Microtissue Array Research Articles

Digital light processing-based bioprinting of microtissue hydrogel arrays using dextran-induced aqueous emulsion ink

As a microscale three-dimensional bionic model, a microtissue array is a promising method for disease modeling and drug screening. Digital light processing (DLP) 3D bio-printing technology with the capabilities of excellent printing speed and resolution shows great potential for fabricating microtissue arrays. However, the outcomes of the microtissue arrays using DLP are limited by the shortage of biomaterials. In this work, we prepare hydrogel microtissues by combining DLP printing technology with biphasic bio-ink consisting of gelatin methacrylate (GelMA) and dextran. GelMA/ dextran is mixed to form bio-inks with different volume ratios, which can achieve high-precision printing of various patterns. Due to the presence of dextran, the bio-inks form the internal porous structure of the hydrogel in the printed patterns. The pore size is related to the proportion of dextran in bio-inks, and the pore size of hydrogel becomes smaller with the increase of dextran ratio. In addition, the adipose-derived stem cells (ADSCs) cells encapsulated in the porous hydrogels can maintain a high survival rate of 98.01% after 3 days of culture, and the larger pore size in the hydrogels promotes cell migration. Combining the DLP printing system with the porous hydrogel allows multiple micro-size geometric pattern hydrogels to be printed simultaneously to form microtissue arrays. In this study, a new bio-ink suitable for microtissue arrays is proposed, and a simple, accurate, and high-throughput biological manufacturing method for microtissue arrays is developed, thus providing a valuable tool for drug screening and tissue engineering.

A Microwell Device for the Efficient Generation of Arrays of Microtissues and Humanized Bone Marrow Micro-Ossicles

(1) Background: There are no high-throughput microtissue platforms for generating bone marrow micro-ossicles. Herein, we describe a method for the assembly of arrays of microtissues from bone marrow stromal cells (BMSC) in vitro and their maturation into bone marrow micro-ossicles in vivo. (2) Methods: Discs with arrays of 50 microwells were used to assemble microtissues from 3 × 105 BMSCs each on a nylon mesh carrier. Microtissues were cultured in chondrogenic induction medium followed by hypertrophic medium in an attempt to drive endochondral ossification, and then they were implanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, where they were remodeled into bone marrow micro-ossicles. Mice were transplanted with 105 human umbilical cord blood CD34+ cells. (3) Results: Micro-ossicles contained more human CD45+ cells, but fewer human CD34+ progenitor cells than mouse marrow. Human hematopoietic progenitor cells cycle rapidly at non-physiological rates in mouse marrow, and reduced CD34+ cell content in micro-ossicles is consistent with the notion that the humanized niche better controls progenitor cell cycling. (4) Conclusions: Assembling microtissues in microwells, linked by a nylon membrane carrier, provides an elegant method to manufacture and handle arrays of microtissues with bone organ-like properties. More generally, this approach and platform could aid bridging the gap between in vitro microtissue manipulation and in vivo microtissue implantation.

Abstract 15136: Matrix Mechanics Regulate Engineered Myocardial Microtissue Organization and Contractility

Introduction: The mechanical function of the myocardium is dictated by cardiomyocytes (CMs) and the surrounding fibrous extracellular matrix (ECM). Due to limitations in existing approaches for engineering myocardial tissue, how CMs sense and respond to mechanical microenvironmental changes remains understudied. Here, we established a method for creating arrays of cardiac microtissues with highly tunable mechanical features by integrating synthetic, fibrous matrices and iPSC-CMs. Through real-time contractility readouts enabled, we explored how biomechanical cues influence engineered cardiac tissue assembly and function. Methods: Arrayed cardiac microtissues were generated by selectively photo-crosslinking electrospun dextran vinyl sulfone fiber matrices onto pairs of microfabricated PDMS cantilevers. Subsequently, iPSC-CMs were patterned onto matrices using a microfabricated seeding mask. Matrix and cantilever stiffnesses were tuned by adjusting photoinitiator concentrations and cantilever geometry, respectively. Results: In tissues contracting against soft cantilevers (0.41 N/m), tissue contractile stress decreased with increasing matrix stiffness (E = 0.68 to 17.3 kPa). We also observed an increase in contractile stress on soft and aligned matrices, compared to non-aligned matrices. However, tissues contracting against stiffer cantilevers (1.2 N/m) exerted similar contractile stresses independent of matrix alignment. This may be explained by the enhanced alignment of myofibrils in non-aligned matrices contracting against stiffer posts. The size of vinculin-rich cell-ECM adhesion decreased on stiff matrices. Vinculin also localized to z-discs forming organized costameres on soft matrices. Tissue response to mavacamten suggests that β-cardiac myosins are required for costamere formation and myofibril maturation. Conclusions: We developed a new microfabrication strategy to create tunable cardiac microtissues and examined how biomechanical cues affect tissue assembly and function. We found that matrix stiffness, matrix alignment, and tissue constraint distinctly affect iPSC-CM function and structure, potentially due to differential influences on costamere formation and myofibrillar assembly.

