Co-culture Platform Research Articles

A microfluidic hanging drop-based spheroid co-culture platform for probing tumor angiogenesis.

Co-culturing of embryoid bodies (EBs) and tumor spheroids (TSs) allows mimicking tumor angiogenesis in vitro. Here, we report a microfluidic hanging drop-based spheroid co-culture device (μ-CCD) that permits the generation and co-culturing of EBs and TSs using a simple manual operation procedure and setup. In brief, uniform-sized EBs and TSs can be generated on the device in eight pairs of hanging droplets from adjacent microfluidic channels, followed by the confrontation of EB and TS pairs by merging the droplet pairs to culture the EB-TS spheroids to investigate tumor-induced angiogenic sprouting. The physical parameters of the device were optimized to maintain the long-term stability of hanging droplets for up to ten days. The mouse embryonic stem cell line ES-D3 and breast cancer cell lines MDA-MB-231 and MCF-7 were used to generate EBs, invasive TSs, and non-invasive TSs respectively. Confocal imaging results showed that the vessel percentage area and total vessel length which are linked to tumor angiogenesis increased after 6 days of co-culturing. An anti-angiogenesis drug testing on the co-cultured EB-TS spheroids was also demonstrated in the device. The μ-CCD provides a simple yet high-efficiency method to generate and co-culture cell spheroids and may also be useful for other applications involving spheroid co-culturing.

Characterization of Patterned Microbial Growth Dynamics in Aqueous Two-Phase Polymer Scaffolds.

Microbial growth confinement using liquid scaffolds based on an aqueous two-phase system (ATPS) is a promising technique to overcome the challenges in microbial-mammalian co-culture in vitro. To better understand the potential use of the ATPS in studying these complex interactions, the goal of this research was to characterize the effects of bacteria loading and biofilm maturation on the stability of a polyethylene glycol (PEG) and dextran (DEX) ATPS. Two ATPS formulations, consisting of 5% PEG/5% DEX and 10% PEG/10% DEX (w/v), were prepared. To test the containment limits of each ATPS formulation, Escherichia coli MG1655 overnight cultures were resuspended in DEX at optical densities (ODs) of 1, 0.3, 0.1, 0.03, and 0.01. Established E. coli colonies initially seeded at lower densities were contained within the DEX phase to a greater extent than E. coli colonies initially seeded at higher densities. Furthermore, the 10% PEG/10% DEX formulation demonstrated longer containment time of E. coli compared to the 5% PEG/5% DEX formulation. E. coli growth dynamics within the ATPS were found to be affected by the initial bacterial density, where colonies of lower initial seeding densities demonstrate more dynamic growth trends compared to colonies of higher initial seeding densities. However, the addition of DEX to the existing ATPS during the growth phase of the bacterial colony does not appear to disrupt the growth inertia of E. coli. We also observed that microbial growth can disrupt ATPS stability below the physical carrying capacity of the DEX droplets. In both E. coli and Streptococcus mutans UA159 colonies, the ATPS interfacial tensions are reduced, as suggested by the loss of fluorescein isothiocyanate (FITC)-DEX confinement and contact angel measurements, while the microbial colony remained well defined. In general, we observed that the stability of the ATPS microbial colony is proportional to polymer concentrations and inversely proportional to seeding density and culture time. These parameters can be combined as part of a toolset to control microbial growth in a heterotypic co-culture platform and should be considered in future work involving mammalian-microbial cell interactions.

Control of osteoblast arrangement by osteocyte mechanoresponse through prostaglandin E2 signaling under oscillatory fluid flow stimuli

Anisotropic collagen/apatite microstructure is a prominent determinant of bone tissue functionalization; in particular, bone matrix modulates its anisotropic microstructure depending on the surrounding mechanical condition. Although mechanotransduction in bones is governed by osteocyte function, the precise mechanisms linking mechanical stimuli and anisotropic formation of collagen/apatite microstructure are poorly understood. Here we developed a novel anisotropic mechano-coculture system which enables the understanding of the biological mechanisms regulating the oriented bone matrix formation, which is constructed by aligned osteoblasts. The developed model provides bone-mimetic coculture platform that enables simultaneous control of mechanical condition and osteoblast-osteocyte communication with an anisotropic culture scaffold. The engineered coculture device helps in understanding the relationship between osteocyte mechanoresponses and osteoblast arrangement, which is a significant contributor to anisotropic organization of bone tissue. Our study showed that osteocyte responses to oscillatory flow stimuli regulated osteoblast arrangement through soluble molecular interactions. Importantly, we found that prostaglandin E2 is a novel determinant for oriented collagen/apatite organization of bone matrix, through controlling osteoblast arrangement.

Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production

BackgroundPlants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years, and the production of valuable plant compounds using microorganisms such as Escherichia coli or yeast cells has been realized. However, the development of complicated pathways in a single cell remains challenging. Additionally, microbial cells may experience toxicity from the bioactive compounds produced or negative feedback effects exerted on their biosynthetic enzymes. Thus, co-culture systems, such as those of E. coli–E. coli and E. coli-Saccharomyces cerevisiae, have been developed, and increased production of certain compounds has been achieved. Recently, a co-culture system of Pichia pastoris (Komagataella phaffii) has gained considerable attention due to its potential utility in increased production of valuable compounds. However, its co-culture with other organisms such as E. coli, which produce important intermediates at high concentrations, has not been reported.ResultsHere, we present a novel co-culture platform for E. coli and P. pastoris. Upstream E. coli cells produced reticuline from a simple carbon source, and the downstream P. pastoris cells produced stylopine from reticuline. We investigated the effect of four media commonly used for growth and production of P. pastoris, and found that buffered methanol-complex medium (BMMY) was suitable for P. pastoris cells. Reticuline-producing E. coli cells also showed better growth and reticuline production in BMMY medium than that in LB medium. De novo production of the final product, stylopine from a simple carbon source, glycerol, was successful upon co-culture of both strains in BMMY medium. Further analysis of the initial inoculation ratio showed that a higher ratio of E. coli cells compared to P. pastoris cells led to higher production of stylopine.ConclusionsThis is the first report of co-culture system established with engineered E. coli and P. pastoris for the de novo production of valuable compounds. The co-culture system established herein would be useful for increased production of heterologous biosynthesis of complex specialized plant metabolites.

Optimization and Validation of a Novel Three-Dimensional Co-Culture System in Decellularized Human Liver Scaffold for the Study of Liver Fibrosis and Cancer.

Simple SummaryThis study aims to overcome the current methodological limitations in discovering new therapeutic targets. Therefore, we optimized and validated a co-culture system using decellularized human liver three-dimensional (3D) scaffolds obtained from healthy and cirrhotic human livers for anti-fibrotic and anti-cancer dual drug screening. Both platforms mimic the naturally healthy and physio-pathological microenvironment and are able to recapitulate the key cellular and molecular events leading to liver fibrogenesis and cancer. This study demonstrates the differences between single versus co-cultures and the usage of human-derived liver 3D ECM scaffolds from healthy and cirrhotic livers. As lead compounds, we used Sorafenib and Regorafenib, first- and second-line drugs, and identified two different drug-induced mechanisms depending on the 3D ECM microenvironment. The 3D ECM scaffolds may represent innovative platforms for disease modeling, biomarker discovery, and drug testing in fibrosis and primary cancer.The introduction of new preclinical models for in vitro drug discovery and testing based on 3D tissue-specific extracellular matrix (ECM) is very much awaited. This study was aimed at developing and validating a co-culture model using decellularized human liver 3D ECM scaffolds as a platform for anti-fibrotic and anti-cancer drug testing. Decellularized 3D scaffolds obtained from healthy and cirrhotic human livers were bioengineered with LX2 and HEPG2 as single and co-cultures for up to 13 days and validated as a new drug-testing platform. Pro-fibrogenic markers and cancer phenotypic gene/protein expression and secretion were differently affected when single and co-cultures were exposed to TGF-β1 with specific ECM-dependent effects. The anti-fibrotic efficacy of Sorafenib significantly reduced TGF-β1-induced pro-fibrogenic effects, which coincided with a downregulation of STAT3 phosphorylation. The anti-cancer efficacy of Regorafenib was significantly reduced in 3D bioengineered cells when compared to 2D cultures and dose-dependently associated with cell apoptosis by cleaved PARP-1 activation and P-STAT3 inhibition. Regorafenib reversed TGF-β1-induced P-STAT3 and SHP-1 through induction of epithelial mesenchymal marker E-cadherin and downregulation of vimentin protein expression in both co-cultures engrafting healthy and cirrhotic 3D scaffolds. In their complex, the results of the study suggest that this newly proposed 3D co-culture platform is able to reproduce the natural physio-pathological microenvironment and could be employed for anti-fibrotic and anti-HCC drug screening.

