A microfluidic co-culture model for investigating colonocytes-microbiota interactions in colorectal cancer.

Changes in the abundance of certain bacterial species within the colorectal microbiota correlate with colorectal cancer (CRC) development. While carcinogenic mechanisms of single pathogenic bacteria have been characterized in vitro, limited tools are available to investigate interactions between pathogenic bacteria and both commensal microbiota and colonocytes in a physiologically relevant tumor microenvironment. To address this, we developed a microfluidic device that can be used to co-culture colonocyte spheroids and colorectal microbiota. The device was used to explore the effect of Fusobacterium nucleatum, an opportunistic pathogen associated with colorectal cancer development in humans, on colonocyte gene expression and microbiota composition. F. nucleatum altered the transcription of genes involved in cytokine production, epithelial-to-mesenchymal transition, and proliferation in colonocytes in a contact-independent manner; however, most of these effects were significantly diminished by the presence of commensal microbiota. Interestingly, F. nucleatum significantly altered the abundance of multiple bacterial clades associated with mucosal immune responses and cancer development in the colon. Our results highlight the importance of evaluating the potential carcinogenic activity of pathogens in the context of a commensal microbiota, and the potential to discover novel inter-species microbial interactions in the CRC microenvironment.

Single-cell calcium monitoring of Caco-2 cell co-cultured with intestinal microbiome through carbon fiber based potentiometric microelectrode.

The Caco-2 cells were used as intestinal epithelial cell model to illustrate the hyperuricemia (HUA) mechanism under the co-culture of the imbalanced intestinal microbiome in this work. The uric acid (UA) concentration in the HUA process was monitored, and could be up to 425 µmol/L at 8 h co-cultured with the imbalanced intestinal microbiome. Single-cell potentiometry based on ion-selective microelectrode was used to study extracellular calcium change, which is hypothesized to play an important role in the UA excretion. The potential signal of the calcium in the extremely limited microenvironment around single Caco-2 cell was recorded through the single-cell analysis platform. The potential signal of sharp decrease and slow increase followed within a few seconds indicates the sudden uptake and gradually excretion process of calcium through the cell membrane. Moreover, the value of the potential decrease increases with the increase of the time co-cultured with the imbalanced intestinal microbiome ranging from 0 to 8 h. The Ca2+ concentration around the cell membrane could decrease from 1.3 mM to 0.4 mM according to the potential decrease of 27.0 mV at the co-culture time of 8 h. The apoptosis ratio of the Caco-2 cells also exhibits time dependent with the co-culture of the imbalanced intestinal microbiome, and was 39.1 ± 3.6 % at the co-culture time of 8 h, which is much higher than the Caco-2 cells without any treatment (3.9 ± 2.9 %). These results firstly provide the links between the UA excretion with the apoptosis of the intestinal epithelial cell under the interaction of the imbalanced intestinal microbiome. Moreover, the apoptosis could be triggered by the calcium signaling.

Establishment of a novel microfluidic co-culture system for simultaneous analysis of multiple indicators of gefitinib sensitivity in colorectal cancer cells.

The therapeutic effect of gefitinib on colorectal cancer (CRC) is unclear, but it has been reported that stromal cells in the tumor microenvironment may have an impact on drug sensitivity. Herein, we established a microfluidic co-culture system and explored the sensitivity of CRC cells co-cultured with cancer-associated fibroblasts (CAFs) to gefitinib. The system consisted of a multichannel chip and a Petri dish. The chambers in the chip and dish were designed to continuously supply nutrients for long-term cell survival and create chemokine gradients for driving cell invasion without any external equipment. Using this system, the proliferation and invasiveness of cells were simultaneously evaluated by quantifying the area of cells and the migration distance of cells. In addition, the system combined with live cell workstation could evaluate the dynamic drug response of co-cultured cells and track individual cell trajectories in real-time. When CRC cells were co-cultured with CAFs, CAFs promoted CRC cell proliferation and invasion and reduced the sensitivity of cells to gefitinib through the exosomes secreted by CAFs. Furthermore, the cells that migrated out of the chip were collected, and EMT-related markers were determined by immunofluorescent and western blot assays. The results demonstrated that CAFs affected the response of CRC cells to gefitinib by inducing EMT, providing new ideas for further research on the resistance mechanism of gefitinib. This suggests that targeting CAFs or exosomes might be a new approach to enhance CRC sensitivity to gefitinib, and our system could be a novel platform for investigating the crosstalk between tumor cells and CAFs and understanding multiple biological changes of the tumor cells in the tumor microenvironment.

