Bioprocess Engineering and Supporting Technologies
Biotechnology and Bioengineering
DOI 10.1002/bit.26270
Automated microfluidic sorting of mammalian cells labeled with
magnetic microparticles for those that efficiently express and secrete a
protein of interest†
Xuan Droz1*, Niamh Harraghy1*, Etienne Lançon1*, Valérie Le Fourn2, David Calabrese2,
Thierry Colombet3, Pascal Liechti3, Amar Rida3, Pierre-Alain Girod2 and Nicolas Mermod1+
1. Institute of Biotechnology and Department of Fundamental Microbiology, University of
Lausanne, and Center for Biotechnology UNIL-EPFL, Lausanne, Switzerland
2. Selexis SA, Geneva, Switzerland
3. Spinomix SA, Ecublens, Switzerland
* These authors contributed equally to this study
+ Correspondence to: Prof. N. Mermod, Laboratory of Molecular Biotechnology, station 6, EPFL,
CH-1015 Lausanne, Switzerland. Tel. No. + 41 21 693 61 51; Fax No. + 41 21 693 76 10; E-mail:
[email protected]
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
[10.1002/bit.26270]
Additional Supporting Information may be found in the online version of this
article.
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This article is protected by copyright. All rights reserved
Received August 5, 2016; Revised January 5, 2017; Accepted February 15, 2017
Abstract
We developed a method for the fast sorting and selection of mammalian cells expressing and
secreting a protein at high levels. This procedure relies on cell capture using an automated
microfluidic device handling antibody-coupled magnetic microparticles and on a timed release of the
cells from the microparticles after capture. Using clinically compatible materials and procedures, we
show that this approach is able to discriminate between cells that truly secrete high amounts of a
protein from those that just display it at high levels on their surface without properly releasing it. When
coupled to a cell colony imaging and picking device, this approach allowed the identification of CHO
cell clones secreting a therapeutic protein at high levels that were not achievable without the cell
sorting procedure. This article is protected by copyright. All rights
Introduction
Constructing mammalian cell lines efficiently expressing and secreting a therapeutic protein has been
greatly improved by the construction of more efficient DNA vectors and engineered cell lines
(Harraghy et al. 2015). However, a major bottleneck in the development of state-of-the-art
engineered cell lines is the heterogeneity in cell growth, stability and productivity of the transfected
cell population. The majority of cells in a transfected population show a low to average level of protein
production, with a minority showing high productivity (Browne and Al-Rubeai 2007). Therefore,
screening for high-producing clones from such high heterogeneous population of cells is of high
importance for recombinant therapeutic protein production.
Manual screening of cell lines for high producers using Limiting Dilution Cloning (LDC) is
a widely used technique due to its basic process and low cost (Puck and Marcus 1955).
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The LDC method consists of an initial selection of a population of stably transfected cells using a
selection marker such as drug resistance or reporter gene expression (Wagman et al. 1974); (Helman
et al. 2014; Kim et al. 2012). This is followed by dispensing a low density cell suspension into microtiter plates at an average density of less than one cell per well, so as to obtain monoclonal populations
derived from a single cell. The protein production level of such cell populations is then determined to
identify the cell clones with the highest expression level. To isolate the cell clone of interest,
thousands of individual clonal populations must usually be isolated and analyzed, during multiple
rounds of LDC procedures, which makes the entire procedure both time consuming and labor
intensive (Cervino et al. 2008).
To improve the screening throughput and automate the process, fluorescence-activated cell sorting
(FACS) systems have been adapted to the isolation of mammalian cells according to their protein
surface expression (Kuhne et al. 2014). For instance, it has been previously shown that placing CHO
cells at 20°C or 4°C transiently slows down protein secretion, so that the secreted proteins are
transiently displayed on the cell surface for up to 24 hours (Brezinsky et al. 2003; Pichler et al. 2011;
Sen et al. 1990). Consequently, a fluorescent antibody that binds the secreted protein can be used
to label the expressing cells, and those that display the highest amounts of the secreted protein
displayed at their surface can be sorted by flow cytometry.
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A variation on this theme couples an antibody that recognizes the secreted protein of interest with
magnetic particles, so as to immobilize secreting cells using magnets (Liu et al. 2009; Radbruch et
al. 1994). This has been adapted to the preparative separation of cells displaying a given protein
from those that do not express it (Carroll and Al-Rubeai 2005; Miltenyi et al. 1990; Thomas et al.
1992). However, one drawback of these various approaches is that they detect the cellular surface
protein display rather than the effective protein secretion rate, which do not necessarily correlate
(Leno et al. 1991; Meilhoc et al. 1989). Furthermore, these approaches can be difficult to adapt to
the sterile and controlled processes required to produce proteins for clinical use, for instance when
they do not rely on single use vessels to handle the sorted cells.
