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\begin{document}

\title{Mixing and Oxygen Transfer Characteristics of a Microplate Bioreactor
with Surface-Attached Microposts}



\author[1]{Justin Fisher}%
\author[2]{Travis  Gurney}%
\author[3]{Jay  Fisher}%
\author[2]{Brittany  Mason}%
\author[1]{William. J. Kelly}%
\affil[1]{Villanova University}%
\affil[2]{Redbud Labs}%
\affil[3]{Redbud Labs }%


\vspace{-1em}



  
  \date{June 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Bioprocess optimization for cell-based therapies is a resource heavy
activity. To reduce the associated cost and time, it is advantageous to
carry out process development in small volume systems, with the caveat
that such systems be predictive for process scaleup. The transport of
oxygen from the gas phase into the culture medium, characterized using
the volumetric mass transfer coefficient, k\textsubscript{L}a, has been
identified as a critical parameter for predictive process scaleup. In
both large- and small-scale bioreactors, k\textsubscript{L}a is
controlled via mixing, with the method employed dependent upon the size
of the reactor. However, existing microplate bioreactor platforms,
beneficial for their low working volumes and throughput and automation
capabilities, struggle to achieve desired k\textsubscript{L}a for
mammalian cell cultures. Here, we describe the development and testing
of a 96-well microplate with integrated Redbud Posts to provide mixing
and thus enhanced k\textsubscript{L}a. Mixing characteristics were
investigated, with actuating Redbud Posts shown (visually) to increase
convective transport while producing enhanced k\textsubscript{L}a,
providing means to mimic macroscale mammalian cell growth conditions at
the microscale. Improved cell growth rates with mixing was demonstrated
for two cell types, indicating the potential for this technology to play
a valuable role in early stage bioprocess development and optimization.%
\end{abstract}%



\sloppy


\textbf{1 Introduction}

Traditionally, bioreactor performance is quantified by considering the
critical transport processes and the biochemical conversion process
itself {[}1{]}. Since most industrial bioprocesses are aerobic, oxygen
is a limiting nutrient for cell growth and metabolic production; thus,
oxygen transport into the liquid phase is a limiting factor in
bioreactor and overall process performance {[}2{]}. It is therefore
critical to maintain adequate transport of oxygen from the gas phase
into the culture medium. Commonly, characterization of bioreactor oxygen
transport is presented as the volumetric mass transfer coefficient,
k\textsubscript{L}a which can be used to compare oxygen transfer
performance from the microscale to industrial volumes {[}2,3{]} and is
an established criterion for predictive process scale-up {[}4,5{]}.

With the recent development of cell-based therapies to treat a wide
range of unmet clinical needs, pharmaceutical companies have significant
pressure to introduce new products to the market as quickly as possible
{[}6{]}. When developing new therapies, early stage process development
and optimization requires extensive screening of culture conditions to
maximize cell growth and viability, with thousands of experiments
optimized across hundreds of variables and the best performing
conditions transferred to downstream production. This screening process
is time consuming and expensive, due to the high cost of media
components and often reduced cell growth of the cellular therapy cell
types {[}7{]}. As a result, it is advantageous to implement a scaled
development process wherein testing of the broadest parameter space
takes place in the smallest possible volumes with promising candidates
then transferred to larger volumes for additional testing.

Microplates present an ideal format for early process development due to
their low working volumes (\selectlanguage{greek}µ\selectlanguage{english}L-nL) and throughput and automation
capabilities. However, the utility of microplates for high throughput
process development is limited due to poor control of oxygen transfer or
k\textsubscript{L}a (h\textsuperscript{-1}) in these formats. Ideally,
it would be possible to control the k\textsubscript{L}a in microplates
to match those seen in larger volume mammalian cell culture systems,
such as the Sartorius ambr\selectlanguage{ngerman}® 15/250 (15-250mL) and Cytiva's Wave Cellbag
(2L-200L). For the former, k\textsubscript{L}a value ranges are of 0.18
- 7.90 h\textsuperscript{-1} and 2.15 -11.52 h\textsuperscript{-1} are
predicted while the latter can achieve k\textsubscript{L}a values that
range from 1-60+ h\textsuperscript{-1} {[}8,9{]}.

Mechanical shaking is the only commercially deployed solution for
microplate mixing of cultures, and its utility is limited. Microplate
shaking {[}10{]} induces splashing motion by overcoming surface tension.
Splashing is an inertial effect, and as microplate well size decreases,
the inertia of the droplet decreases, while surface tension remains the
same. Thus, smaller wells require greater shaking energy (amplitude,
frequency) to achieve the same mixing performance. For 96 well plates
this critical threshold has been observed to be approximately 800 RPM
(for a 3mm shake amplitude) {[}10,11{]}. Achieving the desired results
from mechanical shaking is surprisingly challenging. While
k\textsubscript{L}a in a conventional bioreactor improves smoothly with
increasing impellor speed {[}12{]}, shaking performance depends on
whether the shaking is in-phase or out-of-phase. Increased frequency has
little impact on out-of-phase mixing, and the harmonic frequency of
microwells varies dramatically with well size and shape {[}11{]}. Due to
these limitations, as well as concerns related to shear stress induced
damage at high shake frequencies, commercially available incubator
shakers are not marketed for use with mammalian cell culture. Shake-less
methods such as acoustic mixing exists, but adoption has been limited by
concerns about heating and scalability. In short, gentle microplate
agitation remains a major unmet need for bioprocess discovery and
development, especially for mammalian cell culture.

