Production and harvesting of A549 cell-derived extracellular matrix-comparing different isolation buffers and deposited layers

Cellular and molecular mechanisms (English)

Minghui Li
University of Groningen, University Medical Center Groningen
BEKIJK PROGRAMMA
 
10 april 15:25 - 15:30 (Lobby)
Introduction: The extracellular matrix (ECM) is an important extracellular microenvironment that can directly or indirectly regulate cell proliferation, adhesion, migration, and differentiation. The ECM plays a key role in the field of regenerative medicine. Cell-derived ECM offers advantageous biophysical and biochemical properties. However, the production of cell-derived ECM is still being investigated. Therefore, in this study, we aimed to optimize a method for harvesting cell-derived ECM through comparison of different isolation buffers, and investigate whether the amount of ECM harvested increased if cells were reseeded on the deposited ECM derived from A549 cell culture.
Methods: 3 x 105 A549 cells were seeded in medium (RPMI 1640) in T25 flasks (n=3). At confluence, cells were lysed with 0.016N NH4OH, and washed with HBSS. Two mL of 2M Urea or 0.05M Tris Hydrochloride buffer were used to harvest the first layer of ECM, respectively. Alternatively, A549 cells were reseeded on the ECM layer. At confluence, the second layer of ECM was harvested and cells were reseeded in the same way. Lastly, a third layer of ECM was harvested and the total concentration of the harvested proteins were detected using a BCA assay.
Results: The protein concentration of the first layer of ECM, isolated using Urea or Tris Hydrochloride was 28.45 ± 5.17 μg/mL, 59.65 ± 7.78 μg/mL, respectively. The total protein concentration in the second layer with Urea and Tris Hydrochloride was 30.05 ± 2.37 μg/mL, 72.02 ± 9.42 μg/mL, respectively. For the third layer of ECM, the total protein with Urea was 31.27 ± 3.74 μg/mL, while Tris Hydrochloride was 54.54 ± 3.65 μg/mL.
Conclusion: The total protein concentration of the second layer was higher than the first andthe third layer of ECM. Therefore, the better way to harvest more ECM from cell culture flasks may be with the second layer. The observed lower protein concentration of the third layer ECM, may be explained by the fact that parts of ECMwere still remaining on the flask. The total protein concentration of ECM with Tris Hydrochloride buffer was higher than Urea. Therefore, Tris Hydrochloride buffer may be better to get more ECM from cell culture flasks.

Acknowledgements:JKB holds a Rosalind Franklin Fellowship co-funded by the University of Groningen and the European Union.
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