To measure the effective protein secretion rate, new methods have been proposed that involve
retention of the secreted recombinant protein in the vicinity of the cell that secretes it, followed by
protein quantification. For example, the ClonePix FL robotic system involves the cells being plated in
methylcellulose-containing semi-solid medium supplemented by a conjugated fluorescent antibody
that binds the secreted protein, thereby forming a fluorescent secretion complex, or “halo of
secretion” in the vicinity of each single-cell derived colony. The fluorescent intensity measured
around individual colonies is related to the amount of the protein released, which can be used to
estimate the secretion rate of the recovered clones. This system can also be used to select single
colonies for expansion (Caron et al. 2009; Holmes and Al-Rubeai 1999; Hou et al. 2014; Nakamura
and Omasa 2015). In another approach, the cells are distributed in an array chip integrated in a
stand-alone automated device equipped with a high-precision motorized micromanipulator
(Yoshimoto et al. 2013). The cells of interest can be detected at the single-cell level using a
fluorescence microscopy equipped with a CCD camera. In this assay, non-toxic lipid-labeled
antibodies confine the antibody close to the cell and bind the nascently secreted proteins
on the cell surface, thereby allowing cell selection based on their fluorescence level (Kida
et al. 2013). Despite their advantages, these single cell analysis systems remain
expensive, and they have a low throughput relative to alternative approaches such as
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FACS. Thus, a large number of cell batches must be screened to yield a relatively small number of
candidate cell clones. Therefore, there is a general need to devise an automated procedure for the
rapid screening of large polyclonal populations of cells secreting a protein of interest, to select those
that secrete the protein of interest at the highest levels, in conditions that are compatible with clinical
use of the cells and/or secreted protein.
Here we describe a new platform for cell sorting based on the magnetic labeling of the surface protein
of interest. The cell sorting is based on the level of expression of the surface protein and is performed
in a sterile and single-use closed microfluidic cartridge, operated within a fully automated and
compact device. This step is followed by a second sorting to recover cells that are quickly released
from the magnetic microparticles, and which therefore exhibit a fast protein secretion rate. We show
that this approach allows the efficient enrichment of cell populations secreting very high levels of a
recombinant protein. When combined with a single cell selection system (i.e. ClonePix FL), the
proposed approach allows the recovery of higher producing cells than can be obtained with ClonePixmediated selection alone. Thus, we report herein a new methodology that greatly facilitates the
identification of the most favorable cell clones from large polyclonal populations based on the
magnetic labeling of the cells and an automated cell selection process that employs sterile, singleuse cell handling vessels.
Materials and Methods
MagCell System
Cell selection was performed using the MagCell system (Spinomix SA, Switzerland) developed
during this study. It consists of a fully automated platform that uses a sterile and single-use cartridge
for sample processing (Fig. 1). This cartridge consists of a plastic fluidic unit, where the
sample, reagents (washing buffer and magnetic microparticles) as well as the cell and
waste recovery vessels are integrated and processed in a fully closed format. Within the
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cartridge, the magnetic particles are mixed and retained in a chamber of
50 µL volume that is in
fluidic communication with the different liquid storage tubes (Figs. 1B and C). For processing, the
cartridge is plugged in the MagCell device, which is mainly composed of a fluidic manifold on which
the active fluidic components (pump and valves) are mounted and connected to the cartridge through
the respective pores as shown in Fig. 1C. A schematic illustration of the MagCell cartridge process
and of the MagPhase technology is provided in video_1 of the supporting information. A micro gear
pump is used to circulate the different liquids in the fluidic circuit. Four solenoidplungers act as valves.
When activated, the plunger presses at a specific point on a soft elastomer layer under the cartridge.
This will clog the corresponding channel and prevent the liquid from flowing through it when the pump
is running. Only one valve can be open at a time to ensure good
operation.
Four magnetic poles positioned around the microfluidic reaction chamber handle the magnetic
microparticles when the cartridge is operated in the MagCell device. These magnetic poles, made of
copper wire wound around ferromagnetic cores, are part of an electromagnetic circuit and can be
electrically controlled independently of each other. To achieve an effective and homogenous mixing
of the magnetic microparticles and sorted cells, MagCell relies on two types of magnetic
microparticles with different physical characteristics: the first type are ferromagnetic microparticles
(Chemicell, SiMAG/CF-DNA, 1.0 µm) composed of cobalt – iron oxide, and the second type are
superparamagentic microparticles (Dynabeads MyOne Streptavidin T1, ThermoFisher, #65601)
composed of iron oxide. In MagCell, the ferromagnetic microparticles are used as “carrier
microparticles” for mixing and separating the superparamagentic microparticles that are, in turn, used
as functionalized “affinity microparticles” that bind the secreted protein and capture the cells. The
affinity microparticles are coated with a specific protein (e.g., streptavidin or Protein A) that interact
in turn with a (biotinylated) antibody that specifically recognizes the protein displayed on
the cell surface.
The physical mechanism underlying the methodology is based on the difference in the
dynamic behavior of magnetic responsive microparticles having different coercive fields
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(i.e., the characterization value of the B – H magnetic hysteresis curve of any magnetic material,
which defines the intensity of the external applied magnetic field required to reduce the magnetization
of that particle to zero after the magnetization of the sample has been driven to saturation), under
the application of an external time-varied magnetic field, e.g., a rotating magnetic field. Effective
mixing of ferromagnetic microparticles in a microfluidic system using a time-varied magnetic field can
be achieved by the formation of columnar-like structures that can be controlled by varying the
magnetic field oscillation frequency with time (Rida and Gijs 2004a; Rida and Gijs 2004b). Under
these conditions, strong mixing of the microparticles was demonstrated in a microfluidic flow
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setting. Furthermore, by using a “rotating magnetic field”, ferromagnetic microparticles will strongly
rotate around the chamber and form a homogenous “dynamic fog” suspension of microparticles (Rida
and Gijs 2004a). allowing optimal exposure and enhanced mixing of the particle surfaces with the
surrounding liquid medium (Martin et al. 2009; Rida (2013) US Patent N° US2013217144; Rida
(2016) US Patent N° US2016018303).