This research reports the development and characterization of a novel
microliter scale culture plate with novel surface-attached agitation
methodology. The microplate is designed for high throughput process
development applications due to its operation in a 96-well culture plate
with culture volume of 150-300 \selectlanguage{greek}µ\selectlanguage{english}L allowing for adequate sampling for
bioanalysis. Evaporation of culture media during operation, which has
been shown to be an issue in small volume culture reactors {[}13{]}, is
compensated for by operation in a high humidity environment. The oxygen
transfer performance of the microreactor is benchmarked against
conventional small-scale culture systems.

\textbf{2 Materials and methods}

\textbf{2.1 Microplate Bioreactor Design}

Microscale mixing is achieved by integrating Redbud Lab's MXR, a novel
microfluidic mixing technology consisting of 1 x
10\textsuperscript{5}Redbud Posts per cm\textsuperscript{2}, as surface
attached impellers in each well (Figure 1A). Redbud posts are
approximately 50 \selectlanguage{greek}µ\selectlanguage{english}m tall and 4 \selectlanguage{greek}µ\selectlanguage{english}m in diameter and are made using a
flexible polymer doped with magnetic material. When a magnetic field is
applied, the posts experience a torque that works to align their
dominant axis with the magnetic field, effectively creating an array of
micro-stir bars with one end attached to the surface. The magnetic
fields used to drive Redbud Posts are generated by a Redbud Stage
(Redbud Labs, RBS-CUST-MWP) modified to accept a multiwell plate (Figure
1B.). The stage provides control over the actuation rate of the posts
and has been modified further for compatibility with an incubator for
environmental controls (temperature, humidity, \%CO2, etc.).

Multi-well chips (Redbud Labs, MXR-CUST-MWP(-96)) designed to evaluate
planar mixing were prepared using RBL-posts affixed to an acrylic
substrate (RBL proprietary method). Biocompatible adhesive spacers were
cut with CO\textsubscript{2} laser to model standard 96-well XY
dimensions. An acrylic lid with fluid delivery ports was affixed atop
the spacer to seal the wells resulting in 4 identical chambers with 16
\selectlanguage{greek}µ\selectlanguage{english}L volumes (6.73 mm well diameter, and 0.4 mm spacer height

\textbf{2.2 Planar (XY) Mixing Evaluation}

To demonstrate the mixing produced by active posts at the macroscale, we
have developed planar and axial mixing assays wherein a colored liquid
is added to a chamber/well containing a clear liquid and the time
required to mix the two is calculated. Each well within the microwell
plate was filled with a clear 1\% solution of Triton X-100 (Thermo
Scientific 85111). Afterward, a diluted blue food dye solution was
prepared (McCormick) and a volume of 4 \selectlanguage{greek}µ\selectlanguage{english}L was delivered via pipet to the
well. The 4-well chip was then placed over a 1-inch drive magnet (120
mT). At this time, the drive system was engaged at the pre-determined
rpm (3000, 6000, 9000), or left off in the case of diffusion. Videos of
the four independent mixing wells driven simultaneously with identical
drive conditions were captured using LCD digital microscope. The data
collected via mixing video analysis was analyzed using the relative
mixing index (RMI) as outlined in Hashmi, 2014 {[}14{]}. A computer
program was written to pull in each frame of a video, select the
appropriate ROI, and perform the analysis for each well in the frame.
For this study, the threshold for a mixed state was set at an RMI of
0.85. The resulting graphs show the average RMI for each independent
drive speed evaluation (n=4).

\textbf{2.3 Vertical Transport Mixing Evaluation}

Multi-well plates (96-well) were designed to evaluate vertical transport
in cell culture plates. Plates were prepared using RBL-posts affixed to
an acrylic substrate (RBL proprietary method). Biocompatible adhesive
spacers were cut with CO\textsubscript{2} laser to model 96-well XY
dimensions. Using these spacers, the RBL posts were bonded to the
underside of clear no-bottom 96-well microplates (Greiner Bio One
655000), creating an open-well chamber with RBL posts covering the floor
of the wells. Each well was first filled with 200 \selectlanguage{greek}µ\selectlanguage{english}L clear 1\% solution
of Triton X-100 (Thermo Scientific 85111) and then the multi-well plate
was placed over a 1-inch drive magnet (120 mT). Afterward, a 7.5\selectlanguage{greek}µ\selectlanguage{english}L
volume of dilute blue food dye was delivered via pipet along the well
periphery and allowed to settle. At this time, the drive system was
engaged at the appropriate rpm (3000, 6000, 9000) and real-time videos
were captured using LCD digital microscope. Quantitative analysis of the
axial transport mixing videos was performed by determining the number of
pixels the indicator dye traveled from the established baseline
boundary. These values were calculated at each selected time interval (1
min.) and the distance traveled was reported in millimeters.