Drug Evaluation Based on a Multi-Channel Cell Chip with a Horizontal Co-Culture.

We developed a multi-channel cell chip containing a three-dimensional (3D) scaffold for horizontal co-culture and drug toxicity screening in multi-organ culture (human glioblastoma, cervical cancer, normal liver cells, and normal lung cells). The polydimethylsiloxane (PDMS) multi-channel cell chip (PMCCC) was based on fused deposition modeling (FDM) technology. The architecture of the PMCCC was an open-type cell chip and did not require a pump or syringe. We investigated cell proliferation and cytotoxicity by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-dphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and analysis of oleanolic acid (OA)-treated multi-channel cell chips. The results of the MTT and LDH assays showed that OA treatment in the multi-channel cell chip of four cell lines enhanced chemoresistance of cells compared with that in the 2D culture. Furthermore, we demonstrated the feasibility of the application of our multi-channel cell chip in various analysis methods through Annexin V-fluorescein isothiocyanate/propidium iodide staining, which is not used for conventional cell chips. Taken together, the results demonstrated that the PMCCC may be used as a new 3D platform because it enables simultaneous drug screening in multiple cells by single point injection and allows analysis of various biological processes.

Dynamic flow priming programs allow tuning up the cell layers properties for engineered vascular graft.

Tissue engineered vascular grafts (TEVG) are potentially clear from ethical and epidemiological concerns sources for reconstructive surgery for small diameter blood vessels replacement. Here, we proposed a novel method to create three-layered TEVG on biocompatible glass fiber scaffolds starting from flat sheet state into tubular shape and to train the resulting tissue by our developed bioreactor system. Constructed tubular tissues were matured and trained under 3 types of individual flow programs, and their mechanical and biological properties were analyzed. Training in the bioreactor significantly increased the tissue burst pressure resistance (up to 18 kPa) comparing to untrained tissue. Fluorescent imaging and histological examination of trained vascular tissue revealed that each cell layer has its own individual response to training flow rates. Histological analysis suggested reverse relationship between tissue thickness and shear stress, and the thickness variation profiles were individual between all three types of cell layers. Concluding: a three-layered tissue structure similar to physiological can be assembled by seeding different cell types in succession; the following training of the formed tissue with increasing flow in a bioreactor is effective for promoting cell survival, improving pressure resistance, and cell layer formation of desired properties.

Adapted Protocol for Saccharibacteria Cocultivation: Two New Members Join the Club of Candidate Phyla Radiation.

The growing application of metagenomics to different ecological and microbiome niches in recent years has enhanced our knowledge of global microbial biodiversity. Among these abundant and widespread microbes, the candidate phyla radiation (CPR) group has been recognized as representing a large proportion of the microbial kingdom (>26%). CPR are characterized by their obligate symbiotic or exoparasitic activity with other microbial hosts, mainly bacteria. Currently, isolating CPR is still considered challenging for microbiologists. The idea of this study was to develop an adapted protocol for the coculture of CPR with a suitable bacterial host. Based on various sputum samples, we tried to enrich CPR (Saccharibacteria members) and to cocultivate them with pure hosts (Schaalia odontolytica). This protocol was monitored by TaqMan real-time quantitative PCR (qPCR) using a system specific for Saccharibacteria designed in this study, as well as by electron microscopy and sequencing. We succeeded in coculturing and sequencing the complete genomes of two new Saccharibacteria species, "Candidatus Minimicrobia naudis" and "Candidatus Minimicrobia vallesae." In addition, we noticed a decrease in the CT values of Saccharibacteria and a significant multiplication through their physical association with Schaalia odontolytica strains in the enriched medium that we developed. This work may help bridge gaps in the genomic database by providing new CPR members, and in the future, their currently unknown characteristics may be revealed. IMPORTANCE In this study, the first TaqMan real-time quantitative PCR (qPCR) system, targeting Saccharibacteria phylum, has been developed. This technique can specifically quantify Saccharibacteria members in any sample of interest in order to investigate their prevalence. In addition, another easy, specific, and sensitive protocol has been developed to maintain the viability of Saccharibacteria cells in an enriched medium with their bacterial host. The use of this protocol facilitates subsequent studies of the phenotypic characteristics of CPR and their physical interactions with bacterial species, as well as the sequencing of new genomes to improve the current database.

Novel N-cadherin antagonist causes glioblastoma cell death in a 3D bioprinted co-culture model.

