Uspostava kulture i biološko oslikavanje organoida mišjeg tankog crijeva

master's thesis

• Title (english): Establishment and bioimaging of mouse small intestine organoids culture
• Author: Snježana Kodba
• Mentor: Iva Marija Tolić (mentor)
• Committee member: Iva Marija Tolić (predsjednik povjerenstva)
• Committee member: Ana Galov (član povjerenstva)
• Committee member: Ana Previšić (član povjerenstva)
• Committee member: Inga Urlić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2020-09-24, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Organoidi su novo područje u molekularnoj biologiji koje ima veliki potencijal u temeljnim i primijenjenim istraživanjima. Minijaturni organi uzgajani in vitro značajno nalikuju svojim nativnim dvojnicima u svim fiziološkim, 3D strukturalnim i anatomskim karakteristikama. Već je razvijeno više tipova organoida, uključujući ljudske i mišje organoide tankog crijeva. U ovom radu, ja sam uspješno uzgojila i održala u kulturi organoide mišjeg tankog crijeva. Nakon optimizacije, uzgojeni su organoidi mogli neodređeno dugo rasti i propagirati se u kulturi. Preciznija karakterizacija anatomije i morfologije organoida napravljena je nakon imunofluorescencijskog bojanja i biološkog oslikavanja. Nadalje, razvila sam novi protokol za imunofluorescencijsko bojanje cističnih, šupljih tipova organoida. U odnosu na literaturno dostupne protokole, novi protokol predstavlja poboljšanja u brzini izvedbe i broju koraka koji štede vrijeme i novac. Također, rezultati su pokazali da organoidi mišjeg tankog crijeva mogu dolaziti u različitim oblicima pri čemu veličina stanica unutar organoida ovisi o točnoj poziciji stanice u organoidu te o njihovom proliferacijskom statusu. Na kraju, stopa proliferacije organoida mišjeg tankog crijeva pada s povećanjem broja dana koje su organoidi proveli u kulturi. Zaključno, uspješno je razvijen protokol za izolaciju i uzgoj složenog sustava organoida iz mišjeg tankog crijeva koji ima potencijal brze optimizacije za korištenje na organoidima drugih organskih sustava.

Abstract (english)

A novel field arising in molecular biology, organoids, have a great potential in basic and applied research. Mini organs grown in a dish remarkably resemble their native counterparts in almost every aspect of their physiological, 3D structural and anatomical properties. Many different types of organoids have yet been developed, including mouse and human small intestine organoids. In this thesis, I have successfully established and maintained a culture of mouse small intestine organoids. After some optimization was done, the organoids were able to keep growing and differentiating indefinitely. More precise characterization of their anatomy and morphology was made after immunofluorescent staining and bio-imaging. I have created a novel protocol for immunostaining of cystic, hollow types of the organoids which reduces the time and money consumed. The results have revealed different shapes of small intestine organoids and variation in cell size depending on the position and proliferation status. The proliferation rate, however, decreases as the days in culture increase. To conclude, a novel protocol for isolation and propagation of complex organoid system was successfully developed from mouse small intestine. However, it has potential for fast optimization and adjustment for other types of mouse and human organoids.

• Keywords: organoidi; tanko crijevo; miš; mikroskopija
• Keywords (english): organoids; small intestine; mouse; microscopy
• Language: croatian
• URN:NBN: urn:nbn:hr:217:490054
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2022-09-24
• Created on: 2020-10-02 09:04:13