Application Note
Microcarrier Mixing in
PadReactor® Mini Bioreactor
USD 3012
Introduction
The need for microcarriers in animal cell culture applications has been enhanced by the demand to increase
production capacity. In many cases, the productivity yields per cell for recombinant proteins or viruses are low
and require large volumes of production. Therefore, microcarriers have been used to allow the scale-up of
anchorage-dependent cells in bioreactors. Cells attach to the carriers and can generate mono- or multi-layers
depending on the geometry of beads (small spheres vs. macroporous structures). Some biotech companies are
currently able to reach high cell densities depending the microcarrier concentrations and batch sizes. Hence,
this increases the production capacities and eases the separation of the cells from the supernatant at harvest
compared to suspension cell culture. Today, many applications using microcarriers have been established for
the production of viral vaccines (e.g. Influenza, Poliovirus), recombinant proteins (e.g. Interferon) or monoclonal
antibodies using different types of cell lines (CHO, MDCK, VERO, Hybridomas). Moreover, microcarriers appear
to be the key solution for large scale production for cell therapy.
Within the range of its PadReactor systems, Pall Life Sciences has introduced the PadReactor Mini bioreactor,
a scaled-down model perfectly suited for feasibility studies, research, process development and small volume
production of proteins, cells and viruses (Figure 1). The gentle mixing, linked to an efficient gas transfer, allows
the PadReactor Mini system to host suspension and anchorage-dependent cell cultures (e.g. on microcarriers).
Critical cell culture parameters such as pH, DO and temperature are controlled and monitored by the PadReactor
Mini controller.
For optimal use, it is recommended to put microcarriers in complete or homogeneous suspension (Figure 2).
This ensures entire microcarrier surface availability for cell anchorage and growth.
Stirring speed must be determined by considering:
• The complete suspension of microcarriers
• Low shear stress
• Good medium mixing
Stirring speed impacts cell growth rate and productivity indirectly. Too low of a speed allows settling of microcarriers. Too high of a speed can damage cells or detach them from the microcarriers. Collagen-coated and Dextran-based microcarrier distributions and integrities inside the PadReactor Mini bioreactor have been assessed
for several mixing speeds and microcarrier concentrations.
Figure 1
PadReactor Mini system
2
Figure 2
Illustration of microcarrier suspension in PadReactor Mini bioreactor (A: partial suspension; B: complete
suspension; C: homogeneous suspension)
A
B
C
Method
This study was carried out in the PadReactor Mini bioreactor fitted with a 16 L biocontainer. Collagen-coated
plastic (Solohill, cat #: C102-1521; Diameter: 125 – 212 µm; density: 1.026) and Dextran-based (Diameter:
147 – 248 µm; density: 1.030) carriers were selected as microcarrier models. Working volumes and microcarrier
concentrations were selected to screen the mixing efficiency of the system on both types of beads. Tested
conditions are listed in Table 1. Bioreactor working volumes tested in this study were 5, 9 and 13 L, where 5
and 13 L represent the minimal and maximal working volumes in the PadReactor Mini biocontainer (equivalent
to 20 to 80% of the total volume).
The size of hydrated dextran microcarriers is related to osmolality. To control for size, the study was performed
with a liquid having an osmolality close to a standard cell culture medium. Medium-like solution, composed of
96 mM NaCl, 42 mM NaHCO3, 2.5 mM NaH2PO4 and 0.04 mM Red Phenol, was identified as the best solution.
Table 1
Conditions tested for the microcarrier homogeneity inside the PadReactor Mini bioreactor.
Type of microcarrier
Working volume (L)
Microcarrier concentration (g/L)
Dextran-based
5
9
13
9
3
3&6
3, 6 & 12
11.65, 35.00 & 58.25
Collagen-coated
Stirring and Sampling
At the beginning of each experiment, microcarriers were allowed to settle. Paddle rotation was started at the
required speed and samples were taken after 15 minutes and at least 12 hours, to check that no settling over
time occurred. For each sampling step, one 11 mL sample was taken at the top of the bag and a second one
at the bottom, both with a graduated pipette.
Sample Analysis
The final goal was to compare microcarrier concentrations at the top and at the bottom of the PadReactor Mini
biocontainer. To do so, each sample was inserted into a graduated cylinder. Once the microcarriers were settled,
their volume was measured (Figure 3). Heterogeneity between the bottom and the top of the bag was calculated
as follows:
% heterogeneity (H) =
2 x (VolBottom – VolTop)
(VolBottom – VolTop)
X 100
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Precision of obtained data was not calculated. Heterogeneity <5% was identified as not significant. For this
reason, conclusions were made as followed:
• % = 0 to 5%: Top and bottom concentrations are equivalent. Fully Homogeneous suspension state is reached
(considering the precision range of 5%). (Figure 2).
• % = 5 to 10%: Top and concentrations are very close. Considering the precision, we can estimate the
suspension as homogeneous.
• 100% > % > 10: The difference of beads from bottom and top is big and does not give a good suspension.
• % = 100%: All the microcarriers are settled down at the bottom of the bag.
