Three-dimensional Microtissues Research Articles

Flushing out the scattering: fast on-chip microfluidic clearing of microtissues

By generating three-dimensional microtissues or organoids that better mimic the in vivo cellular functions than the two-dimensional cell cultures, the organ-on-a-chip technologies have been playing an increasingly important role in fundamental biology and drug discovery (1). The fast-growing on-chip microfluidic field is accompanied by the high demand for on-site imaging of the microtissues’ morphology, function, and molecular signatures. Clearly, to image the three-dimensional microtissues, the imaging technique should ideally provide volumetric information, together with microscopic resolutions for cellular or even subcellular analysis (2). Although many imaging modalities possess three-dimensional imaging capability, only optical microscopy, primarily the confocal microscopy and multi-photon microscopy, has the required spatial resolution and molecular/chemical sensitivity, and thus is widely used with on-chip microfluidic systems (3). However, the biological tissues are notoriously unfriendly to photons, with strong optical scattering resulted from the inhomogeneous distribution of subcellular structures, including cell membranes, nuclei, and mitochondria (4). High-resolution optical microscopy often suffers from the strong optical scattering of tissues, leading to a shallow imaging depth of a few hundred micrometers.

Three-dimensional microtissues as an in vitro model for personalized radiation therapy.

This paper describes the use of 3D microtissues as an intermediate model between the 2D cell culture and the animal model to assess radiation-induced cellular and DNA damage in the context of personalized radiation therapy. An agarose microwell array was used to generate 3D microtissues with controlled size and shape. The microtissues were exposed to X-ray radiation of various doses, and the radiation damage to cells was examined using a variety of techniques with different end points. Damage to cell membranes and reduction in metabolic activity were examined with the MTT assay and dye inclusion assay. DNA damage was tested with the micronucleus assay, γ-H2AX immunostaining, and HaloChip assay. 3D microtissues exposed to X-rays are smaller compared to unexposed ones in extended cultures, indicating that X-ray radiation can retard the growth of cells in 3D microtissues, where cells at the outer shells of microtissues can prevent further damage to those inside.

3D is not enough: Building up a cell instructive microenvironment for tumoral stroma microtissues.

This work concerns the engineering of tumor tissue in vitro. Tumor models serve as biological equivalent to study pathologic progression and to screen or validate the drugs efficacy. Tumor tissue is composed by malignant cells surviving in a microenvironment, or stroma. Stroma plays a pivotal role in cancer progression. Current in vitro models, i.e. spheroids, can't replicate the phenomena related to the tumor stroma remodeling. For this reason, to better replicate the tumor physiology in vitro that include functional and morphological changes, a novel 3D cancer model is proposed.

Multi-analyte biosensor interface for real-time monitoring of 3D microtissue spheroids in hanging-drop networks.

Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities. Here we present the integration of enzyme-based multi-analyte biosensors into a multi-tissue culture platform for ‘body-on-a-chip’ applications. The microfluidic platform is based on the technology of hanging-drop networks, which is designed for the formation, cultivation, and analysis of fluidically interconnected organotypic spherical three-dimensional (3D) microtissues of multiple cell types. The sensor modules were designed as small glass plug-ins featuring four platinum working electrodes, a platinum counter electrode, and an Ag/AgCl reference electrode. They were placed directly into the ceiling substrate from which the hanging drops that host the spheroid cultures are suspended. The electrodes were functionalized with oxidase enzymes to enable continuous monitoring of lactate and glucose through amperometry. The biosensors featured high sensitivities of 322±41 nA mM−1 mm−2 for glucose and 443±37 nA mM−1 mm−2 for lactate; the corresponding limits of detection were below 10 μM. The proposed technology enabled tissue-size-dependent, real-time detection of lactate secretion from single human colon cancer microtissues cultured in the hanging drops. Furthermore, glucose consumption and lactate secretion were monitored in parallel, and the impact of different culture conditions on the metabolism of cancer microtissues was recorded in real-time.

A review of the three-dimensional cell culture technique: Approaches, advantages and applications.

