Diferencijacija ljudskih endocervikalnih stanica uzgojenih u 3D staničnoj kulturi

master's thesis

• Title (english): Differentiation of human endocervical cells grown in 3D cell culture
• Author: Nikolina Bradarić
• Mentor: Marija Ćurlin (mentor)
• Mentor: Romana Gračan (sumentor)
• Committee member: Romana Gračan (predsjednik povjerenstva)
• Committee member: Sunčica Bosak (član povjerenstva)
• Committee member: Sofia Ana Blažević (član povjerenstva)
• Committee member: Ana Galov (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2020-09-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Endocervikalne kripte izlučuju cervikalnu sluz čija se biokemijska, biofizička i funkcionalna svojstva mijenjaju ovisno o fazi menstruacijskog ciklusa, stoga je za istraživanje sekrecijske aktivnosti endocerviksa potrebno koristiti odgovarajući eksperimentalni model. Razvoj 3D kultura stanica omogućio je uzgoj visokodiferenciranih i funkcionalnih endocervikalnih stanica s jasno definiranom polarnošću, međustaničnim spojevima i sposobnošću izlučivanja mucina. Za postizanje uspješne 3D kulture, potrebno je optimizirati specifične uvjete. U dvije uzastopne kultivacije ispitivane su optimalne eksperimentalne postavke za uzgoj stanične linije humanih epitelnih endocervikalnih stanica End1/E6E7 u 3D kulturi primjenom transwell podloga koje omogućuju rast stanica na granici „medij - zrak“. Ispitivane su različite koncentracije i vrijeme izlaganja međustaničnoj tvari, različito vrijeme izlaganja ß-estradiolu i vrijeme inkubacije u kojem stanice postižu potpunu diferencijaciju i funkcionalnost. Rast, diferencijacija i funkcionalnost stanica se procjenjivala citološkim, citokemijskim, imunocitokemijskim metodama i elektronskim mikroskopom. Ovim radom postignuti su optimalni uvjeti za rast, diferencijaciju i funkcionalizaciju endocervikalnih stanica u 3D kulturi. Prvi znakovi diferencijacije i funkcionalizacije pojavili su se šesti dan kultivacije, a potpuna funkcionalnost postignuta je do 18. dana. Ujednačenost proizvodnje mucina u stanicama omogućila je primjenu postignute 3D kulture u pokusu izlaganja stanica ß-estradiolu. Izložene stanice pojačale su stvaranje nepropusnih i pričvrsnih spojeva, te smanjile količinu mucina unutar stanica.

Abstract (english)

Endocervical crypts secrete cervical mucus whose biochemical, biophysical and functional properties change depending on the phase of the menstrual cycle, so it is required to use an appropriate experimental model to investigate the secretory activity of the endocervix. The development of 3D cell cultures has enabled the cultivation of highly differentiated and functional endocervical cells with clearly defined polarity, intercellular junctions, and mucin secretion ability. To achieve a successful 3D culture, it is necessary to optimize specific conditions. In two consecutive cultivations, the optimal experimental settings for culturing the human epithelial endocervical cell line End1 / E6E7 in 3D culture were examined using transwell insert that allows cells growth at the "medium - air" interface. Different concentrations and times of exposure to extracellular matrix were examined, as well as different exposure time to ß-estradiol and incubation time at which cells achieve complete differentiation and functionality. Cell growth, differentiation and functionality were assessed by cytological, cytochemical, immunocytochemical methods and electron microscopy. With this thesis optimal conditions for growth, differentiation and functionalization of endocervical cells in 3D culture were established. The first signs of differentiation and functionalization appeared on the sixth day of cultivation, and full functionality was achieved by the 18th day. The uniformity of mucin production in the cells enabled the application of the established 3D culture in the experiment of cell exposure to ß-estradiol. Exposed cells improved the formation of tight and adherent junctions and reduced the amount of mucin within the cells.

• Keywords: 3D kultura stanica; endocervikalne stanice; međustanična tvar; transwell; mucini; klaudin 4; međustanični spojevi
• Keywords (english): 3D cell culture; endocervical cells; extracellular matrix; transwell; mucins; claudin 4; intercellular junctions
• Language: croatian
• URN:NBN: urn:nbn:hr:217:876496
• Study programme: Title: Experimental Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-10-06 11:16:28