\textbf{2.4 Oxygen Mass Transfer Coefficient Measurement}

Dissolved Oxygen (DO) measurement was performed using OXR50 Retractable
Fiber Oxygen Microsensors (Pyroscience, Aachen, Germany), along with a
standard optical temperature (Pyroscience, Aachen, Germany), which were
connected to a FireStingO2 optical oxygen meter (Pyroscience, Aachen,
Germany). Signals were analyzed using FireSting Logger software
(Pyroscience, Aachen, Germany). Experiments at each condition were
performed in triplicate.

The oxygen mass transfer coefficient, k\textsubscript{L}a, for the
microreactor was determined using the dynamic gassing out method
(without cells) as described in Dunn \& Einsele, 1975 {[}15{]}.
Initially, the microbioreactor was deoxygenated by continuously sparging
with nitrogen gas until the dissolved oxygen concentration dropped to
near zero. Air was then sparged into the system, and the increase in
dissolved oxygen concentration was measured until the system returned to
a steady-state oxygen concentration. During the reoxygenation step, the
rate of at which the dissolved oxygen concentration changes is governed
by the following equation (1) where
C\textsubscript{L}\textsuperscript{*} is the oxygen saturation
concentration in the liquid phase at the gas-liquid interface
{[}4,16{]}.

\(\frac{dC_{L}}{\text{dt}}=\ k_{L}a(C_{L}^{*}-C_{L})-Q_{O2}X\) (1)

Thus, equation (1) can be integrated from time t\textsubscript{1} to
t\textsubscript{2} and k\textsubscript{L}a can be determined from the
slope of the graph of\(ln(\frac{C_{L0}-C_{L}}{C_{L0}-C_{s}})\ vs\ t\) where C\textsubscript{s} is the
final steady-state dissolved oxygen concentration.

\textbf{2.5 Chinese Hamster Ovary (CHO) Cell Expansion}

CHO-S cells (Gibco R80007) were cultured in Freestyle CHO expression
medium (Gibco 12651014) supplemented with 8 mM L-glutamine (Gibco
25030081), 0.1\% Kolliphor P188 (Sigma 15759), and 0.5X Pen/Strep (Gibco
15140122). Cells (passage 3-7) were transferred from active culture in
125-mL shake flask (DWK Life Sciences WPFPC0125S) and seeded in RBL
bioreactor 96-well microplate at a concentration of 150,000 cells/mL per
well with a volume of 300 \selectlanguage{greek}µ\selectlanguage{english}L. The plates were then sealed with a
breathable rayon-acrylate film (Thermo Scientific 241205) to prevent
evaporative loss during culture. Drive system was set to duty-cycle (10
min. ON, 50 min. OFF) at a selected rate (6000 RPM) and placed inside a
cell culture incubator at 37\textsuperscript{o}C with a humidified
atmosphere at 8\% CO\textsubscript{2}.~ Total protein values for each
culture condition at the end of a 72-h culture period were determined by
Bradford protein assay kit (Thermo Scientific 23246) following complete
cell lysis. The resulting protein values were reported as cell
concentration (cells/mL) after comparison to standard protein curve
obtained from known CHO-S cell counts.

\textbf{2.6 CHO Cell Lysis and Determination of Cell Number}

Upon completion of a 72 hr growth cycle, the RBL drive system was
disengaged and cells were allowed to settle. Growth media was removed
from the culture wells and transferred to labeled 0.5mL tubes. The
media/cell supernatant was centrifuged at 1000 rpm for 5 min. to pellet
any intact suspended cells remaining in growth media. At this time, a
volume of 100 \selectlanguage{greek}µ\selectlanguage{english}L Triton X-100 (1\%) was added to each well containing
the settled cells and gently mixed by pipet to disassociate any large
clusters. This plate was left undisturbed for 10 min. to ensure complete
lysis. Meanwhile, the supernatant/media was removed from 0.5mL tubes
while being careful not to disturb the pellet. The lysed cell solution
(100 \selectlanguage{greek}µ\selectlanguage{english}L) was transferred to its corresponding 0.5mL tube, vortexed (2-3
pulses), and allowed to incubate for another 5 min. to complete lysis of
any residual cells from media suspension. After this period, 0.5mL tubes
were centrifuged at 5,000 rpm for 5 min. to spin down any cell debris
and leaving soluble protein in supernatant. A volume of 10 \selectlanguage{greek}µ\selectlanguage{english}L from the
supernatant solution was transferred to a clear 96-well plate for
protein analysis. The detergent compatible Bradford protein assay was
run according to manufacturer recommended microplate procedures and read
at 595 nm using multi-mode plate reader (BioTek Synergy LX). In order to
relate soluble protein absorbance values to cell count, lysate prepared
from cell populations with known concentrations ranging from 0 to 1 X
10\^{}6 cells/mL was evaluated for protein content. A standard curve of
soluble protein to cell count was generated and cell populations are
reported as cells/mL.

