With the aim of understanding and recapitulating cellular interactions of hepatocytes in their physiological microenvironment and to generate an artificial 3D in vitro model, a co-culture system ...using 3D extrusion bioprinting was developed. A bioink based on alginate and methylcellulose (algMC) was first shown to be suitable for bioprinting of hepatocytes; the addition of Matrigel to algMC enhanced proliferation and morphology of them in monophasic scaffolds. Towards a more complex system that allows studying cellular interactions, we applied core-shell bioprinting to establish tailored 3D co-culture models for hepatocytes. The bioinks were specifically functionalized with natural matrix components (based on human plasma, fibrin or Matrigel) and used to co-print fibroblasts and hepatocytes in a spatially defined, coaxial manner. Fibroblasts acted as supportive cells for co-cultured hepatocytes, stimulating the expression of certain biomarkers of hepatocytes like albumin. Furthermore, matrix functionalization positively influenced both cell types in their respective compartments by enhancing their adhesion, viability, proliferation and function. In conclusion, we established a functional co-culture model with independently tunable compartments for different cell types via core-shell bioprinting. This provides the basis for more complex in vitro models allowing co-cultivation of hepatocytes with other liver-specific cell types to closely resemble the liver microenvironment.
liver models allow the investigation of the cell behavior in disease conditions or in response to changes in the microenvironment. A major challenge in liver tissue engineering is to mimic the ...tissue-level complexity: besides the selection of suitable biomaterial(s) replacing the extracellular matrix (ECM) and cell sources, the three-dimensional (3D) microarchitecture defined by the fabrication method is a critical factor to achieve functional constructs. In this study, coaxial extrusion-based 3D bioprinting has been applied to develop a liver sinusoid-like model that consists of a core compartment containing pre-vascular structures and a shell compartment containing hepatocytes. The shell ink was composed of alginate and methylcellulose (algMC), dissolved in human fresh frozen plasma. The algMC blend conferred high printing fidelity and stability to the core-shell constructs and the plasma as biologically active component enhanced viability and supported cluster formation and biomarker expression of HepG2 embedded in the shell. For the core, a natural ECM-like ink based on angiogenesis-supporting collagen-fibrin (CF) matrices was developed; the addition of gelatin (G) enabled 3D printing in combination with the plasma-algMC shell ink. Human endothelial cells, laden in the CFG core ink together with human fibroblasts as supportive cells, formed a pre-vascular network in the core in the absence and presence of HepG2 in the shell. The cellular interactions occurring in the triple culture model enhanced the albumin secretion. In conclusion, core-shell bioprinting was shown to be a valuable tool to study cell-cell-interactions and to develop complex tissue-like models.
One of the key challenges in osteochondral tissue engineering is to define specified zones with varying material properties, cell types and biochemical factors supporting locally adjusted ...differentiation into the osteogenic and chondrogenic lineage, respectively. Herein, extrusion-based core-shell bioprinting is introduced as a potent tool allowing a spatially defined delivery of cell types and differentiation factors TGF-β3 and BMP-2 in separated compartments of hydrogel strands, and, therefore, a local supply of matching factors for chondrocytes and osteoblasts. Ink development was based on blends of alginate and methylcellulose, in combination with varying concentrations of the nanoclay Laponite whose high affinity binding capacity for various molecules was exploited. Release kinetics of model molecules was successfully tuned by Laponite addition. Core-shell bioprinting was proven to generate well-oriented compartments within one strand as monitored by optical coherence tomography in a non-invasive manner. Chondrocytes and osteoblasts were applied each in the shell while the respective differentiation factors (TGF-β3, BMP-2) were provided by a Laponite-supported core serving as central factor depot within the strand, allowing directed differentiation of cells in close contact to the core. Experiments with bi-zonal constructs, comprising an osteogenic and a chondrogenic zone, revealed that the local delivery of the factors from the core reduces effects of these factors on the cells in the other scaffold zone. These observations prove the general suitability of the suggested system for co-differentiation of different cell types within a zonal construct.
