Kontrola kvalitete stanične terapije autolognim keratinocitima kod zacjeljivanja teških opeklina

master's thesis

• Title (english): Quality control of cell therapy with autologous keratinocytes for severe burn wound healing
• Author: Ivona Arić
• Mentor: Marija Zekušić (mentor)
• Mentor: Inga Urlić (sumentor)
• Committee member: Inga Urlić (predsjednik povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Committee member: Sofia Ana Blažević (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-02-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Lijekovi za naprednu terapiju (engl. Advanced Therapy Medicinal Products, ATMP) pomažu pri liječenju opeklina i rana koje teško zacjeljuju. U Banci tkiva i stanica u Kliničkom bolničkom centru Sestre milosrdnice uzgaja se i klinički primjenjuje autologna suspenzija keratinocita. Duboke dermalne opekline (II B°) i subdermalne opekline (III°) nemaju sposobnost spontanog zacjeljivanja zbog nedostataka keratinocitnih matičnih stanica. Nakon biopsije kože, enzimatskom digestijom se odvaja epidermis od dermisa, a zatim se izoliraju pojedinačni keratinociti i zasijavaju na 3T3 stanice tretirane mitomicinom C koje služe kao hranjiva podloga. Keratinociti se uzgajaju u plastičnim bočicama u mediju koji je obogaćen serumom i faktorima rasta. Klinički se primjenjuju u šprici sa dvokomponentnim fibrinskim ljepilom. Neophodna je kontrola kvalitete tijekom uzgoja koja podrazumijeva provjeru ukupnog broja keratinocita, postotak vijabilnih stanica i klonogeničnosti (holokon, meroklon, paraklon). Udio ostatnih 3T3 stanica u suspenziji određuje se metodom protočne citometrije. U staničnoj terapiji se imunocitokemijskim metodama provjerava prisutnost epitelnih stanica bazalnog porijekla (CK14) te matičnih (p63), proliferacijskih (Ki-67) i diferenciranih (CK19) stanica. Mikrobiološkim kontrolama se dokazuje sterilnost svih staničnih medija te se mjeri koncentracija bakterijskih endotoksina i provjerava odsutnost Mycoplasma spp. Kontrola kvalitete osigurava efikasnu i sigurnu primjenu autolognih keratinocita kao ATMP.

Abstract (english)

Advanced therapy medicinal products (ATMPs) help treat burns and wounds that are difficult to heal. An autologous keratinocyte suspension is cultivated at the Tissue and Cell Bank in Sestre milosrdnice University Hospital Center. Deep dermal burns (II B°) and subdermal burns (III°) do not have the ability to heal spontaneously due to keratinocyte stem cell deficiencies. After skin biopsy, enzymatic digestion separates the epidermis from the dermis and then individual keratinocytes are isolated and seeded onto mitomycin C treated 3T3 cells used as a nutrient medium. Keratinocytes are cultivated in flasks in a serum and growth factors enriched medium. Clinically, they are applied by a syringe using fibrin glue. A quality control during cultivation in necessary and it includes keratinocyte number count, viable cells percentange and clonogenicity (holoclon, meroclone, paraclone). A number of the rest of 3T3 cells in the suspension is determined using flow cytometry. In the cell therapy, immunocytochemic methods are used for determining the presence of basal epithelial cells (CK14) and stem (p63), proliferation (Ki-67) and differential (CK19) cells. Microbiological controls prove the sterility of all cell mediums as well as measure the concentration of bacterial endotoxins and check for Mycoplasma spp. absence. Quality control ensures efficient and safe autologous keratinocytes usage as ATMP.

• Keywords: tkivno inženjerstvo; lijek za napredne terapije; matične stanice; opekline
• Keywords (english): tissue engineering; advanced therapy; stem cells; burns
• Language: croatian
• URN:NBN: urn:nbn:hr:217:091888
• 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: 2023-02-25
• Created on: 2021-03-08 13:59:13