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Browsing by Subject "Bioreactor"

Now showing 1 - 7 of 7
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    Bioreactor Conditioning for Accelerated Remodeling of Fibrin-Based Tissue Engineered Heart Valves
    (2015-05) Schmidt, Jillian
    Fibrin is a promising scaffold material for tissue engineered heart valves, as it is completely biological, allows for engineered matrix alignment, and is able to be degraded and replaced with collagen by entrapped cells. However, the initial fibrin matrix is mechanically weak, and extensive in vitro culture is required to create valves with sufficient mechanical strength and stiffness for in vivo function. Culture in bioreactor systems, which provide cyclic stretching and enhance nutrient transport, has been shown to increase collagen production by cells entrapped in a fibrin scaffold, accelerating strengthening of the tissue and reducing the required culture time. In the present work, steps were taken to improve bioreactor culture conditions with the goal of accelerating collagen production in fibrin-based tissue engineered heart valves using two approaches: (i) optimizing the cyclic stretching protocol and (ii) developing a novel bioreactor system that permits transmural and lumenal flow of culture medium for improved nutrient transport. The results indicated that incrementally increasing strain amplitude cyclic stretching with small, frequent increments in strain amplitude was optimal for collagen production in our system. In addition, proof of concept studies were performed in the novel bioreactor system and increased cellularity and collagen deposition near the lumenal surface of the tissue were observed.
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    Constructing a Smaller Permeameter to Test the Hydraulic Properties of Bioreactor Bed Media
    (2021-05) Parsi, Armon L
    Bioreactors are a wall of organic filter material that remove nitrate from agricultural tile drainage water through microbial anaerobic respiration, removing it from our freshwater resources. Permeameters can be used to analyze the hydraulic capabilities of these media and optimize their performance, allowing less nitrates to contaminate freshwater sources.
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    Development of a completely biological tissue engineered heart valve.
    (2009-04) Syedain, Zeeshan Hayder
    In the United States alone, over 100,000 heart valve replacement procedures are performed each year, with approximately 45% of patients below age 65. While current mechanical and bioprosthetic heart valves are viable options, they have several limitations. The most significant limitation is for pediatric patients, since neither of these valve types grow and remodel with the patient. Tissue engineering provides a methodology to create functional heart valves that can grow and remodel similar to native tissue once implanted. Several tissue engineering approaches have been proposed using decellularized native scaffolds, synthetic biopolymers, and biological polymers seeded with cells. Fibrin provides a scaffold to create tissue-engineered heart valves (TEHV) that are completely biological with an environment permissive for extracellular matrix (ECM) deposition. Previous research in our lab has demonstrated the feasibility of creating a fibrin-based TEHV with neonatal human dermal fibroblast (nHDF) that yields valve leaflets with structural and mechanical anisotropy similar to native leaflets. However, the TEHV had sub-optimal tensile mechanical properties and was thus unable to withstand physiological forces. The development of tissue can be accelerated by both chemical and mechanical stimulus. Previously, fibrin based TEHV were cultured with chemical stimulus in the form of growth factors supplemented in the culture medium resulting in improved ECM deposition by the cells; however, no mechanical stimulation was applied. Prior research in both our lab and by other researchers has shown cyclic stretching with constant strain amplitude is a method to stimulate remodeling of biological scaffolds seeded with cells. Initial experiments were conducted to evaluate the effect of cyclic stretching on fibrin-based tubular constructs seeded with porcine valve interstitial cells (PVIC) and nHDF. Cyclic stretching with 10% constant strain amplitude applied for 3 weeks led to modest improvement in tensile properties of the tubular constructs. We hypothesized that long-term cyclic stretching, as was used in this study, could induce cellular adaptation, minimizing the benefits of cyclic stretching. This hypothesis was tested in subsequent experiments using tubular constructs cultured with incremental strain amplitude cyclic stretching, with an average strain of 10% for 3 weeks. Both PVIC and nHDF seeded constructs exhibited a 2-fold improvement in ultimate tensile strength (UTS) and collagen density over samples conditioned with constant strain amplitude strteching. To verify that this was the result of a cellular response, phosphorylation of extracellular signal-regulated kinase (ERK) was measured by western blot. At 5 weeks, the phosphorylated ERK was 255% higher in incremental cyclic strained samples compared to constant strain