteers and were expanded in the presence of IL-2, IL-15, and zoledronate for 2 weeks. Cytotoxicity was measured using the lactate dehydrogenase (LDH) assay in vitro and by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) in vivo. RESULTS: The cytotoxicity of expanded HI γδ T cells against Huh7 cells was associated with a higher pyrophosphate expression in Huh7 cells compared to SNU398 cells. In contrast, the cytotoxicity of HI γδ T cells against SNU398 cells depended on NKG2D. HI γδ T cells expressed less PD-1 than PB γδ T cells. The cytotoxicity of HI γδ T cells against Du145 and PC3 prostate cancer cells was also associated with pyrophosphate expression in these cells, as well as NKG2D and DNAM-1. CONCLUSION: The expression levels of phospho-antigen in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules also contribute to their cytotoxicity. γδ T cells are attractive candidates for cancer immune cell therapy.

Optimization of 3D-aggregated spheroid model (3D-ASM) for selecting high efficacy drugs.

Various three-dimensional (3D) cell culture methods have been developed to implement tumor models similar to in vivo. However, the conventional 3D cell culture method has limitations such as difficulty in using an extracellular matrix (ECM), low experimental reproducibility, complex 3D cell culture protocol, and difficulty in applying to high array plates such as 96- or 384-plates. Therefore, detailed protocols related to robust 3D-aggregated spheroid model (3D-ASM) production were optimized and proposed. A specially designed wet chamber was used to implement 3D-ASM using the hepatocellular carcinoma (HCC) cell lines, and the conditions were established for the icing step to aggregate the cells in one place and optimized ECM gelation step. Immunofluorescence (IF) staining is mainly used to simultaneously analyze drug efficacy and changes in drug-target biomarkers. By applying the IF staining method to the 3D-ASM model, confocal microscopy imaging and 3D deconvolution image analysis were also successfully performed. Through a comparative study of drug response with conventional 2D-high throughput screening (HTS), the 3D-HTS showed a more comprehensive range of drug efficacy analyses for HCC cell lines and enabled selective drug efficacy analysis for the FDA-approved drug sorafenib. This suggests that increased drug resistance under 3D-HTS conditions does not reduce the analytical discrimination of drug efficacy, also drug efficacy can be analyzed more selectively compared to the conventional 2D-HTS assay. Therefore, the 3D-HTS-based drug efficacy analysis method using an automated 3D-cell spotter/scanner, 384-pillar plate/wet chamber, and the proposed 3D-ASM fabrication protocol is a very suitable platform for analyzing target drug efficacy in HCC cells.

Cell Proliferation Receptor-Enhanced 3D High-Throughput Screening Model for Optimized Drug Efficacy Evaluation in Breast Cancer Cells.

A higher correlation of epidermal growth factor receptor (EGFR)-targeting drugs has been reported with the use of the cell proliferation receptor-enhanced three-dimensional high-throughput screening model (CPRE 3D-HTS model) compared with two-dimensional (2D) cell-based HTS. A greater expression of differential human EGFR 2 (HER2) protein between HER2-positive and HER2-negative cell lines was observed in breast cancer (BC) cell lines cultured using the CPRE 3D-HTS model compared with 2D-cultured cells. When using 2D-cultured cells, properties such as the expression of the cell proliferation receptor are lost as the cells attach to the bottom of the well plate. In an effort to solve this problem, the CPRE 3D-HTS model expressing high cell proliferation receptors was optimized by the selection of alginate as the extracellular matrix. Results from the use of the CPRE 3D-HTS model showed higher drug resistance with increased expression of drug resistance-related proteins. Of particular interest, a higher correlation of HER2-targeted drugs was observed with the use of the CPRE 3D-HTS model. In order to validate this higher correlation of target drugs observed in the CPRE 3D-HTS model, the results of Western blot analysis and high content imaging analysis were analyzed, which confirmed that 3D-cultured BC cell lines showed a greater difference in the expression of HER2-positive and HER2-negative BC cell lines than 2D-cultured cells. Thus, the use of CPRE 3D-HTS using a 384-pillar plate resulted in increased accuracy when screening HER2-targeted drugs in BC, and it is a very useful platform for analyzing the efficacy of targeted drugs by enhancing the expression of HER2.

