Periodic Reporting for period 2 - UroPrint (Urinary bladder bioprinting for fully autologous transplantation)
Reporting period: 2022-09-01 to 2024-02-29
A number of conditions, including trauma, inflammation, incontinence, overactive bladder, renal impairments and neurological disorders (like spinal cord injury or spina bifida) require bladder augmentation (cystoplasty). For almost a century now, the majority of cystoplasties utilize bowel segments (enterocystoplasty). Functional incompatibilities between the intestinal epithelium and the bladder urothelium, and the contact of urine with the intestinal epithelium lead to numerous complications affecting the majority of patients, thus compromising the quality of life while burdening the health care systems. Bladder cancer (BC) affects millions of people worldwide with a substantial portion undergoing cystectomy. These patients need urine diversion and are facing two options: an external incontinent diversion (urostomy or ileal conduit) or an internal continent neo-bladder, using a bowel segment. Both have several side effects and complications affecting the quality of life of the patient throughout lifetime. This has fueled efforts to develop engineered bladder tissue. The development of 3D-printing technology opened a new era in regenerative medicine by enabling the printing of living cells (bioprinting) with or without the use of matrices (bioinks) with confined spatial distribution (geometry/density) that is impossible to achieve with current seeding procedures. The financial burden of treating side-effects of enterocystoplasty as performed today together with the societal burden associated with those side-effects, indicate that novel solutions are needed. The vision of the UroPrint consortium is to laser print fully functional immunocompatible urothelial tissue ex vivo and in vivo for bladder augmentation and replacement. This vision is enabled by combining and advancing a number of achievements in the fields of optics and laser technologies, materials, engineering and micro-instrumentation, and experimental surgery.
The first step for the project materialization is to integrate all ex vivo cell processes under GMP compliance, staring with the isolation and expansion of urothelial cells and smooth muscle cells from primary bladder specimens. These specimens come from patients with benign prostate hyperplasia and undergo intravesical prostatectomy (hyperplastic prostate is accessed through a hole in the bladder wall, which allows the surgeon to obtain a small biopsy). Initial experiments relied on commercially available primary cells. We have established protocols for cell isolation and expansion. Through media testing and optimization, GMO-compliant conditions were identified for smooth muscle cells. Urothelial cells are less robust in growing under defined media lacking pituitary cell extract. Cells from several patients were obtained, expanded and biobanked.
In parallel, urothelial and smooth muscle cells of mouse and human origin were mixed or printed onto or within PLMA aiming to generate engineered explants for transplantation. SMCs grow better onto stiff PLMA whereas urothelial cells grown inside forming organoids in less stiff PLMA. ICCS and BRFAA have collaborated to optimize printing conditions (fluence and wavelength for printing and polymerization of PLMA). BRFAA and ICCS have printed PLMA bioinks with mouse urothelial and smooth muscle cells and grown them successfully. Urothelial cells form organoids inside PLMA while SMCs grow very well on top of stiff PLMA surfaces.
For the development of a prototype for elasticity and permeability measurements, device specifications (explant size, stretching length, permeability assay, etc.) have been defined. A high level and a detailed design have been prepared. Some of the components have been selected and tested. Optics11 has obtained PLMA from META for initial tests. Importantly, initial permeability tests have been successful. This is an important development, because the permeability testing module is associated with a high risk.
During RP2, we prepared sandwiches of PLMA of different composition containing SMCs and UCs of mouse and human origin. These sandwiches were characterized histologically. Moreover, we assessed the DNA damage induced by green laser and was found minimal. The metrology module was prepared and tested. The elasticity/permeability device was prepared and tested with native and denuded mouse intestinal and bladder samples. Ths surgery protocol for transplantation was established. Finally, in vivo denudation of intestinal tissue was performed in live mice.
In parallel, urothelial and smooth muscle cells of mouse and human origin were mixed or printed onto or within PLMA aiming to generate engineered explants for transplantation. SMCs grow better onto stiff PLMA whereas urothelial cells grown inside forming organoids in less stiff PLMA. ICCS and BRFAA have collaborated to optimize printing conditions (fluence and wavelength for printing and polymerization of PLMA). BRFAA and ICCS have printed PLMA bioinks with mouse urothelial and smooth muscle cells and grown them successfully. Urothelial cells form organoids inside PLMA while SMCs grow very well on top of stiff PLMA surfaces.
