Uticaj prisustva ćelija u procesu elektrostatičke ekstruzije alginata

In this study, an attempt was made to assess the effects of cell addition and final concentration on the process of electrostatic extrusion as a method for cell immobilization in alginate microbeads. The electrostatic extrusion process is a complex function of several operating parameters, the system geometry, and the properties of the polymer solution, which is being extruded. The addition of cells results in the formation of a two-phase system, adding to the complexity of the process by the associated phenomenon of two-phase flow. This study especially focused on analyzing the effects of cell presence on each stage of the immobilization process. Specifically, the effects of the cell and alginate concentrations on the resulting microbead size and uniformity were assessed. Under the investigated conditions, microbeads, 50-600 ěm in diameter, were produced and the increase in both alginate and cell concentrations resulted in larger microbeads with higher standard deviations in size. We attempted to rationalize the obtained findings by rheological characterization of the cell-alginate suspensions. H-NMR Spectroscopy of the alginate used in this study revealed a high content (67%) of guluronic residues and GG diad blocks (FGG = 55%). The mole fractions of the MM and GM diad sequences, Fmm and Fgm, were 21 and 12%, respectively. Rheological characterization revealed non-Newtonian, pseudoplastic behavior of the cell-alginate suspensions with an increase in viscosity as the alginate concentration was increased. However, the presence of cells even at high concentrations (5 ÷ 108 and 1 ÷ 109 cell/ml) did not significantly affect the rheological properties of Na alginate solution. Finally, the effects of the alginate and cell concentrations on the gelation kinetics and the dynamic-mechanical behavior of the obtained hydrogels were investigated. A molar ratio of G units to Ca2+ ions of 3.8 : 1 provided complete crosslinking, while an increase in the alginate concentration resulted in prolonged gelation times, but higher strength of the resulting gel. Cell presence decreased the rate of network formation, as well as the strength of the obtained Ca alginate hydrogel.U ovom radu izvršeno je ispitivanje fenomena elektrostatičke ekstruzije. Proces elektrostatičke ekstruzije je vrlo složen, jer obuhvata pojedinačne uticaje različitih fenomenoloških parametara (jako spoljašnje električno polje, mikrohidrodinamičke pojave unutar kapilare, reološko ponašanje polimera), kao i međusobne uticaje ovih faktora. Prisustvo ćelija u sistemu čini ga još složenijim, što do sada fenomenološki nije razjašnjeno. Istraživanja su obuhvatila ispitivanje pojedinih značajnih fenomena koji se javljaju u ovoj metodi imobilizacije ćelija, polazeći od karakteristika samog nosača i suspenzije alginat/ćelije, preko parametara elektrostatičke ekstruzije, do kinetike geliranja alginata. Uz to, posebno je analiziran uticaj prisustva ćelija na sve faze ovog procesa. Detaljno je proučen uticaj promene koncentracije alginata i koncentracije ćelija u polaznoj suspenziji alginat/ćelije na veličinu i uniformnost proizvedenih mikročestica. Pri ispitivanim operativnim uslovima, proizvedene su mikročestice prečnika od 50-600 ěm i porast koncentracije bilo alginata, bilo ćelija uzrokuje nastajanje većih čestica neuniformnije raspodele veličina. U pokušaju da se objasne dobijeni rezultati i ispita reološko ponašanje suspenzije kao jedan od parametara koji utiču na dinamiku dvofaznog strujanja u toku ekstruzije, izvršeno je reološko karakterisanje suspenzija alginat/ćelije. Reološka ispitivanja su pokazala da prisustvo ćelija čak i pri visokim koncentracijama (5x108 i 1x10 ćelija/ml) ne utiče značajno na reološke osobine rastvora natrijum alginata. Na kraju, ispitan je uticaj koncentracije alginata i ćelija na kinetiku geliranja u uslovima ograničenog prisustva Ca jona, s obzirom da imobilizacija izuzetno osetljivih ćelija sisara može da zahteva minimalno izlaganje rastvoru CaCl2. Rezultati su pokazali da molski odnos guluronskih jedinica prema Ca2+ jonima od 3,8:1 omogućuje fizičko umrežavanje i da porast koncentracije alginata uzrokuje duže geliranje, ali i nastajanje jačeg gela. Prisustvo ćelija dovodi do sporijeg geliranja i prouzrokuje nastajanje slabijih hidrogelova što upućuje na zaključak da imobilisani mikroorganizmi uzrokuju nepravilnosti u strukturi mreže