archives@tulane.edu / We have developed a laser-biomaterial interaction-based prototyping platform capable of three fabrication modes: (1) laser direct write of cells, microbeads, and other biomaterials; (2) fabrication of cell encapsulating microspheres (microcapsules); and (3) laser micromachining of substrates. Using this system, we are able to precisely place biomaterials, such as cells, into substrates with spatial constraints from laser micromachining or wholly fabricate scaffolds that are cell laden. This enables fabrication of cocultures in almost any geometry and controlled gradients of chemical factors. In addition, the process is parallelizable, thus allowing for numerous potential bioassay applications. One such assay is a differential system for quantifying multiple outcomes in response to multiple parallel biophysicochemical cues in competition. These novel assays are complex, reproducible, and disposable microenvironments. This document will summarize the control integration developed for Laser Direct Write, a 2D model of laser ablation, with a computational method demonstrating preliminary results. Finally the biofabrication methods discussed are applied to an Organ-on-a-Chip model to develop a fully automated fabrication process. / 1 / samuel sklare

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.

Ductal Carcinoma in Situ (DCIS) does not have a clinically useful indicator of malignancy, and it is often benign, except in 20% of cases. Even more important, it has a cure – removal of the affected breast. DCIS patients overwhelmingly elect for invasive therapies to escape that 20% malignant chance. Overtreatment such as this costs the patients, and it highlights the need for a DCIS model capable of distinguishing the 20% in need of treatment. Some labs have taken steps toward three-dimensional, complex, and biomimetic models of mammary tissues using a variety of endogenous and synthetic gels and 3D printing. We developed FRESH (Freeform Reversible Embedding of Suspended Hydrogels) as the first method capable of 3D printing highly biomimetic shapes from endogenous gels. Utilizing FRESH, we aim to rapid prototype models of mammary duct epithelia that are biomimetic, parametric, and capable of iterative evolution. First, we investigate the principles of 3D printers modified for extruding fluids and construct a comprehensive hardware and software platform for printing gelling fluids. Second, we apply the FRESH method to 3D print collagen and alginate hydrogels, demonstrating patency of printed vascular models, topological fidelity, and the synergistic combination of hydrogel properties in multi-material prints. Finally, we rapid prototype an epithelial monolayer by seeding a 3D printed collagen manifold, and we demonstrate maintenance of the tissue’s geometry across a week of culture. We provide evidence of fidelity in prints such as an epithelial tree printed at 200% scale using unmodified collagen type I, and we investigate the combination of hydrogel properties in multi-material prints by utilizing a second hydrogel (alginate) to reinforce and preserve the fidelity of this collagen tree during handling. Our approach utilizes faster (>40 mm/s), cheaper (

In this thesis, a rapid magnetic printing technique has been developed for contactless, label-free, and scaffold-free printing of three dimensional (3D) cellular structures in vitro. The biological inks (bioinks) used to form these structures were composed of cells suspended in a liquid medium. Development of this technique was based on exploiting the inherent magnetic susceptibility of cells. Since cells and their liquid medium are diamagnetic (negative magnetic susceptibility), a paramagnetic salt hydrate, gadopentatic acid (Gd-DTPA), was added to the liquid medium to increase its magnetic susceptibility. When a magnetic field was applied, the host fluid containing the paramagnetic salt was towards regions of high magnetic field strength, displacing the cells towards regions towards regions of low magnetic field strength. This rapid printing technique using magnetic cell bioinks was first described using whole blood to form various structures including spherical clusters (spheroids), strips, and three-pointed