Cell culture is a core and basic technique in biotechnology and is widely applied in biology, medicine, drug research and development. Traditional two-dimensional cell culture methods have undergone great developments. However, with in-depth basic research, higher requirements are needed to better mimic the in vivo environment to accurately observe cell behavior and explore its mechanisms. To comply with this situation, the three-dimensional cell culture technique emerged and has made profound advances in sustaining inherent cell properties. Here, we briefly review the development of this technique, including the main approaches to form three-dimensional microtissues, and its application and potential for future clinical therapies.

Special Collection: Emerging Concepts in Three-Dimensional Microtissues.

Over the past decade, the concept of tissue engineering has been extended to include technologies that use multicellular aggregates, not only to repair or replace tissue but also as a stand-alone in vitro device (e.g., "organ-on-a-chip") with well-defined biological outputs. The advantage of such systems is that they allow for culture of one or more cell types in three dimensions, which may promote tissue function that is more mimetic of the in vivo state, while allowing high-throughput sample testing and a large degree of control of external culture factors that may lead to more reproducible results than that found in the more complex in vivo environment. While the means used to achieve these devices vary greatly, in this special collection, we focus our attention on formation and use of scaffold-free cellular aggregates (three-dimensional microtissues).

Mass Spectrometric Proteome Analysis of Small Three-Dimensional Microtissues Allows for the Quantitative Description of Toxic Effects of Drugs.

Mass Spectrometric Proteome Analysis of Small Three-Dimensional Microtissues Allows for the Quantitative Description of Toxic Effects of Drugs.

Abstract 1810: Three-dimensional microtissues as phenotypic screening model to identify radiation modifiers for breast cancer

Abstract Among breast cancer patients, radiation therapy resistance is a common problem in treatment. Overcoming the mechanisms responsible for this phenotype is an ongoing challenge. Options include repurposing established chemotherapeutics and the discovery of new radiosensitizing compounds. Three-dimensional (3D) microtissue models offer a physiological environment for efficient preclinical drug testing. We have adopted the 3D microtissue technology (Insphero, AG) to generate highly reproducible mammary cancer microtissues in a 96 well format. Two breast cancer cell lines (T47D and MDA-MB-361) were shown to be able to form 3D spheroids and grow in a reproducible manner over time. Validation of the phenotypic screen using breast cancer 3D microtissues was performed by HER2_shRNA knockdown, exposure to ionizing radiation and a panel of selected chemotherapeutic compounds. HER2_shRNA knockdown and irradiation decelerated growth, as proven by measuring the area of the cell spheroids after treatment. Of ten analyzed chemotherapeutics, vinblastine was the most effective compound, with docetaxel and doxorubicine being less effective in combination with radiation. To investigate the response in a model closer to the in vivo situation we investigated the response of heterotypic 3D microtissues containing both fibroblasts and breast cancer cells. Drug treatment of these heterotypic 3D cultures confirmed treatment with radiation plus vinblastine to be additive in causing breast cancer growth inhibition. Additionally a histological method to embed 3D microtissues in paraffin was established. The expression of the proliferation marker Ki67 and the apoptosis marker active Caspase 3 was evaluated in MDA-MB-361 cells after irradiation and concurrent vinblastine treatment. Such combined treatment caused prominent 3D microtissue damage - defined as extensive staining of active Caspase 3 and less Ki67. This suggests that combining vinblastine and radiation may be an effective radiation sensitizing strategy. Citation Format: Natasa Anastasov, Ines Höfig, Vanja Radulovic, Sabine Richter, Jan Lichtenberg, Jens M. Kelm, Christian Thirion, Michael J. Atkinson. Three-dimensional microtissues as phenotypic screening model to identify radiation modifiers for breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1810. doi:10.1158/1538-7445.AM2015-1810

Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis.