We demonstrate, herein, for the first time a new methodology to manipulate superparamagnetic
particles in a microfluidic chamber using a previously described time varied magnetic field (Rida
(2016) Patent N° US2016018303). The approach consists of using the strong mixing and steering of
ferromagnetic particles under a time varied magnetic field as a driver to disperse and mix
superparamagnetic particles in the microfluidic chamber. Conceptually, in a time-varied magnetic
field, when ferromagnetic microparticles (represented by closed circles) in combination with
superparamagnetic microparticles (represented by open circles) are manipulated using a low
frequency time-varied magnetic field, the two particles tend to agglomerate, as illustrated in the top
drawing of Fig. 2A. This agglomeration, which occurs under a low frequency time-varied magnetic
field, as in a static magnetic field, is due to the fact that the two types of particles will “rotate” in a
synchronized way with the external field polarity variation. Under such conditions, the two particle
types can follow the “slow” field polarity variations, which lead their respective magnetic moment to
be aligned, and therefore the particles attract one another, resulting in aggregation. Within a high
frequency time-varied magnetic field, the magnetic moment of the particles will tend to be
desynchronized with respect to the polarity variation (or rotation) of the external magnetic field due
to the high viscosity torque exerted by the surrounding liquid medium. The high frequency regime will
therefore be characterized by a time “lag” between the magnetic field variation and the magnetic
moment of the particles. This time lag is lower for ferromagnetic microparticles (i.e. particles with a
high coercive field e2, which tend to follow the magnetic field variation more easily). When
ferromagnetic microparticles and superparamagnetic microparticles (which have a lower coercive
field e2) are operated together in a high frequency time-varied magnetic field, the two particles tend
to separate (i.e., desegregate) from each other due to the repulsive magnetic interaction
created by the “lag” between their respective magnetic moment. By further increasing the
frequency of the polarity variation of the external magnetic field, one can reach a
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frequency threshold (f 1) where only the ferromagnetic microparticles can dynamically move in
response to the field variations, while the superparamagnetic microparticles will remain static, with
relatively low or
absent physical movement of the particles. In a liquid environment, however, the superparamagnetic
microparticles will be strongly steered by the movement of the ferromagnetic microparticles, resulting
in a strong mixing and homogenous distribution of the superparamagnetic
microparticles.
Cell lines
Chinese hamster ovary (CHO) cells (CHO-M) growing in suspension and expressing an
immunoglobulin (IgG) were used in this study. Cells secreting and displaying high levels of IgG were
subsequently transfected with an Enhanced Green Fluorescent Protein (EGFP) expression vector
for ease of detection (F206 cells). Cell lines secreting and/or displaying medium or low levels of IgG
were generated by co-transfecting CHO-M cells with IgG heavy and light chain expression vectors,
a puromycin resistant plasmid, and various amounts (5, 50, 500 ng) of the eBFP2 reporter plasmid
(provided by S. Majocchi, see (Majocchi et al. 2014) for details) as previous studies indicated that
the amount of co-transfected reporter plasmid can influence IgG production. (Pick et al. 2002) Cells
were seeded at 3x105 cells per mL in 12-well plates and transfected with 1µg total plasmid DNA using
Fugene HD, with a Fugene:DNA ratio of 3:2. After 48h, the cells were transferred to 25 cm 2 culture
flasks and selection was started. To increase the probability of isolating cell clones with the desired
properties, cells were selected with either 5 or 10 µg/mL of puromycin. After approximately 2 weeks
of selection, the cells were assessed for surface IgG display, BFP fluorescence and IgG production.
The cells were the sorted by fluorescence activated cell sorting (FACS) to obtain clones with sufficient
levels of BFP expression for microscopy and FACS analyses and the required surface
IgG display. Limiting dilution of these polyclonal populations was then performed to obtain
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Cell culture
monoclonal populations. Clones were selected to display a surface IgG profile lesser than-equivalent
to- or greater than- F206, but with lower levels of IgG secretion (Fig. 3).
CHO K1 cells were cultured in Spin tubes (TPP, #87050) in HyClone SFM4CHO cell culture media
(Thermo Scientific, #SH30548.02) supplemented with 1x HT supplement (Gibco, #41065-012) and
8 mM L-glutamine (Gibco, # 25030-024), with or without 5% of Cell Boost 5 (CB5, Thermo
Scientific, #SH30865.01) and 1x Antibiotic Antimycotic solution (Sigma, # A5955). For the selection
of neomycin-resistant cells, Geneticin G418 (Gibco, #10131-027) was added to a final concentration
of 500 μg/ml. For selection of puromycin-resistant cells, Puromycin (Gibco, #A1113802) was added
to a final concentration of 2.5 μg/mL for F206 and C_MF cells, 5 μg/mL for BS2 cells. For Hygromycin
B resistance selection, Hygromycin B (Invitrogen, #10685-010) was added to a final concentration of
250 μg/mL. Cells were maintained under agitation (180 rpm) at 37°C and 5% CO2, in a humidified
incubator (Kühner shaker ISF-4-W) with 85 % relative humidity. Cells were counted every 3 or 4
days, and adjusted to 2x105 or 3x105 cells per mL, respectively, in fresh media. Cell surface staining
and flow cytometry analysis were performed as previously described (Harraghy et al. 2012).