stars. This demonstration verified the printing technique as a safe and non-toxic method. Subsequent studies were performed using a frequently studied human breast cancer cell line, Michigan Cancer Foundation-7 (MCF-7), to develop a thorough protocol using mammalian cells. Here, the printing method was used to form 3D cellular structures on ultra-low attachment (ULA) and 2.5D cellular structures on tissue-culture-treated (TCT) surfaces. These geometries were produced within 6 hours with high reproducibility. The use of a co-culture on TCT surfaces using MCF-7 and human umbilical vein endothelial cells (HUVECs) and on ULA surfaces using MD Anderson metastatic breast-231 (MDA-MB-231) and embryonic mouse fibroblast (3T3) cells demonstrated the observance of unique cellular interactions and improved printing abilities (accelerated time and improved reproducibly) of the structures printed with magnetic inks, respectively. The use of magnetic cell inks in research and clinical settings can accelerate the development of medical innovations such as drug discovery, personalized medicine, and treatment of disease. / Thesis / Doctor of Philosophy (PhD) / In this thesis, a rapid magnetic printing technique has been developed for contactless, label-free, and scaffold-free printing of three dimensional (3D) cellular structures in vitro. The biological inks (bioinks) used to form these structures were composed of cells suspended in a liquid medium. Development of this technique was based on exploiting the inherent magnetic susceptibility of cells. Since cells and their liquid medium are diamagnetic (negative magnetic susceptibility), a paramagnetic salt hydrate, gadopentatic acid (Gd-DTPA), was added to the liquid medium to increase its magnetic susceptibility. When a magnetic field was applied, the host fluid containing the paramagnetic salt was towards regions of high magnetic field strength, displacing the cells towards regions towards regions of low magnetic field strength. This rapid printing technique using magnetic cell bioinks was first described using whole blood to form various structures including spherical clusters (spheroids), strips, and three-pointed stars. This demonstration verified the printing technique as a safe and non-toxic method. Subsequent studies were performed using a frequently studied human breast cancer cell line, Michigan Cancer Foundation-7 (MCF-7), to develop a thorough protocol using mammalian cells. Here, the printing method was used to form 3D cellular structures on ultra-low attachment (ULA) and 2.5D cellular structures on tissue-culture-treated (TCT) surfaces. These geometries were produced within 6 hours with high reproducibility. The use of a co-culture on TCT surfaces using MCF-7 and human umbilical vein endothelial cells (HUVECs) and on ULA surfaces using MD Anderson metastatic breast-231 (MDA-MB-231) and embryonic mouse fibroblast (3T3) cells demonstrated the observance of unique cellular interactions and improved printing abilities (accelerated time and improved reproducibly) of the structures printed with magnetic inks, respectively. The use of magnetic cell inks in research and clinical settings can accelerate the development of medical innovations such as drug discovery, personalized medicine, and treatment of disease.

Knorpeldefekte gelten in der Medizin als besonders schwierig zu beheben, da das avaskuläre und aneurale hyaline Knorpelgewebe nur über sehr begrenzte Selbstheilungskräfte verfügt. Die Entwicklung neuer klinischer Therapien für eine erfolgreiche Regeneration bis hin zum vollständigen Ersatz von beschädigtem oder erkranktem Knorpel stellt daher das Ziel umfangreicher Forschung dar. Darüber hinaus zeichnet sich Knorpel durch eine organisierte, zonale Zell-Matrix-Verteilung und -Dichte aus, die möglichst naturgetreu nachgebildet werden muss, um einen adäquaten Gelenkknorpelersatz zu schaffen. Das dreidimensionale Bioprinting von humanen mesenchymalen Stromazellen (hMSCs) in Hydrogelen ist hierbei ein vielversprechender Ansatz. Es sind jedoch umfangreiche Studien erforderlich, um herauszufinden, wie 3D-Stammzellkonstrukte mit unterschiedlichen Zelldichten und Zell-Zell-Wechselwirkungen in einer gedruckten Hydrogel Matrix interagieren. Deshalb