|
Author
Rubio Hospital, Xavier
|
Under development therapies based on small extracellular vesicles (sEVs) are showing great promise, both for its application as drug delivery vehicles as well as for diagnosis purposes. As sEVs are still a relatively new technology, an established method for its large-scale production has not yet been achieved.
Currently in progress in the GEMAT group of which this work is a part of, are two parallel research lines using sEVs for the treatment of diseases such as cystic fibrosis and non-smallcell lung cancer. For this purpose, two different cell lines HEK293 and BEAS-2B are used, respectively. In this work, a platform for the massive production of sEVs with both cell lines is performed. As an initial step, media screening for both HEK-293 and BEAS-2B cell line was performed. Additionally, as culture media and supplements present nanoparticles in the size range of sEVs, two different methods for particle depletion from serum were assayed: the use of a 100 kDa Amicon Ultracentrifugal Filter and a 100 nm pore-sized polycarbonate filter, with not satisfactory results.
Different platforms and methods were examined for the adaptation of the anchorage dependant cell line BEAS-2B to suspension growth. For its culture in suspension, a microcarrier culture was performed examining three different microcarriers: Cytodex 1, Cytodex 3 and CultiSpher -S. Several elemental processes for the microcarrier culture were setup, the first being the determination of the best suitable microcarrier for this specific culture (BEAS-2B).
Cell count and harvesting methods were examined to obtain adapted protocols. To end this work, cultures at bioreactor scale were run. A bioreactor previously set up for bacterial culture was adapted for the more sensitive animal cell culture and two different cultures were performed: a BEAS-2B culture with Cytodex 3 microcarriers and a HEK-293 culture engineered to express GFP labelled sEVs.
|
|
