From Microscale Devices to 3D Printing: Advances in Fabrication of 3D Cardiovascular Tissues

Abstract

Current strategies for engineering cardiovascular cells and tissues have yielded a variety of sophisticated tools for studying disease mechanisms, for development of drug therapies, and for fabrication of tissue equivalents that may have application in future clinical use. These efforts are motivated by the need to extend traditional 2-dimensional (2D) cell culture systems into 3D to more accurately replicate in vivo cell and tissue function of cardiovascular structures. Developments in microscale devices and bioprinted 3D tissues are beginning to supplant traditional 2D cell cultures and preclinical animal studies that have historically been the standard for drug and tissue development. These new approaches lend themselves to patient-specific diagnostics, therapeutics, and tissue regeneration. The emergence of these technologies also carries technical challenges to be met before traditional cell culture and animal testing become obsolete. Successful development and validation of 3D human tissue constructs will provide powerful new paradigms for more cost effective and timely translation of cardiovascular tissue equivalents.

Keywords: biocompatible materials; heart; printing, three-dimensional; stem cells; tissue engineering.

Figure 1

Utility of the 3D relative to the 2D formats for cardiovascular tissue engineering applications. Red circle indicates the feature only feasible in 3D. Pink, gray and blue circles and their corresponding positions represent features compatible with both 2D and 3D systems, but more ideally achieved in the formats in closest proximity. Note, the overwhelming majority of ideal feature are best achieved in 3D and typically result in a more anatomic and physiologic representation of cardiac tissues. In particular, action potential, abundance of sarcomeric and sarcoplasmic proteins, quality of Frank-Starling behavior, force-frequency relationship, reaction to calcium, isoprenaline and carbachol have been found to be more akin to tissue response when assessed in 3D format.

Figure 2

In vitro testing of cells and tissues may occur in several ways. Microfluidic systems (A) have emerged as a tool for basic science studies of the effect of highly controlled fluid mechanical and solid mechanical forces on single cell types or co-cultures. Microfluidic systems are also gaining favor as a diagnostic tool and a platform for drug development. Organoid cultures (B) are described as organ buds grown in culture that feature realistic microanatomy and are useful as cellular models of human disease. These cultures have found utility in the study of basic mechanisms of organ-specific diseases. Spheroid cultures (C) feature sphere-shaped clusters of a single cell type or co-culture sustained in a gel or a bioreactor in order to interact with their 3D surroundings and are useful in testing drug efficacy and toxicity. (D) Engineered heart tissues are constructed by polymerizing an extracellular matrix-based gel containing cardiac cell types between two elastomeric posts or similar structures allowing auxotonic contraction of cardiomyocytes. This allows to mimic the normal conditions of the heart contracting against the hydrostatic pressure imposed by the circulation. This type of tissue construct has been used for testing toxicity of drugs and basic studies of muscle function and interplay between multiple cardiac cell types.

Figure 3

Bioprinting is usually accomplished using a combination of gel and cells. Laser assisted bioprinting (A) using Laser-induced forward transfer (LIFT) relies on the focused energy of a laser onto an energy absorbing ribbon to induce bioink droplet formation. This technique is advantageous because it avoids the problem of clogging of the bioink nozzle that plagues other bioprinting techniques. Multiphoton excitation-based printing (B), is accomplished via photocrosslinking of proteins or polymers in the focal volume of the laser and excels in its high resolution and ability to polymerize many native proteins that do not form hydrogels spontaneously outside the body. Inkjet printing (C), one of the most common printing techniques, relies upon a vapor bubble or a piezoelectric actuator to displace material to extrude the bioink from a nozzle. Robotic dispensing (D) employs other mechanical means of displacing bioink under robotic control.

Figure 4

Scaffold-free bioprinting employs cell spheroids and does not utilize a gel as a carrier. Robotic dispensing of spheroids (A) typically occurs through a nozzle onto a carrier substrate. Cell spheroids can be delivered to form various shapes including blood vessels (B). Cell spheroids will self-organize, fuse, and begin forming their own extracellular matrix (C). Under appropriate mechanical stimulation, fused spheroids can develop enough mechanical integrity to become suitable for implantation as a load-bearing tissue replacement.