Liofilizacija neuroblastomskih celic in vrednotenje preživetja celic med zamrzovanjem v formulacijah s polioli in trehalozo

Bordon, Gregor (avtor), Planinšek, Odon (mentor), Dhananjay, Jere (komentor)

Proteinski terapevtki so omogočili zdravljenje nekaterih težkih boleznih, ki jih prej ni bilo mogoče zdraviti z zdravilnimi učinkovinami v obliki malih organskih molekul. Danes celična in genska terapija ponuja rešitve na področjih, ki sežejo še dlje od zdravil s proteinskimi biomolekulami. Možnosti, ki jih ponujajo produkti za celične terapije (PCT) pa ne prihajajo brez izzivov, ki so posledica visoke kompleksnosti teh zdravil – proizvodnja, distribucija in skladiščenje so glavni primeri. Hramba PCTjev v večini primerov sloni na krioprezervaciji v tekočem dušiku, ki ima nekaj pomanjkljivosti. Poleg tega, da je drag način hrambe, zahteva da zaposleni v zdravstvu sledijo pogosto zahtevnim protokolom taljenja in ostalim post-produkcijskim postopkom. Ti vključujejo odstranjevanje dimetilsulfoksida (DMSO) iz taljene formulacije. Ta standardni krioprotektant, uporabljen v mnogih PCTjih, zaradi dobre zaščite celic pred nizkimi temperaturami, ni varen za uporabo v ljudeh. Razvoj alternativnega načina shrambe celic pri višji temperaturi kot je uporabljena pri tekočem dušiku in brez uporabe DMSOja, bi izboljšal varnost in stroškovnost učinkovitost teh terapij. Potencialna aternativa temu pristopu bi lahko bili liofilizirani PCTji, kjer bi bile celice v suhem stanju in rekonstituirane pred odmerjanjem. Uspešen proces liofilizacije celic in formulacija zaenkrat še vedno predstavljajo nedosežen izziv. Cilj tega raziskovalnega dela je bil testirati preživetje celic med procesom zamrzovanja in taljenja v različnih formulacijeh ter preveriti, če se jih da liofilizirati. Za ta namen so bile uporabljene SK-N-AS celice kot modelna rakasta adherentna celična linija. Študija je bila na začetku osredotočena na zamenjavo DMSOja s polioli, optimzacijo toničnosti, uporabe dodatkov iz celičnega medija in vpeljavo trehaloze v notranjost celic. Z nekaj formulacijami je bilo doseženo dobro preživetje zamrznitve in taljenja celic, vendar je vizualen pregled liofiliziranih formulacij pokazal slab izgled liofilizatov. V nadaljevanju so bile celice uspešno dvodimenzionalno gojene na površini steklenih vial in tridimenzionalno v matrici hidrogela. Te celične kulture so bile nato zamrznjene pri -80°C in vloga pritrjenosti celic za prezervacijo, je bila raziskana. Hidrogelne matrice so bile zatem liofilizirane in liofilizat je bil vizualno pregledan. Dodatek raztopine za krioprotekcijo k hidrogelu, je negativno vplival na izgled liofilizata. Preliminarni rezultati so pokazali zmožnost hidrogelnih matric za zaščito celic med zamrzovanjem in zelo dobro liofilizabilnost.

• Jezik: Slovenski jezik
• Ključne besede: Celična terapija, razvoj formulacije, shramba, liofilizacija, hidrogeli, zamrzovanje celic.
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2019
• PID: 20.500.12556/RUL-112699
• Datum objave v RUL: 06.11.2019

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Lyophilisation of neuroblasoma cells and evaluation of cell survival during freezing in polyol and trehalose formulations

Izvleček

Protein therapeutics have significantly widened the scope of difficult-to-treat diseases which were not treatable with small molecule drugs. Nowadays, the cell and gene therapy is providing solutions to medical conditions, beyond proteinaceous medicines. Cell therapy products (CTPs) are however, highly complex systems, which face substantial challenges in manufacturing, distribution and storage. The storage is mostly based on cryopreservation in liquid nitrogen, which has several drawbacks. Besides being expensive, it also demands from clinicians to be skilled in often complex thawing protocols and in other post-manufacturing procedures. This involves elimination of dimethylsulfoxide (DMSO) from thawed formulations. DMSO is currently a standard cryoprotectant, utilized in numerous CTPs due to its good protection against low temperature, even though it is not safe in humans. Development of an alternative preservation model with cells stored at temperatures above those of liquid nitrogen for long periods, without the use of DMSO, would make these therapies much safer and cost efficient. A potential alternative could be freeze dried CTPs, where the cells are in dried formed and are reconstituted before administration. However, a successful formulation and process, that would provide sufficient cell recovery after freeze drying, is yet to be developed. The aim of this study was to screen the cell survival of freeze-thawing process in a number of formulations and establish, if it’s possible to lyophilize them. For this purpose, SK-N-AS cells were used as a model adherent cancer cell line. The research firstly focused on the DMSO replacement with polyols, optimization of tonicity, testing the effect of cell medium supplements and introduction of trehalose intracellularly. A good post-thaw cell survival was obtained with several designed formulations, however the visual inspection showed a poor appearance of freeze dried products. Secondly the cells were successfully cultured in 2D on the glass vial surface and in 3D hydrogel scaffolds. Cultures were then freeze-thawed and the function of cell attachment in cell preservation at -80°C storage was tested. Next, the lyophilisability of cell scaffolds was evaluated by visual inspection. Addition of polyol/trehalose cryoprotectant formulations to the scaffold negatively impacted the lyophilization of the product. Preliminary results showed some protective ability of hydrogels and a very good lyophilisability.

• Ključne besede: Cell therapy, formulation development, storage, lyophilization, hydrogels, cell freezing.