Periodic Reporting for period 1 - DOMES (Directed Orchestration of Microfluidic Environments for guided Self-organisation)
Período documentado: 2022-06-01 hasta 2023-11-30
Congenital anomalies of the kidneys and urinary tracts (CAKUT) constitute approximately 20 to 30% of all identified birth defects. CAKUTs are the most common cause of pediatric chronic kidney disease (CKD). Although genetic disorders play a major role in CAKUT, approximately 70% of all congenital diseases cannot directly be linked to a specific genetic mutation. In such cases, environmental factors play an important role in manifestation of these anomalies. During kidney development, the ureteric bud (UB) undergoes repeated rounds of branching and simultaneously induces the metanephric mesenchyme to form preliminary nephrons around its UB branch tips. This fundamental process, called branching morphogenesis, establishes a spatial architecture of the mature kidney and urinary tract. Any disturbances in this process, caused by maternal/fetal exposure to teratogens and environmental toxicants, leads to the spectrum of abnormalities in CAKUT. Studying the impact of potential teratogens on developing kidneys is challenging due to obstacles in obtaining a substantial number of embryonic kidneys and difficulties in ex vivo culturing. In vitro models that mimic kidney development are essential to provide better insights in disease modeling. UB organoids, characterized by their tree-like tubular structures, accurately mimic the branching morphogenesis and spatial architecture of the kidney. Current UB organoid models have some challenges that hamper their widespread application. In the following, we highlight our approach to overcome them:
• Our project aims at the development of a commercial microfluidic platform, which is easy to use even in non-specialized laboratories. The DOMES product provide a new toolbox to position in vitro UB models in specific confinements and to expose them to biochemical and biophysical factors in a controlled manner. This unique approach proves advantageous for the medium- to high-throughput quantification of image-based parameters such as whole-organoid morphology and growth rate.
• Our novel in vitro model for kidney branching morphogenesis fabricated from thin, transparent polymer films enabling high-resolution imaging and a real-time assessment of the effect of chemicals on this intricate process.
• Current UB organoid models rely on conventional static cultures, with regular media exchanges performed manually by researchers. Unlike in vivo, where the flow of both nutrients and teratogens is constant in flow, static cultures cannot mimic the fluctuating exposure of in vitro UB organoids to possible chemical compounds. To address this challenge, we established a microfluidic platform for a continuous and active delivery of soluble factors, which also facilitates the administration of drugs with a short half-life.
Objectives
The overall aim is screening of ureteric bud related teratogens with four main objectives:
Objective 1. Design and fabrication of a microfluidic DOMES product family to position budding and branching UB organoids in microporous compartments
Objective 2. Development of a reliable protocol to generate PSCs-derived ureteric bud organoids in the porous compartments (milliwells) of the DOMES product family
Objective 3. Assess the influence of a small library of chemical compounds on UB branching morphogenesis
Objective 4. Development of a microphysiological DOMES system which enables the dynamic culture and control of local concentrations of soluble factors
• Our project aims at the development of a commercial microfluidic platform, which is easy to use even in non-specialized laboratories. The DOMES product provide a new toolbox to position in vitro UB models in specific confinements and to expose them to biochemical and biophysical factors in a controlled manner. This unique approach proves advantageous for the medium- to high-throughput quantification of image-based parameters such as whole-organoid morphology and growth rate.
• Our novel in vitro model for kidney branching morphogenesis fabricated from thin, transparent polymer films enabling high-resolution imaging and a real-time assessment of the effect of chemicals on this intricate process.
• Current UB organoid models rely on conventional static cultures, with regular media exchanges performed manually by researchers. Unlike in vivo, where the flow of both nutrients and teratogens is constant in flow, static cultures cannot mimic the fluctuating exposure of in vitro UB organoids to possible chemical compounds. To address this challenge, we established a microfluidic platform for a continuous and active delivery of soluble factors, which also facilitates the administration of drugs with a short half-life.
Objectives
The overall aim is screening of ureteric bud related teratogens with four main objectives:
Objective 1. Design and fabrication of a microfluidic DOMES product family to position budding and branching UB organoids in microporous compartments
Objective 2. Development of a reliable protocol to generate PSCs-derived ureteric bud organoids in the porous compartments (milliwells) of the DOMES product family
Objective 3. Assess the influence of a small library of chemical compounds on UB branching morphogenesis
Objective 4. Development of a microphysiological DOMES system which enables the dynamic culture and control of local concentrations of soluble factors
Methodology
The research described involves a multi-step methodology for studying the differentiation of PSCs into UB organoids and assessing the effects of chemical compounds on their development in a new microfluidic platform (DOMES)
Step 1. Fabrication of the polymer film-based milliwell array
Polymer film-based milliwells were created by thermoforming using newly developed microstructured moulds and a custom-built setup.
