A monolayer differs significantly from the multicellular nature and three- dimensional growth of a tumor. Tumors are often modeled with a multicellular aggregate of cancer cells referred to as a spheroid embedded in a collagen gel. Although embedded spheroids in research are widespread, the scope of experiments performed is often limited to observations of growth. Therefore in this thesis, we developed methods to use an embedded spheroid model to study drug response, secondary cell types, and injury. Tumor associated macrophages are critical as stromal components intimately involved with the progression, invasion, and metastasis of cancer. To mimic clinically observed TAM localizations, two tumor cell/macrophage models are described. Macrophages are incorporated as a heterospheroid, a spheroid containing tumor cells and macrophages or diffusely seeded in the collagen surrounding a spheroid. The inclusion of macrophages as a heterospheroid changes the metabolic profile, indicative of synergistic growth, which is not observed in the diffuse model. The macrophages in the heterospheroid secrete cytokines that promote tumor cell growth and indicate Tam-like differentiation. In summary, macrophages incorporated in a heterospheroid are exposed to increased tumor cell contact, hypoxia and metabolic gradients, which promote TAM- like characteristics. To investigate the effect of spheroid culture on chemotherapeutic efficacy, we evaluated the response of our spheroid model compared to cells diffusely seeded in collagen or a traditional monolayer. The spheroid model contains two populations, a core, a dense aggregate of cells, and a periphery, cells that have grown into the surrounding collagen. The core demonstrates chemoresistance, compared to cells in the spheroid periphery, diffusely seeded in collagen and monolayer. Preliminary research indicates that the core is associated with a higher percentage of chemoresistant cancer stem cells Cancer is described as a wound that does not heal due to the presence of inflammation. To study the effect of a wound on a growing tumor, we developed a model for removing half of the spheroid and monitoring subsequent growth. Invasion into the collagen indicates increased growth and a microarray was performed to investigate relative changes in expression. In conclusion, we have demonstrated the utility of embedded spheroids in several cancer research applications. / 2018-06-21T00:00:00Z

Multicellular spheroids have emerged as a promising tool for drug delivery, cancer therapy, and tissue engineering. Compared to 2D monolayers, spheroids provide a more realistic representation of the 3D cellular environment, enabling better understanding of the signaling cascades and growth factors involved in vivo. The formation of in vitro spheroids involves the aggregation of several cells that proliferate to grow into larger spheroids. Biophysical cues provide crucial information for the cells to assemble into 3D structures. We used suspended fiber networks to demonstrate a new way to form and spatially pattern spheroids comprised of human pericytes. We show that fiber architecture (aligned vs. crosshatched), diameter (200, 500, and 800 nm), and contractility influence spheroids in their spontaneous formation, growth, and maintenance, and report a dynamic trade of cells between adjacent spheroids through remodeled fiber networks. We found that aligned fiber networks promoted spheroid formation independent of fiber diameter, while large-diameter crosshatched networks abrogated spheroid formation, promoting growth of 2D monolayers. Thus, a mixture of diameters and architectures allowed for spatial patterning of spheroids and monolayers within a single system. We further quantified various dynamic interactions and describe the forces involved during spheroid formation, cell efflux from spheroids, and show the loss and recovery of spheroid forces with pharmacological perturbation of Rho-associated protein kinase (ROCK). Thus, we develop new insights on the dynamics of spheroids using suspended fiber networks of varying diameters and architectures, with the potential to connect matrix biology with developmental, disease, and regenerative biology. / Master of Science / In recent years, studies involving multicellular spherical aggregates or 'spheroids' have gained popularity since they capture the 3D cellular environments seen within the body more realistically when compared to 2D cell culture systems (such as monolayers) traditionally used for biological studies. These spheroids resemble organs and tissues in terms of their structure and function better and are increasingly being studied for an array of applications such as drug delivery, cancer therapy, as implants and in tissue regeneration and tissue engineering. The cellular microenvironment consists of fibrous proteins of varying diameter arranged in various geometric patterns, which can influence the growth and culture of spheroids. Here, we use our Spinneret-Based Tunable Engineered Parameters (STEP) technique to fabricate fibrous networks with precise control over fiber diameter and architecture and study how biophysical cues can influence the formation and culture of spheroids. Using aortic pericytes, we show that fiber architecture (aligned vs. crosshatched) and diameter (200, 500, and 800 nm) can control how pericytes aggregate into either 2D monolayers or 3D spheroids. We study the effect of each of these parameters to show that stiffer, denser fibers are robust networks which the cells refrain from remodeling, and thus lead to monolayers while more compliant and sparser networks are easily remodeled to promote spheroid formation. Thus, we spatially pattern a mixture of 3D spheroids and 2D monolayers in a single system by varying the parameters at different regions. We quantify various interactions such as spheroid formation, spheroid merging, spheroid migration, cell efflux from spheroids and the dynamic contractile forces exerted on the matrix by spheroids during these interactions. We also show that contractility has a major role in spheroid formation and to maintain their structure and look at the changes in the gene expressions associated with contractility during the formation and growth of spheroids. Thus, we develop new knowledge in controlling the growth of pericytes into 2D and 3D structures and show that our fiber networks can be an essential platform for studying spheroids.

