Over the past decades, many studies have been conducted to generate in vitro tissue systems that help understanding tissue development and disease progression. Hydrogel scaffolds have been frequently used in creating such models. Self-assembling peptide hydrogels are functional in providing the cells a scaffold that supports cell proliferation, however, organoid and lumen formation remains a challenge. Hydrogels can be synthesized and modified based on the essential physiological properties, which can be achieved by altering the chemical composition of the initial material. Thus, in this study, we test the effect of the laminin-derived IKVAV motif on ultrashort self-assembling peptide in relation to cell proliferation and lumen formation in colorectal cancer stem cells. Further, we test the printability of the modified peptide. The modification of ultrashort peptide serves the purpose of providing signals to direct cell adhesion, differentiation, and lumen formation. One particular combination of peptides showed the formation of colorectal organoids containing lumen of outperforming characteristics as compared to the others, also in 3D bioprinting.

No description available.

Die Photobiotechnologie nutzt photosynthesegetriebene Bioprozesse zur nachhaltigen Synthese von Wertstoffen und Energieträgern. Diese Bioprozesse rücken vor allem durch die stoffliche Nutzung von CO2 als Kohlenstoff- und Licht als regenerative Energiequelle in den Fokus von Forschung und Entwicklung. Trotz der enormen Vielfalt von geschätzten 500.000 Algenspezies werden zurzeit nur ca. 15 Mikro- und 220 Makroalgen technisch genutzt. Dieser Umstand ist u.a. dem geringen Prozessverständnis und den spezifischen Anforderungen der photobiotechnologische Prozesse an die technischen Systeme geschuldet. Im Rahmen der vorliegenden Arbeit wurden Kultivierungssysteme für die photosynthetisch aktiven Mikroorganismen Rhodobacter sphaeroides DSM158, Chlamydomonas reinhardtii 11.32b und Chlorella sorokiniana UTEX1230 entwickelt und evaluiert. Die photofermentative Wasserstoffproduktion mittels R. sphaeroides DSM158 erfolgte in einem eigens dafür konzipierten gerührten Halogen-Photobioreaktor durchgeführt. Im Satzbetrieb wurde der Einfluss des volumetrischen Leistungseintrages (P0/VL) und der mittlere Bestrahlungsstärke (I0) untersucht. Es konnte gezeigt werden, dass R. sphaeroides DSM158 bei einer durchschnittlichen I0 von 2250 W m-2 und einem P0/VL von 0,55 kW m-3 im Satzbetrieb eine maximale Wasserstoffproduktionsrate (rH2) von 195 mL L-1 h-1 erzielt. Das Reaktorsystem wurde mittels optischer Ray Tracing Simulation, einer empirischer Simulation der Strahlungsverteilung und Computational Fluid Dynamics (CFD) charakterisiert, um die Prozessbedingungen für R. sphaeroides DSM158 zu analysieren. Der photofermentative Prozess wurde in ein kontinuierliches Verfahren überführt, welches unter optimalen Bedingungen von I0 = 2250 W m-1, einer Durchflussrate von 0,096 h-1 und einem C:N-Verhältnis von ca. 22,5 eine rH2 von 170,5 mL L-1 h-1 lieferte. Für Mikroalgen wurden Kultivierungssysteme für Suspensions- und immobilisierte Kulturen entwickelt und charakterisiert. Zur Kultivierung immobilisierter Mikroalgen wurde die Methode des Green Bioprinting etabliert, die auf der 3D-Bioprinting Technologie des Tissue Engineerings beruht. Bei diesem Verfahren werden Algenzellen über einen Extrusionsprozess in ein strukturiertes Hydrogel eingebettet. In vergleichenden Studien zum Wachstum in Suspensionskulturen konnte gezeigt werden, dass die Hydrogelumgebung ideale Bedingungen für das photoautotrophe Wachstum und die Zellviabilität von C. reinhardtii 11.32b und C. sorokiniana UTEX1230 liefert. Der MicrOLED-Bioreaktor bezeichnet ein miniaturisiertes Flat-Panel-Airlift (FPA)-Bioreaktor-system mit 15 mL Arbeitsvolumen und nichtinvasiver optischer Prozessüberwachung in Bezug auf zellspezifische Parameter (Zelldichte und Fluoreszenz) und Suspensionsparameter (pH, dO2 und dCO2). Hydrodynamische Untersuchungen der miniaturisierten FPA-Kultivierungskammer zeigten vergleichbare und damit skalierbare Eigenschaften zu Labor- und Produktions-FPA-Bioreaktoren. Im Zuge des MicrOLED-Bioreaktors wurden erstmals organische Leuchtdioden für den Einsatz in Photobioreaktoren verwendet und charakterisiert. Die geometrisch komplexen Bioreaktorkomponenten wurden mittels additiver Fertigungstechnologien aus Polyamid hergestellt und erlauben die Integration der optischen Elemente zur Überwachung des Bioprozesses in Echtzeit.

