Functionalization of a porous polymeric matrix with a dextran layer for cell adsorption

Z razvojem regenerativne medicine in tkivnega inženirstva je narasla potreba po razvoju afinitetnih metod izolacije celic. Zaradi specifičnosti in čistosti je glavni kandidat kromatografija. Ozko grlo kromatografije pa še vedno ostaja čiščenje in izolacija večjih količin specifičnih proteinov in določenih populacij celic, predvsem matičnih celic. Konvektivna afinitetna kromatografija je zelo učinkovita metoda za ločevanje velikih biomolekul, kot so večji proteini, virusi, bakteriofagi, DNA in celice. Monoliti imajo značilno porozno strukturo, kjer so pore medsebojno povezane in s tem omogočajo hitro izmenjavo molekul na osnovi konvektivnega prenosa snovi. Pri tem je pomembno, da ne prihaja do nespecifičnih interakcij s površino, kar je še posebej pogosto pri velikih makromolekulah in celicah. To lahko zmanjšamo z uvedbo hidrofilnega sloja. Ena možnost je polietilen glikol (PEG), če pa želimo vezati na tak sloj afinitetne ligande, je boljša alternativa dekstran, ki ima več reaktivnih mest za površinsko imobilizacijo, kar se odraža v visoki gostoti biološko aktivnih molekul. Prav uvedba sloja dekstrana je bil tudi glavni cilj mojega magistrskega dela. Magistersko delo je bilo razdeljeno v tri sklope. V prvem sklopu sem porozne polimerne nosilce kemijsko modificirala z etilendiaminom in karbonildiimidazolom, in s tem pridobila funkcionalne skupine na površini. V drugem sklopu sem se osredotočila na razvoj vezave dekstranskega sloja, ki bi izboljšal kovalentno imobilizacijo biomolekul in zmanjšal nespecifične interakcije celic s površino. Dekstran sem z natrijevim periodatom oksidirala po principu Malapradove reakcije ter ga kovalentno vezala na pore nosilca. Za določitev števila veznih mest sem razvila kvantitativno BCA metodo za določevanje karbonilnih skupin, ki je sicer namenjena določanju količine proteinov. V zadnjem sklopu poskusov pa sem na porozne polimerne nosilce vezala protitelesa proti antigenu CD20 in jih inkubirala v krvnem lizatu pacienta s KLL. Limfocite, ki so se vezali na porozne polimerne nosilce, sem opazovala s svetlobno in fluorescenčno konfokalno mikroskopijo.With the development of regenerative medicine and tissue engineering, the need to develop affinity chromatographic methods has increased. The bottleneck of chromatography, however, still remains the purification and isolation of large quantities of specific biologic mamcromolecules and nanoassemblies, including certain populations of cells, such as stem cells. Convective affinity chromatography is a very effective method for separating large biomolecules such as larger proteins, viruses, bacteriophages, DNA and cells. Monoliths have a characteristic porous structure where the pores are interconnected and thus allow the rapid exchange of molecules on the basis of convective mass transport. A very important challange, particularly connected to large macromolecules and cells, are non-specific interactions. Frequently this issue is solved by introduction of a hydrophilic layer. A common approach is the introduction of polyethylene glycol (PEG), however, for affinity chromatography, a better option is dextran with a higher number of reactive moieties, resulting in higher density of biologically active molecules. The introduction of dextrane layers was the main goal of this master thesis. The master thesis can be divided into three sections. In the first part I successfully prepared small volume porous polymer supports. This was followed by the chemical modification with ethylenediamine and carbonyldiimidazole. In the second set I focused on the development of dextrane layers, that will improve the covalent immobilization of biomolecules. I oxidized the dextran with the sodium periodate according to the principle of the Malaprad reaction and bound it covalently to the developed chromatographic columns. For the purpose of evaluating oxidized dextran, I implemented quantitative BCA methods for the determination of carbonyl groups, originally proposed for determination of proteins. In the last set of experiments, I prepared CDI and epoxy porous polymer supports on which I immobilized antibodies to the CD20 antigen and incubated them in the blood lysate of a patient with KLL. The lymphocytes bound to the porous polymer supports were observed by light and fluorescence confocal microscopy