For the development of a prototype for elasticity and permeability measurements, device specifications (explant size, stretching length, permeability assay, etc.) have been defined. A high level and a detailed design have been prepared. Some of the components have been selected and tested. Optics11 has obtained PLMA from META for initial tests. Importantly, initial permeability tests have been successful. This is an important development, because the permeability testing module is associated with a high risk.
During RP2, we prepared sandwiches of PLMA of different composition containing SMCs and UCs of mouse and human origin. These sandwiches were characterized histologically. Moreover, we assessed the DNA damage induced by green laser and was found minimal. The metrology module was prepared and tested. The elasticity/permeability device was prepared and tested with native and denuded mouse intestinal and bladder samples. Ths surgery protocol for transplantation was established. Finally, in vivo denudation of intestinal tissue was performed in live mice.
UroPrint aims to employ dual laser-assisted bioprinting based on LIFT for ex vivo and in vivo printing of pUC-laden PLMA bioinks in an operating room during surgery. Laser induced forward transfer (LIFT) will be for the first time employed for in vivo printing of bladder. ICCS/NTUA and PhosPrint group hold a PCT on the use two laser beams. Here, we will employ the dual laser beam process for the first time to print the pUCs in PLMA bioink using a 532 nm laser beam, and then to photopolymerize the PLMA using 405 nm. An interferometer system module and appropriate software will be designed and developed by PhosPrint (third party of ICCS/NTUA) to enable precise scanning along the z axis to map tissue anomalies, and to synchronize the laser beam with the high-speed stages. This way, the distance between the donor and the receiver substrate (tissue) will be constantly adjusted and kept fixed at all times during printing. The system will be equipped with a high-power imaging system to allow imaging of printing. In addition, a next generation of human-derived or patient-specific 3D anatomically-shaped scaffold will be engineered for the first time. Human blood-derived platelet lysates (PLs) have recently arisen as a source of highly biocompatible XF biomaterials with intrinsic cell-recruiting and pro-regenerative capacity. These unique features, along with platelet lysates wide availability and low-cost, renders them ideal to be applied in engineering biocomplex multi-tissue assemblies. In UroPrint, PLs will be used to create a portfolio of innovative PLMA biomaterials exhibiting unique and tunable mechanical, bioadhesive, haemostatic and bio-instructive properties that positively influence both tissue integration, tissue vascularization and regenerative efficiency. The biomaterials provided by META are fully biocompatible and biodegradable and will be developed under Good Laboratory Practice conditions. PLMA scaffolds will be functionalized with the bladder proteoglycan GAG, which forms a mucus-like layer protecting urothelial cells from directly contacting the otherwise toxic urine.
In UroPrint, we are developing a novel prototype enabling simultaneous measurements of these properties. The tensile-testing prototype allows stretching of a tissue explant (i.e. intestinal muscle, bladder muscle, scaffolds, printed tissue etc.) and is compatible with standard microscopes. By leveraging the innovative fiber optic interferometry method of O11, we can measure the elasticity of small and soft tissue samples in an incubator- and microscope-compatible device with high resolution. Furthermore, this system will allow direct printing of cells within the testing apparatus to greatly simplify the process and expedite experimentation. This state-of-the-art device will greatly increase throughput and ability in testing bladder grafts, and it will be widely applicable to other tissues or composites as well.
In UroPrint, we are developing a novel prototype enabling simultaneous measurements of these properties. The tensile-testing prototype allows stretching of a tissue explant (i.e. intestinal muscle, bladder muscle, scaffolds, printed tissue etc.) and is compatible with standard microscopes. By leveraging the innovative fiber optic interferometry method of O11, we can measure the elasticity of small and soft tissue samples in an incubator- and microscope-compatible device with high resolution. Furthermore, this system will allow direct printing of cells within the testing apparatus to greatly simplify the process and expedite experimentation. This state-of-the-art device will greatly increase throughput and ability in testing bladder grafts, and it will be widely applicable to other tissues or composites as well.