Die Photobiotechnologie nutzt photosynthesegetriebene Bioprozesse zur nachhaltigen Synthese von Wertstoffen und Energieträgern. Diese Bioprozesse rücken vor allem durch die stoffliche Nutzung von CO2 als Kohlenstoff- und Licht als regenerative Energiequelle in den Fokus von Forschung und Entwicklung. Trotz der enormen Vielfalt von geschätzten 500.000 Algenspezies werden zurzeit nur ca. 15 Mikro- und 220 Makroalgen technisch genutzt. Dieser Umstand ist u.a. dem geringen Prozessverständnis und den spezifischen Anforderungen der photobiotechnologische Prozesse an die technischen Systeme geschuldet. Im Rahmen der vorliegenden Arbeit wurden Kultivierungssysteme für die photosynthetisch aktiven Mikroorganismen Rhodobacter sphaeroides DSM158, Chlamydomonas reinhardtii 11.32b und Chlorella sorokiniana UTEX1230 entwickelt und evaluiert. Die photofermentative Wasserstoffproduktion mittels R. sphaeroides DSM158 erfolgte in einem eigens dafür konzipierten gerührten Halogen-Photobioreaktor durchgeführt. Im Satzbetrieb wurde der Einfluss des volumetrischen Leistungseintrages (P0/VL) und der mittlere Bestrahlungsstärke (I0) untersucht. Es konnte gezeigt werden, dass R. sphaeroides DSM158 bei einer durchschnittlichen I0 von 2250 W m-2 und einem P0/VL von 0,55 kW m-3 im Satzbetrieb eine maximale Wasserstoffproduktionsrate (rH2) von 195 mL L-1 h-1 erzielt. Das Reaktorsystem wurde mittels optischer Ray Tracing Simulation, einer empirischer Simulation der Strahlungsverteilung und Computational Fluid Dynamics (CFD) charakterisiert, um die Prozessbedingungen für R. sphaeroides DSM158 zu analysieren. Der photofermentative Prozess wurde in ein kontinuierliches Verfahren überführt, welches unter optimalen Bedingungen von I0 = 2250 W m-1, einer Durchflussrate von 0,096 h-1 und einem C:N-Verhältnis von ca. 22,5 eine rH2 von 170,5 mL L-1 h-1 lieferte. Für Mikroalgen wurden Kultivierungssysteme für Suspensions- und immobilisierte Kulturen entwickelt und charakterisiert. Zur Kultivierung immobilisierter Mikroalgen wurde die Methode des Green Bioprinting etabliert, die auf der 3D-Bioprinting Technologie des Tissue Engineerings beruht. Bei diesem Verfahren werden Algenzellen über einen Extrusionsprozess in ein strukturiertes Hydrogel eingebettet. In vergleichenden Studien zum Wachstum in Suspensionskulturen konnte gezeigt werden, dass die Hydrogelumgebung ideale Bedingungen für das photoautotrophe Wachstum und die Zellviabilität von C. reinhardtii 11.32b und C. sorokiniana UTEX1230 liefert. Der MicrOLED-Bioreaktor bezeichnet ein miniaturisiertes Flat-Panel-Airlift (FPA)-Bioreaktor-system mit 15 mL Arbeitsvolumen und nichtinvasiver optischer Prozessüberwachung in Bezug auf zellspezifische Parameter (Zelldichte und Fluoreszenz) und Suspensionsparameter (pH, dO2 und dCO2). Hydrodynamische Untersuchungen der miniaturisierten FPA-Kultivierungskammer zeigten vergleichbare und damit skalierbare Eigenschaften zu Labor- und Produktions-FPA-Bioreaktoren. Im Zuge des MicrOLED-Bioreaktors wurden erstmals organische Leuchtdioden für den Einsatz in Photobioreaktoren verwendet und charakterisiert. Die geometrisch komplexen Bioreaktorkomponenten wurden mittels additiver Fertigungstechnologien aus Polyamid hergestellt und erlauben die Integration der optischen Elemente zur Überwachung des Bioprozesses in Echtzeit.