Integration of multiple three-dimensional microtissues into microfluidic networks enables new insights in how different organs or tissues of an organism interact. Here, we present a platform that extends the hanging-drop technology, used for multi-cellular spheroid formation, to multifunctional complex microfluidic networks. Engineered as completely open, 'hanging' microfluidic system at the bottom of a substrate, the platform features high flexibility in microtissue arrangements and interconnections, while fabrication is simple and operation robust. Multiple spheroids of different cell types are formed in parallel on the same platform; the different tissues are then connected in physiological order for multi-tissue experiments through reconfiguration of the fluidic network. Liquid flow is precisely controlled through the hanging drops, which enable nutrient supply, substance dosage and inter-organ metabolic communication. The possibility to perform parallelized microtissue formation on the same chip that is subsequently used for complex multi-tissue experiments renders the developed platform a promising technology for 'body-on-a-chip'-related research.

Micropatterned Cell–Cell Interactions Enable Functional Encapsulation of Primary Hepatocytes in Hydrogel Microtissues

Drug-induced liver injury is a major cause of drug development failures and postmarket withdrawals. In vitro models that incorporate primary hepatocytes have been shown to be more predictive than model systems which rely on liver microsomes or hepatocellular carcinoma cell lines. Methods to phenotypically stabilize primary hepatocytes ex vivo often rely on mimicry of hepatic microenvironmental cues such as cell-cell interactions and cell-matrix interactions. In this work, we sought to incorporate phenotypically stable hepatocytes into three-dimensional (3D) microtissues, which, in turn, could be deployed in drug-screening platforms such as multiwell plates and diverse organ-on-a-chip devices. We first utilize micropatterning on collagen I to specify cell-cell interactions in two-dimensions, followed by collagenase digestion to produce well-controlled aggregates for 3D encapsulation in polyethylene glycol (PEG) diacrylate. Using this approach, we examined the influence of homotypic hepatocyte interactions and composition of the encapsulating hydrogel, and achieved the maintenance of liver-specific function for over 50 days. Optimally preaggregated structures were subsequently encapsulated using a microfluidic droplet-generator to produce 3D microtissues. Interactions of engineered hepatic microtissues with drugs was characterized by flow cytometry, and yielded both induction of P450 enzymes in response to prototypic small molecules and drug-drug interactions that give rise to hepatotoxicity. Collectively, this study establishes a pipeline for the manufacturing of 3D hepatic microtissues that exhibit stabilized liver-specific functions and can be incorporated into a wide array of emerging drug development platforms.

Mesenchymal morphogenesis of embryonic stem cells dynamically modulates the biophysical microtissue niche

Stem cell fate and function are dynamically modulated by the interdependent relationships between biochemical and biophysical signals constituting the local 3D microenvironment. While approaches to recapitulate the stem cell niche have been explored for directing stem cell differentiation, a quantitative relationship between embryonic stem cell (ESC) morphogenesis and intrinsic biophysical cues within three-dimensional microtissues has not been established. In this study, we demonstrate that mesenchymal embryonic microtissues induced by BMP4 exhibited increased stiffness and viscosity accompanying differentiation, with cytoskeletal tension significantly contributing to multicellular stiffness. Perturbation of the cytoskeleton during ESC differentiation led to modulation of the biomechanical and gene expression profiles, with the resulting cell phenotype and biophysical properties being highly correlated by multivariate analyses. Together, this study elucidates the dynamics of biophysical and biochemical signatures within embryonic microenvironments, with broad implications for monitoring tissue dynamics, modeling pathophysiological and embryonic morphogenesis and directing stem cell patterning and differentiation.

Human stem cell-based three-dimensional microtissues for advanced cardiac cell therapies

