Tissue-in-a-Tube: three-dimensional in vitro tissue constructs with integrated multimodal environmental stimulation

Abstract

Three-dimensional (3D) in vitro tissue models are superior to two-dimensional (2D) cell cultures in replicating natural physiological/pathological conditions by recreating the cellular and cell-matrix interactions more faithfully. Nevertheless, current 3D models lack either the rich multicellular environment or fail to provide appropriate biophysical stimuli both of which are required to properly recapitulate the dynamic in vivo microenvironment of tissues and organs. Here, we describe the rapid construction of multicellular, tubular tissue constructs termed Tissue-in-a-Tube using self-assembly process in tubular molds with the ability to incorporate a variety of biophysical stimuli such as electrical field, mechanical deformation, and shear force of the fluid flow. Unlike other approaches, this method is simple, requires only oxygen permeable silicone tubing that molds the tissue construct and thin stainless-steel pins inserted in it to anchor the construct and could be used to provide electrical and mechanical stimuli, simultaneously. The annular region between the tissue construct and the tubing is used for perfusion. Highly stable, macroscale, and robust constructs anchored to the pins form as a result of self-assembly of the extracellular matrix (ECM) and cells in the bioink that is filled into the tubing. We demonstrate patterning of grafts containing cell types in the constructs in axial and radial modes with clear interface and continuity between the layers. Different environmental factors affecting cell behavior such as compactness of the structure and size of the constructs can be controlled through parameters such as initial cell density, ECM content, tubing size, as well as the distance between anchor pins. Using connectors, network of tubing can be assembled to create complex macrostructured tissues (centimeters length) such as fibers that are bifurcated or columns with different axial thicknesses which can then be used as building blocks for biomimetic constructs or tissue regeneration. The method is versatile and compatible with various cell types including endothelial, epithelial, skeletal muscle cells, osteoblast cells, and neuronal cells. As an example, long mature skeletal muscle and neuronal fibers as well as bone constructs were fabricated with cellular alignment dictated by the applied electrical field. The versatility, speed, and low cost of this method is suited for widespread application in tissue engineering and regenerative medicine.

Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges

“…[ 249 ] Several bioprinting‐based strategies have also utilized collagen. [ 242 , 243 , 244 , 245 , 304 ] In addition, collagen I has been shown to interact with and serve as a slow‐release reservoir for bFGF. [ 248 ] All collagens share a triple‐helix structure but differ in amino acid sequence and overall organization.…”

“…The “tissue in a tube” strategy relies on the contraction of a collagen‐based bioink within a tube, leaving a hollow, media‐filled space surrounding the construct, which may be used for perfusion. [ 304 ] This method has been tested successfully with endothelial, epithelial, muscle, osteoblast, and neuronal cells and is suitable for the production of tubular constructs several centimeters in length, although the diameter is limited by the fact that channels are not present in the construct's interior. Molding of scaffolds around linear wire arrays has been used as a strategy for introducing vascular‐like channels, which were demonstrated to improve both fibroblast seeding and oxygen and nutrient delivery within small‐scale constructs.…”

“…Scaffolds derived from a combination of collagen and hydroxyapatite have been investigated for use as bone grafts and can support the adhesion and proliferation of fibroblasts. [ 436 ] Growth of bone tissue or osteogenic cells on decellularized apple [ 326 ] and carrot [ 327 ] scaffolds, PLGA hollow fiber membranes, [ 196 ] soda bread, [ 191 ] RADA16 hydrogels, [ 237 ] collagen hydrogels, [ 304 ] and laser sintered PCL [ 281 ] has also been demonstrated. Engineering of tendon [ 327 , 384 , 437 ] and cartilage [ 438 , 439 ] tissue are also active areas of investigation for biomedical applications.…”

Modelling the central nervous system: tissue engineering of the cellular microenvironment

“…Inclusion of conductive components also enables integrated analysis of neuronal network activity [ 58 ]. Several studies have employed a combination of concurrent biophysical features such as electrical and mechanical stimulation, to produce dynamic culture systems capable of promoting neural alignment and neurite extension [ 88 , 103 ]. Stimuli-responsive hydrogels respond to chemical or physical stimuli, including light, magnetic/electric fields, shear forces, temperature, pH, ions, chemicals, drugs, enzymes etc.…”

“…Multiphase release systems are needed, to ensure sequential and spatiotemporal delivery of biochemical cues similar to naturally occurring tissues in vivo [ 82 ]. Ultimately, development of hydrogels with a combination of multiphase, dynamic and stimuli-responsive properties is the ideal approach to modelling complex living tissues in vitro [ 103 ].…”