The goal of nerve tissue engineering is to promote and guide axon growth across a site of nerve injury without misdirection. Bioengineered tissue scaffolds have been shown to be promising for the regeneration of damaged peripheral nerves. Schwann cells play a pivotal role following nerve injury by forming aligned “bands of Büngner” that promote and guide axon regeneration into the distal nerve segment. The incorporation of living Schwann cells into various hydrogels has therefore been urged during the fabrication of tissue engineered nerve scaffolds. The aim of this research is to characterize biomaterials suitable for 3D bioplotting of nerve repair scaffolds. Here a novel technique of scaffold fabrication has been optimized to print alginate-based three-dimensional tissue scaffolds containing hyaluronic acid and living Schwann cells. Alginate/hyaluronic acid scaffolds were successfully fabricated with good printability and cell viability. Addition of the polycation polyethyleneimine (PEI) during the fabrication process stabilized the structure of alginate through the formation of a polyelectrolyte complex and had a significant influence on the degree of swelling, degradation rate, mechanical property, and release kinetics of incorporated protein within the scaffolds. A preliminary in vivo study showed the feasibility of implanting 3D printed alginate/hyaluronic acid scaffolds as nerve conduits in Sprague-Dawley (SD) rats with resected sciatic nerves. However alginate/hyaluronic acid scaffolds were found to be unsuitable for axonal regeneration. Further in vitro culture of Schwann cells was performed in collagen type-I, fibrin, fibrin/hyaluronic acid, and their combination with alginate. It was found that Schwann cells had more favorable cell morphology in fibrin/hyaluronic acid or collagen without alginate. Schwann cell proliferation and alignment were better in fibrin/hyaluronic acid. Therefore fibrin/hyaluronic acid is more ideal than most other hydrogel formulations for use in the bioprinting of nerve repair tissue engineering scaffolds, which incorporate cellular elements. As Schwann cells also align along the long axis of the printed fibrin/hyaluronic acid strands, 3D bioprinting of multiple layers of crosslinked fibrin strands can be used to fabricate a nerve conduit mimicking the bands of Büngner.

The interactions between cells and their microenvironment influence their morphological features and regulate important cellular processes. To understand deleterious neurological disorders such as Parkinson’s disease, there is an immense need to develop efficient in vitro 3D models that can recapitulate complex organs such as the brain. Ultrashort self- assembling peptides offer a revolutionary tool for generating tunable and well-defined 3D in vitro neural tissues capable of recreating complex cellular characteristics, and tissue-level responses. Herein, we describe the use of ultrashort self-assembling peptide-based scaffolds for the development of functional 3D neuronal models including an in vitro model for Parkinson’s disease. Both primary mouse embryonic dopaminergic neurons and human dopaminergic neurons derived from human embryonic stem cells were found biocompatible in our peptide-based models. Using microelectrode arrays, we recorded spontaneous activity in dopaminergic neurons encapsulated within these 3D peptide scaffolds for more than 1 month without a decrease in signal intensity. In addition, we demonstrate a 3D bioprinted model of dopaminergic neurons inspired by the mouse brain using an extrusion-based 3D robotic bioprinting technology. We used our 3D in vitro neuronal models to study the effect of both gabapentin and pregabalin on the development of dopaminergic neurons. Pregabalin and gabapentin are frequently regarded as first-line therapies for a variety of neuropathic pain syndromes, regardless of the underlying cause. Our results showed that both drugs can interfere with the neurogenesis and morphogenesis of ventral midbrain dopaminergic neurons during early brain development. Finally, to gain a better understanding of the influence of cell-cell and cell- matrix interactions on cellular behavior and function in 3D cultured cells within our peptide-based scaffolds compared to the ones cultured in 2D, we studied the metabolic and transcriptomic profiles of 2D and 3D cultured cells. 2D cultured cells exhibited distinct metabolic and transcriptomic profiles compared to the 3D cultured cells. Advancements in the fields of 3D in vitro modeling, 3D bioprinting, and biomaterials are of extreme value for the development of efficient models suitable for investigating disease-specific pathways, aiding the discovery of novel treatments, and promoting tissue regeneration.

