Browsing by Author "Ungrin, Mark D."
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Item Open Access Cardiac extracellular matrix gel for ESC-derived cardiomyocyte maturation(2020-08-31) Thompson, Madalynn Jade; Rancourt, Derrick E.; Greenway, Steven C.; Krawetz, Roman J.; Ungrin, Mark D.The need for precision medicine has been established and the use of organoids to test individual drug efficacy and develop individual disease models is creating an opportunity for innovation. Human embryonic stem cells (hESCs) are commonly used for these applications but differentiation often results in immature phenotypes that may not be physiologically relevant. While hESCs can be differentiated into any cell type using small molecules, emerging concepts suggest that additional exogenous factors influence differentiation. In situ, stem cells exist in an organ-specific niche which contains extracellular matrix (ECM) proteins that have important paracrine effects to promote the maturity. This project aimed to investigate whether either of two alternative preparations of organ-specific ECM matrices support would offer improved support for tissue-specific differentiation and maturation. ECM matrices from porcine hearts (i.e. cardiogel) were prepared and decellularized either completely or partially. These cardiogel preparations were then studied using mass spectrometry and cytochemical staining before being used to support the maturation of hESCs that had been differentiated into cardiomyocytes (CMs). Moderate decellularization of porcine heart was achieved as shown by histochemical staining and the cardiogels contained variable amounts of important ECM components. However, neither cardiogel preparations appeared to increase the maturity of hESC-derived CMs, as assessed by cellular gene expression after 4 days of culture on cardiogel following differentiation. In conclusion, a protocol to generate cardiogels from porcine heart was achieved but significant batch variability was found. Further optimization may lead to a substrate which promotes cellular maturation and which can be used in conjunction with other technologies to increase cellular yield and homogeneity of resulting cardiac lineages.Item Open Access Development of an extracellular matrix to improve pseudoislet survival for the treatment of Type I diabetes(2019-09-12) Wong, Sarah; Ungrin, Mark D.; Kallos, Michael S.; Rancourt, Derrick E.Throughout the islet transplantation process, many islets die due to removal from their native environment. Studies suggest that select extracellular matrices (ECMs) have the potential to support islet survival throughout transplantation. However, there is currently no overview evaluating ECM interactions with pseudoislets. In this study, three common methods of quantifying cell survival were applied to aggregates encapsulated in thin hydrogel sheets and their effectiveness was evaluated. Next, a Design of Experiments approach was used to evaluate composite hydrogel ECMs as a method of improving the survival of INS1 and primary human pseudoislets post-isolation. Encapsulating INS1 pseudoislets in [alginate] = 2wt/v%, [collagen I] ~ 4mg/mL, and [Matrigel] ~ 4mg/mL improved INS1 survival and proliferation when cultured for 3 days. Similarly, collagen I and Matrigel were found to have a neutral or positive effect on primary human pseudoislet survival when encapsulated in 2 wt/v% alginate gels and cultured for 5 days.Item Open Access Engineering of Gelatin Methacryloyl (GelMA) Hydrogels for Developing Biomimetic Tissue Constructs(2020-10-14) Janmaleki, Mohsen; Sanati Nezhad, Amir; Ungrin, Mark D.; Yipp, Bryan G.; Ronsky, Janet L.; Gaharwar, Akhilesh K.; Khoshnazar, Rahil; Lu, Qingye G.Three-dimensional (3D) cell culture offers a more physiologically-relevant context for disease modeling and drug screening. Hydrogel-based biomaterials support the long-term culture of cells in 3D and enhance memetic cell-cell and cell-matrix interactions. Herein, gelatin methacryloyl (GelMA) hydrogel, a well-known photo-crosslinkable hydrogel, was selected for engineering of different tissue constructs.First, the practicability of imprinting cell topography on GelMA hydrogel was investigated. A novel method was developed to fabricate cell-like niches over the hydrogel’s substrate, and its effects on cytocompatibility and drug susceptibility of breast cancer cells were studied. Second, GelMA hydrogel was tuned in terms of mechanical properties and porosity to facilitate in vitro myelination of dorsal root ganglia (DRG) neurons by Schwann cells (SCs). It was shown that the tuned GelMA enhanced single axonal generation (unlike collagen) and promoted DRGs’ interaction with SCs (unlike PDL). Third, the role of temperature on bioprintability of GelMA bioinks