Development of a 3D bioprinting and standalone bioreactor unit for the production and maintenance of bioscaffolds in vitro
- Authors: Hundling, Jethro Daniel
- Date: 2021-10-29
- Subjects: Bioreactors , Tissue scaffolds , Cell culture , Polyethylene glycol Biotechnology , 3D bioprinting , Poly(ethylene glycol) diacrylate (PEGDA)
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192063 , vital:45192
- Description: The most common method for in vitro cell culture currently is to grow a specific cell type in isolation, in monolayer format, adhered to a 2D culture surface. This brings about many limitations in comparison to in vivo models due to altered cell phenotypes, as caused by the culturing technique itself, and the lack of naturally occurring cell-to-cell interactions. Three dimensional mammalian cell culture technologies have the potential to overcome these limitations, and provide models more representative of natural systems. Unfortunately, the cost and difficulty associated with achieving sustainable and useful 3D mammalian cell culture is still very high, preventing its widespread adoption across scientific platforms. In this research, we investigate the feasibility of developing and producing a visible light-based 3D stereolithographic bioprinter to produce 3D scaffolds for cell culture. Furthermore, we investigate the possibility of developing and implementing a forced perfusion bioreactor system, which would support the produced scaffold and improve longer-term culture conditions. The developed 3D bioprinter, and bioreactor designs were developed and tested alongside Poly (ethylene glycol) diacrylate (PEGDA), a versatile synthetic scaffold material. PEGDA itself was also evaluated for its printability, robustness in culture conditions over time, and its ability to maintain 3D mammalian cell culture. This research showed that both the developed 3D bioprinter, and bioreactor unit were capable of producing and maintaining an easily modifiable PEGDA scaffold, in culture conditions. In addition, the PEGDA formulation developed was shown to allow for the effective and reproducible 3D cell culture conditions over the medium term, with automated media feeding. The research presented here aimed to illustrate a proof of concept that the low-cost development and production of 3D culture scaffold production and maintenance systems was feasible to the scientific research environment. This technology can then be built upon, into a system that would then allow for the broader adoption and investigation of 3D cell culture as a tool within the scientific community. , Thesis (MSc) -- Faculty of Science, Biotechnology Innovation Centre, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Hundling, Jethro Daniel
- Date: 2021-10-29
- Subjects: Bioreactors , Tissue scaffolds , Cell culture , Polyethylene glycol Biotechnology , 3D bioprinting , Poly(ethylene glycol) diacrylate (PEGDA)
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192063 , vital:45192
- Description: The most common method for in vitro cell culture currently is to grow a specific cell type in isolation, in monolayer format, adhered to a 2D culture surface. This brings about many limitations in comparison to in vivo models due to altered cell phenotypes, as caused by the culturing technique itself, and the lack of naturally occurring cell-to-cell interactions. Three dimensional mammalian cell culture technologies have the potential to overcome these limitations, and provide models more representative of natural systems. Unfortunately, the cost and difficulty associated with achieving sustainable and useful 3D mammalian cell culture is still very high, preventing its widespread adoption across scientific platforms. In this research, we investigate the feasibility of developing and producing a visible light-based 3D stereolithographic bioprinter to produce 3D scaffolds for cell culture. Furthermore, we investigate the possibility of developing and implementing a forced perfusion bioreactor system, which would support the produced scaffold and improve longer-term culture conditions. The developed 3D bioprinter, and bioreactor designs were developed and tested alongside Poly (ethylene glycol) diacrylate (PEGDA), a versatile synthetic scaffold material. PEGDA itself was also evaluated for its printability, robustness in culture conditions over time, and its ability to maintain 3D mammalian cell culture. This research showed that both the developed 3D bioprinter, and bioreactor unit were capable of producing and maintaining an easily modifiable PEGDA scaffold, in culture conditions. In addition, the PEGDA formulation developed was shown to allow for the effective and reproducible 3D cell culture conditions over the medium term, with automated media feeding. The research presented here aimed to illustrate a proof of concept that the low-cost development and production of 3D culture scaffold production and maintenance systems was feasible to the scientific research environment. This technology can then be built upon, into a system that would then allow for the broader adoption and investigation of 3D cell culture as a tool within the scientific community. , Thesis (MSc) -- Faculty of Science, Biotechnology Innovation Centre, 2021
- Full Text:
- Date Issued: 2021-10-29
The immobilization of Microcystis aeruginosa PCC7806 on a membrane nutrient-gradostat bioreacator for the production of the secondary metobolites
- Authors: Strong, Peter James
- Date: 2002
- Subjects: Microcystis aeruginosa , Myrocystins , Bioreactors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11083 , http://hdl.handle.net/10948/283 , Microcystis aeruginosa , Myrocystins , Bioreactors