A Microphysiological Cell-Culturing System for Pharmacokinetic Drug Exposure and High-Resolution Imaging of Arrays of 3D Microtissues.

Understanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo–like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions.

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Using the Microwell-mesh to culture microtissues in vitro and as a carrier to implant microtissues in vivo into mice

Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of “pieces” of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ−/− (NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 μm achieving superior microtissue vascularisation relative to mesh with 36×36 μm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation.

Simple, Affordable, and Modular Patterning of Cells using DNA.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Mechanical stretch sustains myofibroblast phenotype and function in microtissues through latent TGF-β1 activation.

Developing methods to study tissue mechanics and myofibroblast activation may lead to new targets for therapeutic treatments that are urgently needed for fibrotic disease. Microtissue arrays are a promising approach to conduct relatively high-throughput research into fibrosis as they recapitulate key biomechanical aspects of the disease through a relevant 3D extracellular environment. In early work, our group developed a device called the MVAS-force to stretch microtissues while enabling simultaneous assessment of their dynamic mechanical behavior. Here, we investigated TGF-β1-induced fibroblast to myofibroblast differentiation in microtissue cultures using our MVAS-force device through assessing α-SMA expression, contractility and stiffness. In doing so, we linked cell-level phenotypic changes to functional changes that characterize the clinical manifestation of fibrotic disease. As expected, TGF-β1 treatment promoted a myofibroblastic phenotype and microtissues became stiffer and possessed increased contractility. These changes were partially reversible upon TGF-β1 withdrawal under a static condition, while, in contrast, long-term cyclic stretching maintained myofibroblast activation. This pro-fibrotic effect of mechanical stretching was absent when TGF-β1 receptors were inhibited. Furthermore, stretching promoted myofibroblast differentiation when microtissues were given latent TGF-β1. Altogether, these results suggest that external mechanical stretch may activate latent TGF-β1 and, accordingly, might be a powerful stimulus for continued myofibroblast activation to progress fibrosis. Further exploration of this pathway with our approach may yield new insights into myofibroblast activation and more effective therapeutic treatments for fibrosis.

Cyclic Stretching of Fibrotic Microtissue Array for Evaluation of Anti-Fibrosis Drugs.

Progression of pulmonary fibrosis, characterized by the deterioration of lung tissue's mechanical properties, is affected by respiratory motion-induced dynamic loading. Since the development of anti-fibrosis drugs faces major hurdles in animal tests and human clinical trials, preclinical models that can recapitulate fibrosis progression under physiologically-relevant cyclic loading hold great promise. However, the integration of these two functions has not been achieved in existing models. Recently we developed static human lung microtissue arrays that recapitulate the progressive changes in tissue mechanics during lung fibrogenesis. In the current study, we integrate the lung microtissue array with a membrane stretching system to enable dynamic loading to the microtissues. The effects of a pro-fibrotic agent and anti-fibrosis drugs were tested under cyclic stretching. Cyclic stretching that mimics respiratory motion was shown to affect the cytoskeletal organization and cellular orientation in the microtissue and cause the increase in microtissue contractility and stiffness. Fibrosis induction using TGF-β1 further promoted fibrosis-related mechanical activity of the lung microtissues. Using this system, we examined the therapeutic effects of two FDA approved anti-fibrotic drugs. Our results showed that Nintedanib was able to fully inhibit TGF-β1 induced force increase but only partially inhibited stretching induced force increase. In contrast, Pirfenidone was able to fully inhibit both TGF-β1 induced force increase and stretching-induced force increase. Together, these results highlight the pathophysiologically-relevant modeling capability of the current fibrotic microtissue system and demonstrated the potential of this system to be used for anti-fibrosis drug screening.