The Reynolds stresses in a three-dimensional turbulent boundary layer were examined using octant analysis. The representative flow was a pressure driven, three-dimensional turbulent boundary layer on the leeside (x/L=0.76-0.78, φ=105°-130°) of a 6:1 prolate spheroid at 10° angle of attack. The Reynolds number for the flow was Re_L=4.2x10⁺⁶. The LDV data of Chesnakas, Simpson, and Madden (1994) were the basis of examination. This data set employed a post-processing technique for refining the radial location of the measurements. A least-squares fit of the Spalding wall law was used to both correct the measurement locations and estimate the wall shear stress. This paper presents a previously unpublished assessment of the technique. Octant analysis was performed on the corrected data under free-stream and wall-collateral coordinates. (The wall-collateral coordinate system is aligned with the mean tangential velocity in the buffer-layer.) The octant analysis led to the development of a structural model that extends the sweep/ejection process to three dimensions. Ejections and sweeps produce w' through the same mechanism that produces u'; they transport fluid across a spanwise velocity gradient. The model's results remain consistent with coordinate rotation. The model also describes the asymmetries that evolve between ejections and sweeps with spanwise fluctuations (w') of opposite sign. These asymmetries cause non-zero u'w' and v'w' in the buffer layer. Comparison of the two coordinate systems reveals that wall-collateral coordinates provides a simpler foundation for octant analysis. The sweep and ejection octants maintain a nearly equal distribution of velocity events throughout the buffer and lower log layers. Also, the spanwise velocity profile monotonically decreases to a constant value at the boundary layer edge, simplifying application of the sweep/ejection model to spanwise fluctuations. Comparison with other 3DTBL experiments suggests that the wall-collateral coordinates are more closely aligned with the quasi-streamwise vortex structures than free-stream coordinates. The octant analysis also reveals structural behavior consistent with the four mechanisms revealed by the direct numerical simulation of Sendstad and Moin (1992). / Master of Science

No description available.

The rotating wall vessel (RWV) bioreactor is a well-established cell culture device for the simulation of microgravity for suspension cells and the generation of spheroids and organoids. The key to the success of these systems is the generation of a delicately maintained fluid dynamics system which induces a solid body rotation capable of suspending cells and other particles in a gentle, low-shear environment. Despite the unique capabilities of these systems, the inherently delicate nature of their fluid dynamics makes the RWV prone to multiple failure modes. One of the most frequently occurring, difficult to avoid, and deleterious modes of failure is the formation of bubbles. The appearance of even a small bubble in an RWV disrupts the crucial laminar flow shells present in the RWV, inducing a high-shear environment incapable of maintaining microgravity or producing true spheroids. The difficulty of eliminating bubbles from the RWV substantially increases the learning curve and subsequent barrier-to-entry for the use of this technology. The objective of this study is to create a novel RWV design capable of eliminating the bubble formation failure mode and to demonstrate the design’s efficacy. The tested hypothesis is: “The addition of a channel capable of segregating bubbles from the fluid body of the RWV will protect its crucial fluid dynamics system while enabling the growth of consistently sized and properly formed cell spheroids, improving ease of use of the RWV and decreasing experimental failure.” / Bioengineering

