Optimizacija mehanske izolacije stromalnih žilnih celic iz človeškega maščobnega tkiva

Jakovljević, Kaja (avtor), Jeras, Matjaž (mentor), Veber, Matija (komentor)

Pri celični terapiji se za popravilo okvar oz. poškodb pogosto uporabljajo matične celice. Od leta 2001 je znano, da mezenhimske matične celice (ASC, ang. adipose-derived stem cells) najdemo tudi v maščobnem tkivu. To sestoji iz adipocitov, ki predstavljajo tretjino celic, in frakcije stromalnih žilnih celic (SVF, ang. stromal vascular fraction), v kateri so prisotne tudi ASC. Uporabljajo se za celjenje ran, kroničnih razjed in fistul, pri kozmetičnih operacijah in za zdravljenje radiacijskih poškodb. Trenutno ASC rutinsko pridobivajo iz SVF z minimalno invazivnim postopkom podkožne lipoaspiracije in sledečo encimsko obdelavo s kolagenazo. Postopek je problematičen, ker so ostanki encima škodljivi za regeneracijo kože in tkiv, poleg tega pa je uporaba encimov pravno strogo regulirana. Zato se kot alternativa proučujejo mehanske metode, med katerimi je osnovna centrifugiranje. V tem magistrskem delu smo raziskali učinkovitost mehanske izolacije stromalnih žilnih celic iz človeškega maščobnega tkiva, ki predstavlja alternativo metodi centrifugiranja. Preverili smo, kako na učinkovitost izolacije vplivata oblika povezovalnega priključka in število potiskov skozi brizgo. Primerjali smo dva priključka različne oblike ter 15, 30 in 60 potiskov. Kontrolni eksperiment je uporabljal zgolj centrifugiranje. Uspešnost postopka smo ocenili z analizo parametrov pridobljenih celic. Merili smo število celic in klonogeni potencial pridobljenih celic. Nato smo preverili še sposobnosti pridobljenih celic za diferenciacijo z gojenjem v adipoindukcijskem in osteoindukcijskem gojišču. Diferenciacijo v adipoindukcijskem gojišču smo ovrednotili z barvanjem z barvilom Oil Red O, ki obarva lipidne vakuole. Diferenciacijo v osteoindukcijskem gojišču smo spremljali z merjenjem koncentracije kalcija, ki so ga celice izločile. Ugotovili smo, da lahko optimiziramo mehanični postopek izolacije SVF. V primerjavi s centrifugiranjem smo dosegli do 93-krat večji izkoristek. Encimska metoda razgradnje pa je imela še vedno 16-krat večji izkoristek od optimizirane mehanske razgradnje. Celice, pridobljene z optimiziranim mehanskim postopkom so sicer imele nekoliko slabše fenotipske lastnosti, a so kljub temu izkazovale enako sposobnost diferenciacije kakor tiste, izolirane z encimsko izolacijo.

• Jezik: Slovenski jezik
• Ključne besede: mezenhimske matične celice iz maščobe (ASC), stromalne žilne celice (SVF), mehanska izolacija, diferenciacijska sposobnost, fenotipske lastnosti
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-127064
• Datum objave v RUL: 15.05.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Optimisation of stromal vascular cells mechanical isolation from human adipose tissue

Izvleček

In cell therapy, stem cells are commonly used to repair damages and treat injuries. Since 2001, adipose-derived stem cells (ASC) have been known to be found in adipose tissue, as well. It consists of adipocytes, which make up a third of the cells, and a stromal vascular fraction (SVF), which also comprises ASC. They are used for healing of wounds, chronic ulcers and fistulas, in cosmetic surgery and for treating radiation injuries. Currently, ASC are routinely obtained from SVF by a minimally invasive process of subcutaneous lipoaspiration, followed by enzymatic processing with collagenase. The process is problematic due to enzyme residues, which are detrimental to skin and tissue regeneration. Additionally, the use of enzymes is strictly regulated. Therefore mechanical methods as alternatives are studied with centrifugation being the most rudimentary. In this thesis, the effectiveness of mechanical isolation of SVF from human adipose tissue as an alternative to the centrifugation method was studied. We tested how the isolation efficiency is affected by the shape of a connecting nozzle and the number of transfers through the syringe. We compared two nozzles of different shapes with 15, 30 and 60 transfers. In the control experiment, only centrifugation was used. The efficiency of the procedure was assessed by analysing the parameters of the obtained cells. The number of the cells and their clonogenic potential were determined. Subsequently, the ability of the cells to differentiate was measured by culturing them in adipoinduction and osteoinduction medium. Their differentiation in the adipoinduction medium was evaluated by Oil Red O, which dyes lipid vacuoles. Their differentiation in the osteoinduction medium was determined by measuring the concentration of calcium, which had been excreted by the cells. We found that the mechanical isolation process can be optimised. Compared to centrifugation, a 93 times higher efficiency was measured. However, the enzymatic degradation method was still 16 times more efficient that the optimised mechanical decomposition. The cells obtained by the optimised mechanical process exhibited slightly poorer phenotypic properties, yet they had the same differentiation ability as the enzymatically isolated cells.

• Ključne besede: adipose-derived stem cells (ASC), stromal vascular fraction (SVF), mechanical isolation, differentiation ability, phenotypic properties.