4 Discussion
We have developed a novel surface agitated 300 µL microbioreactor
suitable for small-scale process development. The oxygen mass-transfer
coefficient, kLa, was characterized to range from 12 to
89 hr-1 which are of similar magnitude as those
commonly found in small scale reactors for mammalian cell culture
[5,20,22]. Mixing evaluation determined the surface agitated
microposts achieved increased planar (XY) mixing when compared to
diffusion alone as well as decreased time to achieve complete mixing as
mixing speed was increased (RMI > 0.85 achieved between 90
to 215s). Evaluation of vertical transport mixing determined that active
mixing achieved vertical homogeneity sooner than diffusion alone,
supported by an increased distance traveled in active mixed wells than
those achieved in a diffusion alone system. Static microplate small
scale mammalian cell process development technologies have insufficient
oxygen transport capabilities due to complications arising from
mechanical agitation of static microplate cultures [22–24]. Thus,
the developed microplate bioreactor’s microenvironment overcomes such
complications, and has sufficient convective flow of nutrients, which
more closely resembles that of larger scale stirred tank bioreactors, as
opposed to diffusional transport.
When compared to previously described microbioreactor’s [3,5,21,25],
our results consistently follow reported trends whereby increased
agitation speed leads to an increased kLa, as well as
increased fill volume leads to a decreased kLa. However,
the increase in kLa in large volume systems often
reported occurs with agitation speeds an order of magnitude lower than
those reported in this study. There are certainly differences in
systems, with the reviewed microbioreactor being mixed via
surface-attached microposts, which are agitated via a magnetic drive
system. Further, in our system there are approximately 1 x
105 Redbud Posts per cm2 therefore,
agitation rate may not directly reflect the overall mixing paradigm
within the culture vessel. Therefore, rather than a comparison in
systems performed based on comparing trends in increased agitation
speed, a more useful comparison may be to compare trends based on
increasing power per unit volume values. While power input is certainly
a function of the diameter of impeller, it may be useful to consider
rather how the magnetic strength translates to power input within the
fluid and the efficiency in the conversion of energy within the fluid
when determining the overall power input within this culture vessel.
In our novel microbioreactor, oxygen is supplied throughout the culture
via oxygen diffusion into the liquid phase at the surface gas-liquid
interface. Therefore, the observed improvement in kLa
values in the microbioreactor at higher magnetic rotational speeds can
be attributed primarily to better mixing and distribution of dissolved
oxygen in the liquid phase, thus indicating the microbioreactor has an
increased oxygen transport capability. Further, the observed
kLa values are of the same magnitude as those commonly
found in mammalian cell culture, thus there is confidence the
microbioreactor is suitable for expanding mammalian cells [8,9].
Preliminary results demonstrated that, in both mammalian cell types,
higher cell yield was achieved in our actively mixed vessel than static
cultures, indicating that the convective flow of nutrients achieved in
the microbioreactor promotes increase cell growth. In the CHO cultures,
there was an observed greater cell growth in both vessels, which
contained RBL posts regardless of the presence of agitation, compared to
a standard 96-well plate. Potentially, this is due to the presence of
RBL posts acting as additional support for cell growth, allowing for
cells to adhere to posts during static culture, thus creating sections
of plate composing of bilayers rather than a monolayer found in standard
96 well plates.
In the T cell cultures, there was an observed greater cell yield when
mixed, regardless of cytokine condition, than the unmixed conditions.
This observed increase, while certainly due to increased oxygen transfer
into the liquid; may also be due to improved convective transport of
nutrient (including oxygen) into the T cell-bead aggregates that form.
Further, there was an observed significant decline in cell yield
following seed until day 4, which is hypothesized to be due to cells
being cultured straight from cryopreservation, thus having a significant
adaptation phase. Additionally, decline observed following day 6 of
culture indicates the need for culture medium feed and potential further
expansion. Further, presence of magnetic beads required for T cell
activation alongside magnetic field for agitation led to posts knockdown
(caused by magnetic bead interaction with posts) further optimization is
necessary of culture expansion conditions including agitation speed and
medium feed.
Across both mixed and unmixed, the 110 condition had higher daily cell
yield than the 000 condition, indicating the 110 condition, which
contained higher IL-2 & IL-7 concentrations, is more suitable for cell
culture than that of the 000 condition interleukin concentrations. The
common T cell expansion factor, IL-2 has been extensively researched to
evaluate effects of varying levels on cell expansion [26–28].
However, Jaleco et al., 2003 [29] found that IL-2 and IL-7 act
together to promote proliferation, and is significantly more effective
than using IL-2 or IL-7 alone. Further, due to IL-2 and IL-7 acting
through one of the same pathways, higher levels of both cytokines (such
as those found in the 110 condition) would act to enhance the
effectiveness of both IL-2 and IL-7 in tandem [30], which supports
the findings reported here.
The novel bioreactor developed achieves mixing and oxygen transport
capabilities extending beyond those found in static small-scale culture
and preliminary cell culture results suggest enhanced cell yield. Based
on these results, the microenvironment found in this system is suitable
for cell culture and is more closely representative of that in a
traditional stirred tank bioreactor for mammalian cell culture and has
potential for use in small scale process development applications.