The aim of subproject A01 is the systematic investigation and optimization of adhesive, microstructural as well as microscopic and macroscopic mechanical properties of biofabricated hierarchical tissue analogues based on alginate dialdehyde (ADA). This also includes optimizing the temporal degradation of the alginate matrix and its replacement by a cell-generated extracellular matrix. Our long-term goal is to achieve tight control over the temporal changes in adhesive, mechanical and structural properties and the associated cell-biological behavior in alginate-based biofabricated tissues.
A02 | Hyaluronic acid based hydrogel platform with multi-functional cross-linkers for the controlled differentiation of mesenchymal stem cells
Hyaluronic acid (HyA) represents a major component of the human extracellular matrix, however, so far highly versatile HyA-based materials for biofabrication are lacking. The overall aim of this project is, to develop a HyA-based bioink platform utilizing branched multi-functional cross-linkers (PEG, polyglycidol) that enables the controlled differentiation of mesenchymal stem cells. The modification with peptides and growth factors will add further biofunctionality to the printed 3D constructs in order to improve chondrogenic and adipogenic differentiation of the stem cells. For the long term, this work aims to generate bioinks for the development of clinically relevant tissue models.
This project aims at developing a synthetic, cell-compatible and modular bioink-platform. Diblock copolymers based on poly(2-oxazoline)s will be deployed exhibiting suitable thermo- and shearresponsive behavior. This novel bioink platform will be modified by a dual-crosslinking mechanism to (i) guarantee mechanical stability after printing and (ii) to modulate the stiffness of the ink during cell-culture experiments.
A04 | Expansion of the biofabrication window using 2.5D scaffolds made from (AB)n-segmented copolymers
The aim of the project is the expansion of the biofabrication window of bioinks. By using 2.5D scaffolds – a structurally stable substrate – for the bioprinting process a better precision and reproducibility of the bioink depostion can be achieved. For this purpose, new (AB)n-segmented copolymers with hydrophilic soft segments and supramolecular hard segments are developed, which can be processed by Melt Electrospinning Writing. By selective swelling with biological media hydrogel scaffolds are formed covering a wide range of Young’s Moduli and enabling different cellular interactions. Until today, this cannot be realized with conventional biofabrication processes.
A06 | Cell-loaded microgels as mechanical protection and controlled microenvironment for cells in bioinks
Aim of this project is the generation of cell-loaded microgels with uniform size and their establishment as additive for bioinks. The microgels shall on the one side act as mechanical protection from shear-forces during printing and thereby extend the biofabrication window towards bioinks with higher viscosity and towards higher shear forces. In addition to that, biochemical functionalization and adjusting microgel degradability shall be used to tailor the microenvironment of the encapsulated cells and control their behavior. Prospectively, this project aims at establishing suspensions of cell-loaded microgels as bioink especially for the simultaneous printing of different cell types.
The subproject deals with the bilateral task of improved shape fidelity and cell viability in biofabrication by applying fiber-reinforced hydrogels as bioinks. Thereby, fiber fragments which can be used both, as cell carriers and for matrix-reinforcement, are produced by electrospinning. Strikingly, the morphology of the fibers plays a significant role for cell behavior (adhesion, proliferation, alignment) as well as for the rheological and mechanical properties of the composite (flow behavior, spreading kinetics, strength), which will be investigated analytically within the project. The obtained results will allow for a systematic improvement of bioinks, which can furthermore be transferred to other materials.
Ultra-soft matrices are challenging for cell culture due to their poor structural properties. However, it is known that they promote excellent interconnection between neurons and create neural networks. This project proposes to use highly resolved 3D-printed structures to mechanically stabilize ultra-soft matrices so that 3D electrophysiology can be performed on neural networks. In line with neural networks, controlled migration of glioblastoma tumor cells through similar ultra-soft matrix/fiber composites will also be investigated so that neurons, astrocytes and tumor cells can be used in the future as an in vitro model for glioblastoma research.
B02 | Pre-endothelialized perfusable microvascular networks for biofabrication of standardized in vitro tissue models.
The aim of this project is biofabrication of perfusable and fully pre-endothelialized microvascular networks, which will be produced by MEW of thermosensitive polyoxazolines and subsequently embedded in cell-containing hydrogels. Particular focus will be placed on biocompatibility of hydrogels (bioinks) and selection of geometries compatible with microscopic techniques. As microvascular endothelium provides an important barrier and functional regulation of physiological processes, we will extensively investigate the efficacy of colonization and network functionality. In longer perspective, these models will be adapted for tissue-specific and tumor research.
Due to the complexity of bioprinted constructs, the supply of the embedded cells with nutrients, as well as maturation of the construct to a functional tissue, tailored conditions must be maintained. The technological focus of TP B03 is on the integration of the bioprinting process in a functional bioreactor system where the bioconstruct and the bioreactor are printed simultaneously. By biofabrication of tissue models based on hydrogel composite materials and by generation of a suitable bioreactor platform, the feasibility of this concept will be shown. Furthermore, functional biofabrication of muscle tissue will demonstrate for the efficient generation of personalized specific 3D construct.