wurde in dieser Arbeit untersucht, ob die mesenchymalen Stromazellen in Form von Einzelzellen oder Sphäroiden durch das Extrusionsdruckverfahren in ihrer Proliferationsfähigkeit und ihrem chondrogenen Differenzierungspotential beeinträchtigt werden. Hierfür wurden in dieser Arbeit sowohl das Zellüberleben als auch Proliferations- und Differenzierungsmarker in gedruckten und nicht gedruckten Proben mit Einzelzellkonzentrationen von 2-20 Millionen Zellen sowie bei Sphäroiden mit ca 4000 Zellen/Sphäroid untersucht. Es konnte gezeigt werden, dass das extrusionsbasierte Druckverfahren keine negativen Auswirkungen auf die Überlebensfähigkeit und die Proliferation der hMSCs hat. Zum Nachweis der chondrogenen Differenzierung wurden mehrere Experimente durchgeführt. Durch die Expression von Typ-II-Kollagen und Aggrecan sowie durch die Quantifizierung von GAG welches zu einem großen Teil in der ECM von Knorpelgewebe zu finden ist, konnte bestätigt werden, dass die mesenchymalen Stromazellen durch den Druckprozess ihr chondrogenes Differenzierungspotential nicht einbüßen. Die beim 3D-Bioprinting auftretenden Scherkräfte scheinen die in-vitro Chondrogenese sogar ohne chemische Stimulation durch TGF-β1 anzustoßen. Außerdem zeigten die Sphäroidgruppen ein höheres chondrogenes Differenzierungspotential als die Einzelzellgruppen. Um dies im Zusammenhang mit dem 3D Extrusionsdruckverfahren zu bestätigen, erscheint es sinnvoll, weitere Versuche mit noch höheren Zellkonzentrationen in Form von Sphäroiden durchzuführen. Zusammenfassend zeigte sich in dieser Arbeit, dass das extrusionsbasierte Druckverfahren in Alginat/Gelatine Hydrogelen keine Zellschädigung verursacht und weder die chondrogene Differenzierung von Einzelzellen noch von Sphäroiden beeinträchtigt. / Cartilage defects are considered particularly difficult to repair in medicine, since avascular and aneural hyaline cartilage tissue has only very limited self-healing capabilities. The development of new clinical therapies for successful regeneration to complete replacement of damaged or diseased cartilage therefore represents the goal of extensive research. In addition, cartilage is characterized by an organized, zonal cell-matrix distribution and density that must be replicated as closely as possible to nature in order to create an adequate articular cartilage replacement. Three-dimensional bioprinting of human mesenchymal stromal cells (hMSCs) in hydrogels is a promising approach in this regard. However, extensive studies are needed to determine how 3D stem cell constructs interact with different cell densities and cell-cell interactions in a printed hydrogel matrix. Therefore, this work investigated whether the mesenchymal stromal cells in the form of single cells or spheroids are affected in their proliferative capacity and chondrogenic differentiation potential by the extrusion printing process. For this purpose, cell survival as well as proliferation and differentiation markers were investigated in this work in printed and non-printed samples with single cell concentrations of 2-20 million cells and in spheroids with approximately 4000 cells/spheroid. It was shown that the extrusion-based printing process had no negative effects on the survival and proliferation of hMSCs. Several experiments were performed to demonstrate chondrogenic differentiation. By expressing type II collagen and aggrecan and quantifying GAG which is largely found in the ECM of cartilage tissue, it was confirmed that the mesenchymal stromal cells do not lose their chondrogenic differentiation potential by the printing process. The shear forces involved in 3D bioprinting appear to trigger in vitro chondrogenesis even without chemical stimulation by TGF-β1. In addition, the spheroid groups showed a higher chondrogenic differentiation potential than the single cell groups. To confirm this in the context of the 3D extrusion printing process, it seems reasonable to perform further experiments with even higher cell concentrations in the form of spheroids. In summary, this work showed that the extrusion-based printing process in alginate/gelatin hydrogels does not cause cell damage and does not affect the chondrogenic differentiation of either single cells or spheroids.