Glioblastoma multiforme (GBM) is a deadly type of brain cancer. There is a need to identify novel therapies for GBM as current treatments only marginally increase survival. Modelling the complexity of cancerous tissues using 3D bioprinted constructs serves as a novel approach for preclinical testing of anticancer drugs. A novel small molecule antagonist of the cell adhesion molecule, N-cadherin (NCAD), (S)-1-(3,4-Dichlorophenoxy)-3-(4-((S)-2-hydroxy-3-(4-methoxyphenoxy)propylamino)piperidin-1-yl)propan-2-ol has shown promise as an anticancer agent. This study investigated the influence of this antagonist on GBM cells bioprinted with astrocytes into 3D constructs. The NCAD antagonist prevented spheroid formation and induced cell death in the 3D model. This is the first demonstration that an NCAD antagonist can cause GBM cell death.

Co-Culturing of Endothelial and Cancer Cells in a Nanofibrous Scaffold-Based Two-Layer System.

Angiogenesis is critical for local tumor growth. This study aimed to develop a three-dimensional two-layer co-culture system to investigate effects of cancer cells on the growth of endothelial cells (ECs). Poly(ε-caprolactone) (PCL) nanofibrous membranes were generated via electrospinning of PCL in chloroform (C-PCL-M) and chloroform and dimethylformamide (C/DMF-PCL-M). We assembled a two-layer co-culture system using C-PCL-M and C/DMF-PCL-M for EC growth in the upper layer with co-cultured cancer cells in the lower layer. In the absence of vascular endothelial growth factor (VEGF), growth of bEND.3 ECs decreased on C/DMF-PCL-M but not on C-PCL-M with time. Growth of bEND.3 cells on C/DMF-PCL-M was enhanced through co-culturing of CT26 cancer cells and enhanced growth of bEND.3 cells was abrogated with anti-VEGF antibodies and sorafenib. However, EA.hy926 ECs displayed steady growth and proliferation on C/DMF-PCL-M, and their growth was not further increased through co-culturing of cancer cells. Moreover, chemical hypoxia in CT26 cancer cells upon treatment with CoCl2 enhanced the growth of co-cultured bEND.3 cells in the two-layer system. Thus, EC growth on the nanofibrous scaffold is dependent on the types of ECs and composition of nanofibers and this co-culture system can be used to analyze EC growth induced by cancer cells.

Application of co-culture technology of epithelial type cells and mesenchymal type cells using nanopatterned structures.

Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments.

Patient-derived small intestinal myofibroblasts direct perfused, physiologically responsive capillary development in a microfluidic Gut-on-a-Chip Model.

The development and physiologic role of small intestine (SI) vasculature is poorly studied. This is partly due to a lack of targetable, organ-specific markers for in vivo studies of two critical tissue components: endothelium and stroma. This challenge is exacerbated by limitations of traditional cell culture techniques, which fail to recapitulate mechanobiologic stimuli known to affect vessel development. Here, we construct and characterize a 3D in vitro microfluidic model that supports the growth of patient-derived intestinal subepithelial myofibroblasts (ISEMFs) and endothelial cells (ECs) into perfused capillary networks. We report how ISEMF and EC-derived vasculature responds to physiologic parameters such as oxygen tension, cell density, growth factors, and pharmacotherapy with an antineoplastic agent (Erlotinib). Finally, we demonstrate effects of ISEMF and EC co-culture on patient-derived human intestinal epithelial cells (HIECs), and incorporate perfused vasculature into a gut-on-a-chip (GOC) model that includes HIECs. Overall, we demonstrate that ISEMFs possess angiogenic properties as evidenced by their ability to reliably, reproducibly, and quantifiably facilitate development of perfused vasculature in a microfluidic system. We furthermore demonstrate the feasibility of including perfused vasculature, including ISEMFs, as critical components of a novel, patient-derived, GOC system with translational relevance as a platform for precision and personalized medicine research.

Human Tumor-Lymphatic Microfluidic Model Reveals Differential Conditioning of Lymphatic Vessels by Breast Cancer Cells.

Breast tumor progression is a complex process involving intricate crosstalk between the primary tumor and its microenvironment. In the context of breast tumor-lymphatic interactions, it is unclear how breast cancer cells alter the gene expression of lymphatic endothelial cells and how these transcriptional changes potentiate lymphatic dysfunction. Thus, there is a need for in vitro lymphatic vessel models to study these interactions. In this work, a tumor-lymphatic microfluidic model is developed to study the differential conditioning of lymphatic vessels by estrogen receptor-positive (i.e., MCF7) and triple-negative (i.e., MDA-MB-231) breast cancer cells. The model consists of a lymphatic endothelial vessel cultured adjacently to either MCF7 or MDA-MB-231 cells. Quantitative transcriptional analysis reveals expression changes in genes related to vessel growth, permeability, metabolism, hypoxia, and apoptosis in lymphatic endothelial cells cocultured with breast cancer cells. Interestingly, these changes are different in the MCF7-lymphatic cocultures as compared to the 231-lymphatic cocultures. Importantly, these changes in gene expression correlate to functional responses, such as endothelial barrier dysfunction. These results collectively demonstrate the utility of this model for studying breast tumor-lymphatic crosstalk for multiple breast cancer subtypes.

