skip to main content


The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 11:00 PM ET on Thursday, May 23 until 2:00 AM ET on Friday, May 24 due to maintenance. We apologize for the inconvenience.

Title: Perfused Organ Cell‐Dense Macrotissues Assembled from Prefabricated Living Microtissues

Engineered tissues usually fall short of physiological cell densities and sizes, resulting in limited functional performance. Viability of large tissues is constrained by inadequate diffusion‐driven nutrient exchange. Methods to form large viable tissues are lacking and are constrained by diffusion‐driven nutrient exchange. Here, the use of the Bio‐Pick, Place, and Perfuse (Bio‐P3) is reported, an integrated biofabrication‐bioreactor platform that semiautomatically and rapidly assembles physiologically cell‐dense macrotissues with 100 million cells while being actively perfused. The Bio‐P3 grips, aligns, and stacks prefabricated, scaffold‐free microtissue parts with integrated lumens on a perfusable build‐platform. Parts spontaneously fuse into one continuous macrotissue with perfusable channels. Customizable microtissues are rapidly prepared up to centimeter‐scale with sustained functional performance. Computational models are developed and experimentally validated to elucidate the effects of perfusion rate and tissue geometry on convective nutrient transport in built macrotissues. It is shown that macrotissues constructed from human hepatocellular microtissues maintain geometry and function (albumin and urea secretion) over 5 days. The Bio‐P3 technology fabricates massive solid tissues with high cell numbers and densities to mimic human physiology for preclinical and clinical applications.

more » « less
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Advanced Biosystems
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    3D microenvironments provide a unique opportunity to investigate the impact of intrinsic mechanical signaling on progenitor cell differentiation. Using a hydrogel‐based microwell platform, arrays of 3D, multicellular microtissues in constrained geometries, including toroids and cylinders are produced. These generated distinct mechanical profiles to investigate the impact of geometry and stress on early liver progenitor cell fate using a model liver development system. Image segmentation allows the tracking of individual cell fate and the characterization of distinct patterning of hepatocytic makers to the outer shell of the microtissues, and the exclusion from the inner diameter surface of the toroids. Biliary markers are distributed throughout the interior regions of micropatterned tissues and are increased in toroidal tissues when compared with those in cylindrical tissues. Finite element models of predicted stress distributions, combined with mechanical measurements, demonstrates that intercellular tension correlates with increased hepatocytic fate, while compression correlates with decreased hepatocytic and increased biliary fate. This system, which integrates microfabrication, imaging, mechanical modeling, and quantitative analysis, demonstrates how microtissue geometry can drive patterning of mechanical stresses that regulate cell differentiation trajectories. This approach may serve as a platform for further investigation of signaling mechanisms in the liver and other developmental systems.

    more » « less
  2. Abstract

    Recent advances in tissue engineering and 3D bioprinting have enabled construction of cell‐laden scaffolds containing perfusable vascular networks. Although these methods partially address the nutrient‐diffusion limitations present in engineered tissues, they are still restricted in both their viable vascular geometries and matrix material compatibility. To address this, tissue constructs are engineered via encapsulation of 3D printed, evacuable, free standing scaffolds of poly(vinyl alcohol) (PVA) in biologically derived matrices. The ease of printability and water‐soluble nature of PVA grant compatibility with biologically relevant matrix materials and allow for easily repeatable generation of complex vascular patterns. This study confirms the ability of this approach to produce perfusable vascularized matrices capable of sustaining both cocultures of multiple cell types and excised tumor fragments ex vivo over multiple weeks. The study further demonstrates the ability of the approach to produce hybrid patterns allowing for coculture of vasculature and epithelial cell‐lined lumens in close proximity, thereby enabling ex vivo recapitulation of gut‐like systems. Taken together, the methodology is versatile, broadly applicable, and importantly, simple to use, enabling ready applicability in many research settings. It is believed that this technique has the potential to significantly accelerate progress in engineering and study of ex vivo organotypic tissue constructs.

    more » « less
  3. Tissue engineering has been largely confined to academic research institutions with limited success in commercial settings. To help address this issue, more work is needed to develop new automated manufacturing processes for tissue-related technologies. In this article, we describe the automation of the funnel-guide, an additive manufacturing method that uses living tissue rings as building units to form bio-tubes. We developed a method based on 96-well plates and a modified off-the-shelf liquid-handling robot to retrieve, perform real-time quality control, and transfer tissue rings to the funnel-guide. Cells seeded into 96-well plates containing specially designed agarose micromolds self-assembled and formed ring-shaped microtissues that could be retrieved using a liquid-handling robot. We characterized the effects of time, cell type, and mold geometry on the morphology of the ring-shaped microtissues to inform optimal use of the building parts. We programmed and modified an off-the-shelf liquid-handling robot to retrieve ring-shaped microtissues from the 96-well plates, and we fabricated a custom illuminated pipette to visualize each ring-shaped microtissue prior to deposit in the funnel guide. Imaging at the liquid-air interface presented challenges that were overcome by controlling lighting conditions and liquid curvature. Based on these images, we incorporated into our workflow a real-time quality control step based on visual inspection and morphological criteria to assess each ring prior to use. We used this system to fabricate bio-tubes of endothelial cells with luminal alignment. 
    more » « less
  4. Abstract

    The ability to replicate the microenvironment of biological tissues creates unique biomedical possibilities for stem cell applications. Current fabrication methods are limited by either the control on feature size and shape, or by the throughput and size of the replicas. Here, a novel platform is reported that combines thermal scanning probe lithography (tSPL) with innovative methodologies for the low‐cost and high‐throughput nanofabrication of large area quasi‐3D bone tissue replicas with high fidelity, sub‐15 nm lateral precision, and sub‐2 nm vertical resolution. This bio‐tSPL platform features a biocompatible polymer resist that withstands multiple cell culture cycles, allowing the reuse of the replicas, further decreasing costs and fabrication times. The as‐fabricated replicas support the culture and proliferation of human induced mesenchymal stem cells, which display broad therapeutic and biomedical potential. Furthermore, it is demonstrated that bio‐tSPL can be used to nanopattern the bone tissue replicas with amine groups, for subsequent tissue‐mimetic biofunctionalization. The achieved level of time and cost‐effectiveness, as well as the cell compatibility of the replicas, make bio‐tSPL a promising platform for the production of tissue‐mimetic replicas to study stem cell‐tissue microenvironment interactions, test drugs, and ultimately harness the regenerative capacity of stem cells and tissues for biomedical applications.

    more » « less
  5. Abstract

    Many cell responses that underlie the development, maturation, and function of tissues are guided by the architecture and mechanical loading of the extracellular matrix (ECM). Because mechanical stimulation must be transmitted through the ECM architecture, the synergy between these two factors is important. However, recapitulating the synergy of these physical microenvironmental cues in vitro remains challenging. To address this, a 3D magnetically actuated collagen hydrogel platform is developed that enables combined control of ECM architecture and mechanical stimulation. With this platform, it is demonstrated how these factors synergistically promote cell alignment of C2C12 myoblasts and enhance myogenesis. This promotion is driven in part by the dynamics of Yes‐associated protein and structure of cellular microtubule networks. This facile platform holds great promises for regulating cell behavior and fate, generating a broad range of engineered physiologically representative microtissues in vitro, and quantifying the mechanobiology underlying their functions.

    more » « less