Step 2. Generation of the PSCs-derived ureteric bud organoids in milliwell arrays
We used established differentiation protocols to generate UB organoids. Gene and protein expression analyses were utilized to validate the expression of UB-specific markers. Subsequently, harvested progenitors were dissociated and seeded on milliwell arrays.
Step 3. Assess the influence of chemical compounds on kidney branching morphogenesis
A panel of compounds with diverse chemical and pharmacological properties was applied to the UB organoids. The treatment duration and concentrations of reagents selected according to their chemical characteristics. Fluorescence imaging data were analyzed for morphological changes.
Step 4. Fabrication of microphysiological system from thin and transparent polymer films
Finally, the study involved the fabrication and optimization of a microphysiological system. Using proprietary microfabrication techniques, a system composed of a central UB compartment, microchannels, and porous walls was established. Computational modeling and flow visualization techniques have been employed to establish chip design and modulate flow parameters. Establishing the microphysiological system has encountered delays primarily due to the ongoing need for optimization. Specifically, the microphysiological system requires further refinement to effectively guide the differentiation and generation of branched structures in UB organoids.
Main Achievements
Our new in vitro model, fabricated from thin and highly transparent polymer films, facilitates high-resolution imaging, thereby enabling the real-time tracking of individual organoids through live-cell time-lapse imaging. This study marks the first-ever teratogenicity assessment of kidney organoids, offering timely and cost-efficient solutions for the discovery of potential kidney disruptors and prevention of CAKUT. We are currently in the process of preparing two open-access papers that will help attract the interest of future collaborators and investors.
The research described involves a multi-step methodology for studying the differentiation of PSCs into UB organoids and assessing the effects of chemical compounds on their development in a new microfluidic platform (DOMES)
Step 1. Fabrication of the polymer film-based milliwell array
Polymer film-based milliwells were created by thermoforming using newly developed microstructured moulds and a custom-built setup.
Step 2. Generation of the PSCs-derived ureteric bud organoids in milliwell arrays
We used established differentiation protocols to generate UB organoids. Gene and protein expression analyses were utilized to validate the expression of UB-specific markers. Subsequently, harvested progenitors were dissociated and seeded on milliwell arrays.
Step 3. Assess the influence of chemical compounds on kidney branching morphogenesis
A panel of compounds with diverse chemical and pharmacological properties was applied to the UB organoids. The treatment duration and concentrations of reagents selected according to their chemical characteristics. Fluorescence imaging data were analyzed for morphological changes.
Step 4. Fabrication of microphysiological system from thin and transparent polymer films
Finally, the study involved the fabrication and optimization of a microphysiological system. Using proprietary microfabrication techniques, a system composed of a central UB compartment, microchannels, and porous walls was established. Computational modeling and flow visualization techniques have been employed to establish chip design and modulate flow parameters. Establishing the microphysiological system has encountered delays primarily due to the ongoing need for optimization. Specifically, the microphysiological system requires further refinement to effectively guide the differentiation and generation of branched structures in UB organoids.
Main Achievements
Our new in vitro model, fabricated from thin and highly transparent polymer films, facilitates high-resolution imaging, thereby enabling the real-time tracking of individual organoids through live-cell time-lapse imaging. This study marks the first-ever teratogenicity assessment of kidney organoids, offering timely and cost-efficient solutions for the discovery of potential kidney disruptors and prevention of CAKUT. We are currently in the process of preparing two open-access papers that will help attract the interest of future collaborators and investors.
Approximately 300.000 newborns die within 28 days of birth due to congenital disorders every year. Current methods of screening congenital anomalies in developing embryos remain limited and lack accurate representation of the in vivo environment. Using our DOMES prototypes, we have identified the teratogenic potential of several chemical compounds for the first time, marking a significant breakthrough in this field. However, to advance this successful model towards a CAKUT-on-chip system, further research is essential. The intricacies of achieving optimal conditions for DOMES have proven more complex and time-consuming than initially anticipated. This optimization process is crucial to ensure the accurate replication of physiological responses and the development of intricate UB-branched structures. Overcoming these challenges is pivotal for the overall success of the system, necessitating additional time and effort to enhance chip conditions for the desired outcomes. Success in this endeavor relies on addressing these optimization hurdles, further research, and dedicating resources to refine and improve the system for broader applications. A careful study of market potential and business opportunities has shown that a service-oriented business case would be more likely to succeed. A CRO construction, such as preclinical drug discovery, involving the modular DOMES devices could fill the current gap of the much-needed alternatives to animal models and provide consistency and standardization in the splintered 3D cell model market.