Characteristics of Human Peripheral Blood γδ T Cells Expanded With Zoledronate.

BACKGROUND/AIM: This study aimed to investigate the characteristics of human peripheral blood γδ T cells, which were expanded ex vivo in the presence of zoledronate (ZOL). MATERIALS AND METHODS: Human peripheral blood cells were cultured with IL-2 and IL-15 in the presence or absence of ZOL, which was added as a phospho-antigen, and their phenotypes were assessed by flow cytometry. Expanded γδ T cells were transduced with CD19 CAR vector, and the cytotoxicity was evaluated in vitro and in vivo by flow cytometry. RESULTS: Ex vivo expansion did not hamper the expression of activating receptors. Interestingly, ZOL promoted the expression of CD226 (DNAM-1), TRAIL, and FAS-L in the Vδ1 subset of γδ T cells. Expanded γδ T cells containing CD19 CAR+ γδ T cells removed B cell lymphoma cells effectively in vivo. CONCLUSION: γδ T cells could be a promising immunotherapeutic for cancer.

Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate.

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-ß2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.

Industrial Applications of Dinoflagellate Phycotoxins Based on Their Modes of Action: A Review.

Dinoflagellates are an important group of phytoplanktons, characterized by two dissimilar flagella and distinctive features of both plants and animals. Dinoflagellate-generated harmful algal blooms (HABs) and associated damage frequently occur in coastal areas, which are concomitant with increasing eutrophication and climate change derived from anthropogenic waste and atmospheric carbon dioxide, respectively. The severe damage and harmful effects of dinoflagellate phycotoxins in the fishing industry have been recognized over the past few decades, and the management and monitoring of HABs have attracted much attention, leaving aside the industrial application of their valuable toxins. Specific modes of action of the organisms' toxins can effectively be utilized for producing beneficial materials, such as Botox and other therapeutic agents. This review aims to explore the potential industrial applications of marine dinoflagellate phycotoxins; furthermore, this review focuses on their modes of action and summarizes the available knowledge on them.

Characterization of a Novel Mannose-Binding Lectin with Antiviral Activities from Red Alga, Grateloupia chiangii.

Lectins have the ability to bind specific carbohydrates and they have potential applications as medical and pharmacological agents. The unique structure and usefulness of red algal lectin have been reported, but these lectins are limited to a few marine algal groups. In this study, a novel mannose-binding lectin from Grateloupia chiangii (G. chiangii lectin, GCL) was purified using antiviral screens and affinity chromatography. We characterized the molecular weight, agglutination activity, hemagglutination activity, and heat stability of GCL. To determine the carbohydrate specificity, a glycan microarray was performed. GCL showed strong binding affinity for Maltohexaose-ß-Sp1 and Maltoheptaose-ß-Sp1 with weak affinity for other monosaccharides and preferred binding to high-mannan structures. The N-terminal sequence and peptide sequence of GCL were determined using an Edman degradation method and LC-MS/MS, and the cDNA and peptide sequences were deduced. GCL was shown to consist of 231 amino acids (24.9 kDa) and the N-terminus methionine was eliminated after translation. GCL possessed a tandem repeat structure of six domains, similar to the other red algal lectins. The mannose binding properties and tandem repeat structure of GCL may confer it the potential to act as an antiviral agent for protection against viral infection.

Expression of Immune Checkpoint Receptors on T-Cells and Their Ligands on Leukemia Blasts in Childhood Acute Leukemia.

