Validacija Endosafe PTS sustava i razvoj postupka testiranja kulture keratinocita i limbalnih stanica na endotoksine

master's thesis

• Title (english): Validation of Endosafe PTS system and practical approach for endotoxin testing of keratinocytes and limbal stem cells
• Author: Valentina Blažević
• Mentor: Višnja Gaurina Srček (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Cell Technology, Application and Biotransformations); Zagreb
• Defense date and country: 2017-07-21, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Stanična terapija keratinocitima i limbalnim stanicama pripada ATMP proizvodima koji se koriste za kliničku primjenu, stoga je potrebno osigurati visoku razinu sigurnosti i kvalitete. Endotoksini su komponente stanične stijenke gram-negativnih bakterija, kod pacijenta mogu izazvati septički šok i smrt. Proizvodi stanične terapije imaju potencijal za uvođenje endotoksina izravno u krv. Svrha ovog rada bila je razvoj postupka testiranja stanične kulture keratinocita i limbalnih stanica na prisutnost endotoksina putem komercijalnog Endosafe PTS sustava koji se temelji na LAL metodi. Prilikom provođenja testiranja na endotoksine razvijen je izračun putem kojeg je moguće definirati prihvatljive vrijednosti koncentracije endotoksina u proizvodu u skladu s smjernicama FDA, Europske farmakopeje 2.6.14. i ISO standardima. Endosafe PTS sustav uspješno je validiran za testiranje otpadnog medija kulture stanica na prisutnost endotoksina unutar kratkog vremenskog roka. Prilikom testiranja javile su se interferencije uzorka s LAL reagensom koje su uspješno prevladane razrjeđivanjem uzorka do MVD vrijednosti koja je određena za svakog pojedinog pacijenta. Nadalje, endotoksini snažno adheriraju na staklenu površinu, te uklanjanje endotoksina predstavlja kritičnu točku prilikom pohrane GM medija. GM medij testiran je na endotoksine tijekom 24 h i 11 dana, u staklenim bocama nije zabilježeno povećanje koncentracije endotoksina.

Abstract (english)

Limbal stem cells and keratinocytes for clinical use are considered as an Advanced Therapy Medicinal Product (ATMP).It is necessary to ensure a high level of safety and quality of the ATMPs. Endotoxins are part of the outer membrane of the cell wall of Gram-negative. In a blood stream, presence of endotoxins can lead to spetic shock and death. Somatic cell therapy products have the potential to introduce endotoxin directly into the blood. The aim of this thesis was to validated a commercial Endosafe PTS system for the determination of endotoxins in complience with EU Pharmacopoeia 2.6.14, FDA guidelines and ISO standards. The Endosafe PTS system has been successfully validated for the testing of cell culture media in a short period of time. During the testing for endotoxins, interference of some sample was successfully overcome by diluting the sample to the MVD value determined for each individual patient. Removal of endotoxin is a critical point at storage GM media because endotoxins strongly adhere to the surface of glass. The GM medium was tested on endotoxins for 24 h and 11 days and there has been no increase in endotoxin concentration in glass containers.

• Keywords: endotoksini; limbalne stanice; keratinociti; Endosafe PTS; validacija
• Keywords (english): endotoxins; limbal cells; keratinocytes; Endosafe PTS; validation
• Language: croatian
• URN:NBN: urn:nbn:hr:159:094300
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Closed access
• Created on: 2017-11-07 13:22:52