Currently, many drugs are preclinically tested on two-dimensional cell cultures. However, this method does not adequately replicate the cellular interactions or diffusion gradient that occur in three-dimensional tissues, leading to poor indicators of how a drug may affect human tissues. The objective of this project was to use bioprinted pancreatic cancer cell cultures as a platform for three-dimensional drug testing. Various bioink formulations of cellulose, gelatin, and alginate were evaluated to determine which provided the best printability and cell viability. A cellulose nanocrystal and alginate hydrogel showed superior printability due to its shear thinning properties. Additionally, initial cell viability was nearly 80%, and it remained above 60% over four days. Use of a custom spinning bioreactor at 50 rpm resulted in no improvements to cell viability. Overall, the system shows potential as a drug testing platform to evaluate the effectiveness of various drug formulations on three-dimensional pancreatic cancer cell cultures.

3D bioprinting is an emerging field with the potential to reform the process of organ transplantation. The ability to 3D print new organs and tissues would supplement the organ donor shortage and decrease the risk associated with organ rejection. One of the current areas of research focuses on printing cells using hydrogels composed of methacrylated compounds as a scaffolding. One of the chemical means of crosslinking the hydrogels is using the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) to crosslink with light. The 3D bioprinter in the lab currently has an attachment for a 365nm lamp, however this is cytotoxic to cells. A 405nm laser was designed to mount on the hot tool of the BioAssemblyBot by Advanced Solutions and flash at a specific frequency when sent a signal from the bioprinter. This tool was then tested to determine effective flash frequencies for crosslinking hydrogels.

Additive manufacturing processes such as 3D printing are booming in the industry due to their high degree of freedom in terms of geometric shapes and available materials. Focusing on patient-specific medicine, 3D printing has also proven useful in the Life Sciences, where it exploits the shape fidelity for individualized tissues in the field of bioprinting. In parallel, the current systems of bioreactor technology have adapted to the new manufacturing technology as well and 3D-printed bioreactors are increasingly being developed. For the first time, this work combines the manufacturing of the tissue and a tailored bioreactor, significantly streamlining the overall process and optimally merging the two processes. This way the production of the tissues can be individualized by customizing the reactor to the tissue and the patient-specific wound geometry. For this reason, a common basis and guideline for the cross-device and cross-material use of 3D printers was created initially. Their applicability was demonstrated by the iterative development of a perfusable bioreactor system, made from polydimethylsiloxane (PDMS) and a lignin-based filament, into which a biological tissue of flexible shape can be bioprinted. Cost-effective bioink-replacements and in silico computational fluid dynamics simulations were used for material sustainability and shape development. Also, nutrient distribution and shear stress could be predicted in this way pre-experimentally. As a proof of functionality and adaptability of the reactor, tissues made from a nanocellulose-based Cellink® Bioink, as well as an alginate-based ink mixed with Me-PMeOx100-b-PnPrOzi100-EIP (POx) (Alginate-POx bioink) were successfully cultured dynamically in the bioreactor together with C2C12 cell line. Tissue maturation was further demonstrated using hMSC which were successfully induced to adipocyte differentiation. For further standardization, a mobile electrical device for automated media exchange was developed, improving handling in the laboratory and thus reduces the probability of contamination. / Additive Fertigungsverfahren wie der 3D-Druck boomen in der Industrie aufgrund ihres hohen Freiheitsgrads in Bezug auf geometrische Formen und verfügbare Materialien. Mit Blick auf die patientenspezifische Medizin hat sich der 3D-Druck auch in den Biowissenschaften bewährt, wo er die Formtreue