BACKGROUND: Possible correlations between the expression of immune checkpoint molecules and prognosis in childhood acute leukemia were investigated. MATERIALS AND METHODS: The expression of programmed-death 1 (PD1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and B- and T-lymphocyte attenuator (BTLA) was determined by flow cytometry on peripheral αß+ and γδ+ T-cells from patients with newly diagnosed acute lymphoblastic leukemia (ALL) (n=9) or acute myeloid leukemia (AML) (n=12), and from healthy volunteers (n=7). The expression of programmed-death ligand 1 (PD-L1), B7-1, B7-2, human leukocyte antigen-ABC (HLA-ABC), and herpesvirus-entry mediator (HVEM) ligands was determined on leukemia blasts. RESULTS: PD1 expression on αß+ and γδ+ T-cells was significantly higher in patients with ALL than in those with AML (p=0.0019 and 0.0239, respectively). CTLA-4 expression was moderately higher on αß+ and γδ+ T-cells in ALL (p=0.077 and 0.077, respectively), whereas HLA-ABC expression was significantly higher in AML blast cells (p=0.0182). The expression of CTLA-4 on γδ+ T-cells and the B7-2 ligand on blasts was higher in patients with high-risk ALL (p=0.02 and 0.02, respectively). In AML, PD1 expression on αß+ T-cells was higher in the intermediate-risk group (p=0.05), whereas HVEM expression was significantly higher in the low-risk group (p=0.02). Expression of CTLA-4 on γδ+ T-cells and PD-L1 on blasts were both associated with poor relapse-free survival outcomes in ALL (p=0.049). CONCLUSION: The higher expression of immune checkpoint molecules, in particular, CTLA-4 and PD-L1 are associated with a poorer prognosis in ALL, suggesting that selective use of the immune checkpoint blockade might improve the clinical outcomes in patients with ALL.

Multiplex quantitative analysis of stroma-mediated cancer cell invasion, matrix remodeling, and drug response in a 3D co-culture model of pancreatic tumor spheroids and stellate cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a stroma-rich carcinoma, and pancreatic stellate cells (PSCs) are a major component of this dense stroma. PSCs play significant roles in metastatic progression and chemoresistance through cross-talk with cancer cells. Preclinical in vitro tumor model of invasive phenotype should incorporate three-dimensional (3D) culture of cancer cells and PSCs in extracellular matrix (ECM) for clinical relevance and predictability. METHODS: PANC-1 cells were cultured as tumor spheroids (TSs) using our previously developed minipillar chips, and co-cultured with PSCs, both embedded in collagen gels. Effects of PSC co-culture on ECM fiber network, invasive migration of cancer cells, and expression of epithelial-mesenchymal transition (EMT)-related proteins were examined. Conditioned media was also analyzed for secreted factors involved in cancer cell-PSC interactions. Inhibitory effect on cancer cell invasion was compared between gemcitabine and paclitaxel at an equitoxic concentration in PANC-1 TSs co-cultured with PSCs. RESULTS: Co-culture condition was optimized for the growth of TSs, activation of PSCs, and their interaction. Increase in cancer cell invasion via ECM remodeling, invadopodia formation and EMT, as well as drug resistance was recapitulated in the TS-PSC co-culture, and appeared to be mediated by cancer cell-PSC interaction via multiple secreted factors, including IL-6, IL-8, IGF-1, EGF, TIMP-1, uPA, PAI-1, and TSP-1. Compared to gemcitabine, paclitaxel showed a greater anti-invasive activity, which was attributed to suppresion of invadopodia formation in cancer cells as well as to PSC-specific cytotoxicity abrogating its paracrine signaling. CONCLUSIONS: Here, we established 3D co-culture of TSs of PANC-1 cells and PSCs using minipillar histochips as a novel tumoroid model of PDAC. Our results indicate usefulness of the present co-culture model and multiplex quantitative analysis method not only in studying the role of PSCs and their interactions with tumor cell towards metastatic progression, but also in the drug evaluation of stroma-targeting drugs.