A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging.

The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.

Abstract 2777: Modelling T Cell-Vasculature co-culture in a high-throughput microfluidic platform

Abstract Recent clinical success of immune checkpoint inhibitors and chimeric antigen receptor T cells has highly increased the attention for the field of immunotherapy. However, identifying responders to these therapies is challenging underscoring the necessity for translational models that increase understanding of tumor-immune responses. In the present study, a co-culture system containing immune cells and vasculature was established. Both are essential components of the tumor microenvironment and very often lacking in in vitro tumor models, highlighting the added value of our co-culture platform. We focused on optimizing endothelial and CD8+ T cell co-cultures and subsequently assessing T cell migration from the endothelial tubes via endothelial sprouts towards various chemoattractants. In order to generate stratified 3D co-cultures, the Mimetas OrganoPlate Graft containing 64 microfluidic culture units was used. The microfluidic units in this platform are composed of two parallel microfluidics channels and a central chamber. The two parallel microfluidic channels were used for generating parallel endothelial tubules, whilst angiogenic factors (S1P, VEGF, bFGF and PMA) were added to the central chamber of the culture unit resulting in a generation of a gradient and sprouting of the endothelial tubes towards the central chamber. Generated sprouts were stable and perfusable. The central chamber is designed for culturing complex microtissues such as spheroids, organoids and explants. Angiogenic endothelial tubules formed vascular beds in presence of added factors within 3-5 days. Once vascular beds were formed, activated and fluorescently labeled CD8+ T cells were loaded in the endothelial tubules and followed in culture for 48 hours. CD8+ T cell migration was observed both via the sprouts as well as by crossing the endothelial barrier, and increased in presence of gradients of CCL2, CCXl12 and CCL9. Highest CD8+ T cell numbers were observed in presence of a gradient generated with a mix of these three chemokines. Therefore, we present a high throughput co-culture system containing angiogenic endothelial tubules and CD8+ T cells. These co-cultures are highly suitable for studying T cell migration, event which precedes the detection and recognition of antigens at the surface of antigen-presenting cells and for interactions with other cells involved in the immune response. In addition, these co-cultures serve as a platform for understanding the interplay between T cell migration and angiogenesis in the tumor microenvironment. Furthermore, we envision that this model will evolve into an immunocompetent patient-derived tumor model that can be used to study immune responses to tumors. Citation Format: Luuk de Haan, Sacha Spelier, Johnny Suijker, Erik Wallinga, Lenie van den Broek, Henriette Lanz, Jos Joore, Paul Vulto, Karla Queiroz. Modelling T Cell-Vasculature co-culture in a high-throughput microfluidic platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2777.

Abstract 2978: Unique 3D organoids autologous TIL coculture platform enables high throughput immune-oncology drug response studies