\textbf{2.7 T Cell Expansion}

CD4\textsuperscript{+} Na\selectlanguage{ngerman}ïve cells were obtained from Astarte Biologics
(Astarte Biologics, Washington, USA) and were cultured in X-VIVO\selectlanguage{english} serum
free T-Cell medium (Lonza, New Jersey, USA). This medium was
additionally supplemented with recombinant human interleukin 2 (IL-2),
recombinant human interleukin 7 (IL-7), and recombinant human
interleukin 15 (IL-15) (Miltenyi Biotech GmbH, Germany) at various
concentrations on Day 3. In the `000' condition, medium was supplemented
with the following interleukin concentrations: IL-2 (1 ng/mL), IL-7 (6
ng/mL), IL-15 (6 ng/mL). In the `110' condition, medium was supplemented
with the following interleukin concentrations: IL-2 (10 ng/mL), IL-7 (36
ng/mL), IL-15 (6 ng/mL). T cells were combined with anti-CD3/anti-CD28
DynaBeads(r) (Gibco, Waltham, MA) in bead:T cell ratio of 1:1. Cells were
then seeded at 166,667 cells per mL in the described surface-attached
microplate with 300 \selectlanguage{greek}µ\selectlanguage{english}L culture medium. The day of seeding is referred to
as Day 0 of the expansion. Cell samples were obtained in the process
daily starting on Day 3 for enumeration.

During expansion, mixed condition was subject to agitation via
surface-attached microposts which were agitated via magnetic motor. The
motor was operated at 4000 rpm 5 minutes on 55 minutes off cycle
continuously throughout the duration of expansion.

\textbf{2.8 Statistical Analysis}

Descriptive statistics showing means, and standard deviations for cell
expansion were calculated using Microsoft\selectlanguage{ngerman}® Excel® data analysis.

\textbf{3 Results}

\textbf{3.1 Investigation of Oxygen Mass Transfer Coefficient in 96-Well
Microreactor}

As previously discussed, k\textsubscript{L}a has been established as a
critical parameter for predictive process scale- up {[}17,18{]} and
optimal bioreactor performance. Figure 2 shows a comparison between the
dissolved oxygen recovery curve (Figure 2A.) and estimated
k\textsubscript{L}a values by equation (2) in a mixed microbioreactor
versus an unmixed microbioreactor as measured by the dynamic gassing
method (Figure 2B.). In this comparison, there was an increased rate of
oxygen recovery following deoxygenation when mixed via Redbud Labs
proprietary micro-posts than via diffusion alone (no mixing). This
increased oxygen recovery corresponded to a calculated
k\textsubscript{L}a value of 42.48 h\textsuperscript{-1} in the mixed
condition when mixed at 7400 rpm versus 21.24 h\textsuperscript{-1} in
the unmixed at a well volume of 150 \selectlanguage{greek}µ\selectlanguage{english}L.

To investigate the magnetic agitated micro-post's capability to improve
oxygen transfer in a 96-well microbioreactor, the k\textsubscript{L}a
was determined in a single well at different magnetic rotational speeds
(0-9000 rpm). For the unmixed control conditions, the observed
k\textsubscript{L}a values ranged from 3.72 to 12.47
h\textsuperscript{-1} decreasing as culture volume increased, a
variation which is consistent with variation seen in similar sized
microtiter plates {[}5{]} and shake flasks {[}19{]}.

In the 96 well plate, higher k\textsubscript{L}a values were obtained as
the magnetic rotational speed was increased (Figure 2C) ranging from the
unmixed k\textsubscript{L}a values to 89.28 h\textsuperscript{-1} (9000
rpm, 200 \selectlanguage{greek}µ\selectlanguage{english}L). At 3500 rpm the observed k\textsubscript{L}a values were
14.64 h \textsuperscript{-1} (150\selectlanguage{greek}µ\selectlanguage{english}L), 8.88 h
\textsuperscript{-1}(200\selectlanguage{greek}µ\selectlanguage{english}L), and 9.12 h \textsuperscript{-1} (300\selectlanguage{greek}µ\selectlanguage{english}L). At
6500 rpm the observed k\textsubscript{L}a values were 34.2
h\textsuperscript{-1}(150\selectlanguage{greek}µ\selectlanguage{english}L), 33.6 h \textsuperscript{-1} (200\selectlanguage{greek}µ\selectlanguage{english}L), and
19.41 h\textsuperscript{-1} (300\selectlanguage{greek}µ\selectlanguage{english}L). At 9000 rpm the observed
k\textsubscript{L}a values were 88.20 h \textsuperscript{-1} (150\selectlanguage{greek}µ\selectlanguage{english}L),
89.28 h\textsuperscript{-1} (200\selectlanguage{greek}µ\selectlanguage{english}L), and 37.20 h
\textsuperscript{-1}(300\selectlanguage{greek}µ\selectlanguage{english}L). This observed variation in
k\textsubscript{L}a with increased rotational speed is comparable to the
change observed with increased shaking frequency in comparable 200 \selectlanguage{greek}µ\selectlanguage{english}L
microtiter plates {[}5,20{]}. Initially as the rotational speed
increased, there is not a substantial increase in k\textsubscript{L}a.
However, there is an observed critical rotational speed, beyond which
k\textsubscript{L}a increases exponentially.

