Automated Suspension Cell
Monitoring in Stirred Bioreactors
Continuous Suspension Cell Culture
Monitoring in Bioreactors
Cell monitoring in large scale suspension cell cultures often relies on
sampling followed by a staining procedure. An estimation of the cell count
and cell viability are traditionally performed once a day using a Trypan-Blue
cell exclusion method or other. These off-line techniques require manual
operations and the stained samples are destroyed after measurement
(creating toxic waste). It often requires weekend work too.
Differential Digital Holographic Microscopy (DDHM) is a new
quantitative imaging technique that allows cell counting as well as cell
viability monitoring in a continuous, label-free set-up. No need for
sampling (eliminating the risk of contamination) or staining, results are
available in nearly real-time and this over the whole run as the OsOne
software plots the cell growth curves live on the screen. Moreover,
OsOne also shows real-time images of the cells, offering to the
experienced eye a convenient way to have a look at the culture.
In this study, we compared the results generated by the iLine-F with 2
reference methods applying Trypan-Blue staining: a) manual counting and
b) the off-line counter Vi-Cell XR (Beckman Coulter). The iLine-F was set to
generate 2 cell count cycles per hour and the culture was left to grow in
batch mode for a total of 13 days.
In addition to the benefits of having a continuous monitoring of the
culture, a correlation factor R² of 0.93 was obtained for the viable cell
density demonstrating that the results achieved with the label-free DDHM
method are in line with current methods applying Trypan-Blue staining.
• Full traceability at single cell level
• Continuous monitoring
• User independent, improving
• No sampling, no staining
• No toxic waste
• Ready for automation
• Increased insight in your culture
• Adds cluster information as
clusters are not disrupted during
• More than just cell density and
viability: up to 16 morphologic
parameters are recorded
Materials & Methods
• iLine-F DDHM device
• BioConnect: autoclavable and
disposable closed-loop interface
with the bioreactor.
• Reusable pump engine with
automated flushing (in case air
bubbles or large aggregates are
detected, patent pending).
• OsOne Acquisition & Analysis
software (developed in-house by
OVIZIO), version 3.8.
• Applikon 3 liters glass bioreactor
controlled by ez-Control.
• CHO cells inoculated at 0.3x106
viable cells/mL in CD-OptiCHO™
medium (Life Technologies),
Jérémie Barbau & Joël Henneghien
OVIZIO Imaging Systems SA/NV, Brussels, Belgium
Contacts: [email protected], [email protected]
§ The availability of full data, per cell, for the whole experiment
allows to envision the use of iLine F for a PAT approach.
Indeed the large amount of data produced can be used to
perform various statistical analysis on the cell population in
order to define and control critical parameters of the process.
§ One possibility is also to link the iLine F with the bioreactor
controller in order to control for instance the feeding, based
on the cell density, or the harvest time after a viral infection,
based on the viability of the culture. There is virtually no
limitation to automate your cell culture process.
§ One next step for us would be to have a central control
server that collects data from several iLine-F simultaneously.
Thus a centralized computer could perform all the calculations
while only the microscope occupies valuable space in the lab.
§ Furthermore, smartphones could connect to the central
control server and cell data could become accessible
§ Finally, an API (Application Programming Interface) is
currently in development in order to integrate into other
Processes Information Management Systems.
§ Very good correlation (R² = 0.93) of the cell density and cell viability compared
to classic staining methods applying sampling and Trypan-Blue staining. This also
proves that the label-free approach is equivalent to extensively validated
§ Availability of full data per cell, for the whole experiment, for validation, QC, audit,
Correlation between Vi-Cell XR and iLine F for cell viability counting
iLine F generated results: (a) growth curve plotting Viability, Total Cell Density (TCD) and Viable Cell Density (VCD) over time
(b) cell viability monitoring, accessible at single cell level (showing a dead cell plotted on the screen and on the dot plot, with below its morphologic data)
(c) results comparative assay: measurements with OsOne are fitting with those performed offline by Vi-Cell XR or by Trypan-Blue manual counting.