samples. nHDF-seeded tubular constructs were also used to optimize the use of transforming growth factor beta (TGF-β). Studies showed that under cyclic stretching conditions, TGF-β has detrimental effects on total collagen deposition and collagen maturation. Western blot analysis showed a decrease in p-ERK signaling in TGF-β treated samples. However, TGF-β use demonstrated a benefit by increasing the elastin content of the tissue constructs. In subsequent experiments, a sequence of cyclic stretching and TGF-β supplementation was used to optimize tensile mechanical properties and elastin content of the engineered tissue. Based on the results with tubular constructs, a novel bioreactor was designed to apply controlled cyclic stretching to the fibrin-based TEHV. Briefly, the valve was mounted on two plastic end-pieces with elastic latex tube placed around TEHV. Using a reciprocating syringe pump, culture medium was cyclically pumped into the bioreactor. The root distension, which was determined by the stiffer latex was used as a control parameter, and in turn stretched the leaflets. A separate flowloop (connected to the bioreactor end-pieces) was used to control nutrient transport to the TEHV. Using an incremental strain amplitude stretching regime, fibrin-based TEHV were conditioned in the bioreactor for 3 weeks. Cyclically stretched valves (CS valve) had improved tensile properties and collagen deposition compared to statically-cultured valves. The mechanical stiffness (modulus) and anisotropy (measured as ratio of leaflet modulus in circumferential to radial directions) in the leaflets was comparable to native sheep pulmonary valve leaflets. Collagen organization/ maturation also improved in CS valves over statically-cultured valves as observed by picrosirius red staining of tissue crosssections. In addition, the CS valve root could withstand pressures of up to 150 mmHg and its compliance was comparable to that of the sheep pulmonary artery at physiological pressures. To assess in vivo remodeling TEHV were implanted in the pulmonary artery of two sheep for 4.5 weeks with the pulmonary valve either left intact or rendered incompetent by leaflet excision. Echocardiography immediately after implantation showed functional coapting leaflets, with normal right heart function. It was also performed just prior to explantation, revealing functional leaflets although with moderate regurgitation in both cases and a partial detachment of one leaflet from the root in one case. The explanted leaflets had thickness and tensile properties comparable the implanted leaflets. There was endothelialization on the lumenal surface of the TEHV root. These preliminary results are unprecedented for a TEHV developed from a biological scaffold; however, many issues remain to be surmounted. In further development of the TEHV with a fibrin scaffold, photo-cross linking of the fibrin gel was utilized as a method to stiffen the matrix, thereby inhibiting excessive cell-induced compaction. Preliminary studies with tubular constructs demonstrated reduced compaction of cross-linked fibrin gel during cyclic stretching with no effect on nHDF proliferation or deposited collagen. In addition, a preliminary investigation using blood outgrowth endothelial cells (BOEC) has been conducted to assess their adhesion to the remodeled TEHV surface. Studies showed BOEC adhesion and proliferation on remodeled fibrin surface creating a confluent layer after 4 days of culture. Successful seeding of sheep BOEC on the TEHV surface prior to implantation would reduce the risk of clotting. Overall, the studies presented in this dissertation advance the development of a completely biological tissue-engineered heart valve. These studies improve our understanding of the role of cyclic stretching in tissue remodeling and have furthered the science of mechnotransduction and tissue remodeling.
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    Electrochemical analysis of Shewanella oneidensis engineered to bind gold electrodes.
    (2011-03) Kane, Aunica L.
    Three-electrode bioreactors can be utilized to examine the mechanisms involved in electron flow from bacteria to insoluble electron acceptors and allow these processes to be analyzed quantitatively. As an electrode, gold is an ideal surface to study the electrophysiology occurring during extracellular respiration; yet previous research has shown that Shewanella is resistant to colonization on gold surfaces. Therefore, the goal of this work was to direct adhesion of Shewanella oneidensis to gold surfaces via cell surface display of a modified E. coli outer membrane protein, LamB, and a gold-binding peptide (5rGBP) to encourage microbe-electrode interaction, improve whole-cell biocatalytic systems, and increase overall current production. Expression of LamB-5rGBP increased the affinity of Shewanella for gold surfaces, but also led to the displacement of certain outer membrane components required for extracellular electron transport. Displacement of these outer membrane proteins decreased the rate at which Shewanella was able to reduce both insoluble iron and riboflavin. Expression of LamB-5rGBP, although effectively increasing attachment to gold, did not greatly increase current production in gold-electrode bioreactors.
