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Isolation and cultivation of metabolically competent alveolar epithelial cells from A/J mice

: Hansen, T.; Chougule, A.; Borlak, J.


Toxicology in vitro 28 (2014), Nr.5, S.812-821
ISSN: 0887-2333
Fraunhofer ITEM ()
A/J mice; AECs isolation and cultivation; characterization of AECs; cytochrome P450 monooxygenases; EROD and testosterone metabolism; gene expression

The A/J mouse strain is used in lung cancer studies. To enable mechanistic investigations the isolation and cultivation of alveolar epithelial cells (AECs) is desirable. Based on four different protocols dispase digestion of lung tissue was best and yielded 9.3 +/- 1.5 x 10(6) AECs. Of these 61 +/- 13% and 43 +/- 5% were positive for AP and NBT staining, respectively. Purification by discontinuous Percoll gradient centrifugation did not change this ratio; however, reduced the total cell yield to 4.4 +/- 1.1 x 10(6) AECs. Flow cytometry of lectin bound AECs determined 91 +/- 7% and 87 +/- 5% as positive for Helix pomatia and Madura pomifera to evidence type II pneumocytes. On day 3 in culture the ethoxyresorufin-O-demethylase activity was 251 +/- 80 pmol/4 h x 1.5 x 10(6) and the production of androstenedione proceed at 243.5 +/- 344.4 pmol/24 h x 1.5 x 10(6) AECs. However, 6-alpha, 6-beta and 16-beta-hydroxytestosterone were produced about 20-fold less as compared to androstenedione and the production of metabolites depended on the culture media supplemented with 2% mouse serum or 10% FCS. Finally, by RT-PCR expression of CYP genes was confirmed in lung tissue and AECs; a link between testosterone metabolism and CYP2A12, 3A16 and 2B9/10 expression was established. Taken collectively, AECs can be successfully isolated and cultured for six days while retaining metabolic competence.