3D bioprinting technology facilitates development of an in vitro biomimetic bladder model

Kate Gessner, MD, PhD

The urinary bladder is a dynamic organ that serves the complex functions of both storing urine and subsequently emptying it in response to neuromuscular signals. To fulfill these functions, the shape and pressure within the bladder change continuously in response to urine filling and emptying dynamics, and these movements affect bladder development and disease. However, many of the existing in vitro models of bladder disease do not mimic this physiologic movement or evaluate its impact on disease development. Additionally, the ability to study how molecular alterations, physiological stimuli, and medications affect bladder compliance is especially reliant on a 3D bladder model mimicking normal bladder movement.

Philip Abbosh, MD, PhD

Recognizing the importance of developing a model of the bladder that incorporates a 3D cellular scaffold and physiological movement of the urinary bladder, scientists have worked to develop in vitro models of the urinary bladder utilizing 3D bioprinting.

What is 3D bioprinting? It is an innovative technology that utilizes 3D printing to produce complex structures comprised of biomaterials, cells, and growth factors. Bioink, a hydrogel that can include living cells, is a material used in 3D bioprinting to create a suitable microenvironment for cellular growth. Bioink can consist of single-agent materials (which are components of extracellular matrix such as fibrin, alginate, and collagen) or decellularized extracellular matrix (dECM). The latter has the advantage of containing tissue-specific extracellular matrix components, recapitulating the structure, complexity, and function of that specific tissue or organ, and can be mixed with cells and printed onto a prepared platform to generate in vitro model systems.

Chae et al combined 3D bioprinting with mechanical movements to develop a biomimetic bladder model comprised of 3 essential components: the bladder tissue–derived dECM (bladder dECM) bioink, human bone marrow–derived mesenchymal stem cells, and a contract-release system for physiological stimulation. The bladder dECM is generated through delamination and decellularization of porcine bladder tissue, which is subsequently further processed into a pre-gel solution. The bladder dECM pre-gel solution is mixed with human bone marrow–derived mesenchymal stem cells and printed onto a sterilized polydimethylsiloxane (PDMS) membrane located within a custom integrated housing platform. This housing platform consists of a baseplate (bottom chamber) and a wall template. To mimic the physiologic movement of the bladder, Chae et al developed a contract-release system, which consists of a programmable syringe pump and syringe attached to the baseplate of the housing platform. When air is injected into the baseplate, the PDMS membrane and attached cells expand, while the removal of air returns the model to baseline position. For membranes testing physiological movement of the bladder, cyclical stretching was performed for 16 hours daily with 8 hours of daily rest.

Following generation of this in vitro bladder model, Chae et al evaluated its cellular ability to maintain viability, proliferate, and form organized muscle fibers. They first assessed cell viability and found that more than 90% of cells were viable over all time points across 10 days. Cell adhesion testing using F-actin staining demonstrated interconnected networks within the bladder dECM matrix. Finally, compared with a collagen-based, single-component bioink, the bladder dECM bioink showed increased cell proliferation (which was perceived to be a positive characteristic). These results overall demonstrate that the bladder dECM bioink provides a conducive environment in which mesenchymal stem cells can establish, proliferate, and form interconnected networks.

To test their hypothesis that mechanical stimulation improves stem cell differentiation into bladder smooth muscle lineage, Chae et al evaluated the impact of physiological stimuli on muscle formation in the in vitro model. Specifically, they measured cellular expression of markers for muscle tissue in tissue models that underwent cyclical stretching (mimicking regular bladder movement) compared with models that did not experience movement. Cells that experienced periodic movement expressed significantly higher levels of muscle lineage markers compared with those without movement and were longitudinally aligned along the axis of movement. This result highlights the role that external stimuli play on stem cell differentiation into the muscle lineage and reinforces the importance of incorporating physiological stimuli into models of the urinary bladder.

What does this all mean? The development of an in vitro bladder model incorporating mechanical stimuli provides a useful model for testing both bladder development and disease. For example, this model could be used to interrogate how altered physiologic bladder movements or specific gene alterations affect cellular organization or viability in the urinary bladder. There are some shortcomings to the model that could be addressed in future iterations. The lamina propria and urothelium are absent from this model and likely share bidirectional intercellular communication signals with the muscular layers of the bladder. Further, the tissue was expanded using compressed air rather than fluid and was stretched by 5% every 10 seconds. In organisms that use a bladder, it is expanded/contracted with liquid urine to a much higher volumetric fold change and occurs over a more protracted time course. However, this is only a very simplified, first-pass model and did recapitulate several key features of the bladder.

In addition to the advances in research studying bladder diseases, the use of 3D bioprinting will continue to revolutionize the scientific and medical fields. This exciting and innovative technology has a wide range of possible applications, including tissue engineering, drug development, and precision medicine.

REFERENCE

Chae S, Kim J, Yi HG, Cho DW. 3D bioprinting of an in vitro model of a biomimetic urinary bladder with a contract-release system. Micromachines (Basel). 2022;13(2):277. doi:10.3390/mi13020277