für individualisierte Gewebe im Bereich des Bioprinting nutzt. Parallel dazu haben sich auch die derzeitigen Systeme der Bioreaktortechnologie an die neue Fertigungstechnologie angepasst, und es werden zunehmend 3D-gedruckte Bioreaktoren entwickelt. In dieser Arbeit werden erstmals die Herstellung des Gewebes und ein maßgeschneiderter Bioreaktor kombiniert, wodurch der Gesamtprozess erheblich gestrafft und beide Verfahren optimal zusammengeführt werden. Auf diese Weise kann die Herstellung der Gewebe individualisiert werden, indem der Reaktor an das Gewebe und die patientenspezifische Wundgeometrie angepasst wird. Aus diesem Grund wurde zunächst eine gemeinsame Basis und Leitlinie für den Geräte- und Materialübergreifenden Einsatz von 3D-Druckern geschaffen. Deren Anwendbarkeit wurde durch die iterative Entwicklung eines perfundierbaren Bioreaktorsystems aus Polydimethylsiloxan (PDMS) und einem Lignin-basierten Filament demonstriert, in das ein biologisches Gewebe mit flexibler Form gedruckt werden kann. Kostengünstige Biotintenalternativen und emph in silico Computational Fluid Dynamics Simulationen wurden für eine materialschonende Formentwicklung verwendet. Nährstoffverteilung und Scherspannung konnten auf diese Weise präexperimentell vorhergesagt werden. Als Beweis für die Funktionalität und Anpassbarkeit des Reaktors wurden Gewebe aus einer Cellink® Bioink auf Nanocellulosebasis sowie einer Tinte auf Alginatbasis, welche mit Me-PMeOx100-b-PnPrOzi100-EIP (POx) gemischt wurde (Alginat-POx-Bioink), erfolgreich zusammen mit C2C12-Zelllinie dynamisch im Reaktor kultiviert. Die Gewebereifung wurde außerdem mit hMSC demonstriert, die erfolgreich zur adipozyten Differenzierung induziert wurden. Zur weiteren Standardisierung wurde ein mobiles elektrisches Gerät für den automatischen Medienwechsel entwickelt, welches die Handhabung im Labor verbessert und damit die Wahrscheinlichkeit einer Kontamination deutlich verringert.

Early-onset diagnostics, or the detection of disease before clinical symptoms arise, has gained traction for its potential to improve patient quality of life and health outcomes. Biosensors, found in point-of-care (POC) devices, facilitate early-onset diagnostics and disease monitoring by addressing the limitations of current diagnostics strategies, which include timeliness, cost-effectiveness, and accessibility. Biosensors often incorporate microarrays within their design to allow for the detection of disease-associated biomolecules, known as biomarkers. Microarrays are composed of capture biomolecules, such as monoclonal antibodies, that are immobilized through either contact or non-contact printing techniques. In the following thesis, we investigated microarray designs within novel biosensing platforms for diagnostic and disease monitoring applications. First, we highlighted the advantages and challenges of implementing different types of biosensors, detection methods, and biomolecule immobilization strategies. Additionally, we proposed a novel 3D microarray incorporating hydrogels composed purely of crosslinked bovine serum albumin (BSA) proteins decorated with capture antibodies (CAbs). Utilizing industry-standard inkjet printing, we developed and optimized a two-step fabrication protocol, by which BSA proteins and CAbs are printed first, followed by the crosslinking agent, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC). Characterization of the unique three-dimensional (3D) microstructure and hydrogel parameters and conducting comparisons with standard two-dimensional (2D) microdots, showed that increasing biosensor surface area led to a 3X increase in signal amplification. The limits of detection (LODs) for cytokine biomarkers were 0.3pg/mL for interleukin-6 (IL-6) and 1pg/mL for tumor necrosis factor receptor I (TNF RI), which were highly sensitive compared to reported LODs from literature. Alongside the investigation of novel printing protocols, proof-of-concepts for multiplex detection and distinguishing clinical patient samples from healthy donors were also presented. Overall, this thesis demonstrated the fabrication and optimization of microarray development shows promise in improving current biosensor designs, allowing for enhanced early-onset disease detection and monitoring. / Thesis / Master of Applied Science (MASc)