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.