Bioprinting Method for Fabricating Vascularized Organoids in Regenerative Medicine Applications

Abstract

Abstract: The present invention discloses a bioprinting method for the fabrication of vascularized organoids, enabling the development of complex, three-dimensional tissues with integrated vascular networks for use in regenerative medicine. By using bioinks containing endothelial and supporting stromal cells, along with biodegradable scaffold materials, the invention enables the creation of tissue structures with precise vascular architecture and functionality. This method provides a scalable and reproducible approach for creating vascularized organoids applicable in tissue transplantation, disease modeling, and high-throughput drug screening.

Specification

Description:Background of the Invention:
In regenerative medicine, the creation of functional tissues and organs for transplantation and disease modeling has been limited by the lack of a vascular network within engineered tissues. Vascularization is crucial for supplying oxygen, nutrients, and removing waste, and without it, larger tissue constructs face issues with cell death and poor integration post-transplantation. Traditional methods of organoid culture have difficulty achieving proper vascularization, thus limiting their functionality and scalability.
Bioprinting technology offers a promising solution by enabling precise spatial deposition of cells and biomaterials to fabricate complex tissue structures with built-in vascular networks. However, existing bioprinting techniques have not yet optimized the creation of vascularized organoids that can support long-term tissue survival and integration in vivo. This invention aims to address these limitations by presenting a bioprinting method that incorporates endothelial cells, supporting stromal cells, and vascularized scaffold materials, which together promote the formation of functional vasculature within the printed tissue.
Summary of the Invention:
The invention provides a bioprinting method for the fabrication of vascularized organoids. Key innovations include:
1. Bioink Composition with Endothelial and Supporting Cells:
o Use of a novel bioink containing endothelial cells and supportive stromal cells, such as fibroblasts or mesenchymal stem cells (MSCs), to promote vascular network formation.
o Customizable bioink formulations for specific tissue types, enabling compatibility with liver, kidney, heart, and other organoid types.
2. Layer-by-Layer Bioprinting for Vascular Structure:
o A layer-by-layer printing method that enables the sequential deposition of bioinks containing vascular and parenchymal (functional tissue) cells, allowing precise control of vascular network architecture.
o Introduction of hollow channels within the tissue structure to mimic blood vessels, later populated by endothelial cells to form a continuous vascular lumen.
3. Scaffold Materials for Structural Support and Biodegradation:
o Use of biodegradable scaffold materials that provide initial structural support and gradually degrade, leaving behind an extracellular matrix (ECM)-like structure conducive to vascularization.
o Scaffold materials are tailored for each organoid type, enabling customization for mechanical and biochemical compatibility with specific tissues.
4. Growth Factors and ECM Components for Enhanced Vascular Maturation:
o Incorporation of growth factors, such as VEGF (vascular endothelial growth factor) and FGF (fibroblast growth factor), and ECM components to enhance the differentiation and stabilization of endothelial cells within the printed vascular network.
o Gradual release of growth factors to promote vascular network maturation and reduce reliance on external supplementation.
5. Maturation and Perfusion Bioreactors:
o Use of specialized bioreactors that provide perfusion and nutrient flow to the printed organoids, supporting vascular maturation and functionality.
o Perfusion bioreactors mimic physiological blood flow, enhancing tissue integration and viability for transplantation or testing applications.
Detailed Description of the Invention:
1. Bioink Composition and Preparation:
The invention uses bioinks containing endothelial cells and supporting stromal cells. Endothelial cells facilitate vascular network formation, while stromal cells, such as fibroblasts or MSCs, provide structural support and secrete ECM components that support tissue integrity.
For example, in a liver organoid, a mixture of hepatic cells, endothelial cells, and MSCs is used. The bioink is enriched with ECM components like collagen, laminin, and hyaluronic acid to improve cell attachment and viability. Additionally, the bioink contains specific growth factors (VEGF, FGF) encapsulated within biodegradable microparticles, allowing a controlled release that promotes vascular differentiation and maturation over time.
2. Layer-by-Layer Bioprinting Process:
The bioprinting method utilizes a layer-by-layer approach to build up the organoid structure. Each layer contains a specific arrangement of parenchymal cells and vascular bioinks. Vascular bioinks are deposited in a pre-defined pattern to form branching vascular channels, while parenchymal bioinks containing tissue-specific cells fill the surrounding space. This sequential deposition enables precise control over the spatial organization of vascular and functional tissue cells.
Hollow channels are created using a sacrificial bioink that is later dissolved, leaving a lumen that can be lined with endothelial cells to form blood vessel-like structures. This provides a pathway for blood or nutrient perfusion, facilitating cell survival and function throughout the organoid.
3. Biodegradable Scaffold Materials:
The invention incorporates a biodegradable scaffold material made from polymers such as polylactic acid (PLA), polyglycolic acid (PGA), or gelatinmethacryloyl (GelMA), depending on the target tissue type. These materials provide structural support during early stages of vascular formation and gradually degrade, allowing the development of a native-like extracellular matrix that supports long-term tissue viability.
The scaffold materials are tailored to each tissue type, providing specific mechanical and biochemical properties that match the target organoid. For example, a cardiac organoid scaffold is designed to match the elasticity of heart tissue, while a kidney organoid scaffold is optimized for filtration functions.
4. Growth Factor and ECM Integration:
Growth factors such as VEGF and FGF are essential for promoting vascular network formation and are incorporated directly into the bioink or scaffold material in the form of microspheres. These microspheres enable a sustained release of growth factors, providing a controlled environment for endothelial differentiation and vascular maturation.
ECM components, including fibronectin and collagen, are also added to the bioink to enhance cell adhesion and support structural integrity. The ECM composition can be adjusted based on the tissue type, allowing the creation of liver, kidney, or cardiac-specific matrices that enhance organoid function.
5. Perfusion Bioreactors for Organoid Maturation:
After printing, the vascularized organoids are placed in perfusion bioreactors that provide a controlled flow of nutrients and oxygen, mimicking physiological conditions. This setup promotes vascular network integration, allowing for further maturation of the vascular structure and the functional parenchymal tissue.
The bioreactors apply gentle perfusion to stimulate blood flow-like conditions, encouraging endothelial cells to align and form tight junctions that mimic capillary structures. These bioreactors can be configured to mimic different physiological conditions, enhancing the specificity of the organoid for various regenerative medicine applications.
, Claims:Claims:
1. A method of bioprinting vascularized organoids comprising:
o The use of bioinks containing endothelial cells and stromal cells.
o Layer-by-layer deposition of bioinks to create a network of vascular channels within a parenchymal matrix.
o Hollow channel formation to serve as blood vessel analogs within the tissue structure.
2. The method of claim 1, wherein growth factors are incorporated into the bioink or scaffold to promote vascular differentiation and network stabilization.
3. The method of claim 1, further comprising a biodegradable scaffold material that supports initial structural integrity and gradually degrades to allow ECM deposition and tissue maturation.
4. A perfusion bioreactor for vascularized organoid maturation, configured to supply nutrients and oxygen and to simulate physiological blood flow.
5. A vascularized organoid fabricated by the method of claim 1, for use in regenerative medicine, drug screening, or disease modeling.

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