We would like to thank NSF CAREER Award #1653193, Arizona Biomedical Research Commission (ABRC) New Investigator Award (ADHS18-198872), and the Flinn Foundation Award for providing funding sources for this project. The hiPSC line, SCVI20, was obtained from Joseph C. Wu, MD, PhD at the Stanford Cardiovascular Institute funded by NIH R24 HL117756. The hiPSC line, IMR90-4, was obtained from WiCell Research Institute55,56.

The authors declare that they have no competing financial interests.

The formation of an in vitro human cardiac tissue model with enhanced cell-cell interactions and biomimetic 3D structure is imperative for basic cardiovascular research and corresponding clinical applications1. This outlined protocol explains the development of 3D human anisotropic cardiac tissue within a microfluidic device, using co-culture of stem cell-derived CMs with connective CFs encapsulated within a collagen hydrogel, serving to model the complex cell composition and structure of...

Tissue engineering approaches have been widely explored, in recent years, to accompany in vivo clinical findings in regenerative medicine and disease modeling1,2. Significant emphasis has been particularly placed on in vitro cardiac tissue modeling due to the inherent difficulties in sourcing human primary cardiac tissue and producing physiologically relevant in vitro surrogates, limiting the fundamental understanding of the complex mechanisms of cardiovascular diseases (CVDs)1,3. Traditional models have often involved 2D monolayer culture assays. However, the importance of culturing cardiac cells within a 3D environment to mimic both the native landscape of the myocardium and complex cellular interactions has been extensively characterized4,5. Additionally, most models produced thus far have included a mono-culture of CMs differentiated from stem cells. However, the heart is comprised of multiple cell types6 within a complex 3D architecture7, warranting the critical need to improve the complexity of the tissue composition within 3D in vitro models to better mimic cellular constituents of the native myocardium.
To date, many different approaches have been explored to produce biomimetic 3D models of the myocardium8. These approaches range from experimental setups&#160;that allow for the real-time calculation of generated force, from mono-culture CMs seeded on thin films (deemed muscular thin films (MTFs))9, to co-culture cardiac cells in 3D hydrogel matrices suspended among free-standing cantilevers (deemed engineered heart tissues (EHTs))10. Other approaches have focused on implementing micromolding techniques to mimic myocardial anisotropy, from mono-culture CMs in a 3D hydrogel suspended among protruding microposts in a tissue patch11, to mono-culture CMs seeded among indented microgrooves12,13. There are inherent advantages and disadvantages to each of these methods, therefore, it is pertinent to utilize the technique that aligns with the intended application and the corresponding biological question.
The ability to enhance the maturation of stem cell-derived CMs is essential for the successful in vitro engineering of adult-like myocardial tissue and translation of subsequent findings to clinical interpretations. To this end, methods to mature CMs have been widely explored, both in 2D and 3D14,15,16. For example, electrical stimulation incorporated in EHTs, forced alignment of CMs with surface topography, signaling cues, growth factors from co-culture, and/or 3D hydrogel conditions, etc., all lead to a change in favor of CM maturation in at least one of the following: cell morphology, calcium handling, sarcomeric structure, gene expression, or contractile force.
Of these models, the approaches that utilize microfluidic platforms retain certain advantages in nature, such as control of gradients, limited cell input, and minimal necessary reagents. Furthermore, many biological replicates can be generated at once using microfluidic platforms, serving to better dissect the biological mechanism of interest and increase the experimental sample size in favor of statistical power17,18,19. Additionally, using photolithography in the microfluidic device fabrication process enables the creation of precise features (e.g., topographies) at the micro- and nano-level, which serve as mesoscopic cues to enhance the surrounding cellular structure and macro-level tissue architecture18,20,21,22 for different applications in tissue regeneration and disease modeling.
We previously demonstrated the development of a novel 3D cardiac tissue on-chip model that incorporates surface topography, in the form of innate elliptical microposts, to align hydrogel-encapsulated co-cultured cardiac cells into an interconnected, anisotropic tissue20. After 14 days of culture, the tissues formed within the microfluidic device are more mature in their phenotype, gene expression profile, calcium handling characteristics, and pharmaceutical response when compared to monolayer and 3D isotropic controls23. The protocol described herein outlines the method for creating this 3D co-cultured, aligned (i.e., anisotropic) human cardiac tissue within the microfluidic device using hiPSC-derived CMs. Specifically, we explain the methods to differentiate and purify hiPSCs towards CMs, supplementation of hCFs with CMs to produce an established co-culture population, insertion of the cell population encapsulated within the collagen hydrogel into the microfluidic devices, and subsequent analysis of the 3D constructed tissues through contractile and immunofluorescent assays. The resultant 3D engineered micro-tissues are suitable for various applications, including fundamental biology studies, CVD modeling, and pharmaceutical testing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.65 mL centrifuge tubes</td>