The present thesis aimed to fabricate a liver sinusoid model by combining different components and techniques to closely mimic the physiological microenvironment of the hepatic cells. Since liver is ...a complex multicellular organ, 3D extrusion bioprinting was employed as well as core-shell 3D bioprinting for creating more complex constructs with relevant physiological microarchitecture to the in vivo liver sinusoid. Figure 1 illustrates the concept of the aimed model fabrication and the combination of components required to achieve this model. To create an initial model, HepG2, a human carcinoma-derived liver cell line was used as a model cell line for hepatocytes. Biocompatible inks based on a printable alginate-methylcellulose (algMC) blend were aimed to be developed for the encapsulation of hepatocytes by functionalization with bioactive molecules to better recapitulate the hepatocytes biochemical microenvironment, supporting cellular functions. Towards tissue complexity, a further aim was to employ core-shell bioprinting to establish a coculture model of hepatocytes and fibroblasts, which acted as supportive cells; by coaxially printing HepG2 encapsulated in the shell with fibroblasts in the core of a single core-shell strand. Different bioinks were investigated as core for the fibroblasts encapsulation, whereby plasma and fibrin were utilized to functionalize the algMC blends with the aim of enhancing the fibroblasts attachment, proliferation and spreading. Moreover, the influence of functionalized core bioinks on the hepatocytes performance and function would be demonstrated, as well as the influence of the coculture with the fibroblasts. As a final step towards integrating vascularized structures in the liver sinusoid model, endothelial cells (EC) were to be cocultured with the supporting fibroblasts in the core of the core-shell constructs to create a triple-culture model with the hepatocytes in the shell. Based on collagen-fibrin matrices, suitable to support angiogenesis, a natural extracellular matrix-like Introduction 5 core bioink, which is independently printable and allows for the printing of stable core-shell vascularized constructs is aimed to be developed. The culture and coculture parameters of the ECs will be optimized and evaluated. Optimization of the shell bioink encapsulating the hepatocytes is aimed to be investigated with the goal of enhancing the HepG2 microenvironment. Printing parameters and crosslinking procedures as well as culture conditions for all the cells were to be optimized for the model. The final triple-culture core-shell printed in vitro model is aimed to characterize the ability of this triple-culture construct to support vascularization by the HUVECs printed in the core, the physiological functions of the hepatocytes printed in the shell bioink, as well as to evaluate the cellular interactions between core and shell compartments. Through engineering and modifying the bioinks which represent the extra-cellular matrix and adjusting the culture conditions for the cells, cell-cell and cell-matrix interactions can be studied in such coculture models, providing new insights towards clinical and therapeutic biomedical applications.:Abbreviations 1
1 Motivation 3
2 Introduction and state of the art 6
2.1 Liver and its microarchitecture 6
2.2 Tissue Engineering 9
2.2.1 Liver Tissue Engineering 9
2.3 From two- to three-dimensional cell cultures 10
2.3.1 2D and 3D hepatocytes coculture models 12
2.4 3D Bioprinting 14
2.4.1 Biomaterial inks and bioinks for 3D bioprinting 15
2.4.2 Core-shell 3D bioprinting 17
2.4.3 3D bioprinting of in vitro liver models 18
3 Materials and methods 20
3.1 Biomaterials for ink preparation 20
3.2 Cell lines used for bioink encapsulation 20
3.3 Cell culture media 21
3.4 3D bioprinting 22
3.5 Characterization assays 23
3.5.1 Rheological and mechanical characterization of the inks and printed scaffolds 23
3.5.2 Characterization of cell viability 23
3.5.3 Characterization of cell metabolic activity 24
3.5.4 Determination of cell number and proliferation 24
3.5.5 Quantitative analysis of hepatocytes functionality 25
3.5.6 Immunostaining 26
3.5.7 Imaging 27
3.5.8 Statistics 28
3.6 Specific experimental procedures and characterizations 28
3.6.1 Bioink development for bioprinting of hepatocytes 28
3.6.1.1 Bioink preparation
3.6.1.2 Bioprinting and crosslinking
