Evaluation of Drug Permeability In Vitro in Microgravity Simulation
- Paper number
IAC-07-A2.4.03
- Author
Ms. Marlise Santos, Pontificia Universidade Catolica do Rio Grande do Sul, Brazil
- Coauthor
Ms. Cynthia Bosquillon, King's College London, United Kingdom
- Coauthor
Mr. Chris Marriott, King's College London, United Kingdom
- Coauthor
Dr Thais Russomano, Microgravity Laboratory/PUCRS, Brazil
- Coauthor
Mr. Felipe Falcao, Microgravity Laboratory/PUCRS, Brazil
- Coauthor
Mr. Ricardo B. Cardoso, Brazil
- Coauthor
Mr. Leo Martinelli, Brazil
- Coauthor
Mr. Ben Forbes, King's College London, United Kingdom
- Year
2007
- Abstract
Limitations in genuine space-flight experimentation opportunities have led to the use of microgravity simulations to study the effect of this environment on cells in vitro. However, no study into how microgravity simulation influences the absorption of drugs across epithelial cell layers has been reported to date. The aim of this work was to evaluate the permeability of monolayers of human airway epithelial cells Calu-3 after exposure to microgravity simulation in a 3D-Clinostat using mannitol as a hydrophilic marker. Calu-3 cells were seeded on 24-well polyester Transwell® cell culture inserts. After 24h, cells were grown an an air-liquid interface and the transepithelial electrical resistance (TER) was thereafter used to monitor the development of the cell layer permeability barrier. After TER values had reached a plateau, some inserts were transferred to a specifically designed microgravity diffusion chamber and rotated in the 3D-Clinostat for 24 or 48 h. In parallel, other inserts were placed into the microgravity diffusion chambers but were not subjected to microgravity simulation. Permeability experiments were then performed in Hank’s Balanced Salt Solution (HBSS) supplemented with 20 mM HEPES under gravity in Transwell® plates 14C-mannitol was added to the apical chamber and after 30, 60, 90 and 120 min incubation at 37ºC under stirred conditions, each monolayer was transferred to a new well containing fresh HBSS/HEPES and samples were taken from the original receiver chamber. Samples were analysed by liquid scintillation . The integrity of the monolayers were verified by TER measurements and inter-group permeability data were compared by an ANOVA two factors > with replication test. After 24 or 48 h of clinostat rotation, all Calu-3 monolayers maintained a TER value which allowed transport experiments to be performed. Incubation of Calu-3 cell monolayers with 14C-mannitol for 120 min did not lead to a significant decrease in TER. No significant difference in 14C mannitol apparent permeability was observed among all samples (p> 0.05). Overall, this work represents preliminary experiments designed to establish a simple methodology for exposing cultured mammalian cells to microgravity on earth with minimum adaption of established tissue culture techniques. These results provide an encouraging basis for further study of drug permeability in the clinostat and the investigation of any effect of microgravity on transepithelial drug transport. The mannitol permeability indicated that the barrier to hydrophilic drugs is unaltered in cell layers that have been subjected to 24 to 48 h of microgravity simulation.
Authors: Santos, M.A.1,2, Bosquillon, C. 2 Marriott, C. 2, Russomano1, T. Falcão1, F.P., Cardoso1, R. B., Martinelli1, L. Forbes, B2. 1: Microgravity Center, PUCRS-Brazil; 2: Department of Pharmacy - Kings College London - UK
- Abstract document