in a two-step crosslinking strategy was investigated. Lowering the temperature can enhance the physical gelation of GelMA and consequently improve filament formation. Results showed that the decrease in the temperature could improve the printability and shape fidelity of the deposited hydrogel, particularly at 15 °C. Time-dependent mechanical testing confirmed higher elastic properties of the collected hydrogel at the lower temperature.Fourth, a hydrogel-based 3D human intracranial aneurysm (IA) model was developed using liquid assisted injection molding. With clinically relevant dimensions and tuned fluidic and matrix properties, the essential endothelium was successfully lined inside the reconstructed IA over pre-cultured smooth muscle cells. Based on the characterized viscoelastic properties of the GelMA hydrogel and with the help of a fluid-structure interaction model, the capability of the IA construct model in predicting the response of the IA to different fluid flow profiles was demonstrated.Finally, using the techniques developed in this thesis, a new approach is suggested to fabricate a fully hydrogel-based platform for tissue engineering and organ-a-chip applications.Item Open Access Key cytoskeletal proteins are upregulated in 2D cell culture in comparison to 3D culture(2020-01) Aslam, Saba; Ungrin, Mark D.; Krawetz, Roman J.; Nezhad, Amir SanatiThe cytoskeleton is the structural framework of the cell, and plays an important role in cell motility, signalling pathways, and cell behaviour. It is composed of various proteins that form a dynamic network of filaments and tubules to mechanically support the cell. Commonly, cytoskeletal proteins such as β-actin are used as controls or references in many laboratory techniques because they are assumed to remain stable across various conditions. However, most of the research to date has been performed on 2D monolayers of cells adhered to plastic. Recently, 3D cell culture techniques have become more feasible and increasingly popular as they better represent the physiological microenvironment found in the body. As more research shifts from 2D to 3D culture, the changes in cytoskeleton due to culture conditions need to be better characterized. To study gene expression, eleven human cell lines were cultured in 2D and 3D, and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed. For analysis, ten control genes were evaluated for stability, and the geometric mean of the three most stable genes was used for normalization. The results show a significant downregulation of several cytoskeletal genes such as β-actin, vimentin, and keratin in 3D compared to 2D. Similarly, key cytoskeletal proteins were also found to be downregulated in 3D using Western blots normalized to total protein. These results demonstrate that the microenvironmental differences between culture conditions can considerably influence cytoskeletal component levels across various cell types. Integrating these findings with the published literature, we suspect that the supra-physiological stiff surface in plastic-adherent culture leads to overexpression of cytoskeletal proteins – presumably also impacting many other pathways and cell behaviour. These findings challenge the assumption that β-actin provides a consistently stable reference gene/protein, emphasize the importance of physiologically relevant culture systems, and highlight the need to select appropriate controls for specific experimental conditions.Item Open Access Long-term co-culture of endothelial and cancer cells within biomimetic microfluidic chips for investigating juxtacrine and paracrine regulatory signaling pathways(2019-01) Soleimani, Shirin; Sanati-Nezhad, Amir; Rancourt, Derrick E.; Ungrin, Mark D.Emerging evidence on how angiocrine factors confer inductive signals to orchestrate a wide range of pathophysiological phenomena (e.g. tumorigenesis, self-renewal of hematopoietic stem cells, regenerative lung alveolization and liver regeneration) lends credence to the concept that endothelial cells are not only the building blocks of vascular networks but also serve as a rich resource of regulatory angiocrine factors. Cancer-associated endothelial cells initiate a series of signaling modes with cancer cells. These signallings determine cancer progression and response to drugs. While it is widely-believed that endothelial cells are only able to affect the cancer cells that are juxtaposed next to them, cells positioned five-cell diameters away from the sinusoid vessels have been shown to be ruled by angiocrine factors. Considering the distance dependency of cancer-endothelium crosstalk, there is a great demand for an in vitro system with high geometrical precision that offers a diverse range of cell-cell distance and cell-cell contact