\textbf{3.2 Influence of Well Fill Volume on k\textsubscript{L}a}

To investigate the influence of the fill volume on k\textsubscript{L}a,
the k\textsubscript{L}a was measured at three well volumes (150, 200,
300 \selectlanguage{greek}µ\selectlanguage{english}L). As seen in Figure 2C, at a fill volume of 150\selectlanguage{greek}µ\selectlanguage{english}L,
k\textsubscript{L}a values ranged from 12.37
h\textsuperscript{-1}(Unmixed), 14.64 h \textsuperscript{-1} (3500 rpm)
34.2 h\textsuperscript{-1} (6500 rpm) to 88.2 h \textsuperscript{-1}
(9000 rpm). At a fill volume of 200 \selectlanguage{greek}µ\selectlanguage{english}L, k\textsubscript{L}a values
ranged from 9.60 h\textsuperscript{-1} (Unmixed), 8.88 h
\textsuperscript{-1} (3500 rpm), 33.6 h \textsuperscript{-1} (6500 rpm)
to 89.28 h\textsuperscript{-1} (9000 rpm). At a fill volume of 300 \selectlanguage{greek}µ\selectlanguage{english}L,
k\textsubscript{L}a values ranged from 3.72 h
\textsuperscript{-1}(Unmixed), 9.12 h\textsuperscript{-1} (3500 rpm),
19.44 h\textsuperscript{-1} (6500 rpm) to 37.2 h \textsuperscript{-1}
(9000 rpm). As fill volume increased, there was a decrease in observed
k\textsubscript{L}a at a given rotational speed, a trend consistent with
comparable sized microreactors {[}5,20,21{]}. The observed trend is
expected, due to k\textsubscript{L}a being volume specific. Further, as
discussed there is a critical rotational speed, beyond which there is an
exponential increase in k\textsubscript{L}a. As demonstrated in Figure
2C, this critical value is dependent on fill volume, whereby the higher
the well fill volume, the higher rotational speed required to reach the
exponential increase in k\textsubscript{L}a.

\textbf{3.3 Planar (XY) Mixing Analysis}

Active mixing images were taken from wells driven by RBL posts at 9,000
rpm. Frames from real-time videos were selected at intervals t=0, 0.5,
1, 1.5, and 2 min. Diffusion images without active mixing (static RBL
posts) were selected from time-lapse series at intervals t=0, 30, 60,
90, and 120 min. As demonstrated in Figure 3A, homogeneity was achieved
quicker when mixed via micropost agitation (t = 2 min) verse diffusion
alone (t = 120 min), indicating micropost agitation offers greater
radial mixing capabilities than diffusion alone.

Relative Mixing Index (RMI) as outlined by Hashmi, 2014 {[}14{]} was
utilized to compare the active mixing profile of three different
rotational mixing speeds: 3000, 6000, and 9000 rpm. As demonstrated in
Figure 3B, complete mixing was achieved sooner (90 s) at the highest
rotational speed (9000 rpm), however the moderate mixing speed (6000
rpm) achieved complete mixing the slowest (215 s).

\textbf{3.4 Vertical Transport Mixing Analysis}

To establish the micropost ability to achieve sufficient axial mixing,
video analysis (Figure 4A) of vertical transport as a function of time
was utilized. Following 10 minutes of active agitation, microwells
achieved complete vertical transport, indicated by presence of dye at
the liquid surface of well; whereas diffusion alone had very little
vertical transport. To further confirm the video analysis, quantitative
analysis of the distance traveled indicated that when compared to
diffusion, active microposts achieve higher levels of vertical
transport, increasing with increasing rotational speeds (Figure 4B).
After 10 minutes of active agitation, at 9000 rpm, transport dye had
traveled the greatest distance (4.3 mm) compared to 2.93 mm (6000 rpm),
1.88 mm (3000 rpm). Under active agitation, transport dye had greater
distance traveled all rotational speeds than that during diffusion alone
(1.23 mm) indicating microbioreactor has increased axial transport for
nutrient transport.

\textbf{3.5 CHO-S Cell Growth Analysis}

CHO-S cells were cultured for 72 hours in three culture vessels:
Standard 96-well plate (Control), Redbud Labs Microplate (No Mixing) and
Redbud Labs Microplate (Mixing @ 6000 rpm). There was an observed
increase in cell growth when cultured in an agitated Redbud Labs vessel
(566,000 cells/mL) compared to both the unmixed Redbud Labs vessel
(499,214 cells/mL) and the standard 96-well plate (432,071 cells/mL)
(Figure 5A). As established, vessels agitated with Redbud Labs
microposts have increased oxygen transport capabilities, thus it is
expected that cell growth would be increased in such vessels. Further,
there was an observed increase in cell growth in both vessels containing
Redbud posts compared to the standard 96-well plate, suggesting post
microenvironment promotes increase cell growth.

