Thermoresponsive magnetic colloidal gels for in vitro cell expansion

Recent studies and clinical trials have shown the potential of cell-based therapies for the treatment of a number of diseases and organ/ tissue damages. However, limited availability of some therapeutically important cells (i.e. adult stem cells) still remain as main challenges in the development of tissue engineering through to the clinic. Healthy cells are required in large numbers to form a tissue-engineered construct and primary cells must therefore be expanded in vitro for both scientific and clinical applications. Various strategies have been developed to expand cells in vitro with increasing emphasis on 3D matrices because it can provide microenvironments which more closely mimic in vivo systems. In this way the inherent difficulties associated with 2D culture such as loss of phenotype could be overcome. Moreover, 3D matrices provide higher surface areas to support expansion of larger cell numbers compared to monolayer culture. Although each 3D method has certain advantages, there is no single technique that can be used to produce material assemblies that address all the fundamental problems linked to 3D cell seeding (penetration into the scaffold), passaging (use of enzymes), and harvesting (cell yield). Recently, thermally reversibly-associating particles have been studied for the growth and support of multiple cell types and for delivery of therapeutic cells. But coupling of thermoresponsive properties to magnetic microspheres would enhance the 3D culture and expansion of multiple cell types, and facilitate rapid recovery of the expanded cell population by simple magnetic separation. In this study, it was proposed that the thermoresponsive properties would allow simple cell seeding at temperatures below the LCST of polymer stabiliser when the suspension is flowing and upon heating to above the LCST cells would be encapsulated and cultured within the particle gels (every cells surrounded by a number of particles, as the size of the particles are much smaller than the cells). The magnetic responsive property would allow efficient and scaffold free cell recovery after expansion without the need for using trypsin or enzymatic treatment. The ‘switchable’ component of reversibly associating colloidal microparticles were prepared via two different strategies. In the first strategy, thermoresponsive PDEGMA was physically adsorbed onto the surface of PS microspheres, whereas, in the second strategy, PDEGMA was chemically grafted from functionalised PCMS microspheres via SI-ATRP. The most simple method i.e. physical adsorption is rapid and can be adapted to many microparticle surfaces but has the drawback of possible desorption of polymer chains during extended use. The chemical grafting method i.e. the formation of covalent bonds between the polymer corona and the microparticle core provides robust and well defined materials but is more complex and time-consuming. In both cases, particle aggregation in their suspensions occurred on increasing the temperature to above the LCST of PDEGMA, but could be reversed by cooling the suspensions back to below the LCST. This confirmed the presence of the thermoresponsive polymer on the surface of the microspheres using both methods (adsorption and grafting). Rheological measurements demonstrated that the viscoelasticity of the prepared particle gels can be tuned, enabling these gels to have the mechanical properties that should facilitate their applications as 3D cell scaffolds for in vitro expansion of cells. Cell culture studies showed that these microparticle based scaffolds can support expansion of clinically relevant cell types (human MSC) and allowed efficient cell recovery after proliferation without the need for using trypsin or enzymatic treatment. Overall, those results suggest that the designed scaffolds had great potential for 3D in vitro cell expansion. The new developed materials have excellent biocompatibility, allow simple and rapid cell seeding and cell recovery after expansion, and possess mechanical strength and stability to support cell growth and proliferation. The materials developed and studied in this thesis may represent a significant contribution to the fields of biomaterials, tissue engineering, 3D cell culture and even bio-separation

GIS Visualization of Solid Waste Disposal Sites and Environmental Impacts in Kurdistan Region-Iraq

The increase in the quantity of municipal solid waste (MSW) has made environmental problems in the Kurdistan Region (KR)-Iraq. Current study illustrated components and generation rates (GR) of MSW in seven different cities of KR. Geographic information system (GIS) was applied to locate MSW disposal sites, components, and generation rate in the cities. The study reported the maximum GR for MSW in Sulaymaniyah City which was 1.20 Kg/Capita/day and the minimum GR for domestic solid waste in Erbil City was 0.65 Kg/Capita/day. In-addition, the amount of organic waste component (OWC) in Erbil, Halabja, Sulaymaniyah, Semel, Duhok, Qaladize, and Ranya Cities were 79.34 %, 58 %, 65 %, 65 %, 79 %, 75.1 %, and 67.05 %, respectively. The average GR and OWC were calculated to be 0.972 Kg/Capita/day and 71.91%, respectively. Consequently, all MSW disposal sites had great impact to the surrounding areas resulting in air, water, and soil contamination