Cardiac stem cell therapy has been proposed as a therapy option to treat the diseased myocardium. However, the low retention rate of transplanted single-cell suspensions remains a major issue of current therapy strategies. Therefore, the concept of scaffold-free cellular self-assembly into three-dimensional microtissues (3D-MTs) prior to transplantation may be beneficial to enhance retention and survival. We compared clinically relevant, human stem cell sources for their ability to generate 3D-MTs with particular regards to formation characteristics, proliferation-activity, viability and extracellular-matrix production. Single-cell suspensions of human bone marrow- and adipose tissue-derived mesenchymal stem cells (hBMMSCs and hATMSCs), Isl1+ cardiac progenitors derived from human embryonic stem cells (hESC-Isl1+ cells), and undifferentiated human induced pluripotent cells (hiPSCs) were characterized before to generate 3D-MTs using a hanging-drop culture. Besides the principal feasibility of cell-specific 3D-MT formation, a detailed head-to-head comparison between cell sources was performed using histology, immunocyto- and histo-chemistry as well as flow cytometry. Round-oval shaped and uniform 3D-MTs could be successfully generated from all cell types starting with a loose formation within the first 24 h that fully stabilized after 3 days and resulting in a mean 3D-MT diameter of 194.56 ± 18.01 μm (hBMMSCs), 194.56 ± 16.30 μm (hATMSCs), 159.73 ± 19.20 μm (hESC-Isl1+ cells) and 120.95 ± 7.97 μm (hiPSCs). While all 3D-MTs showed a homogenous cell distribution, hiPSC-derived 3D-MTs displayed a compact cell formation primarily located at the outer margin. hESC-Isl1+ and hiPSC-derived 3D-MTs maintained their proliferation-activity which was rather limited in the MSC-based 3D-MTs. All four 3D-MT types revealed a comparable viability in excess of 70% and showed a cell-specific expression profile being comparable to their single-cell counterparts. Extracellular matrix (ECM) production during 3D-MT formation was observed for all cell-specific 3D-MTs, with hiPSC-derived 3D-MTs being the fastest one. Interestingly, ECM distribution was homogenous for hATMSC- and hiPSC-based 3D-MTs, while it appeared to be primarily concentrated within in the center of hESC-Isl1+ and hBMMSC-based 3D-MTs. The results of this head-to-head comparative study indicated that 3D-MTs can be successfully generated from hESC-derived Isl1+ cells, hiPSCs and MSC lines upon hanging drop culture. Cell-specific 3D-MTs displayed sufficient viability and instant ECM formation. The concept of 3D-MT in vitro generation prior to cell transplantation may represent a promising delivery format for future strategies to enhance cellular engraftment and survival.

Dose‐dependent functional benefit of human cardiosphere transplantation in mice with acute myocardial infarction

Despite mounting pre-clinical and clinical evidence of the beneficial effects of cell-based therapy, optimal cell dosing and delivery approaches have not been identified. Cardiospheres are self-assembling three-dimensional (3D) microtissues formed by cardiac stem cells and supporting cell types. The ability of cardiospheres to augment cardiac function has been demonstrated in animal models of ischemic cardiomyopathy. In this study, we studied the dose dependence of the benefits of human cardiospheres, delivered via intramyocardial injection, upon cardiac function and ventricular remodelling in SCID mice with acute myocardial infarction. Four doses of cardiospheres were used: 1 × 104, 5 × 104, 1 × 105 and 5 × 105 (expressed as number of plated cardiosphere-forming cells). Acute (24 hr) cell retention rates in all groups were similar. Functional assessment and quantitative heart morphometry indicated benefit from higher cell doses (≥5 × 104) in terms of ejection fraction, infarct size and capillary density. Histological analysis indicated that the dose-dependent benefit was primarily because of indirect effects of transplanted cells. The results provide scalable data on cardiosphere dosing for intramyocardial injection.

On-chip radiation biodosimetry with three-dimensional microtissues

This paper reports an image-based, on-chip microtissue radiation biodosimeter that can simultaneously monitor radiation responses of multiple mammalian cell types. The microtissue chip is fabricated by molding molten agarose gel onto microfabricated patterns to form microwells, and seeding a variety of cell suspensions into different microwells inside the agarose gel. The camera of a mobile phone is used to collect images of an array of microtissues, and the color changes of microtissues upon X-ray irradiation allow accurate determination of cell death, which is related to radiation dose. The images can be transferred wirelessly, allowing the biodosimeter to be used for convenient and field deployable monitoring of radiation exposure.