area for studying and distinguishing juxtacrine and paracrine signallings between cancer and endothelial cells in a spatiotemporal manner. In the present study, a three-layer microfluidic chip consisting of a top and a bottom channel separated via a porous membrane is devised, to enable studying the signalling pathways between cancer and endothelial cells under highly-controlled conditions. The porous membranes are variable in terms of pore size, thickness and porous surface area. However, transition from macroscopic to microfluidic platforms for reconstructing in vitro models brings with it a variety of challenges such as the effect of device material (Tissue culture Polystyrene vs. PDMS), surface coating, cell number/surface area unit, cell seeding method and cell culture media exchange schedule. The present work has tried to provide a comprehensive protocol to supply precisely controlled conditions along the microchannels for the cells to grow, using human umbilical vein endothelial cells, E4OR1 transduced endothelial cells and breast cancer MCF-7 cells.Item Open Access Microscale Tissue Engineering and Contributors of the Cellular Niche(2019-05-31) Al-Ani, Abdullah; Ungrin, Mark D.; Biernaskie, Jeff A.; Kallos, Michael S.; Rancourt, Derrick E.; Hirota, Simon Andrew; Gratzer, Paul F.The behaviour of cells is modulated by their microenvironment or ‘niche’. While cellular therapies offer promising curative solutions for many diseases, the efficacy of transplanted cells is often hampered by a suboptimal microenvironment. One strategy to overcome this limitation is to reconstruct the niche of the cells of interest prior to transplantation. The central aim of this thesis is to develop novel tissue engineering approaches to further understand and reconstitute the cellular niche. While these approaches were specifically validated in the retinal and islet systems, they were also designed to be easily implemented in other biological systems. One project showcases a novel scaffold-free, scalable and injectable retinal pigment epithelium (RPE) microtissue for minimally-invasive transplantation. While RPE transplantation holds great potential to cure various retinal degenerative diseases, cells transplanted as cellular suspension exhibit suboptimal survival and function. Conversely, transplanting RPE as coherent cellular sheets has yielded better outcomes, but they are complex to transplant and produce at large scale. Our RPE microtissues were designed to capture the benefit of both approaches: namely, simplicity of production and transplantation, as well as enhanced performance. We found that our RPE microtissue exhibited superior cellular behaviour in terms of gene expression and in vitro function when compared to standard adherent culture. Another project presents a unique approach to produce transplants with a reconstituted cellular niche. This approach aims to repopulate the niche by incorporating finite amounts and proportions of niche cells into transplantable constructs. Using it enabled us to produce size-controlled pseudoislet constructs that contained various proportions of mesenchymal stem cells (MSCs), fibroblast and endothelial cells, and to quantitatively evaluate their in vitro performance. Further, applying this approach led us to discover more than one favourable condition that yielded improvements in islet cell performance in vitro. While the islets of Langerhans were used for biological validation, the approach was designed to be broadly applicable to various biological systems. In sum, this thesis offers several novel approaches for scientists to better understand and enhance the cellular niche. The simplicity, accessibility and scalability of these approaches render them suitable for both scientific applications and clinical translation.Item Open Access Microscale Tissue Engineering for the Study and Treatment of Diabetes(2018-12-12) Yu, Yang; Ungrin, Mark D.; Huang, Carol T. L.; Rancourt, Derrick E.Islet transplantation is a promising approach to the treatment of insulin-dependent diabetes. However, a major clinical challenge is inefficient survival and engraftment of the transplanted material. This has been associated with insufficient oxygen and nutrition delivery after loss of the endogenous capillaries, and stress induced during islet isolation and culture. Quantitative modelling of oxygen delivery predicts significant advantages for smaller islets, and consistent with this concept, smaller human islets have also performed better than larger ones in clinical settings. In order to understand and overcome these limitations for both research and clinical applications, we have established a microscale tissue engineering approach that is capable of consistently and efficiently generating size-controlled