In vitro pulmonary alveolar type II cell models not an easy task!

Transkript

1 2 nd esi-workshop In vitro pulmonary alveolar type II cell models not an easy task! Sara Signorelli Department of Physiology and Medical Physics, Innsbruck Medical University Fritz-Pregl-Str Str.. 3, Innsbruck, Austria

2 The Alveolus Type I cells About 95 % alveolar surface Flat large cells Efficient exchange of gases between blood and air Type II cells About 5 % of the alveolar surface area Cuboidal in shape Apical microvilli Lamellar bodies production, secretion and reuptake of pulmonary surfactant phospholipids and surfactant proteins (SPA, SPB,SPC and SPD)

3 Mostly used Alveolar Type II Cell Models Primary Cultures Human Rat Mouse Cell lines A549 They lack the full differentiated phenotype of the in vivo ATII cells Over time they transdifferentiate to an ATI- like phenotype There is a need for more suitable alveolar type II cell models!!

4 Transgenic H-2Kb- ts A58 Immorto mouse: It contains the coding sequence of a temperature sensitive form of the simian virus (SV40) T antigen AT II Cells Permissive conditions: Non Permissive conditions: 33 C IFN in the media 37 C Without IFN in the media SV 40 T antigen is expressed Cells proliferation SV 40 T antigen is not expressed Cell stop dividing Cell differentiation? P g m / P L A U m Alkaline phosphatase (ALP) quantification is si v e is si v e P erm P erm N on *

5 SEM

6 Lysotrackergreen (LTG) staining Nile red staining LTG: fluorescent dye (fluororophore linked to a weak base) that accumulate in acidic organelles as the result of protonation Nile Red: fluorescent dye soluble in organic solvents and lipids but insoluble in water

7 Real Time PCR Primers for: : SPA, SPB, SPC and SPD Results: : SpA, SpC and SPD were not detected SpB was mildly expressed Strategies: How can we prevent the dedifferentiation of type II cells in culture? Use a more appropriate nutrient medium Simulate the microenviroment of the alveolus: air liquid interphase (static and perfusion condition) Co-culture with in vivo neighbour cells All the above??

8 DCI Medium L. W. Gonzales et al., 2002 Differentiation of human pulmonary type II cells in vitro by glucocorticoid plus camp Epithelial cells isolated from human fetal lung treated with DCI medium for 4-5 days DCI medium: serum free medium 10 nm dexamethasone 100 M camp analog 100 M isobuthylmethylxanthine A Day 1 B Day 4 Ø DCI C Day 4 + DCI

9 Air-liquid Interface in Static Condition Endothelial compartment 24 well plate Filter insert 4-18 h 2 days air Epithelial compartment (ATII cells) Endothelial compartment (endothelial cells) Epithelial Compartment

10 Air-Liquid interface in Perfusion Condition: the EpiFlow Apparatus ATII cells monolayer Gas in Medium in coverslip Gas out Filter Medium out Endothelial cells momolayer

11 Rat primary cultures of alveolar type II pneumocytes on microporus supports

12 OUTLOOK Immorto mouse tissue freshly isolated cells (passage 0) passaged cells (passage 1, 3...?) grown in DCI medium QPCR SPA, SPB, SPC & SPD lamellar bodies production Surfactant secretion A/L interface - static conditions - perfusion conditions in vitro model more representative of type II cells in vivo

13 Thanks to: Prof. Walter Pfaller Dr. Paul Jennings Colleagues and friends: Andrea Ravasio Acknowledgements Sonia Aydin Nadia Malloth Thomas Lechleitner Judith Lechner Judy Schwabl Edna Nemati