Abstract Background: Cancer progression and response to therapy are governed by complex cellular interactions in the tumor microenvironment (TME). A better understanding of TME and the immune microenvironment requires robust experimental systems modeling patient specific immune interactions. Conventional 2D cultures lack in recapitulating 3D morphological structures and complex immune biology of native human tumors. A vastly heterogenous TME impacts patient treatment response. There is a dearth of 2D or 3D in vitro models that mimic in vivo diversity of TME, including infiltrating immune populations. 3D organoid cultures typically contain neoplastic epithelium and fail to retain syngeneic tumor-infiltrating lymphocytes (TILs) interactions vital for exhibiting a clinically significant drug response. Here we describe patient-derived xenograft (PDX) derived 3D organoid (PDXO) cultures with autologous TILs to avoid alloreactivity and simulate tumor specific immune response. The described model allows the evaluation of TIL infiltration and high throughput screening of immune-modulatory therapies. Methods: Patient-derived xenografts from mice were enzymatically digested to establish PDXO. PDXO were characterized for expression of cancer specific cell-surface markers. TILs were expanded in culture from PBMCs from matching patients and characterized using flow cytometry. Organoids and TILs from the same patient were fluorescently labeled and cocultured for four days, and tumor infiltration and drug cytotoxicity with and without immune-therapeutics was evaluated. CellInsight CX7 high content imaging platform was used to track TILs, and cancer cells, and TIL infiltration was confirmed via immunofluorescence. Luminex technology was used to quantify changes in the cytokine profile upon drug treatment. Results: PDXO were established to mimic in vivo tumor biology. Tumor-specific TILs were expanded and characterized for expression of memory, inhibitory, proliferation, and regulatory T cell markers. TIL infiltration in organoids was observed from day one in culture and maintained over four days. Immunofluorescence for T cell marker CD3 (cluster of differentiation 3) within PDXO confirmed TIL infiltration. Infiltrating TILs and tumor cell killing evaluation using fluorescent dyes via high throughput imaging platform showed disease and patient-specific response. PDXO treatment with immune-modulatory therapeutics led to changes in the secreted levels of cytokines IL2 (interleukin 2), IFNγ (interferon gamma), and TNFα (tumor necrotic factor alpha). Conclusion: Our patient autologous TILs - PDXO culture platform can be used for high throughput drug screening to model immune-therapeutic response with the tumor specific immune microenvironment. Citation Format: Garima Kaushik, Padma Kamineny, Amy Wesa. Unique 3D organoids autologous TIL coculture platform enables high throughput immune-oncology drug response studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2978.

A high-throughput microfluidic bilayer co-culture platform to study endothelial-pericyte interactions

Microphysiological organ-on-chip models offer the potential to improve the prediction of drug safety and efficacy through recapitulation of human physiological responses. The importance of including multiple cell types within tissue models has been well documented. However, the study of cell interactions in vitro can be limited by complexity of the tissue model and throughput of current culture systems. Here, we describe the development of a co-culture microvascular model and relevant assays in a high-throughput thermoplastic organ-on-chip platform, PREDICT96. The system consists of 96 arrayed bilayer microfluidic devices containing retinal microvascular endothelial cells and pericytes cultured on opposing sides of a microporous membrane. Compatibility of the PREDICT96 platform with a variety of quantifiable and scalable assays, including macromolecular permeability, image-based screening, Luminex, and qPCR, is demonstrated. In addition, the bilayer design of the devices allows for channel- or cell type-specific readouts, such as cytokine profiles and gene expression. The microvascular model was responsive to perturbations including barrier disruption, inflammatory stimulation, and fluid shear stress, and our results corroborated the improved robustness of co-culture over endothelial mono-cultures. We anticipate the PREDICT96 platform and adapted assays will be suitable for other complex tissues, including applications to disease models and drug discovery.

Biofilm-Infected Human Clusteroid Three-Dimensional Coculture Platform to Replace Animal Models in Testing Antimicrobial Nanotechnologies.

Microbial biofilms are a major concern in wound care, implant devices, and organ infections. Biofilms allow higher tolerance to antimicrobial drugs, can impair wound healing, and potentially lead to sepsis. There has been a recent focus on developing novel nanocarrier-based delivery vehicles to enhance the biofilm penetration of traditional antibacterial drugs. However, a feasible in vitro human skin model to mimic the biofilm formation and its treatment for clearance have not yet been reported. This study describes the benefits of using an innovative bacterial biofilm-infected keratinocyte clusteroid model for the first time. It paves a new way for testing innovative nanomedicine delivery systems in a rapid and reproducible way on a realistic human cell-based platform, free of any animal testing. Herein, we have developed a novel composite 3D biofilm/human keratinocyte clusteroid coculture platform, which was used to measure biofilm clearance efficiency of nanoparticle (NP)-based therapeutics. We tested this model by treating the biofilm-infected 3D coculture layers by a ciprofloxacin-loaded Carbopol nanogel particles, surface-functionalized by the cationic protease Alcalase. We measured the antibacterial efficiency of the NP treatment on clearing Staphylococcus aureus and Pseudomonas aeruginosa biofilms on the 3D keratinocyte clusteroid/biofilm coculture model. Our experiments showed that these bacteria can infect the 3D layer of keratinocyte clusteroids and produce a stable biofilm. The biofilms were efficiently cleared by treatment with a formulation of 0.0032 wt % ciprofloxacin-loaded in 0.2 wt % Carbopol NPs surface-functionalized with 0.2 wt % Alcalase. Taken together, these promising results demonstrate that our coculture model can be exploited as a novel platform for testing the biofilm-eliminating efficiency of various NP formulations emulating skin and wound infections and could have wider applicability to replace animal models in similar experiments. This 3D cell culture-based platform could help in developing and testing of more effective antibacterial agents for clinical applications of antiplaque dental treatments, implants, infection control, and wound dressings.