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    Enhanced Microbial Sulfate Removal Through a Novel Electrode-Integrated Bioreactor
    (2018-07) Takaki, Daniel
    In northeast Minnesota, elevated levels of sulfate in freshwater systems is a topic of great interest, due to potential adverse impacts to wild rice ecosystems. Sulfate may contribute to methylmercury production and eutrophication in certain conditions. Increased interest has emerged for developing low cost and efficient technologies to treat high levels of sulfate in mining and industrial waste water. The use of biological sulfate reduction is a promising and economically viable plan for maintaining low levels of sulfate and sulfide, but its performance is highly variable. This project developed a sediment bioelectrochemical batch reactor that used a low electrical potential to enhance and sustain biological sulfate reduction by continuously supplying electron donor substrates (electrolytic hydrogen) to sulfate reducing bacteria. The project aims to understand the effect of a low applied voltage on the efficacy of sulfate reduction and iron sulfide formation. Reactors contained creek sediment (Second Creek, MN) and an artificial mine water with a sulfate concentration of ~1000 ppm. The sulfur chemistry in the pore water of the reactors was assessed to determine sulfate reduction, resulting in over 90% reduction in porewater sulfate at the cathode in batch reactors, where electrolytic hydrogen gas was generated at a rate of 4.14 mmol/day. Simultaneously, ferrous iron was released into the reactor via iron electrodissolution and reacted with reduced sulfide ions to form iron sulfide precipitates. This level of hydrogen generation was sustained over a 14-day period and successfully showed that the application of a low voltage to sediment bioreactors is a promising technology to treat sulfate contaminated waste waters. The microbial community structure and relative abundance of different species associated with sulfate reduction were also examined. It was shown that relative abundance of sulfate reducing bacteria, specifically Desulfovibrio, a genus of deltaproteobacteria positively associated with sulfate reduction, which utilize hydrogen as their preferred electron donor, increased throughout batch reactor operation when operated at 2V. Finally, the sediment bioelectrochemical batch reactor served as a proof of concept for the application of low electrical potential to enhance and sustain biological sulfate reduction. The outcomes of this reactor operation laid the groundwork to develop a prototype flow-through bioelectrochemical reactor designed to handle larger volumes of waste water for an extended period of time. Preliminary results from this flow-through reactor demonstrated the ability to generate a constant supply of electrolytic hydrogen used by sulfate reducing bacteria. Through these experiments, recommendations have been made to improve efficacy of flow-through reactors.
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    Generating cells for lung tissue engineering
    (2014-05) Turgut, Aylin
    Decellularization of essential organs such as the lung has become an integral part of regenerative medicine. As the availability of donors is very low reseeding of these decellularized organs with a patient's own cells is a potential therapy for those desperately in need. This way, the risks associated with allogeneic immune rejection are avoided. Some research groups have been successful in reseeding the lung with allogeneic differentiated cells. However, the barrier to presently overcome is to seed with stem cells and ensure these cells differentiate to all the desired cell types of the lung. Another obstacle is obtaining the desired number of cells for recellularization of large organs such as the lung. Scale-up methods using stirred vessel bioreactors with conditions similar to the physiological environment are a desirable alternative to conventional cell culture. In this study, I demonstrate large-scale cell culture in stirred flask bioreactors by facing the challenges of scale-up from 2D to 3D suspension culture. I also show the existence of exosomes in decellularized pig and mouse lung and identify the miRNAs (miRNAs) contained within them. MicroRNAs are becoming increasingly popular research tools as they are known to regulate many essential processes. Exosomes are enriched with miRNAs and can be shuttled between cells, thereby affecting target cell behavior. I utilized the exosomes from decellularized lungs in directed differentiation of induced pluripotent stem cells (iPSCs) to the definitive endoderm (DE) lineage and compared it with conventional differentiation methods. The exosomes had a profound effect on the morphology of the cells which will lead to further studies on exosome-directed differentiation procedures.
