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The establishment, optimisation, and transplantation of a three-dimensional bioprinted syngeneic granuloma model into C57BL/6 mice.
Thesis (PhD)--Stellenbosch University, 2024.
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| Format: | Thesis |
| Language: | en_ZA en_ZA |
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Stellenbosch : Stellenbosch University
2024
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| _version_ | 1867614028444991488 |
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| access_status_str | Open Access |
| author | Mahabeer, Yastika |
| author2 | Du Plessis, Nelita |
| author_browse | Du Plessis, Nelita Mahabeer, Yastika |
| author_facet | Du Plessis, Nelita Mahabeer, Yastika |
| author_sort | Mahabeer, Yastika |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2024. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/130624 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA en_ZA |
| last_indexed | 2026-06-10T12:45:31.220Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2024 |
| publishDateRange | 2024 |
| publishDateSort | 2024 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/130624 The establishment, optimisation, and transplantation of a three-dimensional bioprinted syngeneic granuloma model into C57BL/6 mice. Mahabeer, Yastika Du Plessis, Nelita Nolan, Margaret Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Granuloma Tuberculosis -- Pathogenesis Macrophages Thesis (PhD)--Stellenbosch University, 2024. ENGLISH ABSTRACT: Tuberculosis (TB) is a highly infectious disease, with the characteristic granuloma formation as its hallmark. The structure, composition, function, and maintenance of granulomas are vital aspects to analyse, as they are thought to be the source of Mycobacterium tuberculosis (M. tb) containment or dissemination. Development of methods to enhance immune defences against M. tb is an attractive means to provide protection, however, before such strategies can be developed, an improved understanding of the immune response to the pathogen is warranted. Our investigation uses the C57BL/6 laboratory mouse to analyse the progression of a TB granuloma in vivo. The main aims of this study were to firstly establish and optimise a murine-derived, syngeneic TB granuloma ex vivo with a defined cellular composition using a novel three-dimensional (3D) magnetic bioprinting and levitation cell culture approach, and secondly, to acutely assess the host immune response to a transplanted granuloma in an in vivo environment. To achieve this, cells used for the construction and establishment of the granuloma were harvested from specific pathogen free C57BL/6 mice via a bronchoalveolar lavage to isolate alveolar macrophages, and Magnetic Assisted Cell Sorting to isolate CD14+ and CD3+ cells from the lungs and spleen respectively. Cells were cultured with the assistance of magnetic levitation to establish a spheroid structure, using an open cell sponge matrix as a scaffold. The 3D TB granuloma was surgically transplanted subcutaneously into the lower lumber region of a C57Bl/6 mouse. Granulomas, along with lungs and spleens, were excised 3-, 7-, 14- and 21-days post-transplantation in order to investigate host immune responses as well as mycobacterial control and dissemination characteristics of the complex multidimensional structure at a cellular level. To accomplish this, we employed the use of Luminex, flow cytometry, histopathological analysis, and mycobacterial CFU determination. Overall, our results demonstrate the host-protective nature of the transplanted syngeneic TB granuloma model, with neovascularisation visible 7-days post-transplant, no dissemination of the pathogen in distal organs, and a myriad of pro-inflammatory cytokines present in the structure. In this proof-of-concept exploratory study, we demonstrate both the protective host mechanisms and bacterial responses involved in the mycobacterial granulomatous formation, which is crucial for the TB research field. This novel pipeline will further establish the TB murine model with defined granulomas that could be monitored in vivo allowing for much greater specificity in TB research. AFRIKAANSE OPSOMMING: Tuberkulose (TB) is 'n hoogs aansteeklike siekte, met die kenmerkende granuloomvorming as sy kenmerk. Die