pseudoislets from human donor islets, yielding improved survival and function both in vitro and in vivo. We then combined this platform with advanced statistical methodologies and laboratory automation systems to enable assessment of large numbers of modifying factors (and their interactions) identified from the literature in parallel. This project has yielded a substantial improvement in the consistency and efficacy of islet cell packaging for transplantation, and laid a foundation for rapid transition to the clinic.Item Open Access Novel Culture Systems for Studying Proliferation and Maturation of Spermatogonia in vitro(2022-04-20) Sakib, Sadman; Dobrinski, Ina; Rancourt, Derrick E.; Ungrin, Mark D.The work presented in this dissertation aims to establish two novel culture systems for the proliferation and differentiation of spermatogonia. First, we established a suspension culture system for porcine spermatogonia using stirred suspension bioreactors. We showed that porcine pre-pubertal spermatogonia have a higher degree of proliferation in suspension culture compared to static culture. We also show that while spermatogonia proliferate more under ambient O2 compared to lower O2 tension of 10%, they are also likely to undergo differentiation. Our findings indicate that the higher proliferation of spermatogonia that is observed in suspension culture is partially mediated by the Wnt/ ?-catenin pathway. Other signal transduction pathways may also be activated in the stirred suspension bioreactor. This lays the foundation for future work aimed towards better understanding and improvement of suspension culture for spermatogonia. Second, we established a primate, human, mouse, rat and porcine testicular organoid system using the microwell culture system. We find that these organoids have testis-specific morphology and mimic testicular functions. We also present evidence that the mouse and rat organoids can undergo maturation and promote differentiation of spermatogonia. Our proof-of-principle experiments with ciliobrevin D, mono-2-ethylhexyl phthalate and cadmium chloride indicate that testicular organoids can serve as a platform for modeling testis morphogenesis and reproductive toxicity in vitro. In summary, the organoid platform established for this dissertation opens up an exciting avenue of research to further develop and adapt the system for studying the human male reproductive system in vitro in a tissue specific context.Item Open Access Poly (ε-Caprolactone)-Silk Fibroin Based Functional Repair for Annulus Fibrosus Tears(2019-01-24) Novin, Mana; Duncan, Neil A.; Kallos, Michael S.; Salo, Paul T.; Ungrin, Mark D.; Sen, Arindom; Lü, QingyeIntervertebral disc degeneration with an associated bulged/herniated disc is a significant cause of low back pain. Annulus fibrosus (AF) tears and defects are a major clinical problem with no current treatments available for its closure and repair, resulting in risk of re-herniation. This thesis focuses on the chemical and mechanical characterization of a newly-designed biodegradable poly(ɛ-caprolactone)-silk fibroin (PCL-SF) as a potential candidate for the closure of irregular AF defects through minimally-invasive implantation. Thermoset PCL-SF scaffolds were produced with two concentrations of the PCL-diacrylate macromer solution (40% and 60% w/v) and five PCL:SF ratios (100:0, 90:10, 80:20, 70:30, and 60:40). Chemical characterization of the scaffolds confirmed the effective blending of PCL and SF macromolecules with uniform distribution of SF throughout the scaffolds and formation of β-sheet conformation in SF. Mechanical characterization of the scaffolds showed: (i) highly interconnected pores with pore sizes of 260–265 μm, (ii) tensile moduli and yield strains of 0.22-0.31 MPa and 41-61%, respectively, and (iii) compressive moduli of 0.11- 0.27 MPa. The above-mentioned porosities were within the range that reportedly supports AF cell penetration, adhesion, and accumulation of a collagen I rich extracellular matrix. The tensile moduli of tested scaffolds were in the range of human AF tissue in radial and axial directions. The compressive moduli were slightly less than native AF tissue but approximately an order of magnitude higher than those of other AF repair biomaterials. Additionally, the in vitro biodegradation rate of scaffolds was found to be slow enough to provide mechanical support in the time frame needed for AF regeneration. However, scaffolds were unable to exhibit shape-memory capabilities suitable for self-fitting in AF defects. Further optimization of the scaffold design with respect to shape memory capability for minimally-invasive delivery and self-fitting in AF defect will be required for clinical application.Item Open Access Scaling Cellular