A cost-effective three-dimensional culture platform functionally mimics the adipose tissue microenvironment surrounding the kidney.

There is an increasing interest in studying the crosstalk between tumor-associated adipose tissue and tumor progression. In proximity to the primary site of kidney tumors, perinephric adipose tissue has direct contact with cancer cells when kidney cancer becomes invasive. To mimic the perinephric adipose tissue microenvironment, we applied the liquid overlay-based technique, which cost-effectively generated functional adipocyte spheroids using mesenchymal stem cells isolated from human perinephric adipose tissue. Thereafter, we co-cultured adipocyte spheroids with unpolarized macrophages and discovered an M2 phenotype skew in macrophages. Moreover, we discovered that, in the presence of adipocyte spheroids, M2 macrophages exhibited stronger invasive capacity than M1 macrophages. We further showed that the perinephric adipose tissue sampled from metastatic kidney cancer exhibited high expression of M2 macrophages. In conclusion, the liquid overlay-based technique can generate a novel three-dimensional platform enabling investigation of the interactions of adipocytes and other types of cells in a tumor microenvironment.

A pump-free tricellular blood-brain barrier on-a-chip model to understand barrier property and evaluate drug response.

Disruption of the blood-brain barrier (BBB) leads to various neurovascular diseases. Development of therapeutics required to cross the BBB is difficult due to a lack of relevant in vitro models. We have developed a three-dimensional (3D) microfluidic BBB chip (BBBC) to study cell interactions in the brain microvasculature and to test drug candidates of neurovascular diseases. We isolated primary brain microvascular endothelial cells (ECs), pericytes, and astrocytes from neonatal rats and cocultured them in the BBBC. To mimic the 3D in vivo BBB structure, we used type I collagen hydrogel to pattern the microchannel via viscous finger patterning technique to create a matrix. ECs, astrocytes, and pericytes were cocultured in the collagen matrix. The fluid flow in the BBBC was controlled by a pump-free strategy utilizing gravity as driving force and resistance in a paper-based flow resistor. The primary cells cultured in the BBBC expressed high levels of junction proteins and formed a tight endothelial barrier layer. Addition of tumor necrosis factor alpha to recapitulate neuroinflammatory conditions compromised the BBB functionality. To mitigate the neuroinflammatory stimulus, we treated the BBB model with the glucocorticoid drug dexamethasone, and observed protection of the BBB. This BBBC represents a new simple, cost-effective, and scalable in vitro platform for validating therapeutic drugs targeting neuroinflammatory conditions.

Organization of liver organoids using Raschig ring-like micro-scaffolds and triple co-culture: Toward modular assembly-based scalable liver tissue engineering.

In order to address the remaining issues of fragile structure and insufficient mass transfer faced in modular assembly-based liver tissue engineering, a Raschig ring-like hollowed micro-scaffold was proposed and fabricated using poly-ε-caprolactone with 60% porosity and 11.4 mm2 effective surface area for cell immobilization. The method of cell inoculation, the types of cells for co-culture and the scalability of the proposed hollowed micro-scaffold in perfusion were all investigated to obtain an optimized organoid made of tissue modules. Extracellular matrix was found necessary to establish a hierarchical co-culture, and the triple co-culture of Human Hepatoma Hep G2 cells, liver sinusoid cell line TMNK-1 cells and fibroblasts (Swiss 3T3 cells) was recognized to be the most efficient to obtain higher cell attachment, proliferation and hepatic function. The equipped intersecting hollow channels provided in the micro-scaffold functioned as flow paths to promote mass transfer to the immobilized cells after the modules have been randomly packed into a bioreactor for perfusion culture, and resulted in enhanced albumin production and high cellular viability. Cell density comparable to those found in vivo were obtained in the perfused construct, which also maintained its rigid structure. Those results suggest that modular tissues made with hollowed micro-scaffold-based organoids hold great potential for scaling up tissue engineered constructs towards implantation.

Plug-and-Play In Vitro Metastasis System toward Recapitulating the Metastatic Cascade.