An Immuno-Cardiac Model for Macrophage-Mediated Inflammation in COVID-19 Hearts.

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Review on the Vascularization of Organoids and Organoids-on-a-Chip.

The use of human cells for the construction of 3D organ models in vitro based on cell self-assembly and engineering design has recently increased in popularity in the field of biological science. Although the organoids are able to simulate the structures and functions of organs in vitro, the 3D models have difficulty in forming a complex vascular network that can recreate the interaction between tissue and vascular systems. Therefore, organoids are unable to survive, due to the lack of oxygen and nutrients, as well as the accumulation of metabolic waste. Organoids-on-a-chip provides a more controllable and favorable design platform for co-culture of different cells and tissue types in organoid systems, overcoming some of the limitations present in organoid culture. However, the majority of them has vascular networks that are not adequately elaborate to simulate signal communications between bionic microenvironment (e.g., fluid shear force) and multiple organs. Here, we will review the technological progress of the vascularization in organoids and organoids-on-a-chip and the development of intravital 3D and 4D bioprinting as a new way for vascularization, which can aid in further study on tissue or organ development, disease research and regenerative medicine.

A primary human hepatocyte/hepatic stellate cell co-culture system for improved in vitro HBV replication

Primary human hepatocytes (PHHs) are considered the gold standard for the in vitro study of HBV replication as they directly reflect the metabolism and functionality of the human liver. However, several limitations of this system include PHH donor-to-donor variability, limited life span and low permissiveness to HBV infection, which precludes long-term infection studies and viral passaging. Here, an easy-to-set-up co-culture platform that combines PHH with hepatic stellate cells (HSCs) was developed. This platform does not rely on chemical supplementation to sustain robust HBV replication and viral antigen secretion making it a more physiologically relevant system for in vitro HBV infection studies compared to the traditional short-lived PHH monocultures.

Micropatterned Coculture Platform for Screening Nerve-Related Anticancer Drugs.

Accumulating evidence suggests that the neural microenvironment plays a vital role in the development and metastasis of cancers. The development of drug candidates or drug combinations targeting the neural microenvironment is thus becoming increasingly urgent. However, the low content of conventional drug screening platforms is a bottleneck that limits the drug evaluation process. In this study, we present a micropatterned coculture-based high-content (μCHC) platform by integrating a micropatterned coculture chip with the high-content analysis (HCA) system, for studying the neuron-cancer cell interactions and drug screening (simultaneously detecting 96 kinds of post-drug-treated conditions). We investigate the contribution of neurons on the migration of cancer cells from different tissues and validate the capability of the μCHC system to study the interaction between neurons and cancer cells. Moreover, we test the effects of individual or combinatory agents targeting the neuron or cancer cell on the neuron-cancer cell interactions, which proposes an optimized therapy regime for targeting both nervous and cancerous factors. Our study suggests that the μCHC system is a facile platform for screening drug candidates or drug combinations for clinical cancer therapy with high efficiency and fidelity.

A Fiber Alginate Co-culture Platform for the Differentiation of mESC and Modeling of the Neural Tube.

Alginate hydrogels are a commonly used substrate for in vitro 3D cell culture. These naturally derived biomaterials are highly tunable, biocompatible, and can be designed to mimic the elastic modulus of the adult brain at 1% w/v solution. Recent studies show that the molecular weight of the alginate can affect cell viability and differentiation. The relationship between the molecular weight, viscosity and ratio of G:M monomers of alginate hydrogels is complex, and the balance between these factors must be carefully considered when deciding on a suitable alginate hydrogel for stem cell research. This study investigates the formation of embryoid bodies (EB) from mouse embryonic stem cells, using low molecular weight (LMW) and high molecular weight (HMW) alginates. The cells are differentiated using a retinoic acid-based protocol, and the resulting aggregates are sectioned and stained for the presence of stem cells and the three germ layers (endoderm, mesoderm, and ectoderm). The results highlight that aggregates within LMW and HMW alginate are true EBs, as demonstrated by positive staining for markers of the three germ layers. Using tubular alginate scaffolds, formed with an adapted gradient maker protocol, we also propose a novel 3D platform for the patterned differentiation of mESCs, based on gradients of retinoic acid produced in situ by lateral motor column (LMC) motor neurons. The end product of our platform will be of great interest as it can be further developed into a powerful model of neural tube development.