Assays for surface display of IgG and IgG secretion
Detection of surface IgG was based on the protocol developed by (Brezinsky et al. 2003) as described
in (Harraghy et al. 2012), with the following modifications: an APC-conjugated antibody
(Allophycocyanin (APC)-conjugated AffiniPure Goat Anti-human IgG (Jackson ImmunoResearch)
♯109-135-098) was used for labeling to allow us to detect surface IgG in cells already transfected
with GFP. In addition, the labeling procedure was done in 1.5 mL Eppendorf tubes. We shortened
the protocol (based on findings from (Pichler et al. 2011)) and omitted fixating the cells, as this did
not impact the results as long as the cells were analysed shortly after labelling. Surface IgG display
was assessed by flow cytometery. IgG titers were determined by ELISA as described previously
(Harraghy et al. 2012).
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MagCell-based cell capture
IgG-expressing cells cultivated in SFM4CHO cell culture media (without CB5 feed) were collected at
Day 3 or Day 4 of culture in a 1.5 mL Eppendorf tube by centrifugation at 1000 rpm at room
temperature (RT). Cells were washed twice and they were resuspended to 1x107 cells per mL in a
1x PBS, pH 7.4 solution. Cells were then incubated with a biotinylated anti-human IgG antibody (KPL,
#216-1006 at 1:200 dilution, or Mabtech AB, #3850-6-250 at a 1:300 dilution) at RT for 20 min. After
incubation with the antibody, cells were washed with an equal volume of the PBS, pH 7.4
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solution and resuspended in the same volume of this solution. 1 µL of ferromagnetic microparticles
(Chemicell, SiMAG/CF-DNA, 1.0 µm) and 20 µL of superparamagentic streptavidin coated
microparticles Dynabeads MyOne Streptavidin T1 (Invitrogen, #65601) were first loaded in the mixing
chamber of MagCell cartridge. 1400 µL of 1x PBS, pH 7.4 solution was subsequently placed in the
"WASH solution" tube. Finally, 170 µL (i.e. approximately 1.7x106 cells) of the biotinylated antibodylabeled cell suspension was placed in the "INLET" tube of the MagCell cartridge (Fig. 1C). The loaded
tubes were then screwed on the cartridge (together with the empty “OUTLET” and “WASTE” tubes),
which had been previously sterilized by gamma-irradiation (24K Gray). All manipulations were
performed under sterile conditions in a laminar flow hood. Once loaded, the cartridge was taken out
of the laminar hood and placed onto the MagCell device. The desired cell capture script was launched
on the computer (see below). When the capture process was completed, the cartridge was brought
under the laminar flow hood to remove the outlet tube from the cartridge. Recovered cells and
microparticles were spun down at RT in a microfuge at 1000 rpm for 5 min. The recovered cells and
microparticles were placed in culture in one well of a 96well plate with the culture medium
supplemented with 1x HT, 5% CB5 and 1x Antibiotic Antimycotic solution (Sigma, #A5955), but
without geneticin or puromycin selection. The cells were separated from the microparticles at the
post-capture time point mentioned in the text and figures (e.g., Day 1, 3 and Day 4). This was
performed using a hand-held magnet to immobilize the superparamagnetic microparticles, to recover
only the cells that had spontaneously detached from the microparticles at the chosen time after
MagCell separation. The recovered cells were cultured in SFM4CHO medium supplemented with
CB5 but without antibiotic for 16 days prior to the analysis of the IgG displayed at the surface of the
recovered cells or protein secretion assays. This culture time was used to ensure the absence of
contamination and that operations had been performed in sterile conditions.
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Optimized cell sorting procedure
Five main types of operation modes were designed for the handling of magnetic microparticles by
MagCell, as needed to fit the major steps of the cell capture process (Fig. 2B and C), which were
termed the mixing, capture, separation, release and recovery modes. The parameters and conditions
for handling the microparticles depend on the two types of particles in use, and had to be determined
experimentally by optimizing each step of the MagCell process. In the mixing mode, a high frequency
(> 100 Hz) rotating magnetic field is applied to separate the two types of microparticles (Fig. 2B, step
1), thereby resulting in the homogeneous mixing of the microparticles. Upon increasing the feeding
current of the electromagnet, the rotating magnetic field becomes stronger, the ferromagnetic
microparticles become attracted to the walls of the microfluidic chamber while rotating around the
chamber, thereby resulting in strong movement of the liquid and homogeneous dispersion of the
superparamagnetic
microparticles. To
medium
homogeneously disperse the superparamagnetic microparticles, the electromagnets are activated
using a rotating magnetic field for three consecutive cycles, with a 1s clockwise rotation, followed by
a 1s anticlockwise rotation, and then 10s of clockwise rotation. This homogenous mixing mode is
used for incubating the superparamagnetic microparticles with the cells, to capture expressing cells,
and also during the washing steps, to remove non-expressing cells.