Aggregate Production in a Microwell System(2020-09-14) Kondro, Douglas A.; Ungrin, Mark D.; Kallos, Michael S.; Nezhad, Amir S.Microtissues, in the form of cellular aggregates, offer a promising format for cell-based therapies, such as the treatment of type 1 diabetes with beta cell replacement. One major challenge is producing a sufficient quantity of microtissue for clinical use. For instance, type 1 diabetes requires approximately 1 million cellular aggregates for treatment of a single individual. Static culture methods of producing aggregates, such as the microwell system, offer an accessible format of producing high quality, size-controlled cellular aggregates, however they cannot easily generate the quantity required for a clinical treatment. This thesis scales an existing microwell format by producing a robust, self-contained, multilayered microwell bioreactor capable of generating over 250,000 cellular aggregates in a well plate footprint. The design of the microwell bioreactor consists of a sealed chamber where cells aggregate on thin sheets of microwells in the device. The microwells are made with an oxygen permeable polymer to facilitate gas exchange in the sealed system, that was demonstrated to deliver approximately 4 times the oxygen to cells in comparison with the well plate format. The design process utilizes hot-embossing, allowing for efficient and scalable manufacturing, with a demonstrated cycle time as low as 15 seconds per microwell sheet. The final device is robust, capable of withstanding centrifugation up to 2000 g, and autoclaving sterilization. Further, the protocol for cell culture is simplified as it allows for removal of >97% of media during a media change without disturbing aggregates, and removal of >98% of cellular aggregates after long term culturing. Biological validation and demonstration of clinical utility was performed using 12 donor islet batches. The microwell bioreactor could scale the production of functional pseudoislets in the form of size-controlled cellular aggregates. Pseudoislets were demonstrated to have indistinguishable functional performance in compared to previously published methods. Applicability of this novel system was also demonstrated for other diseases by successfully differentiating pluripotent stem cells and aggregating testicular organoids. The unique design of the microwell bioreactor provides a straightforward tool to enable researchers to immediately scale static culture experiments, allowing for translation of cell-based therapies to clinical use.Item Open Access Semaphorin3f in the maturation of the outer retina(2020-09-25) Mori-Kreiner, Risa; McFarlane, Sarah; Childs, Sarah J.; Guo, Jiami; Ungrin, Mark D.Cells of multicellular organisms have the remarkable ability to coordinate and control dynamic cellular activities in response to changes in their environment. From development into tissue homeostasis, cells communicate with each other through a myriad of intercellular signalling mechanisms. The large family of Semaphorins is a group of well-known extracellular signalling molecules implicated in a wide range of diverse physiological functions. In particular, the expression of secreted Class III Semaphorins (Sema3s) in the retina, not only during development but also in adult tissue, raises interesting questions about their tissue-specific spatiotemporal roles. The vertebrate retina is a highly complex, light-sensitive tissue that lines the back of our eyes. Within the retina, there are two key players that enables our ability to see: the retinal pigment epithelium (RPE) and the photoreceptors. Located in the outermost layer of the retina, the RPE and photoreceptors develop and mature together throughout the lifetime of the organism, forming an interdependent relationship that is highly critical for visual function. This thesis explores, using CRISPR/Cas9-generated loss-of-function mutants (sema3faca304), cell autonomous and non-cell autonomous roles of an RPE-secreted protein, Semaphorin3f (Sema3f), in the zebrafish retina. First, I demonstrate that both cell types, the RPE and photoreceptors, express multiple members of well-known Sema3 receptors, Nrp and PlxnA families. Second, I define a cell autonomous role of Sema3fa in maturing RPE. The loss of Sema3fa does not affect the maturation of the RPE at the transcriptional and morphological level, but does result in the perturbation of appropriate physiological responses to light conditions. Last, I demonstrate the non-cell autonomous role of Sema3fa in the development and specification of maturing photoreceptors. My work elucidates one of the many endogenous roles of Sema3fa as a regulator of the maturing retina to add to the growing literature of Sema signalling in events other than development.