Osteoarthritis (OA) is a progressive cartilage degeneration disease with a complex pathologic mechanism. Although OA has devastating effects on patient quality of life and places a significant burden on the healthcare system, no disease-modifying drugs have been found, and surgical treatment options are often unsustainable. 3D bioprinting is a novel field within tissue engineering that focuses on developing biocompatible constructs that can be implanted to replace an organ or tissue. Such constructs have a great potential to become treatments for OA. Understanding cell mobility within hydrogels could play a vital role in advancing the development of biocompatible constructs. However, due to the novelty of bioprinting, limited research on cell mobility within hydrogels is available. Therefore, this project aims to fill the gap in existing research regarding cell mobility within bioprinted constructs with varying mechanical properties. To achieve this goal, green fluorescent protein-tagged mesenchymal stromal cells (MSCs) were developed to assess progenitor cell mobility in bioprinted hydrogel constructs. Constructs were printed with three zones: hydrogel with embedded chondrocytes or MSCs; hydrogel spacer; and chemoattractant. Designed constructs were bioprinted (BioAssemblyBot, Advanced Solutions) using GelMA:HAMA bioinks containing photoinitiator with varying bioink percentages. Cell viability and directional mobility within constructs were assessed by fluorescence viability assay and time-lapse fluorescence microscopy. The protocol to evaluate cell mobility in bioprinted constructs and optimized bioprinting settings for GelMA:HAMA bioinks were gained through this project. Overall, this project allowed us to fill the gap in existing knowledge regarding MSC and chondrocyte mobility in hydrogels and contribute to developing a novel treatment method for OA.

Cette thèse a pour objectif de développer une méthode de bioimpression 3D de tissus vivants. Ce nouveau champ disciplinaire a pour but la fabrication de tissus grâce à une bioimprimante en s’appuyant sur les principes fondamentaux de l’ingénierie tissulaire. Pour mener à bien ces travaux, une bio-encre spécifique a été formulée à l’aide de biomatériaux naturels afin de répondre aux critères de biocompatibilité, de maintien de la viabilité cellulaire et de support pour la formation d’un réseau cellulaire en trois dimensions. Plusieurs caractérisations ont ainsi pu être réalisées afin de démontrer l’innocuité du procédé de bioimpression 3D sur les cellules utilisées.L’évolution technologique de la bioimprimante utilisée est ensuite présentée en partant d’une technologie open-source pour arriver à l’utilisation d’un bras robotique 6 axes. L’exigence du cahier des charges de cette bioimprimante a évolué au fil des différents prototypes utilisés.La dernière partie de ce travail de thèse présente les résultats de bioimpression de tissus obtenus grâce à de multiples collaborations. Plusieurs tissus seront étudiés et caractérisés : le derme et sa maturation vers une peau totale, le cartilage et la bioimpression de cellules souches mésenchymateuses, un tissu microvascularisé grâce à l’incorporation de cellules endothéliales et pour finir un tissu perfusable en utilisant une approche de culture dynamique en bioréacteur / This thesis focus on the development of a 3D bioprinting process for living tissue. This new field of research, 3D bioprinting, aims to fabricate tissues using a bioprinter based on the tissue engineering fundamentals.To carry out this work, a specific bioink was formulated using natural biomaterials to meet the requirement of biocompatibility, cell viability and support of a three-dimensional cellular network. Several characterizations have been used to demonstrate the cells viability during the 3D bioprinting process.The bioprinter technological evolution is then presented, starting from an open-source technology and ending with the use of a 6-axis robotic arm. The specifications of this bioprinter evolved through different prototypes.The last part of this thesis concerns tissue bioprinting results obtained through multiple collaborations. Several tissues will be studied and characterized: the dermis and its maturation towards a total skin, the cartilage and the mesenchymal stem cells bioprinting, a microvascularized tissue thanks to the incorporation of endothelial cells and finally a perfusable tissue by using a dynamic culture approach in bioreactor