\textbf{3.6 T Cell Growth Analysis}

CD4\textsuperscript{+} T cells were expanded under two different
concentrations of essential interleukins, IL-2, IL-7, and IL-15 (000 or
110) which corresponded to low and high concentrations of each
respectively and were either mixed via surface attached microposts or
unmixed. CD4\textsuperscript{+} T cells were seeded at a concentration
of 166,667 cells/mL and harvested 7 days later. The expansion kinetics
for T cells are shown in Figure 5B. There was an observed peak cell
yield on day 5 for all conditions, indicating the need for culture feed
and potential further activation. When mixed, the 110 condition
experienced the highest cell yield on day 5 (117,500 cells/mL) whereas
the 000 condition had a lower day 5 cell yield (45,000 cells/mL). When
unmixed, the 110 condition had the highest cell yield on day 5 (72,500
cells/mL) whereas the 000 condition had a lower day 5 cell yield (45,000
cells/mL). When mixed, both interleukin concentration sets (000 \& 110)
had higher daily cell yield than their respective unmixed conditions,
until day 7, which saw a decline in all conditions.

\textbf{4 Discussion}

We have developed a novel surface agitated 300 \selectlanguage{greek}µ\selectlanguage{english}L microbioreactor
suitable for small-scale process development. The oxygen mass-transfer
coefficient, k\textsubscript{L}a, was characterized to range from 12 to
89 hr\textsuperscript{-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 \textgreater{} 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 k\textsubscript{L}a, as well as
increased fill volume leads to a decreased k\textsubscript{L}a. However,
the increase in k\textsubscript{L}a 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
10\textsuperscript{5} Redbud Posts per cm\textsuperscript{2} 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 k\textsubscript{L}a
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
k\textsubscript{L}a 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.

\textbf{Acknowledgement}

This work was performed under a Project Award Agreement from the
National Institute for Innovation in Manufacturing Biopharmaceuticals
(NIIMBL) and financial assistance award 70NANB17H002 from the U.S.
Department of Commerce, National Institute of Standards and Technology

\textbf{Conflict of interest}

Travis O. Gurney, Brittany M. Nelson Dr. Jay K. Fisher are employed by
RedBud Labs

\textbf{5 References}

{[}1{]} L\selectlanguage{ngerman}übbert, A., Jørgensen, S. B., Bioreactor performance: a more
scientific approach for practice. \emph{J. Biotechnol.} 2001, \emph{85}
, 187--212.

{[}2{]} Garcia-Ochoa, F., Gomez, E., Bioreactor scale-up and oxygen
transfer rate in microbial processes: An overview. \emph{Biotechnol.
Adv.} 2008, \emph{27} , 153--76.

{[}3{]} Kirk, T, V., Szita, N., Oxygen transfer characteristics of
miniaturized bioreactor systems. \emph{Biotechnol. Bioeng} .
2013,\emph{110} , 1005--19.

{[}4{]} Doran, M. P., Mass Transfer, in: Elsevier Ltd.
(Ed.)\emph{Bioprocess Engineering Principles,} Academic Press, London
2013, pp. 397-444.

{[}5{]} Hermann, R., Lehmann, M., Büchs, J., Characterization of
gas-liquid mass transfer phenomena in microtiter
plates.\emph{Biotechnol. Bioeng.} 2003, \emph{81} , 178--86.

{[}6{]} Harrison, P, R., Medcalf, N., Rafiq, A, Q., Cell
therapy-processing economics: Small-scale microfactories as a stepping
stone toward large-scale macrofactories. \emph{Regen. Med.}
2018,\emph{13} , 159--73.

{[}7{]} Reichen, M,. Macown, J, R., Jaccard, N., Super, A. et al.,
Microfabricated Modular Scale-Down Device for Regenerative Medicine
Process Development. \emph{PLoS. One.} 2012,\emph{7} , e52246.

{[}8{]} Li, X., Scott, K., Kelly, J, W., Huang, Z,. Development of a
Computational Fluid Dynamics Model for Scaling-up Ambr
Bioreactors.\emph{Biotechnol. Bioprocess. Eng.} 2018,\emph{23} ,
710--25.

{[}9{]} Nienow, W, A., Reactor engineering in large scale animal cell
culture. \emph{Cytotechnology.} 2006, \emph{50} , 9--33.

{[}10{]} Klöckner, W., Büchs, J., Advances in shaking
technologies.\emph{Trends. Biotechnol.} 2012, \emph{30} , 307--14.

{[}11{]} Mitre, E., Schulze, M., Cumme, A, G., Rössler, F. et al.,
Turbo-mixing in microplates. \emph{J. Biomol. Screen.} 2007, \emph{12} ,
361--9.

{[}12{]} Xie, M., Xia, J., Zhou, Z., Chu, J. et al., Flow pattern,
mixing, gas hold-up and mass transfer coefficient of triple-impeller
configurations in stirred tank bioreactors. \emph{Ind. Eng. Chem.
Res.}2014, \emph{53} , 5941--53.

{[}13{]} El-Ali, J., Sorger, K, P., Jensen, F, K., Cells on
chips.\emph{Nature} 2006, \emph{442} , 403--11.