In the separation mode, the electromagnets are operated to generate a low frequency ( 1 Hz)
rotating magnetic field that causes the ferromagnetic and superparamagnetic microparticles to
aggregate (Fig. 2B, step 2). By operating this mode for 5 s, the ferromagnetic particles aggregate
with the superparamagnetic particles and the cells bound during the mixing mode. The
electromagnets are then activated using a static high current to generate a strong magnetic field that
will attract the microparticle aggregates to the corners of the chamber (Fig. 2B, step 3). While the
microparticles are separated, fresh solutions (cells in suspension or washing buffers) are
pumped into the separation chamber, while the solution in the chamber, which may now
contain the undesired or non-expressing cells, is expelled.
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In the cell recovery mode, a high frequency (> 100 Hz) rotating magnetic field is applied to separate
the two types of microparticles from each other and a “mixing mode” is operated again as described
above to homogeneously disperse the superparamagnetic particles in the reaction chamber (Fig. 2B,
step 4). The electromagnets are then operated as in the separation mode but with a weaker current
and medium frequency, and a weaker magnetic field is applied. The ferromagnetic particles move
toward the corner of the chamber because of their stronger magnetization, leaving the
superparamagnetic particles dispersed in the chamber. The superparamagnetic microparticles and
captured target cells can then be easily eluted in the recovery tube by pumping air into the reaction
chamber (Fig. 2B, step 5). A video showing the MagCell operation modes and the handling of the
two microparticles in the chamber is provided in the video of the supporting information.
We devised a process to distinguish cells that quickly release the secreted protein (i.e. the high
secretor cells) from the cells that display the secreted protein at their surface without properly
releasing it, and thus that do not efficiently complete the secretion process (i.e. the high displayer
cells, see Results and Discussion section). This was achieved by timing the release of the cells from
the secreted protein and microparticle complexes, in order to isolate cells that quickly release the
secreted protein (and hence the microparticles). The suspension of MagCell-sorted cells and
microparticles were placed into SFM4CHO medium containing L-glutamine and incubated at 37°C,
5% CO2, in a humidified incubator. After 24h of culture, the cells that spontaneously separated from
the microparticles were recovered in the culture supernatant, after the sedimentation of the
microparticles and associated cells at the bottom of the culture dish, thereby selecting for cells having
a fast secretion rate (referred to as Day 1 cells). The remaining microparticles and microparticleassociated slowly- or non-secreting cells were placed in prolonged culture, and the cells that had
more slowly released the secreted protein and thus had dissociated from the microparticles were
recovered after 3 additional days of culture (referred to as Day 4 cells).
Analysis and single-cell cloning of MagCell sorted cell populations
MagCell-sorted cell populations were analyzed and subcloned using ClonePix™ FL
Imager (Molecular Devices Inc, USA). Briefly a semi-solid media was prepared by mixing
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equal parts of liquid concentrate (2x) SFM4CHO medium containing L-glutamine and CloneMatrix©
(Molecular
Devices). This mixture was supplemented with CloneDetect™ FITC reagent containing anti-human
IgG antibody conjugated to FITC. IgG-expressing non-sorted control cell populations or
MagCellenriched IgG-secreting cell populations were used to inoculate this medium at 200 cells/ml
in 6-well plates. Plates were then incubated 10 days at 37°C, 5% CO2, in a humidified incubator. The
growth and IgG secretion of colonies were analyzed using the Clonepix FL Imaging software.
Highlysecreting colonies were picked and transferred to 96-well plates containing culture medium.
Antibody titer was quantified by ELISA, as described above under assays for detection of IgG. The
clonal cell lines of the best producers were then evaluated in Fed-Batch cultivation process.
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Fed-Batch Performance Evaluation
Clonal cell lines were cultivated in 125 ml shake flasks in 20 ml of SFM4CHO Hyclone serum-free
medium and passaged every 3-4 days. Growth and production performances of individual clones
were evaluated in fed-batch cultivation into 50-ml minibioreactor (TPP, Switzerland) at 37°C in 5%
CO2 humidified incubator for 10 days. Cultures were fed on a daily basis with a commercial Hyclone
Feed (ThermoScientific). Viable cell density was determined using the Guava EasyCyte flow
cytometry system (Millipore). IgG titer in cell culture supernatants was measured by ELISA. Cell
density (Cv/ml) and IgG titer values (µg/ml) were plotted at the indicated sampling days. The specific
IgG productivity (secretion rate) of each expressing clone was determined as the slope of IgG
concentration versus integral number of viable cell (IVCD), calculated from day 3 to day 7 of culture
(production phase), and expressed as pg per cell and per day (pcd).
Results and Discussion
The objective of this study was to develop an automated device and protocol to allow the rapid and
efficient capture of mammalian cells that display high amounts of a recombinant therapeutic antibody
at their surface, using a procedure solely reliant on sterile and disposable cell-handling cartridges.