Biophysical model for high-throughput tumor and epithelial cell co-culture in complex biochemical microenvironments

The in vivo tumor microenvironment is a complex niche that includes heterogeneous physical structures, unique biochemical gradients and multiple cell interactions. Its high-fidelity in vitro reconstruction is of fundamental importance to improve current understandings of cell behavior, efficacy predictions and drug safety. In this study, we have developed a high-throughput biochip with hundreds of composite extracellular matrix (ECM) microchambers to co-culture invasive breast cancer cells (MDA-MB-231-RFP) and normal breast epithelial cells (MCF-10A-GFP). The composite ECM is composed of type I collagen and Matrigel which provides a heterogeneous microenvironment that is similar to that of in vivo cell growth. Additionally, the growth factors and drug gradients that involve human epidermal growth factor (EGF), discoidin domain receptor 1 (DDR1) inhibitor 7rh and matrix metalloproteinase inhibitor batimastat allow for the mimicking of the complex in vivo biochemical microenvironment to investigate their effect on the spatial-temporal dynamics of cell growth. Our results demonstrate that the MDA-MB-231-RFP cells and MCF-10A-GFP cells exhibit different spatial proliferation behaviors under the combination of growth factors and drugs. Basing on the experimental data, we have also developed a cellular automata (CA) model that incorporated drug diffusion to describe the experimental phenomenon, as well as employed Shannon entropy (SE) to explore the effect of the drug diffusion coefficient on the spatial-temporal dynamics of cell growth. The results indicate that the uniform cell growth is related to the drug diffusion coefficient, which reveals that the pore size of the ECM plays a key role in the formation of complex biochemical gradients. Therefore, our integrated, biomimetic and high-throughput co-culture platforms, as well as the computational model can be used as an effective tool for investigating cancer pathogenesis and drug development.

Liver spheroids on chips as emerging platforms for drug screening

Liver plays a critical role in drug metabolism and nowadays multi-cellular culture systems aiming at imitating liver-specific morphology and functionality are advancing robustly. Numerous immortalized or stem cell-induced hepatic cell lines have been investigated to enhance the hepatic phenotype in establishing co-culture systems. Owing to the robust progresses of microtechnology and bioengineering, two-dimensional (2D) co-cultures such as sandwich culture and micropatterned co-culture systems have emerged. Controllably arranging the hepatocytes and fibroblasts allows bio-mimic homotypic and heterotypic interactions, and the miniaturized co-culture platform realizes high-throughput and sensitive drug screening. Yet, to address the rapid dedifferentiation and the decreased maintenance of hepatic functions existing in 2D cellular systems, various three-dimensional (3D) and dynamic hepatocyte co-culturing formats have been established to obtain more physiologically relevant liver microsystems and prolonged hepatic functionalities, such as spheroid barcodes, liver organoids, bioengineered hepatic spheroids, and microfluidic perfused liver-on-a-chip. In this review, we first introduce the typical structural color spheroid barcodes which facilitate multiple screening and testing. Then we sketch various advances in liver-imitating cellular co-culture protocols through representative examples. Meanwhile, we summarize current difficulties and propose the prospects of bioengineered liver-on-barcode systems for reliable and high-throughput drug screening.

A novel human arterial wall-on-a-chip to study endothelial inflammation and vascular smooth muscle cell migration in early atherosclerosis.

