Development of a transendothelial shuttle by macrophage modification
| dc.contributor.advisor | Smith, Carine | en_ZA |
| dc.contributor.author | Visser, Johan Georg | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. | en_ZA |
| dc.date.accessioned | 2017-02-17T08:20:07Z | |
| dc.date.accessioned | 2017-03-29T20:57:04Z | |
| dc.date.available | 2017-12-31T03:00:08Z | |
| dc.date.issued | 2016-12 | |
| dc.description | Thesis (MSc)--Stellenbosch University, 2016. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: Background: Targeted stem cell delivery via macrophage modification is a novel and relatively non-invasive therapeutic intervention. Monocytes circulate through the vasculature and infiltrate damaged tissue in response to chemotactic signalling. Here they differentiate into functionally different macrophage phenotypes - the classically activated pro-inflammatory (M1) or alternatively activated anti-inflammatory (M2) phenotypes. The M1 macrophages are able to cross endothelial barriers, while the M2 anti-inflammatory macrophages are unable to transverse endothelium and instead remain tissue associated. The focus of our research was to produce M1 macrophages in vitro that could transverse endothelium while carrying engulfed stem cells, in order to deliver more stem cells in a relatively short time, to any injured tissue to facilitate recovery. Methods: Primary isolated monocytes were cultured with Granulocyte Monocyte Colony-Stimulating Factor, Lipopolysaccharide and Interferon gamma for 6 days to pre-differentiate them into M1 macrophages. Cells were treated with a Wortmannin-Concanamycin A-Chloroquine cocktail to achieve phagosome maturation arrest and thus preserve ingested cells in a viable state. Preservation of engulfed stem cells (simulated with fluorescent latex beads covalently labelled with IgG antibody) was qualitatively and quantitatively determined by flow cytometry and live cell imaging, respectively. Bead-containing macrophages were co-cultured with HUVEC cells in a Transwell system, and exposed to Monocyte Chemoattractant Protein 1 (added to bottom well) to determine migration capacity. Results: Monocytes were differentiated into M1 classically activated macrophages. The majority of these cells (68.67±3.51%) were able to engulf opsonised beads after successful induction of phagosome maturation arrest – a capacity similar to that of untreated cells (61.19±4.68%). Ingested beads were preserved within macrophages for the duration of our protocol (2 hours), determined by retained red antibody signal on beads and perturbed phagosome acidification. 72.86±16.0 phagosome maturation arrested macrophages were able to transverse a HUVEC coated membrane with 8 μm diameter pores (simulated endothelial layer), while only 70.14±12.6 cells per well migrated when carrying a bead cargo. Conclusion: A delivery system capable of engulfing, preserving and delivering cargo was successfully induced. Further optimisation of this technique could lead to translation into a novel method for delivery of stem cells with regard to regenerative medicine and may even be used as a drug delivery system for the treatment of various malignancies. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Agtergrond: Geteikende stamsel aflewering via makrofaag manipulering is 'n nuwe en relatief nie-indringende terapeutiese intervensie. Monosiete sirkuleer deur die bloedvate en infiltreer beskadigde weefsel in reaksie op chemiese seine. Hier kan hulle funksioneel verskillende makrofaag fenotipes vorm - die klassiek-geaktiveerde pro-inflammatoriese (M1) of alternatiewelik geaktiveerde anti-inflammatoriese (M2) fenotipes. Slegs die tipe M1 makrofage is in staat om deur endoteelweefsel te beweeg, terwyl die M2 makrofage in weefsel geleë bly. Die fokus van ons navorsing was om M1 makrofage in vitro te produseer, wat endoteel kan deurdring selfs nadat dit ʼn stamsel ingeneem het, ten einde meer stamselle af te lewer in 'n relatief korter tydperk en so doende beskadigde weefselherstel aan te help. Metodes: Primêre geïsoleerde monosiete is vir 6 dae gedifferensieer deur hul kwekingsmedia aan te vul met granulosiet-monosiet kolonie-stimulerende faktor, lipopolisakkaried en interferon-gamma, om sodoende M1 makrofage te lewer. M1 selle is daarna behandel met 'n kombinasie van Wortmannin, Concanamycin A en Chloroquine om die proses van fagosoomverryping te verhoed en sodoende ingeneemde selle in 'n lewensvatbare toestand te bewaar. Bewaring van gefagositeerde stamselle (nageboots deur gebruik te maak van fluoreserende polistireen krale, gemerk met kovalent-gebonde IgG teenliggame) is kwalitatief en kwantitatief bepaal deur vloeisitometrie en lewende sel mikroskopie onderskeidelik. Makrofage met ingeneemde krale is daarna bo-op HUVEC selle in 'n dubbelput (Transwell) sisteem gekweek. Die kultuur is daarna aan monosiet chemo-aantrekkingsproteïen 1 (bygevoeg in onderste put) blootgestel om migrasie kapasiteit te bepaal. Resultate: Monosiete is suksesvol gedifferensieer na die klassiek geaktiveerde M1 makrofaag fenotipe. Die meerderheid van hierdie selle (68,67 ± 3,51%) was in staat om geӧpsoniseerde krale te verswelg ná suksesvolle verkoming van phagosoom verryping - 'n resultaat soortgelyk aan dié van onbehandelde selle (61,19 ± 4,68%). Opgeneemde krale het binne makrofage behoue gebly vir die tydsduur van ons protokol (2 ure), soos bepaal deur die behoud van die rooi teenliggaamsein op krale. In terme van migrasie, was 72,86 ± 16.0 behandelde makrofage (per reaksie) in staat om deur 'n HUVEC-bedekte (endoteel) membraan met porieë, 8 μm in deursnee (te beweeg, in vergelyking met ʼn soorteglyke 70,14 ± 12.6 selle per reaksie vir die behandelde selle wat krale bevat het. Gevolgtrekking: Data bewys dat ‘n afleweringstelsel, wat in staat is om ‘n vrag in te neem en behoue te laat bly vir aflewering, suksesvol ontwikkel is. Die verdere optimalisering van hierdie tegniek kan lei tot die toepassing daarvan as 'n nuwe terapeutiese modaliteit in regeneratiewe medisyne, vir die aflewering van stamselle en/of farmaseutiese preparate, vir die behandeling van verskeie maligniteite. | af_ZA |
| dc.embargo.terms | 2017-12-31 | |
| dc.format.extent | 149 pages : illustrations | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/101404 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Macrophages -- Activation | en_ZA |
| dc.subject | Delivery of stem cell | en_ZA |
| dc.subject | Stem cell delivery via macrophage modification | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | Development of a transendothelial shuttle by macrophage modification | en_ZA |
| dc.type | Thesis | en_ZA |
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