- Description: A module and an inoculation technique were developed that would allow for the efficient immobilization of Microcystis aeruginosa PCC7806 on a synthetic membrane. A variety of module types, membranes (ceramic, tubular polyethersulfone and externally skinless polyethersulfone capillary membrane), and methods of immobilization (adsorption, pressure filtration and a developed technique that involved drying a cell slurry on a membrane) were assessed. The morphological properties that affected the immobilization of Microcystis aeruginosa PCC7806, as well as the effects of immobilization upon cell morphology were assessed. Cells in the stationary growth phase, which had a well-developed extra-cellular polysaccharide layer and no gas vesicles, were optimal for immobilization. Microcystin production under immobilized conditions was assessed under different nitrate concentrations, light intensities, biofilm thickness and immobilization times. Additional work included assaying for Microcystin production of two airlift-grown cultures under a high light intensity and complete nutrient deprivation and the inoculation of a ceramic membrane. An immunological technique was used to elucidate where toxin production was greatest within a biofilm immobilized upon an externally skinless polyethersulfone capillary membrane. The externally skinless polyethersulfone capillary membrane was evaluated to assess homogeneity and the physical differences between membrane batches that led to the erratic, incomplete biofilm formation, as a biofilm of a constant thickness could not be immobilized. Microcystis aeruginosa PCC7806 was exposed to a variety of solvents in order to permeabilize the cyanobacteria, as that would have enabled a truly continuous extraction process for the metabolite. FDA hydrolysis had to be optimized in order to use it as an indicator of cell viability. In addition a single-step extraction of Microcystin was attempted using live bacteria. A capillary membrane module, containing the externally skinless polyethersulfone capillary membrane, inoculated using pressure filtration, was the most efficient combination to establish a biofilm. Cells that were no longer actively dividing and that lacked buoyancy displayed superior immobilization to cells that were actively dividing and buoyant. The immobilized cells did produce Microcystin but in much lower concentrations to cells grown in an airlift culture. Biofilms grown with a higher nitrate concentration, a lower biofilm thickness and a lower light intensity had a higher specific microcystin content, while biofilms with a higher nitrate concentration a lower light intensity and a longer growth period displayed the a greater toxin production per mm2 of membrane. Microcystin occurred at its highest concentration in cells just above the pore opening. The diffusion of nutrients occurred relatively quickly to the outside layers of the biofilm, with a true gradient being established laterally from these nutrient veins that were above the pores. Permeabilization of the cells proved unsuccessful, as cells that remained viable did not release the intracellular compound into the surrounding medium.
- Full Text:
- Date Issued: 2002
- Authors: Strong, Peter James
- Date: 2002
- Subjects: Microcystis aeruginosa , Myrocystins , Bioreactors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11083 , http://hdl.handle.net/10948/283 , Microcystis aeruginosa , Myrocystins , Bioreactors
- Description: A module and an inoculation technique were developed that would allow for the efficient immobilization of Microcystis aeruginosa PCC7806 on a synthetic membrane. A variety of module types, membranes (ceramic, tubular polyethersulfone and externally skinless polyethersulfone capillary membrane), and methods of immobilization (adsorption, pressure filtration and a developed technique that involved drying a cell slurry on a membrane) were assessed. The morphological properties that affected the immobilization of Microcystis aeruginosa PCC7806, as well as the effects of immobilization upon cell morphology were assessed. Cells in the stationary growth phase, which had a well-developed extra-cellular polysaccharide layer and no gas vesicles, were optimal for immobilization. Microcystin production under immobilized conditions was assessed under different nitrate concentrations, light intensities, biofilm thickness and immobilization times. Additional work included assaying for Microcystin production of two airlift-grown cultures under a high light intensity and complete nutrient deprivation and the inoculation of a ceramic membrane. An immunological technique was used to elucidate where toxin production was greatest within a biofilm immobilized upon an externally skinless polyethersulfone capillary membrane. The externally skinless polyethersulfone capillary membrane was evaluated to assess homogeneity and the physical differences between membrane batches that led to the erratic, incomplete biofilm formation, as a biofilm of a constant thickness could not be immobilized. Microcystis aeruginosa PCC7806 was exposed to a variety of solvents in order to permeabilize the cyanobacteria, as that would have enabled a truly continuous extraction process for the metabolite. FDA hydrolysis had to be optimized in order to use it as an indicator of cell viability. In addition a single-step extraction of Microcystin was attempted using live bacteria. A capillary membrane module, containing the externally skinless polyethersulfone capillary membrane, inoculated using pressure filtration, was the most efficient combination to establish a biofilm. Cells that were no longer actively dividing and that lacked buoyancy displayed superior immobilization to cells that were actively dividing and buoyant. The immobilized cells did produce Microcystin but in much lower concentrations to cells grown in an airlift culture. Biofilms grown with a higher nitrate concentration, a lower biofilm thickness and a lower light intensity had a higher specific microcystin content, while biofilms with a higher nitrate concentration a lower light intensity and a longer growth period displayed the a greater toxin production per mm2 of membrane. Microcystin occurred at its highest concentration in cells just above the pore opening. The diffusion of nutrients occurred relatively quickly to the outside layers of the biofilm, with a true gradient being established laterally from these nutrient veins that were above the pores. Permeabilization of the cells proved unsuccessful, as cells that remained viable did not release the intracellular compound into the surrounding medium.
- Full Text:
- Date Issued: 2002
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