3.6.1.3 Rheological and mechanical characterization
3.6.1.4 Biological characterization
3.6.2 Coculture of hepatocytes (HepG2) and fibroblasts (NIH 3T3) in core-shell bioprinted scaffolds
3.6.2.1 Bioink preparation
3.6.2.2 Core-shell bioprinting and crosslinking
3.6.2.3 Rheological and mechanical characterization
3.6.2.4 Biological characterization
3.6.3 Vascularization of bioprinted HepG2-laden constructs
3.6.3.2 Collagen : Fibrin (CF)-based core bioinks preparation and characterization 32
3.6.3.3 Optimization of shell bioink for HepG2 encapsulation 34
3.6.3.4 Influence of shell bioink on endothelial tube formation in the core 35
3.6.3.5 Transwell experiment for analysis of triple-cultures 35
3.6.3.6 Core-shell bioprinting of triple-culture in vitro liver sinusoid model 37
4 Results and Discussion 38
4.1 Bioink development for encapsulation of hepatocytes by 3D bioprinting 38
4.2 Spatially defined pattern of hepatocyte-fibroblast co-culture in a core-shell bioprinted system 46
4.2.1 Establishment of core-shell bioprinting 47
4.2.2 Simultaneous embedding of HepG2 and NIH 3T3 cells in core-shell strand scaffolds 49
4.2.3 Functionalization of the core bioink – enhancing fibroblast network formation 51
4.2.4 Influence of the microenvironment on expression of hepatic marker proteins in the core-shell bioprinted co-culture system 56
4.3 Vascularization strategies of an in vitro bioprinted liver sinusoid model 61
4.3.1 Preliminary investigation of prevascular-tube formation in fibrin gels 63
4.3.2 Optimization of culture conditions for HUVECs pre-vascular network formation 65
4.3.2.1 Collagen : Fibrin composite gels 65
4.3.2.2 Optimization of CF network density and influence of supportive cells on HUVECs pre-vascular network formation 70
4.3.3 Core-shell bioprinting: development of a core bioink to support formation of pre-vascular structures 75
4.3.3.1 Collagen : fibrin-based ink development for printability 75
4.3.3.2 Formation of pre-vascular structures in 3D bioprinted CFG core 83
4.3.4 Optimization of the shell bioink: HepG2 encapsulation in Plasma vs. Matrigel functionalized algMC 85
4.3.5 Vascularization in different shell biomaterial inks 94
4.3.6 Triple-culture of HepG2, HUVECs and NHDFs: analysis of shell and core bioinks in transwell-coculture 100
4.3.7 Core-shell bioprinting of a triple-culture 3D in vitro liver sinusoid model 111
5 Conclusions and Outlook 120
5.1 Bioink development for hepatocytes encapsulation 120
5.2 Establishment of core-shell bioprinting to fabricate a liver sinusoid model 120
5.3 Coculture of hepatocytes with supportive fibroblasts 121
5.4 Establishment of a vascularized liver sinusoid model 121
5.4.1 Optimization of culture conditions for endothelial cells 121
5.4.2 Development of CF-based printable bioink for endothelial cells encapsulation in the core 122
5.4.3 Optimizing hepatocytes encapsulation bioink 124
5.4.4 Establishment of the complex triple-culture liver sinusoid model 124
5.5 Outlook and future prospects 125
Summary 129
Zusammenfassung 132
References 135
List of Figures 157
List of Tables 159
List of publications
Organic photovoltaic cells are a promising technology for generating renewable energy from sunlight. These cells are made from organic materials, such as polymers or small molecules, and can be ...lightweight, flexible, and low-cost. Here, we have created a novel mixture of magnesium phthalocyanine (MgPc) and chlorophenyl ethyl diisoquinoline (Ch-diisoQ). A coating unit has been utilized in preparing MgPc, Ch-diisoQ, and MgPc-Ch-diisoQ films onto to FTO substrate. The MgPc-Ch-diisoQ film has a spherical and homogeneous surface morphology with a grain size of 15.9 nm. The optical absorption of the MgPc-Ch-diisoQ film was measured, and three distinct bands were observed at 800-600 nm, 600-400 nm, and 400-250 nm, with a band gap energy of 1.58 eV. The current density-voltage and capacitance-voltage measurements were performed to analyze the photoelectric properties of the three tested cells. The forward current density obtained from our investigated blend cell is more significant than that for each material by about 22%. The photovoltaic parameters (Voc, Isc, and FF) of the MgPc-Ch-diisoQ cell were found to be 0.45 V, 2.12 μA, and 0.4, respectively. We believe that our investigated MgPc-Ch-diisoQ film will be a promising active layer in organic solar cells.