Effects of Trauma Center Establishment on the Clinical Characteristics and Outcomes of Patients with Traumatic Brain Injury : A Retrospective Analysis from a Single Trauma Center in Korea.

OBJECTIVE: To investigate the effects of trauma center establishment on the clinical characteristics and outcomes of trauma patients with traumatic brain injury (TBI). METHODS: We enrolled 322 patients with severe trauma and TBI from January 2015 to December 2016. Clinical factors, indexes, and outcomes were compared before and after trauma center establishment (September 2015). The outcome was the Glasgow outcome scale classification at 3 months post-trauma. RESULTS: Of the 322 patients, 120 (37.3%) and 202 (62.7%) were admitted before and after trauma center establishment, respectively. The two groups were significantly different in age (p=0.038), the trauma location within the city (p=0.010), the proportion of intensive care unit (ICU) admissions (p=0.001), and the emergency room stay time (p<0.001). Mortality occurred in 37 patients (11.5%). Although the preventable death rate decreased from before to after center establishment (23.1% vs. 12.5%), the difference was not significant. None of the clinical factors, indexes, or outcomes were different from before to after center establishment for patients with severe TBI (Glasgow coma scale score ≤8). However, the proportion of inter-hospital transfers increased and the time to emergency room arrival was longer in both the entire cohort and patients with severe TBI after versus before trauma center establishment. CONCLUSION: We confirmed that for patients with severe trauma and TBI, establishing a trauma center increased the proportion of ICU admissions and decreased the emergency room stay time and preventable death rate. However, management strategies for handling the high proportion of inter-hospital transfers and long times to emergency room arrival will be necessary.

A Simple Method for Removal of the Chlamydomonas reinhardtii Cell Wall Using a Commercially Available Subtilisin (Alcalase).

The algal cell wall is a potent barrier for delivery of transgenes for genetic engineering. Conventional methods developed for higher plant systems are often unable to penetrate or remove algal cell walls owing to their unique physical and chemical properties. Therefore, we developed a simple transformation method for Chlamydomonas reinhardtii using commercially available enzymes. Out of 7 enzymes screened for cell wall disruption, a commercial form of subtilisin (Alcalase) was the most effective at a low concentration (0.3 Anson units/mL). The efficiency was comparable to that of gamete lytic enzyme, a protease commonly used for the genetic transformation of C. reinhardtii. The transformation efficiency of our noninvasive method was similar to that of previous methods using autolysin as a cell wall-degrading enzyme in conjunction with glass bead transformation. Subtilisin showed approximately 35% sequence identity with sporangin, a hatching enzyme of C. reinhardtii, and shared conserved active domains, which may explain the effective cell wall degradation. Our trans-formation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.

Induction of Recombinant Lectin Expression by an Artificially Constructed Tandem Repeat Structure: A Case Study Using Bryopsis plumosa Mannose-Binding Lectin.

Lectin is an important protein in medical and pharmacological applications. Impurities in lectin derived from natural sources and the generation of inactive proteins by recombinant technology are major obstacles for the use of lectins. Expressing recombinant lectin with a tandem repeat structure can potentially overcome these problems, but few studies have systematically examined this possibility. This was investigated in the present study using three distinct forms of recombinant mannose-binding lectin from Bryopsis plumosa (BPL2)-i.e., the monomer (rD1BPL2), as well as the dimer (rD2BPL2), and tetramer (rD4BPL2) arranged as tandem repeats. The concentration of the inducer molecule isopropyl ß-D-1-thiogalactopyranoside and the induction time had no effect on the efficiency of the expression of each construct. Of the tested constructs, only rD4BPL2 showed hemagglutination activity towards horse erythrocytes; the activity of towards the former was 64 times higher than that of native BPL2. Recombinant and native BPL2 showed differences in carbohydrate specificity; the activity of rD4BPL2 was inhibited by the glycoprotein fetuin, whereas that of native BPL2 was also inhibited by d-mannose. Our results indicate that expression as tandem repeat sequences can increase the efficiency of lectin production on a large scale using a bacterial expression system.