abstract: Cellular assays are the backbone of biological studies - be it for tissue modeling, drug discovery, therapeutics, or diagnostics. Two-dimensional (2D) cell culture has been deployed for several decades to garner physiologically relevant information and predict data before the cost-intensive animal testing. Although 2D techniques have been valuable for cellular assays, they have a colossal limitation - they do not adequately consider the natural three-dimensional (3D) microenvironment of the cells. As a result, they sometimes provide misleading statistics. Therefore, it is important to develop a 3D model that predicts cellular behaviors and their interaction with neighboring cells and extracellular matrix (ECM) in a more realistic manner. In recent biomedical research, various platforms have been modeled to generate 3D prototypes of tissues, spheroids, in vitro that could allow the study of cellular responses resembling in vivo environments, such as matrices, scaffolds, and devices. But most of these platforms have drawbacks such as lack of spheroid size control, low yield, or high cost associated with them. On the other hand, Amikagel is a low cost, high-fidelity platform that can facilitate the convenient generation of tumor and stem cell spheroids. Furthermore, Amikabeads are aminoglycoside-derived hydrogel microbeads derived from the same monomers as Amikagel. They are a versatile platform with several chemical groups that can be exploited for encapsulating the spheroids and investigating the delivery of bioactive compounds to the cells. This thesis is focused on engineering novel 3D tumor and stem cell models generated on Amikagel and encapsulated in Amikabeads for proximal delivery of bioactive compounds and applications in regenerative medicine. / Dissertation/Thesis / Z-stacks of confocal images of spheroids encapsulated in Amikabeads (compilations of sections) / Masters Thesis Bioengineering 2020

<p>Positron Emission Tomography (PET) has gained an important roll in clinical for diagnosis, staging and prognosis of a range of cancer types. Utilization of PET for monitoring and evaluation of cancer treatment is an attractive but almost new concept. The proper choice of PET-tracer as a biomarker for treatment follow-up is crucial. The important characteristic for a suitable tracer is its ability to reflect the response to a treatment at an early stage, before any morphologically changes occurs. It would be an advantage to screen a battery of PET tracers in a preclinical model and introduce a few potential tracers in clinical trial. </p><p>The most conventional pre-clinical approach in PET-oncology utilizes xenografts in mice or rats and requires a large number of subjects. It would be a great advantage to introduce a less demanding but still reliable preclinical method for a more efficient planning of studies in animal model and then in human trials. </p><p>The Multicellular Tumour Spheroid (MTS) system represents an intermediary level between cells growing as monolayer and solid tumours in experimental animals or patients. It mimics the growth of naturally occurring human tumours before neovascularization and appears to be more informative than monolayer and more economical and more ethical than animal models.</p><p>The aim of this work was to establish, refine and evaluate the application of MTS model as a preclinical approach in PET oncology. The vision was to introduce a preclinical method to probe and select PET tracer for treatment monitoring of anticancer drugs, which can hopefully be applied for optimization in breast cancer treatment.</p><p>In this thesis, a number of basic experiments were performed to explore the character of 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (FDG) uptake in MTS. FDG as the most established PET tracer was an obvious initial option for the evaluation of the model. For further assess-ment, we studied effects on FDG uptake in MTS treated with five routinely used chemother-apy agents. For association of PET tracer uptake to size change of MTS, we developed a reliable and user-friendly method for size determination of MTS. The next step was to apply the MTS model to screen PET tracers for analysis of early response of chemotherapy in breast cancer. Finally the method was utilized for translational imaging exemplified with a new chemotherapy agent.</p><p>The results were encouraging and the MTS model was introduced and evaluated as a preclini-cal tool in PET oncology. The method was implicated to in vitro quickly assess a therapy profile of existing and newly developed anticancer drugs in order to investigate the effects of candidate drugs on tumour-growth, selection of appropriate PET tracer for treatment monitor-ing and finally understanding relation between growth inhibition and biomarkers as part of translational imaging activities.</p>