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    Scalable culture systems for expansion and directed differentiation of rat multipotent adult progenitor cells.
    (2010-05) Park, Yonsil
    Advances in stem cell science have stimulated the prospect of stem cells as therapeutics. For the translation of stem cell research to technology, robust and efficient expansion and differentiation is essential. Rat multipotent adult progenitor cells (rMAPCs) are a type of adult stem cells isolated from the rodent bone marrow. MAPCs can be expanded in vitro without obvious senescence, and are capable of differentiating into cell types of mesoderm, endoderm, and ectoderm in vitro. They are typically maintained surface adherent at low cell densities of 100-300 cells/cm2 in order to maintain their broad potency, which would make scale-up for further clinical applications cumbersome. In this study, we explored different cultivation methods to investigate the feasibility of scalable culture systems for rMAPCs: (1) high density 2D culture, stirred bioreactor culture as (2) 3D aggregates and (3) on microcarriers. Culturing cells under hypoxic condition (5% O2) during the isolation, has yielded rMAPC expressing high levels of the embryonic stem cell specific transcription factor Oct4, which is associated with their greater potency. First, the effect of oxygen tension and cell density on the growth rate and potency of MAPC were examined. MAPC exhibited an increased growth rate at hypoxic conditions (5%) than at normoxic conditions (21%). Furthermore, when inoculated at a cell density of 1000 cells/cm2, MAPC exhibited a small but significant increase in growth rate compared to cells seeded at 300 cells/cm2 at both oxygen levels, though the difference was more pronounced under hypoxic conditions. The Oct4 mRNA or protein expression level and the ability of MAPC to differentiate towards endothelium- and hepatocyte-, and neuroectoderm-like cells were shown to be unaffected by cultivation at a higher cell density and/or oxygen tension for 48 days (1000 cells/cm2; 21% O2; with subculturing every 48 hr). The results provide evidence that MAPC isolated under hypoxic conditions and expressing high levels of Oct4 can be readily cultured at a higher cell density without any apparent loss of potency. Encouraged by MAPCs ability to grow at high density, we explored aggregate formation of MAPC for cell expansion as well as differentiation. Culture in aggregates may be an ideal method to allow large scale expansion, if combined with bioreactor cultures. Time lapse microscopy revealed three stages during the initial period of aggregate formation: agglomeration, compaction, and expansion. Compared to cells from adherent culture, significantly more cells from 3D culture are in G0/G1 phase and fewer in S phase suggesting a partial restriction in cell proliferation possibly due to spatial restriction in aggregates. There was no significant difference in Oct4 level and aggregate size when aggregation was at 5% or 21% O2 after 4 day culture. However, aggregation at 21% O2 increased the percent of cells in G0/G1 and increased expression of early differentiation markers such as Flk1 and Afp. Cultivation of MAPC aggregates in stirred bioreactor lead to a 70-fold expansion in six days with final cell densities of about 106 cells/ml. Importantly, the MAPC aggregates recovered from stirred bioreactors could be differentiated to hepatocyte-like cells that expressed Albumin, Aat, Tat transcripts and also secreted albumin and urea.The cells expressed several mature hepatocyte-lineage genes and asialoglycoprotein receptor-1 (ASGPR-1) surface protein, and secreted albumin and urea. Lastly, the experience with MAPC microcarrier culture was extended to human embryonic stem cell (hESC) microcarrier culture. hESCs as small clumps were attached to Matrigel-coated microcarriers and expanded 10-fold during 4 day static culture. The level of pluripotency-related genes, OCT4 and SOX2, were maintained compared to day 0 cells. The cells expanded on microcarriers underwent hepatic differentiation to increase hepatic genes such as AFP and ALBUMIN. Both aggregate and microcarrier cultivation methods for scalable expansion combined with differentiation can potentially be used to generate large numbers of MAPC and MAPC-derived differentiated cells. These culture systems thus offer the potential of large-scale expansion and differentiation of stem cells in a more controlled bioreactor environment.