Functional Expression and Characterization of the Recombinant N-Acetyl-Glucosamine/N-Acetyl-Galactosamine-Specific Marine Algal Lectin BPL3.

Lectins, characterized by their carbohydrate-binding ability, have extensive practical applications. However, their industrial use is limited due to impurity. Thus, quality-controlled production of recombinant lectin is necessary. In this study, the algal lectin BPL3 (Bryopsis plumosa lectin 3) was successfully produced using a bacterial expression system, BL21(DE3), with an artificial repeated structure (dimeric construct). Recombinant dimeric BPL3 (rD2BPL3) was confirmed by LC-MS/MS spectrometry. Expression efficiency was greater for the construct with the repeat structure (rD2BPL3) than the monomeric form (rD1BPL3). Optimal conditions for expression were 1 mM IPTG at 20 °C. Recombinant lectin was purified under denaturing conditions and refolded by the flash dilution method. Recombinant BPL3 was solubilized in 1× PBS containing 2 M urea. rD2BPL3 showed strong hemagglutination activity using human erythrocyte. rD2BPL3 had a similar sugar specificity to that of the native protein, i.e., to N-acetyl-glucosamine (GlcNAc) and N-acetyl-galactosamine (GalNAc). Glycan array results showed that recombinant BPL3 and native BPL3 exhibited different binding properties. Both showed weak binding activity to α-Man-Sp. Native BPL3 showed strong binding specificity to the alpha conformation of amino sugars, and rD2BPL3 had binding activity to the beta conformation. The process developed in this study was suitable for the quality-controlled large-scale production of recombinant lectins.

Complete mitochondrial genome of Cheilio inermis (Labriformes, Labridae).

In the present report, we describe the first sequencing and assembly of the complete mitochondrial genome of Cheilio inermis. The mitochondrial genome of C. inermis, with 16,494 bp in length, has the typical vertebrate mitochondrial gene arrangement. It contains 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region. All the tRNA genes typically formed a cloverleaf secondary structure. Phylogenetic analysis using mitochondrial genomes of 11 species showed that C. inermis formed monophyletic group with other Labridae species.

Pitch-tunable pillar arrays for high-throughput culture and immunohistological analysis of tumor spheroids.

Tumor spheroids are multicellular, three-dimensional (3D) cell culture models closely mimicking the microenvironments of human tumors in vivo, thereby providing enhanced predictability, clinical relevancy of drug efficacy and the mechanism of action. Conventional confocal microscopic imaging remains inappropriate for immunohistological analysis due to current technical limits in immunostaining using antibodies and imaging cells grown in 3D multicellular contexts. Preparation of microsections of these spheroids represents a best alternative, yet their sub-millimeter size and fragility make it less practical for high-throughput screening. To address these problems, we developed a pitch-tunable 5 × 5 mini-pillar array chip for culturing and sectioning tumor spheroids in a high throughput manner. Tumor spheroids were 3D cultured in an alginate matrix using a twenty-five mini-pillar array which aligns to a 96-well. At least a few tens of spheroids per pillar were cultured and as many as 25 different treatment conditions per chip were evaluated, which indicated the high throughput manner of the 5 × 5 pillar array chip. The twenty-five mini-pillars were then rearranged to a transferring pitch so that spheroid-containing gel caps from all pillars can be embedded into a specimen block. Tissue array sections were then prepared and stained for immunohistological examination. The utility of this pitch-tunable pillar array was demonstrated by evaluating drug distribution and expression levels of several proteins following drug treatment in 3D tumor spheroids. Overall, our mini-pillar array provides a novel platform that can be useful for culturing tumor spheroids as well as for immunohistological analysis in a multiplexed and high throughput manner.