Cartilage tissue engineering has been emerging as a promising therapeutic approach, where engineered constructs or scaffolds are used as temporary supports to promote regeneration of functional cartilage tissue. Hybrid constructs fabricated from cells, hydrogels, and solid polymeric materials show the most potential for their enhanced biological and mechanical properties. However, fabrication of customized hybrid constructs with impregnated cells is still in its infancy and many issues related to their structural integrity and the cell functions need to be addressed by research. Meanwhile, it is noticed that nowadays monitoring the success of tissue engineered constructs must rely on animal models, which have to be sacrificed for subsequent examination based on histological techniques. This becomes a critical issue as tissue engineering advances from animal to human studies, thus raising a great need for non-invasive assessments of engineered constructs in situ. To address the aforementioned issues, this research is aimed to (1) develop novel fabrication processes to fabricate hybrid constructs incorporating living cells (hereafter referred as “construct biofabrication”) for cartilage tissue regeneration and (2) develop non-invasive monitoring methods based on synchrotron X-ray imaging techniques for examining cartilage tissue constructs in situ. Based on three-dimensional (3D) printing techniques, novel biofabrication processes were developed to create constructs from synthetic polycaprolactone (PCL) polymer framework and cell-impregnated alginate hydrogel, so as to provide both structural and biological properties as desired in cartilage tissue engineering. To ensure the structural integrity of the constructs, the influence of both PCL polymer and alginate was examined, thus forming a basis to prepare materials for subsequent construct biofabrication. To ensure the biological properties, three types of cells, i.e., two primary cell populations from embryonic chick sternum and an established chondrocyte cell line of ATDC5 were chosen to be incorporated in the construct biofabrication. The biological performance of the cells in the construct were examined along with the influence of the polymer melting temperature on them. The promising results of cell viability and proliferation as well as cartilage matrix production demonstrate that the developed processes are appropriate for fabricating hybrid constructs for cartilage tissue engineering. To develop non-invasive in situ assessment methods for cartilage and other soft tissue engineering applications, synchrotron phase-based X-ray imaging techniques of diffraction enhanced imaging (DEI), analyzer based imaging (ABI), and inline phase contrast imaging (PCI) were investigated, respectively, with samples prepared from pig knees implanted with low density scaffolds. The results from the computed-tomography (CT)-DEI, CT-ABI, and extended-distance CT-PCI showed the scaffold implanted in pig knee cartilage in situ with structural properties more clearly than conventional PCI and clinical MRI, thus providing information and means for tracking the success of scaffolds in tissue repair and remodeling. To optimize the methods for live animal and eventually for human patients, strategies with the aim to reduce the radiation dose during the imaging process were developed by reducing the number of CT projections, region of imaging, and imaging resolution. The results of the developed strategies illustrate that effective dose for CT-DEI, CT-ABI, and extended-distance CT-PCI could be reduced to 0.3-10 mSv, comparable to the dose for clinical X-ray scans, without compromising the image quality. Taken together, synchrotron X-ray imaging techniques were illustrated promising for developing non-invasive monitoring methods for examining cartilage tissue constructs in live animals and eventually in human patients.

In this diploma thesis we are discussing about using 3D bioprinting in tissue engineering. We are discribing using biomaterials for construction scaffolder and aplication stem cells in 3D bioprinting. Last section of theoretical part deals with very often used techniques of 3D bioprinting and we are focused on extrusion technique. In the practical part we propose a method for print vasculars structures. We realized prototype of print head, her design and 3D printing of individual parts. To mechanical part we create a control system for printing control. At the end we visualize the organization of the cells using program modules.