Objective To assess seminal BAX and BCL2 gene and protein expressions in infertile men with varicocele (Vx). Materials and Methods A total of 111 men were investigated and divided into the following ...groups: healthy fertile men (n = 20), fertile men with Vx (n = 16), infertile oligoasthenoteratozoospermic men without Vx (n = 29), and infertile oligoasthenoteratozoospermic men with Vx (n = 46). They were subjected to history taking, clinical examination, and semen analysis. In their seminal plasma, BAX and BCL2 gene and protein expressions were estimated. Results The mean level of seminal BAX gene and protein was significantly decreased, and the mean level of seminal BCL2 gene and protein was significantly increased in fertile men compared with fertile men with Vx and in infertile men without Vx compared with infertile men with Vx. The mean level of seminal BAX gene and protein were significantly increased in men associated with bilateral Vx compared with men associated with unilateral Vx and in cases with Vx grade III compared with Vx grade I and II cases. Seminal BAX demonstrated significant negative correlation with sperm concentration, sperm motility, and sperm normal forms. Seminal BCL2 demonstrated significant positive correlation with sperm concentration, sperm motility, and sperm normal forms and significant negative correlation with seminal BAX. Conclusion Seminal BAX is significantly increased and seminal BCL2 is significantly decreased in men associated with Vx. Seminal BAX is significantly increased in men associated with bilateral Vx compared with unilateral Vx and in cases with Vx grade III compared with Vx grade I and II cases. Seminal BAX demonstrates significant negative correlation with sperm concentration, sperm motility, and sperm normal forms, whereas seminal BCL2 demonstrates significant reverse positive correlations.
Recently, scientists have shown interest in utilizing biochar made from natural sources to enhance various photovoltaic technologies. In this study, Zinc-Bis-8-hydroxyquinoline (Zn-Hq2) was mixed ...with 10 % biochar obtained from red sea microalgae (Chlorophyta) using a microwave combustion process. This mixture was then used as a novel photoactive layer in a solar cell. The structural properties of Zn-Hq2@BC were analyzed by XRD, FTIR, HRTEM, and SEM. The analysis showed that Zn-Hq2@BC had evenly distributed nano-rods within the BC nanopores network, with widths ranging from 26.94 to 30.90 nm and lengths ranging from 136.43 to 192.38 nm. When measuring dark current density-voltage, it was found that Zn-Hq2@BC/n-Si showed better-rectifying characteristics compared to pristine Zn-Hq2/n-Si, with a higher rectification ratio. The results also showed that the current density and voltage at the maximum power point increased to 5.63 mA/cm2 and 0.45 V, respectively, due to the activation of the biochar. When exposed to light, the addition of approximately 10 % biochar resulted in a 15 % increase in fill factor and a 92 % increase in power conversion efficiency. This is because biochar helps introduce extra charge carriers into the material, thus improving charge transport and reducing recombination losses. These findings reveal the positive effects of microalgae-derived biochar and indicate potential applications of Zn-Hq2 in solar cells.
PDF