Microfluidic-based tumor models that mimic tumor culture environment have been developed to understand the cancer metastasis mechanism and discover effective antimetastatic drugs. These models successfully recapitulated key steps of metastatic cascades, yet still limited to few metastatic steps, operation difficulty, and small molecule absorption. In this study, we developed a metastasis system made of biocompatible and drug resistance plastics to recapitulate each metastasis stage in three-dimensional (3D) mono- and co-cultures formats, enabling the investigation of the metastatic responses of cancer cells (A549-GFP). The plug-and-play feature enhances the efficiency of the experimental setup and avoids initial culture failures. The results demonstrate that cancer cells tended to proliferate and migrate with circulating flow and intravasated across the porous membrane after a period of 3 d when they were treated with transforming growth factor-beta 1 (TGF-ß1) or co-cultured with human pulmonary microvascular endothelial cells (HPMECs). The cells were also observed to detach and migrate into the circulating flow after a period of 20 d, indicating that they transformed into circulating tumor cells for the next metastasis stage. We envision this metastasis system can provide novel insights that would aid in fully understanding the entire mechanism of tumor invasion.

Unravelling Receptor and RGD Motif Dependence of Retargeted Adenoviral Vectors using Advanced Tumor Model Systems.

Recent advances in engineering adenoviruses are paving the way for new therapeutic gene delivery approaches in cancer. However, there is limited knowledge regarding the impact of adenoviral retargeting on transduction efficiency in more complex tumor architectures, and the role of the RGD loop at the penton base in retargeting is unclear. To address this gap, we used tumor models of increasing complexity to study the role of the receptor and the RGD motif. Employing tumor-fibroblast co-culture models, we demonstrate the importance of the RGD motif for efficient transduction in 2D through the epithelial cell adhesion molecule (EpCAM), but not the epidermal growth factor receptor (EGFR). Via optical clearing of co-culture spheroids, we show that the RGD motif is required for transduction via both receptors in 3D tumor architectures. We subsequently employed a custom-designed microfluidic model containing collagen-embedded tumor spheroids, mimicking the interplay between interstitial flow, extracellular matrix and adenoviral transduction. Image analysis of on-chip cleared spheroids indicated the importance of the RGD motif for on-chip adenoviral transduction. Together, our results show the interrelationship between receptor characteristics, the RGD motif, the 3D tumor architecture and retargeted adenoviral transduction efficiency. The findings are important for the rational design of next-generation therapeutic adenoviruses.

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells' behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

In Vitro Conversion of Activated T Cells into Stem Cell Memory-Like T Cells.

Adoptive T cell therapy is an attractive strategy in tumor immunotherapy. The transfer of in vitro expanded tumor-associated antigen (TAA)-specific T cells from patients may effectively destroy the original tumor cells. One of the limitations is a rapid acquisition of tolerant (anergy, deletion, dysfunctional, and/or exhausted) phenotypes. We and others found that stem cell memory T (TSCM) cells are strongly resistant to tolerance, showing strong expansion and persistence in vivo and providing long-lasting antitumor effects. We previously established that phenotypically TSCM cells (iTSCM) can be induced using a simple coculture of activated T cells with OP9 stroma cells expressing a Notch ligand. Here, we describe a defined protocol for generating human iTSCM cells, including reagents, culture setting, and procedure.

In Vitro Differentiation of T Cells: From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

In this chapter, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from non-T cells, followed by the differentiation of the T-cell lineage. Derivation of T cells from PSCs involves three steps: induction of PSCs to hematopoietic progenitor cells (HPCs), differentiation of HPCs into progenitor T cells, and maturation of progenitor T cells into mature T cells (CD8 single-positive (SP) or CD4 SP).

Redifferentiation of Adaptive Naïve-Like CTL from T-Cell-Derived iPSC.

In this chapter, we describe redifferentiation procedures from iPSCs to CD8αß+ cytotoxic T cells in 10 T1/2 and OP9/DL1 feeder condition. iPSC used here is derived from T-cell clone (T-iPSC), which has lost naïve phenotype and acquired exhaustion/senescence phenotype during cloning process (Note 1). On the other hand, redifferentiated T cells (T-iPSC-Ts) reacquire naïve phenotype (CD45RA+CD45RO-CCR7+CD62L+), which are reportedly critical for in vivo persistence of infused T cells and greatly affect therapeutic efficacy of adoptive immunotherapy. Indeed, T-iPSC-Ts exhibit much superior proliferative capacity while retaining equivalent effector function compared to parental T-cell clones. Here, we demonstrate the methodology to produce naïve-like T-iPSC-Ts, which could be potent cell source for adoptive immunotherapy.