Engineering anisotropic 3D tubular tissues with flexible thermoresponsive nanofabricated substrates

Abstract

Tissue engineering aims to capture the structural and functional aspects of diverse tissue types in vitro. However, most approaches are limited in their ability to produce complex 3D geometries that are essential for tissue function. Tissues, such as the vasculature or chambers of the heart, often possess curved surfaces and hollow lumens that are difficult to recapitulate given their anisotropic architecture. Cell-sheet engineering techniques using thermoresponsive substrates provide a means to stack individual layers of cells with spatial control to create dense, scaffold-free tissues. In this study, we developed a novel method to fabricate complex 3D structures by layering multiple sheets of aligned cells onto flexible scaffolds and casting them into hollow tubular geometries using custom molds and gelatin hydrogels. To enable the fabrication of 3D tissues, we adapted our previously developed thermoresponsive nanopatterned cell-sheet technology by applying it to flexible substrates that could be folded as a form of tissue origami. We demonstrated the versatile nature of this platform by casting aligned sheets of smooth and cardiac muscle cells circumferentially around the surfaces of gelatin hydrogel tubes with hollow lumens. Additionally, we patterned skeletal muscle in the same fashion to recapitulate the 3D curvature that is observed in the muscles of the trunk. The circumferential cell patterning in each case was maintained after one week in culture and even encouraged organized skeletal myotube formation. Additionally, with the application of electrical field stimulation, skeletal myotubes began to assemble functional sarcomeres that could contract. Cardiac tubes could spontaneously contract and be paced for up to one month. Our flexible cell-sheet engineering approach provides an adaptable method to recapitulate more complex 3D geometries with tissue specific customization through the addition of different cell types, mold shapes, and hydrogels. By enabling the fabrication of scaled biomimetic models of human tissues, this approach could potentially be used to investigate tissue structure-function relationships, development, and maturation in the dish.

Introduction

Tissues throughout the body possess complex three-dimensional (3D) structures with many degrees of organization and function. For example, the vasculature, like many other tissues, is organized by stratification of several layers of different cell types that perform complementary functions to modulate blood pressure and tissue perfusion [1,2]. The endothelial cells in the lining of the blood vessel's lumen are oriented parallel to the direction of blood flow, whereas the surrounding smooth muscle cells that encircle the endothelium are aligned perpendicularly. Similar patterns of differential organization are observed in the helical fiber organization of the myocardium in the heart and in the radial fan patterns seen in the trapezius and pectoral muscles of the trunk. The function of each of these tissues is highly dependent upon their structure and 3D geometry, and when their organization is compromised by disease it can be detrimental or potentially fatal [[3], [4], [5]].

To study tissue function and their associated diseases, advancements have been made in tissue engineering to recapitulate tissue micro- and macroenvironments in vitro. For example, cell-dense cardiac tissue patches made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can mimic action potential conduction velocities close to those of adult cardiac tissues [[6], [7], [8]]; vascular grafts have been made from cell-deposited matrix and then decellularized before implantation [[9], [10], [11]]; and bioprinting with cellularized-inks (or bioinks) has enabled fabrication of intricate 3D tissue-specific structures with corresponding function [[12], [13], [14], [15]]. A challenge facing each of these approaches is that tissues often have complex 3D geometries, including curved surfaces and hollow lumens. Such structures have been difficult to recreate in vitro due to limitations of available fabrication techniques. Specifically, there are few fabrication approaches that allow for production of curved 3D geometries while also having control over spatial organization at the cell-layer level. The ability to recapitulate these structures would impart function that better mimics native tissues and organs.

To address this need, our group has previously established a nanofabrication technique to pattern sheets of organized cells and stack them to create multi-layered tissue patches using a novel gel-casting technique in conjunction with thermoresponsive substrates [16,17]. In the present study, we sought to improve upon this technology by introducing flexible substrates and custom molds to enable the fabrication of organized 3D tissue structures. We found that multiple cell types could be patterned to form an intact monolayer with a uniform orientation in the direction of the nanotopography. Each monolayer was lifted from the surface through temperature-mediated release provided by the thermoresponsive poly (N‐isopropylacrylamide) (pNIPAM) functional layer. Multiple organized monolayers were stacked onto a single flexible film and were folded into a cylindrical shape, as a form of tissue origami, where the organized cell layers could be casted into a free-standing 3D tubular tissue. We demonstrated the diverse application of this technology by fabricating tubular tissues with curved surfaces from three musclecell types: smooth, skeletal, and cardiac. This approach enabled patterning of all three cell types in 3D multilayered tissues with circumferential alignment that was maintained for two weeks in culture. Additionally, with application of electrical field stimulation, skeletal myotubes assembled functional sarcomeres that could contract, and cardiac tubes could be paced for over one month. This flexible patterned film technology can be readily adapted to fabricate tissues with other complex geometries by changing the shape of the flexible film and custom mold, producing more biomimetic tissues for the study of development and disease.