Mechanistic understanding of atherosclerosis is largely hampered by the lack of a suitable in vitro human arterial model that recapitulates the arterial wall structure, and the interplay between different cell types and the surrounding extracellular matrix (ECM). This work introduces a novel microfluidic endothelial cell (EC)-smooth muscle cell (SMC) 3D co-culture platform that replicates the structural and biological aspects of the human arterial wall for modeling early atherosclerosis. Using a modified surface tension-based ECM patterning method, we established a well-defined intima-media-like structure, and identified an ECM composition (collagen I and Matrigel mixture) that retains the SMCs in a quiescent and aligned state, characteristic of a healthy artery. Endothelial stimulation with cytokines (IL-1β and TNFα) and oxidized low-density lipoprotein (oxLDL) was performed on-chip to study various early atherogenic events including endothelial inflammation (ICAM-1 expression), EC/SMC oxLDL uptake, SMC migration, and monocyte-EC adhesion. As a proof-of-concept for drug screening applications, we demonstrated the atheroprotective effects of vitamin D (1,25(OH)2D3) and metformin in mitigating cytokine-induced monocyte-EC adhesion and SMC migration. Overall, the developed arterial wall model facilitates quantitative and multi-factorial studies of EC and SMC phenotype in an atherogenic environment, and can be readily used as a platform technology to reconstitute multi-layered ECM tissue biointerfaces.

Novel in vitro microfluidic platform for osteocyte mechanotransduction studies.

Osteocytes are the major mechanosensing cells in bone remodeling. Current in vitro bone mechanotransduction research use macroscale devices such as flow chambers; however, in vitro microfluidic devices provide an optimal tool to better understand this biological process with its flexible design, physiologically relevant dimensions and high-throughput capabilities. This project aims to design and fabricate a multi-shear stress, co-culture platform to study the interaction between osteocytes and other bone cells under varying flow conditions. Standard microfluidic design utilizing changing geometric parameters is used to induce different flow rates that are directly proportional to the levels of shear stress, with devices fabricated from standard polydimethylsiloxane (PDMS)-based softlithography processes. Each osteocyte channel (OCY) is connected to an adjacent osteoclast channel (OC) by 20-μm perfusion channels for cellular signaling molecule transport. Significant differences in RANKL levels are observed between channels with different shear stress levels, and we observed that pre-osteoclast differentiation was directly affected by adjacent flow-stimulated osteocytes. Significant decrease in the number of differentiating osteoclasts is observed in the OC channel adjacent to the 2-Pa shear stress OCY channel, while differentiation adjacent to the 0.5-Pa shear stress OCY channel is unaffected compared with no-flow controls. Addition of zoledronic acid showed a significant decrease in osteoclast differentiation, compounding to effect instigated by increasing fluid shear stress. Using this platform, we are able to mimic the interaction between osteocytes and osteoclasts in vitro under physiologically relevant bone interstitial fluid flow shear stress. Our novel microfluidic co-culture platform provides an optimal tool for bone cell mechanistic studies and provides a platform for the discovery of potential drug targets for clinical treatments of bone-related diseases.

Demonstration of re-epithelialization in a bioprinted human skin equivalent wound model

Wound healing is life-dependent feature for animal survival. After a skin insult, keratinocytes, fibroblasts, and immune cells are recruited to restore the protective skin function. Immediately after injury, soluble fibrinogen is cleaved by thrombin and polymerize to insoluble fibrin clot. Immune cells infiltrate into the fibrin clot to activate the inflammatory response. Surrounding keratinocytes in contact with the fibrin clot migrate to the wounded area meanwhile distant keratinocyte proliferate. However, in vitro wound models have important limitations related to wound fibrinogen polymerization and immune cell recruitment. This study expands previous in vitro 3D skin equivalent co-culture platforms to include the installation of a physiologically relevant fibrin provisional matrix. MethodHere, we developed a fibrin provisional matrix installed into a wound facsimile of a bioprinted human skin equivalent (HSE). A mixture of plasma-derived fibrinogen-containing factor XIII, fibronectin, thrombin, and macrophages (an FPM “bioink”) was extruded into the wound site. The surrounding in vitro tissue culture became a source of keratinocytes to achieve wound closure by a re-epithelialization process signaled by the FPM. ResultsAn in vitro analog of wound closure and re-epithelialization by keratinocytes occurred over the FPM after a normal migration initiation at 3 days. ConclusionThis co-culture model was shown to temporally synchronize a re-epithelization process for initiation of keratinocyte migration from a surrounding tissue and the migration process over the top of an FPM. A future study of the analogous subepithelial healing pathway is envisioned using the same in vitro bioprinted tissue study platform for co-culture of keratinocytes, melanocytes, fibroblasts, endothelial cells, and macrophages using more specialized FPMs.