struktuur, samestelling, funksie en instandhouding van granulome is noodsaaklike aspekte om te ontleed, aangesien hulle vermoedelik die bron van Mycobacterium tuberculosis inperking of verspreiding is. Ontwikkeling van metodes om immuunverdediging teen M. tuberculosis te verbeter is 'n aantreklike manier om beskerming te bied, maar voordat sulke strategieë ontwikkel kan word, is dit noodsaaklik om 'n beter begrip omtrent die immuunrespons op die patogeen te ontwikkel. In hierdie ondersoek maak ons gebruik die C57BL/6 laboratorium muis om die vordering van 'n TB-granuloom in vivo te ontleed. Die hoofdoelwite van hierdie studie was om eerstens 'n muis-gebaseerde, singeneiese TB-granuloom met 'n gedefinieerde sellulêre samestelling te vesg en te opmaliseer deur gebruik te maak van 'n nuwe driedimensionele (3D) magneese biodruk- en levitasie-selkultuurbenadering, en tweedens, om akuut te assesseer die gasheer se immuunrespons op 'n oorgeplante granuloom in 'n in vivo omgewing. Om dit te bereik, is selle van spesifieke patogeenvrye C57BL/6 muise geoes via 'n brongoalveolêre spoeling om alveolêre makrofage te isoleer, en Magneese Ondersteunde Selsortering geimplimenteer om CD14+ en CD3+ selle van onderskeidelik die longe en milt te isoleer . Selle is gekweek met behulp van magneese levitasie om 'n sferoïede struktuur te vesg, met behulp van 'n oopsel sponsmatriks as 'n ondersteuning struktuur. Die 3D TB granuloom is chirurgies onder die vel van `n ontvanger C57Bl/6 muis oorgeplant . Granulome, tesame met longe en milte, is 3-, 7-, 14- en 21-dae na-oorplanng uitgesny om sodoende die immuun respons sowel as mikobakteriele beheer- en verspreidingseienskappe van die komplekse muldimensionele struktuur op sellulêre vlak te ondersoek. Om dit te bereik, het ons gebruik gemaak van Luminex, vloeisitometrie, histopatologiese analise en mikobakteriese CFU-bepaling gebruik. Oor die algemeen toon ons resultate dat daar in die omgewing van die oorgeplante singeneiese TB-granuloommodel, neovaskularisasie sigbaar word ongeveer 7 dae na-oorplanng. Ons data wys dat geen verspreiding van die patogeen na distale organe plaasvind nie en dat daar 'n magdom pro-inflammatoriese sitokiene teenwoordig in die struktuur. In hierdie bewys-van-konsep verkennende studie, demonstreer ons beide die beskermende gasheermeganismes en bakteriële reaksies betrokke by die mikobakteriële granulomaese vorming, wat deurslaggewend is vir die TB-navorsingsveld. Hierdie nuwe pyplyn sal die TB-muurmodel verder vesg met gedefinieerde granulome wat in vivo gemonitor kan word, wat baie groter spesifisiteit in TB-navorsing moontlik maak. Masters 2024-02-06T09:50:07Z 2024-04-27T00:23:17Z 2024-02-06T09:50:07Z 2024-04-27T00:23:17Z 2024-02 Thesis https://scholar.sun.ac.za/handle/10019.1/130624 en_ZA en_ZA Stellenbosch University x, 140 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Granuloma Tuberculosis -- Pathogenesis Macrophages Mahabeer, Yastika The establishment, optimisation, and transplantation of a three-dimensional bioprinted syngeneic granuloma model into C57BL/6 mice. |
| title | The establishment, optimisation, and transplantation of a three-dimensional bioprinted syngeneic granuloma model into C57BL/6 mice. |
| title_full | The establishment, optimisation, and transplantation of a three-dimensional bioprinted syngeneic granuloma model into C57BL/6 mice. |
| title_fullStr | The establishment, optimisation, and transplantation of a three-dimensional bioprinted syngeneic granuloma model into C57BL/6 mice. |
| title_full_unstemmed | The establishment, optimisation, and transplantation of a three-dimensional bioprinted syngeneic granuloma model into C57BL/6 mice. |
| title_short | The establishment, optimisation, and transplantation of a three-dimensional bioprinted syngeneic granuloma model into C57BL/6 mice. |
| title_sort | establishment optimisation and transplantation of a three dimensional bioprinted syngeneic granuloma model into c57bl 6 mice |
| topic | Granuloma Tuberculosis -- Pathogenesis Macrophages |
| url | https://scholar.sun.ac.za/handle/10019.1/130624 |
| work_keys_str_mv | AT mahabeeryastika theestablishmentoptimisationandtransplantationofathreedimensionalbioprintedsyngeneicgranulomamodelintoc57bl6mice AT mahabeeryastika establishmentoptimisationandtransplantationofathreedimensionalbioprintedsyngeneicgranulomamodelintoc57bl6mice |