Aim of this project is the biofabrication of artificial vascular structures with correct hierarchical organization and wall morphology consisting of intima, media and adventitia. This shall be reached by the exploitation of the cellular plasticity of vascular wall-resident stem cells (VW-SCs). These cells have been shown to possess the necessary cellular plasticity to deliver such structures. By the printing of VW-SC loaded bioinks into self-healing support gels, the use of vessel wall specific matrix components and perfusion of the structures, we aim on a medium to long term to biofabricate complex and hierarchically organized vascular structures.
B05 | Glycoengineering as a tool to control the behavior of mesenchymal stem cells in biofabrication processes
The modification of the microenvironment of cells by using glycosyltransferases / glycosidases and synthetic sugar structures represents a new tool for the development of bioinks as well as for the transient modification of cell surfaces. Bioinks will be functionalized by glycoengineering in such a way that cells selectively adhere and differentiate. On the other hand, the extracellular glycocalyx will be modified and / or a protective polysaccharide layer will be added to protect cells from shear forces arising during bioprinting. The long-term goal is to modify cells and bioinks by glycoengineering in such a way that the post-production behavior of cells is optimized in biofabricated constructs.
B06 | Reporter constructs and reporter cells for the investigation of cellular stress reply and signal transduction during biofabrication
The goal of B06 is to understand which stresses cells experience during the bioprinting process and in the maturing biofabricates, and how these stresses affect cell behavior. Using lentiviral, fluorescent reporter constructs and reporter cells we will monitor various stress parameters in different cell types in response to bioplotting and inkjet printing conditions, specific integrin-ligation and matrix-stiffness in alginate based biofabricates. We will correlate the stress responses with the activities of specific integrin-dependent signaling processes in order to identify, analyze and modulate molecular mechanisms that determine the cellular response to stresses in alginate based biofabricates.
B07 | Development of a micro particle sensor system to establish correlations between mechanical stress and cell functionality during biofabrication
Hydrodynamic forces during printing can cause enduring damage to living cells, their strength is however largely unknown. Here we will develop sensor particles (microcapsules, microgels) which in combination with computer models will close this knowledge gap. The sensor particles will give insight into the mechanical deformation while the closely corresponding simulations will allow us to calculate resulting mechanical stresses. Finally, by comparing with living cells we will be able to establish correlations between deformation, stress and cell damage during bioprinting.
B08 | Time-resolved biophotonics approach cellular signaling, cell-matrix interactions and matrix remodeling mechanisms in biofabricated constructs
In this optical engineering and bioprocess engineering project we aim to develop new systems to study in situ maturation and regulation of cell-matrix junctions of biofabricated constructs over long time periods. To this end, (i) we will advance high resolution/depth penetrating multiphoton- and light sheet technology towards a 2-photon single plane illumination microscopy (2P-SPIM), and (ii) design and engineer precision positioned mobile mini-bioreactors. We will validate these systems for selected bioconstructs within the consortium regarding (iii) visualization of 3D spatial patterning of focal adhesion proteins between cell and matrix as well as matrix production during long-term maturation of constructs.
Heart diseases are a major socioeconomic burden. Despite considerable progress in the prevention and minimization of heart damage, the prevalence of heart insufficiency increases continuously. The aim of this subproject is the reproducible production of human cardiac substitute tissue based on a scaffold with adjustable mechanical properties from spider silk bioink and cardiac cells using 3D printing technology. The long-term goal is the treatment of heart diseases.
C02 | Biofabrication of a 3D model for the functional investigation of the stromal parameters on the behavior of breast cancer cells
Project C02 aims at the biofabrication of a complex tumor-stroma model to specifically study the behavior of breast cancer cells as a function of stromal parameters. To achieve this goal, innovative 3D printing technologies will be applied to combine breast cancer and stromal cells in bioink matrices with independently controllable physicochemical features. This modular approach allows us to recapitulate relevant aspects of the tumor microenvironment and to gain important mechanistic insights into how tumor-stroma interactions contribute to tumorigenesis.
C03 | Analysis of tumor dormancy and progression in biofabricated and in vivo vascularized 3D models
Using biofabrication methods a 3D tumor model will be developed, serving for the investigation of different aspects of tumor progression in a controlled manner both in vitro and in the vascularized in vivo AV loop model. In particular, with the establishment of a such highly-defined biofabricated melanoma tumor model essential tumor properties, such as tumor dormancy and metastasis, but also the influence of the tumor microenvironment can be analyzed and specifically modified and controlled.
C04 | Biofabrication of cellularized and by the AV-loop vascularized tissue container for the transplantation of cells producing therapeutic proteins
Aim of the project is the development of a broadly applicable transplantable tissue container which enables the in vivo production of recombinant therapeutic proteins, e.g. antibodies. For this a container shell will be filled with two cell doted matrix cylinders structured with tubes. The cylinders contains a bed in which a AV loop can be placed which sprouts into the tube system to ensure the vascularization of the container and producer cell maintenance. Container vascularization in the bioreactor, container immune tolerance and container evaluation in pre-clinical models are the subjects of future funding periods.