Permeabilnost izbranih učinkovin skozi spontano transformirano Caco-2 celično linijo s sposobnostjo hitre vzpostavitve integritete

Kristan, Jerca (author), Žakelj, Simon (mentor)

Monosloj celične linije Caco-2 se pogosto uporablja kot in vitro model za določanje humane instestinalne absorpcije. Referenčni protokol predvideva 21±1 dni, da celice po nasajanju na filtre dosežejo ustrezno stopnjo diferenciacije in ustvarijo konfluenten monosloj, ki se nato lahko uporabi za permeabilnostni poskus. Slabost Caco-2 celic je zato ravno izredno dolg čas kultivacije. V celičnem laboratoriju Fakultete za farmacijo se je z naraščajočim številom pasaž pojavil sev celične linije Caco-2, ki je začel izkazovati lastnosti pospešene diferenciacije in sposobnost pospešene vzpostavitve integritete, zaradi česar doseže ustrezno konfluenten monosloj veliko prej. V naši nalogi smo ta sev ovrednotili po genotipizaciji s strani ATCC, ki je sev prepoznala kot avtentično Caco-2 celično linijo; izoblikovali smo protokol za gojenje, s katerim smo želeli ugotoviti, kako gostota nasaditve celic na inserte vpliva na hitrost deljenja in čas do vzpostavitve integritete. To smo preverjali z merjenjem TEER ter s preverjanjem permeabilnosti modelnih spojin. Po tem protokolu smo 5. dan od nasaditve celic na filtre za gojenje dobili pripravljen monosloj za meritve permeabilnosti in primerjavo z referenčnimi, nespremenjenimi Caco-2 celicami. Na podlagi smernic FDA smo izoblikovali tri skupine modelnih spojin, ki so omogočale sočasno kvantifikacijo v akceptorskih raztopinah, z njimi pa smo ovrednotili ustreznost ločevanja med dobro in srednje ter slabo permeabilnimi spojinami. Primerjali smo pridobljene rezultate med referenčnim in testiranim sevom, ki je izkazoval sposobnost hitre vzpostavitve integritete ter potrdili, da testirani sev hitreje doseže konfluenten monosloj, uporaben za določanje permeabilnosti; ugotovili smo, da ima testiran sev na podlagi merjenja TEER in prepustnosti FITC-dekstranov ustrezno celično integriteto, da dobro ločuje dobro permeabilnime spojine od srednje in slabo permeabilnih ter da izraža dobro korelacijo med humano absorpcijo in vivo ter permeabilnostjo učinkovin in vitro.

• Language: Slovenian
• Keywords: celice Caco-2, permeabilnost, absorpcija učinkovin, integriteta, Lucifer Yellow
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-142485
• Publication date in RUL: 11.11.2022

Secondary language

• Language: English
• Title: Permeability of selected substances through a self-transforming Caco-2 cell line with rapid integrity development

Abstract

The Caco-2 cell line monolayer is widely used as an in vitro model of human intestinal absorption. The reference protocol predicts 21±1 days for the cells to reach an appropriate level of differentiation after seeding on the filters and to create a confluent monolayer, which can then be used for the permeability assay. The disadvantage of the Caco-2 cell line is therefore a very long cultivation time. In the cell laboratory of the Faculty of Pharmacy, with an increasing number of passages, a strain of the Caco-2 cell line appeared, which began to show the properties of accelerated differentiation and rapid integrity development, as a result of which it reaches a properly confluent monolayer much earlier. In our thesis, we evaluated this strain after genotyping by ATCC, which recognized the strain as an authentic Caco-2 cell line; we created a culture protocol, with which we determined how the density of seeding of the cells on the inserts affects the rate of proliferation and the time for integrity development. This was verified by measuring the TEER and by checking the permeability of model compounds. According to this protocol, on the 5th day after planting the cells on the culture filters, a prepared monolayer was obtained for permeability assay and comparison with a monolayer obtained by the reference protocol. Based on the FDA guidelines, we created three groups of model compounds that allowed simultaneous quantification in acceptor solutions, and with them, we evaluated the adequacy of the separation of high (HPM), medium (MPM), and low (LPM) permeable compounds. We compared the obtained results between the reference strain and the tested strain, which demonstrated rapid integrity development, and confirmed that the tested strain quickly reaches a confluent monolayer and is useful for determining permeability; we found that the tested strain, based on the measurement of TEER and permeability of FITC-dextrans, has adequate cell integrity, it has a good ability to differentiate between molecules with high permeability (HPM) of those with medium (MPM) and low (LPM) permeability, and that it shows a good correlation between human absorption in vivo and the permeability of active substances in vitro.

• Keywords: Caco-2 cells, permeability, drug absorption, integrity, Lucifer Yellow