{[}14{]} Hashmi, A., Xu, J., On the Quantification of Mixing in
Microfluidics. \emph{J. Lab. Autom.} 2014, \emph{19} , 488--91.

{[}15{]} Dunn, J, I., Einseleb, A., Oxygen Transfer Coefficients by the
Dynamic Method. \emph{J. appl. Chem. Biotechnol.} 1975, \emph{25} ,
707-720.

{[}16{]} Tribe, A, L. Briens, L, C., Margaritis, A., Determination of
the volumetric mass transfer coefficient (kLa) using the dynamic ``gas
out-gas in'' Method: Analysis of errors caused by dissolved oxygen
probes. \emph{Biotechnol. Bioeng.} 1995, \emph{46} , 388--92.

{[}17{]} Islam, S, R., Tisi, D., Levy, S, M., Lye, J, G., Scale-up of
Escherichia coli growth and recombinant protein expression conditions
from microwell to laboratory and pilot scale based on matched k
La.\emph{Biotechnol. Bioeng.} 2008, \emph{99} , 1128--39.

{[}18{]} Islam, S, R., Tisi, D., Levy, S, M., Lye, J, G., Framework for
the Rapid Optimization of Soluble Protein Expression in Escherichia coli
Combining Microscale Experiments and Statistical Experimental
Design.\emph{Biotechnol. Prog.} 2007, \emph{23} , 785--93.

{[}19{]} Wittmann, C., Kim, M, H., John, G., Heinzle, E.,
Characterization and application of an optical sensor for quantification
of dissolved O2 in shake-flasks. \emph{Biotechnol. Lett.} 2003,\emph{25}
, 377--80.

{[}20{]} John, T, G., Klimant, I., Wittmann, C., Heinzle, E., Integrated
optical sensing of dissolved oxygen in microtiter plates: A novel tool
for microbial cultivation. \emph{Biotechnol. Bioeng.} 2003, \emph{81} ,
829--36.

{[}21{]} Duetz, A, Q., Microtiter plates as mini-bioreactors:
miniaturization of fermentation methods. \emph{Trends. Microbiol.}
2007,\emph{15} , 469--75.

{[}22{]} Betts, I, J., Baganz, F., Microbial Cell Factories Miniature
bioreactors: current practices and future opportunities. \emph{Microb.
Cell. Fact.} 2006, \emph{5,} 21.

{[}23{]} Micheletti, M., Barrett, T., Doig, S., Baganz, F. et al., Fluid
mixing in shaken bioreactors: Implications for scale-up predictions from
microlitre-scale microbial and mammalian cell cultures. \emph{Chem. Eng.
Sci.} 2006, \emph{61} , 2939--49.

{[}24{]} Büchs, J., Introduction to advantages and problems of shaken
cultures. \emph{Biochem. Eng. J.} , 2001, 7, 91--88.

{[}25{]} Zhang, Z., Szita, N., Boccazzi, P., Sinskey, J, A., Jensen, F,
K., A well-mixed, polymer-based microbioreactor with integrated optical
measurements. \emph{Biotechnol. Bioeng.} 2006, \emph{93} , 286--96.

{[}26{]} Litvinova, S, L., Sokhonevich, A, N., Gutsol, A, A., Kofanova,
A, K., The influence of immunoregulatory cytokines IL-2, IL-7, and IL-15
upon activation, proliferation, and apoptosis of immune memory T-cells
in vitro. \emph{Cell. Tissue. Biol.} 2013, \emph{7} , 539--44.

{[}27{]} Ganusov, V, V., Milutinovi\selectlanguage{polish}ć, \selectlanguage{english}D., De Boer, J, R., IL-2 Regulates
Expansion of CD4 + T Cell Populations by Affecting Cell Death: Insights
from Modeling CFSE Data. \emph{J. Immunol.} 2007, \emph{179} , 950--7.

{[}28{]} Deenick, K, E., Gett, V, A., Hodgkin, D, P., Stochastic Model
of T Cell Proliferation: A Calculus Revealing IL-2 Regulation of
Precursor Frequencies, Cell Cycle Time, and Survival. \emph{J.
Immunol.}2003, \emph{170} , 4963--72.

{[}29{]} Jaleco, S., Swainson, L, Dardalhon, V., Burjanadze M, et al.,
Homeostasis of Naive and Memory CD4 + T Cells: IL-2 and IL-7
Differentially Regulate the Balance Between Proliferation and
Fas-Mediated Apoptosis. \emph{J. Immunol.} 2003, \emph{171} , 61--8.

{[}30{]} Rochman, Y., Spolski, R., Leonard, J, W., New insights into the
regulation of T cells by \selectlanguage{greek}γ\selectlanguage{english}c family cytokines. \emph{Nat. Rev.
Immunol.}2009, \emph{9} , 480--90.