Thermoresponsive magnetic colloidal gels for in vitro cell expansion

Recent studies and clinical trials have shown the potential of cell-based therapies for the treatment of a number of diseases and organ/ tissue damages. However, limited availability of some therapeutically important cells (i.e. adult stem cells) still remain as main challenges in the development of tissue engineering through to the clinic. Healthy cells are required in large numbers to form a tissue-engineered construct and primary cells must therefore be expanded in vitro for both scientific and clinical applications. Various strategies have been developed to expand cells in vitro with increasing emphasis on 3D matrices because it can provide microenvironments which more closely mimic in vivo systems. In this way the inherent difficulties associated with 2D culture such as loss of phenotype could be overcome. Moreover, 3D matrices provide higher surface areas to support expansion of larger cell numbers compared to monolayer culture. Although each 3D method has certain advantages, there is no single technique that can be used to produce material assemblies that address all the fundamental problems linked to 3D cell seeding (penetration into the scaffold), passaging (use of enzymes), and harvesting (cell yield). Recently, thermally reversibly-associating particles have been studied for the growth and support of multiple cell types and for delivery of therapeutic cells. But coupling of thermoresponsive properties to magnetic microspheres would enhance the 3D culture and expansion of multiple cell types, and facilitate rapid recovery of the expanded cell population by simple magnetic separation. In this study, it was proposed that the thermoresponsive properties would allow simple cell seeding at temperatures below the LCST of polymer stabiliser when the suspension is flowing and upon heating to above the LCST cells would be encapsulated and cultured within the particle gels (every cells surrounded by a number of particles, as the size of the particles are much smaller than the cells). The magnetic responsive property would allow efficient and scaffold free cell recovery after expansion without the need for using trypsin or enzymatic treatment. The ‘switchable’ component of reversibly associating colloidal microparticles were prepared via two different strategies. In the first strategy, thermoresponsive PDEGMA was physically adsorbed onto the surface of PS microspheres, whereas, in the second strategy, PDEGMA was chemically grafted from functionalised PCMS microspheres via SI-ATRP. The most simple method i.e. physical adsorption is rapid and can be adapted to many microparticle surfaces but has the drawback of possible desorption of polymer chains during extended use. The chemical grafting method i.e. the formation of covalent bonds between the polymer corona and the microparticle core provides robust and well defined materials but is more complex and time-consuming. In both cases, particle aggregation in their suspensions occurred on increasing the temperature to above the LCST of PDEGMA, but could be reversed by cooling the suspensions back to below the LCST. This confirmed the presence of the thermoresponsive polymer on the surface of the microspheres using both methods (adsorption and grafting). Rheological measurements demonstrated that the viscoelasticity of the prepared particle gels can be tuned, enabling these gels to have the mechanical properties that should facilitate their applications as 3D cell scaffolds for in vitro expansion of cells. Cell culture studies showed that these microparticle based scaffolds can support expansion of clinically relevant cell types (human MSC) and allowed efficient cell recovery after proliferation without the need for using trypsin or enzymatic treatment. Overall, those results suggest that the designed scaffolds had great potential for 3D in vitro cell expansion. The new developed materials have excellent biocompatibility, allow simple and rapid cell seeding and cell recovery after expansion, and possess mechanical strength and stability to support cell growth and proliferation. The materials developed and studied in this thesis may represent a significant contribution to the fields of biomaterials, tissue engineering, 3D cell culture and even bio-separation

Lactoferrin-loaded alginate microparticles to target Clostridioides difficile infection

Some forms of bovine lactoferrin (bLf) are effective in delaying Clostridioides difficile growth and preventing toxin production. However, therapeutic use of bLf may be limited by protein stability issues. The objective of this study was to prepare and evaluate colon-targeted, pH-triggered alginate microparticles loaded with bioactive bLf and to evaluate their anti-C. difficile defence properties in vitro. Different forms of metal-bound bLf were encapsulated in alginate microparticles using an emulsification/internal gelation method. The microparticles were coated with chitosan to control protein release. In vitro drug release studies were conducted in pH-simulated gastrointestinal conditions to investigate the release kinetics of encapsulated protein. No significant release of metal-bound bLf was observed at acidic pH; however, on reaching simulated colonic pH, most of the encapsulated lactoferrin was released. The application of bLf (5mg/mL) delivered from alginate microparticles to human intestinal epithelial cells (hIECs) significantly reduced the cytotoxic effects of toxins A and B as well as bacterial supernatant on Caco-2 and Vero cells, respectively. These results are the first to suggest that alginate-bLf microparticles show protective effects against C. difficile toxin-mediated epithelial damage and impairment of barrier function in hIECs. The future potential of lactoferrin-loaded alginate microparticles against C. difficile deserves further study

Professor František Kadeřávek (on the occasion of his seventieth birthday)

We report the synthesis of thermo-responsive polymer brushes with Upper Critical Solution Temperature (UCST)-type behaviour on glass to provide a new means to control cell attachment. Thermoresponsive poly(N-acryloyl glycinamide)-stat-poly(N-phenylacrylamide) (PNAGAm-PNPhAm) brushes with three different monomer ratios were synthesized to give tunable phase transition temperatures (Tp) in solution. Surface energies of surface-grafted brushes of these polymers at 25, 32, 37 and 50 C were calculated from contact angle measurements and atomic force microscopy (AFM) studies confirmed that these polymers were highly extended at temperatures close to Tp in physiologically-relevant media. Importantly, NIH-3T3 cells were attached on the collapsed PNAGAm-PNPhAm brush surface at 30 C after 20 h incubation, while release of cells from the extended brushes was observed within 2 h after the culture temperature was switched to 37 C. Furthermore, the changes in cell attachment followed changes in the Lewis base component of surface energy. The results indicate that, in contrast to the established paradigm of enhanced cell attachment to surfaces where polymers are above a Lower Critical Solution Temperature (LCST), these novel substrates enable detachment of cells from surfaces at temperatures above a UCST. In turn these responsive materials open new avenues for the use of polymer-modified surfaces to control cell attachment for applications in cell manufacture and regenerative medicine