Surface and Bulk Stresses Drive Morphological Changes in Fibrous Microtissues

Engineered fibrous tissues consisting of cells encapsulated within collagen gels are widely used three-dimensional in vitro models of morphogenesis and wound healing. Although cell-mediated matrix remodeling that occurs within these scaffolds has been extensively studied, less is known about the mesoscale physical principles governing the dynamics of tissue shape. Here, we show both experimentally and by using computer simulations how surface contraction through the development of surface stresses (analogous to surface tension in fluids) coordinates with bulk contraction to drive shape evolution in constrained three-dimensional microtissues. We used microelectromechanical systems technology to generate arrays of fibrous microtissues and robot-assisted microsurgery to perform local incisions and implantation. We introduce a technique based on phototoxic activation of a small molecule to selectively kill cells in a spatially controlled manner. The model simulations, which reproduced the experimentally observed shape changes after surgical and photochemical operations, indicate that fitting of only bulk and surface contractile moduli is sufficient for the prediction of the equilibrium shape of the microtissues. The computational and experimental methods we have developed provide a general framework to study and predict the morphogenic states of contractile fibrous tissues under external loading at multiple length scales.

Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping

During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.

3158 Sunitinib-Induced Cardiotoxicity in an Engineered Cardiac Microtissue Model

OBJECTIVES/SPECIFIC AIMS: The aims of this study are threefold. Firstly, we are examining the effects of increased in vitro afterload (a proxy for hypertension) on human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) response to sunitinib in a durable and dynamic cardiac microtissue culture system. Secondly, we are exploring effects of repeat exposure and recovery of both sunitinib and afterload throughout the lifetime of the hiPSC-CM microtissue. Finally, we are assessing methods to prevent and treat sunitinib induced cardiotoxicity. Primary outcomes for this study are commonly utilized metrics of cardiotoxicity: degree of caspase activation, electrophysiology benchmarks for minimum voltage threshold and maximum capture rate, and microtissue force generation. METHODS/STUDY POPULATION: HiPSC-CMs are cultured and matured as 3D cardiac microtissues (CMTs) on a microtissue array. After maturation, cells are exposed to sunitinib doses of 0µM, 0.5µM, 1µM or 5µM for 12 hours. Concurrently with sunitinib dosing, increases in microtissue array stiffness are created with application of an external magnetic field. Afterload spring constants are fixed at pre-determined physiologic values ranging from 0.5µN/µm, to 5µN/µm. For Aim 1: Half of the CMTs are harvested at 8 hours after sunitinib dosing to conduct the caspase 3/7 assay, and the remainder are examined for 3 days following drug exposure to track temporal changes in electrophysiology and force generation. For Aim 2: After CMT maturation, 12-hour exposures to sunitinib are repeated three times at a fixed dose, with doses separated by one week. Concurrently with sunitinib dosing, increases or decreases in microtissue stiffness are created by changing the strength of an applied external magnetic field to create “ramp up” or “ramp down” stiffness conditions. Caspase assay and contractility metrics are measured at each timepoint. For Aim 3: Experimental conditions are conducted as described in Aim 1. Prior to the introduction of sunitinib, either carvedilol or an AMP-kinase activator is added to the CMT culture media at physiologic concentrations. Primary outcomes are examined as in Aim 1. RESULTS/ANTICIPATED RESULTS: Aim 1: We hypothesize that increases in microtissue afterload, synchronized with sunitinib exposure will augment sunitinib toxicity in cardiomyocytes resulting in elevations of caspase 3/7 activity and minimum voltage capture as well as decreases in maximum capture rate and maximum force generation. Aim 2: We hypothesize that repeat exposures to both sunitinib and to increases in afterload will augment sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Additionally, we hypothesize that decreases in afterload will decrease effective sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Aim 3: We hypothesize that exposure to an AMP-kinase activator but not carvedilol will decrease the effects of sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. DISCUSSION/SIGNIFICANCE OF IMPACT: The use of small molecule, targeted chemotherapeutic agents is increasingly common. Many of these agents cause cardiotoxic side effects, the mechanisms of which are incompletely understood. Our lab has developed a novel 3D tissue engineering platform capable of supporting durable in vitro cardiac microtissues that experience dynamic alterations in their biomechanical load. By using this platform to examine the cardiotoxic effects of sunitinib, insight into treatment and prevention of this common problem will be developed.