\textbf{Figures}

\textbf{** Attached as separate files**}

\textbf{Figure legends}

\textbf{Figure 1.} Microplate Bioreactor Design. \textbf{A} . MXR is an
array of Redbud Posts (50 \selectlanguage{greek}µ\selectlanguage{english}m length, 4 \selectlanguage{greek}µ\selectlanguage{english}m diameter) molded from a single
film. The film is mounted on an acrylic backing for rigidity and topped
with a laser cut double sided adhesive to define the chamber mask. The
assembled ``chip'' can be integrated into No Bottom Microplates by
peeling off the adhesive liner covering the mask and mounting under the
microplate. \textbf{B.} RedBud Stage modified to accept multiwell
microplate bioreactor. The stage provides control over the actuation
rate of posts and is compatible with incubator for environmental
control.

\textbf{Figure 2.} Oxygen Mass Transfer Coefficient Evaluation in
Microplate Bioreactor. \textbf{A} . Comparison of Dissolved Oxygen
Profile demonstrating an increased rate of oxygen recovery following
deoxygenation when mixed via Redbud Labs microposts than via diffusion
alone (no mixing). \textbf{B.} Linear (by logarithmic scale) Prediction
of k\textsubscript{L}a between Unmixed and Mixed (7400 rpm) microplate
reactors at a culture volume of 150 \selectlanguage{greek}µ\selectlanguage{english}L corresponding to
k\textsubscript{L}a values of 42.48 h\textsuperscript{-1} (Mixed) and
21.24 h\textsuperscript{-1} for diffusion alone. \textbf{C} .
k\textsubscript{L}a as a function of magnet rotational speed in the
microbioreactor for three culture volumes (150 \selectlanguage{greek}µ\selectlanguage{english}L, 200 \selectlanguage{greek}µ\selectlanguage{english}L, 300 \selectlanguage{greek}µ\selectlanguage{english}L). At
150 \selectlanguage{greek}µ\selectlanguage{english}L, k\textsubscript{L}a values ranged from 12.37
h\textsuperscript{-1} (Unmixed), 14.64 h \textsuperscript{-1} (3500 rpm)
34.2 h \textsuperscript{-1} (6500 rpm) to 88.2 h
\textsuperscript{-1}(9000 rpm). At 200 \selectlanguage{greek}µ\selectlanguage{english}L, k\textsubscript{L}a values
ranged from 9.60 h\textsuperscript{-1} (Unmixed), 8.88 h
\textsuperscript{-1} (3500 rpm), 33.6 h \textsuperscript{-1} (6500 rpm)
to 89.28 h \textsuperscript{-1}(9000 rpm). At 300 \selectlanguage{greek}µ\selectlanguage{english}L,
k\textsubscript{L}a values ranged from 3.72 h\textsuperscript{-1}
(Unmixed), 9.12 h\textsuperscript{-1} (3500 rpm), 19.44 h
\textsuperscript{-1} (6500 rpm) to 37.2 h \textsuperscript{-1}(9000
rpm).

\textbf{Figure 3.} Planar (XY) Mixing Analysis. \textbf{A} . Comparison
of planar mixing rates in wells driven by RBL posts (active mixing)
versus diffusion alone (static RBL posts). Homogeneity was achieved in
agitated wells in 2 minutes, verse 120 minutes in the wells mixed via
diffusion alone. \textbf{B} . Average Relative Mixing Index (Outlined by
Hashmi, 2014 {[}14{]}) of the active mixing wells (3000, 6000, 9000 rpm)
over time. Wells are considered fully mixed when they achieve a RMI of
0.85. At 3000 rpm, complete mixing was achieved after 180 s, whereas at
6000 rpm complete mixing was achieved after 215 s and at 9000 rpm
complete mixing was achieved after 90 s.

\textbf{Figure 4.} Vertical Transport Mixing Analysis. \textbf{A} .
Video Evaluation of Vertical Transport. Images displayed are selected
frames from real-time video at timepoints t=0,2.5, 5, 7.5, and 10 min.
One well (shown left) includes active RBL posts, whereas the well on the
right has static RBL posts to establish diffusion baseline. The RBL
drive magnet was placed below the wells, operating at 9000 rpm.
\textbf{B} . Vertical distance traveled comparison between active
agitation (3000, 6000, 9000 rpm) and diffusion alone. After 10 minutes,
at 9000 rpm dye traveled 4.23 mm compared to 2.93 mm (6000 rpm), 1.88 mm
(3000 rpm) and 1.23 mm (diffusion alone).

\textbf{Figure 5.} Evaluation of Mammalian Cell Growth in
Microbioreactor. \textbf{A.} Comparison of Chinese Hamster Ovary (CHO)
growth results following 72 hr expansion in three culture vessels. When
expanded in the presence of active Redbud posts, increased CHO cell
growth was observed (566,000 cells/mL) compared to both standard 96-well
plate (432,071 cells/mL) and the unmixed Redbud posts (499,214
cells/mL). \textbf{B.} Comparison of CD4\textsuperscript{+} Cell growth
results in active (mixed) Redbud posts or static (unmixed) Redbud posts
at two cytokine levels (110, 000). Highest cell yield was observed on
day 5 where 110 condition had higher cell yields (117,500 cells/mL
mixed, 72,500 cells/mL unmixed) compared to 000 condition (45,000
cell/mL mixed, 45,000 cells/mL unmixed).\selectlanguage{english}
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