Biomedical Engineering Reference
In-Depth Information
40.
J Biosci Bioeng
. 2007 Jan;
103(1):50-9. Cholesterol delivery
to NS0 cells: challenges and
solutions in disposable linear
low-density polyethylene-based
bioreactors. Okonkowski J,
Balasubramanian U, Seamans C,
Fries S, Zhang J, Salmon P,
Robinson D, Chartrain M. Merck
Research Laboratories,
Bioprocess R&D, PO Box 2000,
RY80Y-105, Rahway, NJ 07065.
We report the successful cultivation of cholesterol-
dependent NS0 cells in linear low-density
polyethylene (LLDPE) Wave Bioreactors when
employing a low ratio of cyclodextrin to the
cholesterol additive mixture. While cultivation of
NS0 cells in Wave Bioreactors was successful when
using a culture medium supplemented with fetal
bovine serum (FBS), cultivation with the same
culture medium supplemented with cholesterol-lipid
concentrate (CLC), which contains lipids and
synthetic cholesterol coupled with the carrier
methyl-beta-cyclodextrin (mbetaCD), proved to be
problematic. However, it was possible to cultivate
NS0 cells in the medium supplemented with CLC
when using conventional cultivation vessels such as
disposable polycarbonate shake flasks and glass
bioreactors. A series of experiments investigating the
effect of the physical conditions in Wave Bioreactors
(e.g., rocking rate/angle, gas delivery mode) ruled
out their likely influence, while the exposure of the
cells to small squares of Wave Bioreactor film
resulted in a lack of growth as in the Wave
Bioreactor, suggesting an interaction between the
cells, the CLC, and the LLDPE contact surface.
Further experiments with both cholesterol-
independent and cholesterol-dependent NS0 cells
established that the concurrent presence of mbetaCD
in the culture medium and the LLDPE film was
sufficient to inhibit growth for both cell types. By
reducing the excess mbetaCD added to the culture
medium, it was possible to successfully cultivate
cholesterol-dependent NS0 cells in Wave Bioreactors
using a cholesterol-mbetaCD complex as the sole
source of exogenous cholesterol. We propose that the
mechanism of growth inhibition involves the
extraction of cholesterol from cell membranes by the
excess mbetaCD in the medium, followed by the
irreversible adsorption or entrapment of the
cholesterol-mbetaCD complexes to the LLDPE
surface of the Wave Bioreactor. Controlling and
mitigating these negative interactions enabled the
routine utilization of disposable bioreactors for the
cultivation of cholesterol-dependent NS0 cell lines in
conjunction with an animal-component-free
cultivation medium.