Quantification of the Kinetics and Extent of Self-Sorting in Three Dimensional Spheroids

The self-sorting of cells into distinct compartments in three-dimensional (3D) microtissues is a process critical to developmental biology, cancer metastasis, and tissue engineering. Although self-sorting has been studied since the 1950s, little quantitative data exist that describe this dynamic process. Here, we describe a recently developed assay designed to quantify the extent and kinetics of self-sorting in 3D. Mixtures of fluorescently labeled normal human fibroblasts (NHF) and hepatocyte (H35) cells were fluorescently labeled, red and green respectively, and seeded onto micro-molded non-adhesive hydrogels. The cells self-assembled into a spheroid and self-sorted with NHFs forming the central core and H35s forming the outer shell. A time course of fluorescent images was used to analyze the ratio of red (NHFs) and green (H35s) fluorescence in concentric hollow cylinders throughout a spheroid and was statistically compared with the fluorescent ratio of the perfectly sorted spheroid. We found that NHFs and H35s, at a 1:1 ratio, sorted to a final extent of 88±3% at an initial rate of 0.36±0.06% per minute and reached 50% self-sorted at 2.7±0.3 h. Studies with varying ratios of NHFs and H35s show that self-sorting and self-assembly are coincident in time when the proportion of NHFs are varied over a 6-fold range (14% to 85%). This method can, thus, be used to characterize the sorting behavior of additional pairs of cells, the effect of drugs, and growth factors that may change the kinetics of the process, and bring an understanding to the cellular mechanisms which control self-sorting.

Quantification of the forces driving self-assembly of three-dimensional microtissues

In a nonadhesive environment, cells will self-assemble into microtissues, a process relevant to tissue engineering. Although this has been recognized for some time, there is no basis for quantitative characterization of this complex process. Here we describe a recently developed assay designed to quantify aspects of the process and discuss its application in comparing behaviors between cell types. Cells were seeded in nonadhesive micromolded wells, each well with a circular trough at its base formed by the cylindrical sidewalls and by a central peg in the form of a right circular cone. Cells settled into the trough and coalesced into a toroid, which was then driven up the conical peg by the forces of self-assembly. The mass of the toroid and its rate of upward movement were used to calculate the cell power expended in the process against gravity. The power of the toroid was found to be 0.31 ± 0.01 pJ/h and 4.3 ± 1.7 pJ/h for hepatocyte cells and fibroblasts, respectively. Blocking Rho kinase by means of Y-27632 resulted in a 50% and greater reduction in power expended by each type of toroid, indicating that cytoskeletal-mediated contraction plays a significant role in the self-assembly of both cell types. Whereas the driving force for self-assembly has often been viewed as the binding of surface proteins, these data show that cellular contraction is important for cell-cell adhesion. The power measurement quantifies the contribution of cell contraction, and will be useful for understanding the concerted action of the mechanisms that drive self-assembly.

Encapsulated Arrays of Self-Assembled Microtissues: An Alternative to Spherical Microcapsules

Micro-encapsulation and immuno-isolation of allogenic and xenogenic tissues and cells is a promising method for the treatment of a variety of metabolic disorders. Many years have been spent optimizing spherical microcapsules, yet micro-encapsulation has not achieved its full clinical potential. As an alternative to spherical microcapsules, this study presents an alginate-encapsulated array of self-assembled three-dimensional (3D) microtissues. Monodispersed HepG2 cells were seeded onto a micro-molded agarose gel. Cells settled to the bottom of the mold recesses and self-assembled 3D microtissues (n = 822) within 24 h. This array of densely packed microtissues was encapsulated in situ using alginate. When separated from the agarose micro-mold, the encapsulated array had HepG2 microtissues in close proximity to its surface. This surface could be further modified by a simple dipping process. Microtissue size, viability, and albumin secretion were all controllable by the number of cells seeded onto the original agarose micro-mold, and microtissue shape and spacing were controllable by the design of the micro-mold. This approach to encapsulation and the use of self-assembled/self-packing 3D microtissues offers new design possibilities that may help to address certain limitations of conventional microcapsules.