The procedure was based on the labeling of IgG-secreting CHO cells with antibodies recognizing the
secreted protein, conjugated to a biotin molecule that can be captured by avidinconjugated magnetic
microparticles for cell sorting. The magnetically-labeled cells were handled within an automated
device termed MagCell, using cell-compatible, sterile, single-use microfluidic cartridges, that were
specifically designed for this purpose during the course of this study (Fig. 1A). The cartridge is
comprised of three reservoirs, which contain the cell suspension, the washing and elution buffer, and
the waste, respectively. A fourth tube allows the recovery of the selected cells (Fig. 1B and C). The
cartridge contains flexible membrane-based valves that are operated by the MagCell device to
specifically open or close the microfluidic connections between the reservoirs and the
reaction chamber. Micro-filters (0.2 micron) shield the air vents from potential external
contamination. The MagCell device is controlled by a user graphic interface coupled to a
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computer and through which a process script with different process control parameters can be
loaded.
The programmed steps act to control the system, for instance the closing and opening of the valves,
and operate a pump that generates a vacuum within the cartridge to move solutions in to or out of
the separation chamber. The programmable steps also regulate the magnetic fields generated by the
four electromagnets around the cartridge chamber, so as to control the mixing dynamics or
separation of the particles (see Materials and Methods and the Suppl video_1). The automated cell
capture process involves the incubation of the cells of interest with a biotinylated antibody that binds
the secreted protein at the cell surface (Fig. 2C). The cell suspension is then loaded in the cartridge
chamber, where the biotinylated antibody-displaying cells are captured by the avidin-conjugated
microparticles. After several rounds of washes, the retained cells are then pumped out of the chamber
into the recovery vessel, where they are recovered and placed in culture medium for recovery and
release from the magnetic particles, which occurs when the displayed surface protein is released
from the cell surface.
Manual magnetic cell capture protocols are typically based on superparamagnetic microparticles,
due to their low remanence, which allows easy resuspension of the microparticles and avoids
aggregation of the microparticles, which may impair cell recovery and viability. However, the handling
and mixing of superparamagnetic microparticles is difficult within a microfluidic chamber. In contrast,
ferromagnetic particles are easily handled in a microfluidic environment using timevaried magnetic
fields, but these particles tend to aggregate due their remanent magnetization. In fact, as verified
experimentally in preliminary experiments, these aggregates trap the bound cells, and
thereby interfere with cell viability. Furthermore, the cells were not efficiently released from
the bead aggregates placed in culture (data not shown). Thus, to overcome these issues,
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we developed a novel cell handling approach that consists of the use of the strong mixing of
ferromagnetic particles under a time varied magnetic field as a driver to disperse or retain
functionalised superparamagnetic particles in the microfluidic chamber.
The cell capture principle is based on the control of the movement of the cell-capturing
superparamagnetic microparticles by the ferromagnetic ones, as illustrated by the open and closed
circles of Fig. 2A, respectively. Various operation modes were developed to mediate specific steps
of the cell sorting approach (see Materials and Methods). The electromagnets can be activated
consecutively at a high frequency rotating magnetic field having a moderate magnetic intensity, to
provide the mixing and cell capture modes, where the two types of microparticles are separated, and
the cell-capturing superparamagnetic microparticles are homogeneously mixed within the chamber
(Fig 2A, high frequency magnetic field, and Step 1 of Fig. 2B). Another operation mode (capture
mode), which is performed at a lower frequency, allows the superparamagnetic microparticles and
associated cells to aggregate with the ferromagnetic microparticles, thereby facilitating their
separation within the chamber (Step 2 and 3 of Fig. 2B). During the washing steps that remove
unbound cells, the superparamagnetic microparticles are again homogenously dispersed using the
mixing mode (Fig. 2B, step 1), after which these microparticles together with the captured cells are
released from the immobilized ferromagnetic microparticles and then eluted (Step 4 and 5 of Fig.
2B). Thanks to this automated process control, these operations were adapted and optimized for
cultured cell handling and sorting, so as to rapidly and selectively sort populations of millions of cells
within minutes, to preferentially recover the protein-secreting ones.
In order to test and optimize MagCell for the capture of cells that secrete a recombinant protein at
high levels, four reference cell lines were generated to express a therapeutic antibody (IgG) as an
example of a secreted protein of clinical interest, as described in Materials and Methods. These
reference populations were cell clones that differed in either their surface antibody display or level of
antibody secretion or both. The cell clones were labeled by the transfection of reporter
gene vectors, to enable us to easily distinguish high and low IgG secreting cells (by
microscopy or flow cytometry) without the need to assay IgG production. F206 cells
represent a desired clone, as they display and secrete IgG at very high levels, and they
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were labeled by the stable transfection of an Enhanced Green fluorescent protein (EGFP) expression
vector. Other reference cells (BS2, BLC and BHB cells) were selected to have less favorable
properties, and they were labeled by the expression of a blue fluorescent protein (BFP), so as to
easily distinguish them from the F206 EGFP-expressing cell clone that secretes the same therapeutic
IgG, but at higher levels. The BFPlabeled BHB reference cells accumulated very high levels of the
secreted protein at their surface (Fig. 3A), but they did not efficiently release the secreted protein into
the culture medium when compared to the F206 cells, as the amount of secreted protein released
into the cell culture supernatant remained low (Fig. 3B). Consistently, the BHB reference cells have
a lower secretion rate than F206, and therefore a low specific productivity (Fig. 3C). The
characterization of various clones indicated that the transient display of a secreted protein does not
necessarily correlate with the actual secretion rate, as indicated by the titers and secretion rates of
the various reference cell clones. This is exemplified by comparing the BS2, BLC and BHB
populations that display distinct levels of the IgG on their surface, but secrete it at comparable low
levels (Fig. 3A-C). This observation was an important consideration when developing the MagCell
device and optimizing the procedures to selectively isolate cells that both express high IgG levels, as
indicated by the cell surface display, and efficiently secrete the therapeutic protein.
