Engineering vascularised and aligned tissues using ultrasound cell patterning

Over the last 25 years, scientists have developed methods for growing tissues (e.g. muscle, cartilage), which can be used to replace diseased or injured tissue, or as a tool for testing drugs. Unfortunately, these laboratory-grown tissues do not perform as well as natural tissue. One of the main reasons for this is that it is very difficult to recreate the particular organization of natural tissues. For example, muscle is made up of bundles of aligned cells; an organization that allows it to contract and relax in the same direction. We have shown that we can partially recreate this organization using a technique known as "ultrasound cell patterning". Here, we use ultrasound to remotely move cells into parallel lines; this is very quick (< 5 min) and has very little affect on the health of the cells. We first used ultrasound to pattern lines of muscle cells, which were then set into a collagen gel to preserve the cell pattern. We grew these muscle gels in the laboratory for seven days to form muscle with bundles of aligned muscle cells; just like in natural muscle. These early results are very promising, but there are several ways that it can be improved:
(1) In the early results, we showed that muscle cells could be patterned in 2D lines, however, muscle is made of 3D fibres. The first aim of this work will be to add another ultrasound wave to create 3D tubes of muscle cells, so that we can grow 3D muscle.
(2) Currently we can only do the cell patterning once, per tissue. The second aim of this work will be to develop a way of patterning cells layer by layer. This will allow us to add blood vessels, to supply oxygen, in between layers of muscle.
(3) The early work used mouse muscle cells, which are a poor imitation of human muscle. A better option is "human induced pluripotent stem cells," a new type of cell that can be used to make muscle cells. The third aim of this work would be to use these cells to grow patterned muscle.
The optimised system will be used in the fourth aim of this research; to engineer laboratory-grown patterned muscle for pharmaceutical drug testing. Specifically, we will study a genetic disease known as muscular dystrophy, which causes muscle wastage and degeneration. The final aim of this work is to develop these tools as a platform technology that can be used to grow other organized tissues. This includes cartilage, tendon, ligament and heart tissue.

It is imperative that bioengineering strategies incorporate structural organization into in vitro grown tissue. For example, cardiac contraction is mediated by the synchronous beating of aligned cardiomyocytes, while the anisotropic mechanical properties of musculoskeletal tissues (e.g. ligament, tendon, cartilage, skeletal muscle) are defined by the directional organization of cells and matrix fibres. It is technically challenging, however, to achieve such fine control over tissue microstructure. The resolution of bioprinting is insufficient to fabricate detailed tissue architecture, while materials-based approaches can be methodologically inflexible and difficult to translate into 3D. The aim of this research is to develop a new, versatile strategy, in which the structural elements of complex tissues are reproduced using ultrasound patterning. Ultrasound standing waves present negligible cytotoxicity and can be used to manipulate bulk cell populations over large areas without any pre-labelling, specialised media or complex equipment. Our preliminary results have shown that ultrasound fields can be used to align myoblasts into parallel lines. In this Fellowship, an advanced acoustic set-up will be developed to engineer 3D tissues with hierarchical and multicellular cell patterning. These patterning methodologies will be developed using cell lines derived from human induced pluripotent stem cells, to ensure biological relevance for the real-world applications (disease models, drug screens, tissue grafts). The patterned tissue constructs will be fully characterised using confocal reflectance microscopy, gene expression analysis, immunohistochemistry, Raman mapping, electron microscopy and spatial transcriptomics. The validated tissues will then be applied predominantly for the engineering of vascularised, aligned muscle, but will be developed as platform technologies to enable application towards different organised tissue structures.