Evaluation of the Calu-3 cell line for drug permeability determination

Pljuča omogočajo izmenjavo plinov ter nas ščitijo pred vstopom tujkov. Poleg tega pa zaradi velike površine, prepustnosti in možnosti neinvazivne aplikacije predstavljajo zanimivo mesto za sistemsko ali lokalno dostavo zdravilnih učinkovin. S tega vidika so pljuča precej manj raziskana od prebavnega trakta, zato jih moramo najprej natančno okarakterizirati. Celični modeli, med katere uvrščamo tudi celice Calu-3, predstavljajo učinkovit in dostopen sistem, s katerim lahko dobro spoznamo način prehajanja učinkovin skozi pljučni epitelij. Način gojenja celic vpliva na njihovo rast in končno podobo celičnega monosloja. Celice smo gojili v obogatenem mediju, ki omogoča manjše dodatke seruma, s čimer dosežemo manjšo intervariabilnost in lažje primerjamo rezultate permeabilnosti med seboj. Celice smo gojili tri tedne na dva načina. Eden je bil klasičen način gojenja celičnih kultur, pri katerem je medij obdajal celice s spodnje in zgornje strani (liquid-liquid), drugi pa specifičen za celične kulture epitelija dihalnega trakta, pri katerem medija na apikalni strani ni bilo (air-liquid). Med gojenjem smo z opazovanjem celic pod invertnim mikroskopom preverili, ali so celice prerasle celotno površino membrane in ali je prišlo do mehanskih poškodb celic. Z merjenjem transepitelijske električne upornosti in uporabo paracelularnega markerja permeabilnosti lucifer rumeno smo potrdili integriteto celičnega sloja. Permeabilnost smo merili v apikalno–bazolateralni smeri in obratno. Pri FITC-dekstranih je bil trend manjšanja permeabilnosti z večanjem mase primerljiv s tistim, o katerem poročajo drugi avtorji. Permeabilnost vseh dekstranov je bila očitno manjša od drugih testiranih spojin, s čimer smo še dodatno potrdili, da je integriteta celičnega monosloja Calu-3 primerna. Z merjenjem permeabilnosti majhnih lipofilnih spojin smo ugotovili, da večji logP korelira z večjo permeabilnostjo. Testiranje permeabilnosti v obeh smereh, apikalno–bazolateralni in bazolateralno–apikalni, je bilo pomembno predvsem za določanje aktivnega transporta. Prisotnost in aktivnost P-glikoproteina smo dokazali z rodaminom 123 kot modelnim substratom in s selektivnimi zaviralci. Prenašalca PEPT2 z oksacilinom kot modelnim substratom nismo mogli z gotovostjo dokazati. Kljub nekaterim razlikam, do katerih pride pri različnih načinih gojenja, smo ugotovili, da sta v primeru nanašanja raztopin učinkovin oba primerna za študije permeabilnosti.Human airways enable exchange of gasses and act as protective interface against. In addition, they also represent an interesting site for local or systemic drug delivery due to large absorptive surface area, leaky epithelium, and non-invasive route of application. Since our knowledge about airways as a potential drug delivery route is not as extensive as that of the gastrointestinal tract, we need to thoroughly characterize them first. Cell models, such as Calu-3 cells, are efficient and affordable systems, with which we can learn a lot about drug permeability through lung epithelium. The way of cultivating cells affects their growth and structure of cell monolayer. We cultivated cells in nutrient-rich medium with minimal serum addition, which normally results in lower intervariability and easier comparison of permeability coefficients among all cell monolayers. Epithelial models were cultivated three weeks on two different conditions – liquid-liquid and air-liquid interface, the latter being specific for lung cell cultures. During cultivation cells were monitored under invert microscope to check, whether cells outgrew whole membrane surface and whether they were damaged. Measurements of transepithelial electrical resistance and permeability of Lucifer Yellow were used to evaluate the integrity of the cell layer. Permeability was measured in apical to basolateral and basolateral to apical side. Permeability coefficients of FITC-dextrans decreased with higher molecular masses. This trend was also observed by other authors. Permeability of FITC-dextrans was significantly lower than the permeability of other tested compounds, which confirmed the suitable integrity of Calu-3 monolayer. Permeability coefficients of small lipophilic molecules increased with higher logP values. Measuring of permeability in both directions, apical to basolateral and basolateral to apical, was important in the determination of active transport. The presence and activity of transporter P-glycoprotein were confirmed with Rhodamine 123 as a model substrate and its selective inhibitors. The presence of transporter PEPT2 was not confirmed with oxacillin. We concluded that when solutions are being tested both culturing conditions are appropriate for permeability measurements