We first tested the ability of MagCell to enrich the F206 cells when mixed with the parental CHO cells
that do not express any IgG. MagCell enriched the F206 cells by 2-fold relative to the nonexpressing
cells, which yielded a 2 to 6-fold enrichment of the F206 cells relative to the parental cells, depending
on the input cell composition (Suppl Fig. 1). We then tested the ability of MagCell to enrich F206 cells
from cells secreting the same IgG, but at lower rates, namely the IgGexpressing BS2, BLC and BHB
reference cells. When F206 cells were mixed with the medium or high IgG-displaying BS2 or BLC
cells as the input sample, MagCell achieved a 2-fold enrichment of F206 high secretor
cells (Fig. 4A and 4B). However, when F206 cells were mixed with the very high displayer
BHB cells, MagCell did not significantly enrich the F206 cells (Fig. 4C). This correlated
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well with the fact that BHB cells display a much higher amount of IgG at their surface than F206 cells,
despite their lower secretion rate (Fig. 3A and C). Thus, we concluded that in a polyclonal population
of high, medium and low IgG secreting cells, MagCell could selectively enrich for high secreting cells
among medium or low producer cells, but that it would not fully remove those that display the protein
at very high levels without efficiently secreting it. Thus, it prompted us to design an additional sorting
step to specifically select the cells that have faster secretion kinetics.
To specifically and efficiently enrich the secreting cells from those that just display the recombinant
protein at their surface but do not release it, the MagCell cell sorting process was followed by a
second sorting step relying on a timed release of the cells from the magnetic microparticles (Fig.
2D). The MagCell sorting process was performed on a polyclonal population of cells resulting from
the stable transfection of the IgG expression vectors, with the aim of assessing the diversity of cells
and properties (secretion rate and surface display) that MagCell may recover from heterogeneous
cell populations. After MagCell sorting, the superparamagnetic microparticles and associated cells
recovered from the MagCell microfluidic cartridge were placed in culture medium, and the cells that
had spontaneously released the magnetic particles upon the completion of the protein secretion
process were recovered in the culture supernatants after either one or four days of culture (Fig. 2D).
This was performed to selectively select the cells that have a faster secretion rate, such that they are
fully released from the attached secreted IgG and associated microparticles one day after MagCell
sorting (Day 1 cells). In contrast, cells that remained associated at day 1 post sorting, but dissociated
from the microparticles between day 1 and day 4 post sorting, were selected as slowly secreting cells
(Day 4 cells).
The population recovered from the superparamagnetic microparticles after one day of culture was
enriched in cells displaying medium or high levels of the antibody at their surface relative to the
unsorted population control, whereas the cells released from the microparticles at day 4 were not
significantly enriched relative to the input polyclonal cell population (Fig. 5A). Furthermore,
the cells that had released the magnetic microparticles after 1 day of culture secreted the
therapeutic antibody at rates that were more than 2.5-fold higher than those of input cells,
while the cells eluted at day 4 had lower secretion rates (Fig. 5B). Similar findings were
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made when sorting F206 cells from the non-expressing parental cells, indicating that this approach
may even select faster secreting cell variants from clonal populations (Suppl. Fig. 2). This indicated
that the cells most rapidly released from the superparamagnetic microparticles after recovery from
MagCell sorting were those that secrete the recombinant protein at higher rates. In contrast, the cells
released at a later time likely retained the protein at their surface for longer, thus explaining their poor
secretion
To assess the ability of this process to sort highly productive cells, MagCell-sorted polyclonal
populations were analyzed using a single cell colony imaging and clone picking device, the ClonePix
FL system. A sterile and GMP-compatible MagCell cell sorting was performed on a polyclonal
population of cells transfected to stably express the therapeutic Trastuzumab IgG. (Le
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Fourn et al. 2014). The MagCell-sorted cells were plated in methylcellulose-containing semi-solid
medium supplemented by a FITC-conjugated antibody that binds secreted human IgGs, thereby
forming a fluorescent secretion complex, or “halo of secretion” in the vicinity of each single-cell
derived colony (Caron et al. 2009). The fluorescent intensity measured around individual colonies is
related to the release of the therapeutic protein from the cell surface, which can be used to estimate
the secretion rate of the recovered clones (Supplementary Fig. 3). Ranking of individual colonies
according to their secretion halo intensity indicated that the 60% of the cells that poorly secrete the
IgG had been removed from the population that released the microparticles after one day of culture
(Fig. 6A, day 1 cells).