Microcarrier integrity was inspected under a microscope with a cell culture dish. Broken beads vs. total beads
were counted for a specific area on the plate. Integrity of the microcarriers was calculated as followed:
% Microcarriers integrity =
(Total beads – Broken beads)
Total beads
X 100
Figure 3
Graduated cylinder with 11 mL of microcarrier solution in DPBS
Results and Interpretations
Solohill® Collagen-coated microcarriers (Figure 4)
• For a working volume of 9 L
– With 11.65 g/L of beads, a suspension is obtained from 40 RPM (H = 5.4%). This suspension is fully
homogeneous at 50 RPM (H ≈ 0% which is below the precision rate of 5%).
– With 35.00 g/L of beads, microcarriers are in complete suspension at 40 RPM (H = 7.4%). Full homogeneity
is achieved with a stirring speed of 60 RPM (H ≈ 0%).
– With 58.25 g/L of beads, are in complete suspension at 50 RPM (H = 6.7%). Hence, homogeneous
distribution of microcarriers is obtained at 60 RPM (H = 4.8%).
Figure 4
Heterogeneity of Collagen-coated microcarriers after 15 minutes mixing from bottom and top of the PadReactor
4
Mini bioreactor (*represents the stirring speed where the microcarriers are in suspension (H≤5%)).
Dextran-based microcarriers (Figure 5)
Percentage of concentration diference between
bottom and top (%)
30.0
Collagen-coated microcarriers heterogeneity in PadReactor Mini
25.0
20.0
15.0
10.0
5.0
50 RPM
*
60 RPM
40 RPM
40 RPM
*
30 RPM
35 RPM
40 RPM
50 RPM
60 RPM
70 RPM
80 RPM
60 RPM
*
50 RPM
0.0
8L - 11.65 g/L
8L - 35.00 g/L
8L - 58.25 g/L
-5.0
Tested conditions
• For a working volume of 13 L (maximum working volume)
– With 10 g/L of carriers, a suspension is obtained from 70 RPM (H = 5.2%). This suspension is fully
homogeneous at 90 RPM (H = 1.1% which is below the precision rate of 5%).
– With 6 g/L of carriers, microcarriers are fully homogeneous starting from 40 RPM (H = 4.7%) and seems
to improve with increase of stirring speed of 60 RPM (H = 1.3%).
– With 3 g/L of carriers, are in complete suspension at 40 RPM (H = 8.0%). Hence, homogeneous distribution
of microcarriers is obtained at 80 RPM (H = 3.2%).
• Working volume: 9 L
– With 6 g/L of carriers, full homogeneity is achieved with a stirring speed of 40 RPM (H = 2.5%).
– With 3 g/L of carriers, microcarriers are in suspension from 30 RPM (H = 5.3%), while the distribution
of microcarriers is fully homogeneous at 40 RPM (H = 0.6%).
• Working volume: 5 L (minimum working volume)
– With 3 g/L of carriers, homogeneous distribution of microcarriers is obtained at 40 RPM (H = 0.6%).
Figure 5
Heterogeneity of Dextran-based microcarriers after 15 minutes mixing from bottom and top of the PadReactor
Mini bioreactor (*represents the stirring speed where the microcarriers are in suspension (H≤5%)).
• Microcarrier integrity
Dextran-based microcarriers heterogeneity in PadReactor Mini
Percentage of concentration diference between
bottom and top (%)
30.0
25.0
20.0
30 RPM
40 RPM
50 RPM
60 RPM
70 RPM
80 RPM
90 RPM
15.0
10.0
5.0
40 RPM
70RPM *
40 RPM
*
90RPM 60 RPM
*
80 RPM *
40 RPM
30 RPM
*
40 RPM
*
40 RPM
0.0
-5.0
13L 10 g/L
13L 6 g/L
13L 3 g/L
9L 6 g/L
9L 3 g/L
5L 3 g/L
Tested conditions
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During these settlement tests, both types of microcarriers were sampled and analyzed under a microscope to
quantify their integrity. After more than 15 days of mixing of Dextran-based carriers (agitation speed set to keep
carrier in homogenous suspension), less than 2% of the microcarriers were broken. This observation was even
lower (<1%) for the Collagen-coated plastic carriers. These data demonstrate PadReactor Mini bioreactor
delivers a good mixing efficiency while reducing the shear stress effect.
Conclusions
This study describes the mixing efficiency of diverse concentrations of Collagen-coated plastic and Dextranbased microcarriers in the PadReactor Mini bioreactor at different working volumes (Figure 4 and 5). It showed
that the system is able to get a complete suspension of microcarriers within its stirring speed range (from 0 to
100 rpm), which is the right suspension state for efficient cell anchorage and growth. It also demonstrates the
specificity of each microcarrier regarding the stirring speed to apply to maintain these beads in suspension. Using
two types of microcarriers having similar diameters and densities does not imply similar suspension behavior.
Indeed, identical stirring speed was used to maintain wide concentrations of the two carriers, depicting the
important dependence of these results on the type of microcarrier in use. For this reason, Pall Life Sciences
recommends operators characterize mixing efficiency of the PadReactor Mini bioreactor with their specific type
of microcarrier used for their application.
Finally, it is important to highlight that the impact of cell growth and sparger bubbling on microcarrier mixing
was not considered in this study. Depending on its importance, stirring speed should be adjusted to maintain
microcarriers in suspension.
The study results show that the PadReactor Mini system is suitable for adherent cell culture on microcarriers.
In addition, the study provides PadReactor Mini system users with tools for microcarrier cultures for different
working volumes and microcarrier concentrations. Information contained in this document was used to perform
a 13 L working volume adherent cell culture in the PadReactor Mini system.
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