Positron Emission Tomography (PET) has gained an important roll in clinical for diagnosis, staging and prognosis of a range of cancer types. Utilization of PET for monitoring and evaluation of cancer treatment is an attractive but almost new concept. The proper choice of PET-tracer as a biomarker for treatment follow-up is crucial. The important characteristic for a suitable tracer is its ability to reflect the response to a treatment at an early stage, before any morphologically changes occurs. It would be an advantage to screen a battery of PET tracers in a preclinical model and introduce a few potential tracers in clinical trial. The most conventional pre-clinical approach in PET-oncology utilizes xenografts in mice or rats and requires a large number of subjects. It would be a great advantage to introduce a less demanding but still reliable preclinical method for a more efficient planning of studies in animal model and then in human trials. The Multicellular Tumour Spheroid (MTS) system represents an intermediary level between cells growing as monolayer and solid tumours in experimental animals or patients. It mimics the growth of naturally occurring human tumours before neovascularization and appears to be more informative than monolayer and more economical and more ethical than animal models. The aim of this work was to establish, refine and evaluate the application of MTS model as a preclinical approach in PET oncology. The vision was to introduce a preclinical method to probe and select PET tracer for treatment monitoring of anticancer drugs, which can hopefully be applied for optimization in breast cancer treatment. In this thesis, a number of basic experiments were performed to explore the character of 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (FDG) uptake in MTS. FDG as the most established PET tracer was an obvious initial option for the evaluation of the model. For further assess-ment, we studied effects on FDG uptake in MTS treated with five routinely used chemother-apy agents. For association of PET tracer uptake to size change of MTS, we developed a reliable and user-friendly method for size determination of MTS. The next step was to apply the MTS model to screen PET tracers for analysis of early response of chemotherapy in breast cancer. Finally the method was utilized for translational imaging exemplified with a new chemotherapy agent. The results were encouraging and the MTS model was introduced and evaluated as a preclini-cal tool in PET oncology. The method was implicated to in vitro quickly assess a therapy profile of existing and newly developed anticancer drugs in order to investigate the effects of candidate drugs on tumour-growth, selection of appropriate PET tracer for treatment monitor-ing and finally understanding relation between growth inhibition and biomarkers as part of translational imaging activities.

No description available.

<p>High-throughput processing technologies hold critical position in biomedical research. These include screening of cellular response based on phenotypic difference and production of homogeneous chemicals and biologicals for therapeutic applications. The rapid development of microfluidics technology has provided an efficient, controllable, economical and automatable processing platform for various applications. In particular, emulsion droplet gains a lot of attention due to its uniformity and ease of isolation, but the application of water-in-oil (W/O) single emulsion is hampered by the presence of the oil phase which is incompatible with aqueous phase manipulation and the difficulty in modifying the droplet environment.</p><p>This thesis presents the development of a double emulsion (DE) droplet platform in microfluidics and two applications: (1) high-throughput screening of synthetic gene and (2) production of multicellular spheroids with adjustable microenvironment for controlling stem cell differentiation and liver tissue engineering. Monodisperse DE droplets with controllable size and selective permeability across the oil shell were generated via two microfluidics devices after optimization of device design and flow rates. </p><p>Next, bacterial cells bearing synthetic genes constructed from an inkjet oligonucleotide synthesizer were encapsulated as single cells in DE droplets. Enrichment of fluorescent signals (~100 times) from the cells allowed quantification and selection of functionally-correct genes before and after error correction scheme was employed. Permeation of Isopropyl β-D-1-thiogalactopyranoside (IPTG) molecules from the external phase triggered target gene expression of the pET vector. Fluorescent signals from at least ~100 bacteria per droplet generated clearly distinguishable fluorescent signals that enabled droplets sorting through fluorescence-activated cell sorting (FACS) technique.</p><p>In addition, DE droplets promoted rapid aggregation of mammalian cells into single spheroid in 150 min. Size-tunable human mesenchymal stem cells (hMSC) spheroids could be extracted from the droplets and exhibited better differentiation potential than cells cultured in monolayer. The droplet environment could be altered by loading matrix molecules in it to create spheroid-encapsulated microgel. As an example, hMSC spheroid was encapsulated in alginate or alginate-RGD microgel and enhanced osteogenic differentiation was found in the latter case.</p><p>Lastly, the capability of forming spheroids in DE droplet was applied in liver tissue engineering, where single or co-culture hepatocyte spheroids were efficiently produced and encapsulated in microgel. The use of alginate-collagen microgel significantly improved the long-term function of the spheroid, in a manner similar to forming co-culture spheroids of hepatocytes and endothelial progenitor cells at a 5 to 1 ratio. The hepatocyte spheroid encapsulated in microgel could be useful for developing bioartificial liver or drug testing platform or applied directly for hepatocyte transplantation.</p> / Dissertation