Fibrotic microtissue array to predict anti-fibrosis drug efficacy

Fibrosis is a severe health problem characterized by progressive stiffening of tissues which causes organ malfunction and failure. A major bottleneck in developing new anti-fibrosis therapies is the lack of in vitro models that recapitulate dynamic changes in tissue mechanics during fibrogenesis. Here we create membranous human lung microtissues to model key biomechanical events occurred during lung fibrogenesis including progressive stiffening and contraction of alveolar tissue, decline in alveolar tissue compliance and traction force-induced bronchial dilation. With these capabilities, we provide proof of principle for using this fibrotic tissue array for multi-parameter, phenotypic analysis of the therapeutic efficacy of two anti-fibrosis drugs recently approved by the FDA. Preventative treatments with Pirfenidone and Nintedanib reduce tissue contractility and prevent tissue stiffening and decline in tissue compliance. In a therapeutic treatment regimen, both drugs restore tissue compliance. These results highlight the pathophysiologically relevant modeling capability of our novel fibrotic microtissue system.

Fabrication of In Vitro Cancer Microtissue Array on Fibroblast-Layered Nanofibrous Membrane by Inkjet Printing.

In general, a drug candidate is evaluated using 2D-cultured cancer cells followed by an animal model. Despite successful preclinical testing, however, most drugs that enter human clinical trials fail. The high failure rates are mainly caused by incompatibility between the responses of the current models and humans. Here, we fabricated a cancer microtissue array in a multi-well format that exhibits heterogeneous and batch-to-batch structure by continuous deposition of collagen-suspended Hela cells on a fibroblast-layered nanofibrous membrane via inkjet printing. Expression of both Matrix Metalloproteinase 2 (MMP2) and Matrix Metalloproteinase 9 (MMP9) was higher in cancer microtissues than in fibroblast-free microtissues. The fabricated microtissues were treated with an anticancer drug, and high drug resistance to doxorubicin occurred in cancer microtissues but not in fibroblast-free microtissues. These results introduce an inkjet printing fabrication method for cancer microtissue arrays, which can be used for various applications such as early drug screening and gradual 3D cancer studies.

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Lung Microtissue Array to Screen the Fibrogenic Potential of Carbon Nanotubes.

Due to their excellent physical and chemical characteristics, multi-wall carbon nanotubes (MWCNT) have the potential to be used in structural composites, conductive materials, sensors, drug delivery and medical imaging. However, because of their small-size and light-weight, the applications of MWCNT also raise health concerns. In vivo animal studies have shown that MWCNT cause biomechanical and genetic alterations in the lung tissue which lead to lung fibrosis. To screen the fibrogenic risk factor of specific types of MWCNT, we developed a human lung microtissue array device that allows real-time and in-situ readout of the biomechanical properties of the engineered lung microtissue upon MWCNT insult. We showed that the higher the MWCNT concentration, the more severe cytotoxicity was observed. More importantly, short type MWCNT at low concentration of 50 ng/ml stimulated microtissue formation and contraction force generation, and caused substantial increase in the fibrogenic marker miR-21 expression, indicating the high fibrogenic potential of this specific carbon nanotube type and concentration. The presented microtissue array system provides a powerful tool for high-throughput examination of the therapeutic and toxicological effects of target compounds in realistic tissue environment.

Quick and easy microfabrication of T-shaped cantilevers to generate arrays of microtissues

Over the past decade, a major effort was made to miniaturize engineered tissues, as to further improve the throughput of such approach. Most existing methods for generating microtissues thus rely on T-shaped cantilevers made by soft lithography and based on the use of negative SU-8 photoresist. However, photopatterning T-shaped microstructures with these negative photoresists is fastidious and time-consuming. Here we introduce a novel method to quickly generate T-shaped cantilevers dedicated to generation of cellular microtissues, based on the use of positive photoresist. With only two layers of photoresist and one photomask, we were able to fabricate arrays of microwells in less than 3h, each containing two T-shaped cantilevers presenting either a rectangular or a circular geometry. As a proof of concept, these arrays were then replicated in poly(dimethylsiloxane) and microtissues composed of NIH 3T3 fibroblasts encapsulated in collagen I were generated, while the two cantilevers simultaneously constrain and report forces generated by the microtissues. Immunostainings showed longitudinally aligned and elongated fibroblasts over the whole microtissue after 8days of culture. The method described here opens the potential to quick prototyping platforms for high-throughput, low-volume screening applications.