			<td>VWR</td>
			<td>87003-290</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm Biopsy punch</td>
			<td>VWR</td>
			<td>95039-090</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mm Biopsy punch</td>
			<td>VWR</td>
			<td>95039-088</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Falcon tubes</td>
			<td>VWR</td>
			<td>89039-670</td>
			<td></td>
		</tr>
		<tr>
			<td>18x18mm coverslips</td>
			<td>VWR</td>
			<td>16004-308</td>
			<td>The coverslips should be No.1, to allow for high magnification imaging</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>ThermoFisher</td>
			<td>101176-014</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well flat botttom tissue-culture plates</td>
			<td>VWR</td>
			<td>82050-844</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 minus insulin</td>
			<td>LifeTech</td>
			<td>A1895602</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 plus insulin</td>
			<td>LifeTech</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>VWR</td>
			<td>10188-030</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, rat tail</td>
			<td>Corning</td>
			<td>47747-218</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12</td>
			<td>ThermoFisher</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>ThermoFisher</td>
			<td>21600069</td>
			<td></td>
		</tr>
		<tr>
			<td>E8</td>
			<td>ThermoFisher</td>
			<td>A1517001</td>
			<td>can also be made in house</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>VWR</td>
			<td>45001-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FGM3</td>
			<td>VWR</td>
			<td>10172-048</td>
			<td></td>
		</tr>
		<tr>
			<td>GFR-Matrigel</td>
			<td>VWR</td>
			<td>47743-718</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>VWR</td>
			<td>10152-212</td>
			<td></td>
		</tr>
		<tr>
			<td>hESC-Matrigel</td>
			<td>Corning</td>
			<td>BD354277</td>
			<td></td>
		</tr>
		<tr>
			<td>IPA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IWP2</td>
			<td>Sigma</td>
			<td>I0536-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>VWR</td>
			<td>82003-820</td>
			<td></td>
		</tr>
		<tr>
			<td>MTCS</td>
			<td>Sigma</td>
			<td>440299-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S5881-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>VWR</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (150x15mm)</td>
			<td>VWR</td>
			<td>25384-326</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (60x15mm)</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma</td>
			<td>P3532-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>ThermoFisher</td>
			<td>MT10040CM</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 minus glucose</td>
			<td>VWR</td>
			<td>45001-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafers (100mm)</td>
			<td>University Wafer</td>
			<td>1196</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium lactate</td>
			<td>Sigma</td>
			<td>L4263-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8 2075</td>
			<td>Microchem</td>
			<td>Y111074 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8 Developer</td>
			<td>ThermoFisher</td>
			<td>NC9901158</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard Elastomer</td>
			<td>Essex Brownell</td>
			<td>DC-184-1.1</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>VWR</td>
			<td>82050-856</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>ThermoFisher</td>
			<td>12604021</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.5%)</td>
			<td>ThermoFisher</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Stem Cell Technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>EVG620 Aligner</td>
			<td>EVG</td>
		</tr>
		<tr>
			<td>Plasma cleaner PDC-32G</td>
			<td>Harrick Plasma</td>
		</tr>
		<tr>
			<td>Zeiss AxioObserver Z1 microscope</td>
			<td>Nikon</td>
		</tr>
		<tr>
			<td>Leica SP8 Confocal microscope</td>
			<td>Leica</td>
		</tr>
	</tbody>
</table>

developing 3d organized human cardiac tissue within a microfluidic platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

To obtain a highly purified population of CMs from hiPSCs, a modified version involving a combination of the Lian differentiation protocol33 and Tohyama purification steps34 is used (refer to Figure 1A for experimental timeline). The hiPSCs need to be colony-like, ~85% confluent, and evenly spread throughout the culture well 3-4 days after passage, at the onset of CM differentiation (Figure 1B). Specifically, on Da...

Watch this Scientific Journal Video about Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform at JoVE.com

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

developing-3d-organized-human-cardiac-tissue-within-microfluidic

Research

JoVE Journal

Bioengineering

4.8K Views.  Arizona State University. The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

Video: Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...