Methods, techniques, equipment

Our laboratories at the Department of Pharmacology and Toxicology of Charles University, Faculty of Pharmacy are well equipped and enable us to employ in vivo as well as in vitro approaches and common methods of molecular biology in our research.  Most of our laboratories, including cell culture laboratory are radioactivity- and GMO-accredited and provide us with good technical background and key instruments, including several biohazard laminar flows, CO2 incubators, microscopes, various centrifuges, deep freezers, cell line cryopreservation facility, spectrophotometer and fluorimeter (Tecan Genios Plus plate reader),  iCycler, and the Accuri C6 flow cytometer.

1) Dual perfusion of the rat placenta  

In our lab, we use the method of dually perfused rat term placenta to investigate the functional expression of placental drug transporters. In brief, one uterine horn is excised and submerged in heated Ringer´s saline. A catheter is then inserted into the uterine artery proximal to the blood vessel supplying a selected placenta and connected with the peristaltic pump. Krebs perfusion liquid containing 1% dextran is brought from the maternal reservoir. The uterine vein is ligated behind the perfused placenta and cut so that maternal solution can leave the perfused placenta. The selected fetus is separated from the neighboring fetuses by ligatures. The umbilical artery is catheterized using a 24-gauge catheter connected to the fetal reservoir and perfused. The umbilical vein is catheterized in a similar manner, and the selected fetus is removed.

Two types of perfusion experiments are used in our lab, i.e. open-circuit and closed-circuit (recirculation) perfusion systems:

dual perfusion of the rat placentaOpen-circuit perfusion system is often employed to study maternal-to-fetal and fetal-to-maternal clearances of drugs at various concentrations. In this experimental setup, the compound is added to either maternal (maternal-to-fetal studies) or fetal (fetal-to-maternal studies) reservoir immediately after successful surgery. Fetal effluent is sampled and transplacental clearance calculated.

 

 

 

 

Schematic depiction of dual perfusion of the placenta to study the effect of placental transporters on mother to fetus transport of drugsClosed-circuit (recirculation) perfusion system is employed to investigate the potential of drug transporters to remove their substrates from the fetal circulation. In this experimental setup, both maternal and fetal sides of the placenta are infused with equal concentrations of the compound and the fetal perfusate is recirculated. This experimental setup ensures steady concentration on the maternal side of the placenta and enables investigations of maternal/fetal concentration ratio at equilibrium; any net transfer of the substrate implies transport against a concentrate gradient and is evidence for active transport.

 




2) In vitro functional assays

In our experimental work we employ cellular models of trophoblast cells of human (BeWo) and rat (HRT-1) placenta as well as other common human cell lines (e.g. HCT-8, Caco-2, HepG2).  To investigate the interactions (substrate specificity or inhibitory potency) of drugs with human drug transporters, we take the advantage of canine MDCK cell lines transfected or transduced by a vector carrying human gene encoding expression of particular ABC or SLC transporters. Using these cellular models, the tested drugs can be evaluated for their ability to be transported by the membrane protein and/or to inhibit the transporters´ activity.

We particularly perform:

  • Accumulation/efflux studies followed by subsequent analysis using flow cytometer, fluorimeter or radioactivity counter to study inhibitory potency of tested substance to particular transporter.
  • Transport experiments across the polarized cell monolayers cultured on cell inserts. In this experimental setup, novel substrates of studied transporters can be revealed as their transport across the cellular monolayer is accelerated in the transporter driven direction.
  • ATPase assays on transporter expressing membrane vesicles help us predict interactions of novel drugs with ATPase site of drug efflux transporter.
  • Combination studies based on the combination index method of Chou-Talalay are used to evaluate the quantitative definition for additive effect, synergism or antagonism of two anticancer drugs applied in combination.

3) Expression assays

  • Gene expression assays - in our laboratory we routinely employ quantitative RT-PCR method
  • Protein expression assays - for quantification and visualization of changes in expression of studied transport proteins we use the following methods:

Western blotting

 

Analysis of membrane transporters by flow cytometry

 

Immunofluorescence microscopy

Visualisation of BCRP transporter in BeWo choriocarcinoma cells

Visualisation of BCRP transporter (E) placental alkaline phosphatase (F) and nuclei in BeWo choriocarcinoma cells (G) (Ceckova et al., Clin Exp Pharmacol Phys, 2006)

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