When focusing on the cell clones displaying the most intense secretion halo, there was a 3-fold
increase of highly secreting cells in the population eluted at Day 1, when compared to unsorted
control cells (Fig. 6B). Overall, the MagCell sorting procedure thus allowed enrichment of cells
releasing the IgG at levels that could not be reached from the analysis of the unsorted population or
from cells released at day 4. Given that around 7000 colonies were analyzed from the later
populations, whereas only 3000 cells were recovered and analyzed from the Day 1 population, we
concluded that the process yielded a greater than 23-fold enrichment of cells that secrete the
recombinant proteins at very high levels.
The ClonePix FL device was also used to select and pick single colonies for expansion. The 10
colonies from each population that yielded the most intense secretion halo were picked and placed
in culture to directly assess their secretion rates (Fig. 6C). The most highly secreting cells were
obtained from the Day 1 cells, which secreted 2.6-fold more IgG compared to unsorted control cells.
The 10 clones picked from the sorted and Day 1-eluted cells were assessed in fed batch
cultures for the accumulation of the secreted antibody in the culture medium, yielding 4
clones which secreted the antibody at levels greater than 4 g/L in shaken culture flasks,
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with titers ranging from 4.2 to 5.2 g/L (Fig. 5D). Only one culture surpassing 4 g/L of the IgG was
obtained from the top 10 clones from the cells recovered at day 4, or from the control input cells (4.0
and 5.1 g/L, respectively, data not shown). Overall, we concluded that the MagCell device can
efficiently sort cells to enrich those that secrete a therapeutic protein at higher levels from a
heterogeneous polyclonal population, that the procedure can be successfully adapted to the sterile
conditions required for the generation of cell lines that can produce recombinant proteins in GMPcompatible conditions for clinical use, and that very high protein titers can be obtained from
monoclonal population cultures, as needed to produce protein therapeutics.
Conclusions
In this study, we designed a novel device and protocols that allow the sorting of magnetically labeled
cells that efficiently secrete a protein of interest, using procedures that can be implemented for
production of pharmaceutical proteins. Prior attempts to automate magnetic sorting of cells by
integrating the magnetic bead incubation steps with appropriate mixing in microfluidic devices have
been limited by the properties of magnetic microparticles. Although superparamagnetic particles are
well suited for manual cell separation, due to their low magnetic remanence and fast separation, their
handling and efficient mixing is very difficult to achieve in a microfluidic setting. Our new twomicroparticle handling methodology uses ferromagnetic microparticles as a driver for an effective
homogenous mixing and fast separation of superparamagnetic microparticles under an applied timevaried magnetic field. This methodology is combined with microfluidics to provide a fully automated
system (MagCell) that allows the easy automatic execution of all cell separation steps (i.e.
capture, washing and recovery) within a closed and sterile disposable cartridge.
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Another well-known obstacle to the capture of cells that display a protein of interest, whether using
magnetic particles or staining the cell surface with fluorescent agents, is that a high protein display
is not indicative of efficient secretion, as illustrated in this study. Thus, the cells that display high
levels of the protein at their surface, because they do not release it in the culture medium, may be
preferentially recovered. Here we show that the secretion rate can be estimated from the timing of
the separation between the eluted cells and bound magnetic microparticles, as the cells released
first are those that secrete at the highest rates. This provides a novel and convenient approach to
sort cells on the basis of their secretion rate.
Finally, we show that this approach improves and facilitates the selection of cell clones that produce
a therapeutic protein at high levels. Colony imaging and/or picking devices such as the one used in
this study are convenient to image and pick cell colonies, but they are typically limited by their
throughput. For instance, a limited number of cells can be imaged in soft agar plates, as overloading
increases the likelihood of obtaining non-clonal and heterogeneous populations where one colony
results from 2 founder cells. Use of the MagCell-based cell sorting, as developed in this study, not
only increases the productivities of the cells that can be identified, but it also increases the likelihood
of obtaining truly monoclonal populations, as only the most productive, and thus fewer cells need to
be analyzed to obtain the prized high performing cell clone. Although we illustrate this using a
therapeutic IgG, we expect this process to be of more general use for the selection of select cells on
the basis of their display and/or secretion of any type of protein for which an antibody can be obtained.
Acknowledgements
This work was supported by grants of the Commission for Technology and Innovation of the Swiss
Confederation, Selexis SA, Spinomix SA, and by the University of Lausanne. The funding
agency had no role in study design, data collection and analysis, decision to publish, or
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preparation of the manuscript. We would also like to acknowledge the expert technical support of
Laurie Gerard, and Audrey Berger for assistance in preparing the figures.
Conflict of interest statement
Some of the authors are employed by- and/or own shares of Selexis SA, a company that uses
proprietary technology to generate therapeutic-producing CHO cell lines, or of Spinomix SA, a
company that sells microfluidic equipment.
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Figure legends
Fig. 1 MagCell device and cartridges for the sorting of protein-secreting cells. The optimized
MagCell device (A) and sterile single-use cartridges (B) are pictured, as used for the sorting of live
cells secreting a protein at very high levels. (C) Schematic representation of the components of the
cell sorting cartridge.
Fig 2. MagCell cell separation procedure. (A) Principles of the operation of a mix of ferromagnetic
(closed circles) and superparamagnetic (grey circles) microparticles. The magnetic moment of the
microparticles, as well as the time-varied magnetic field generated by electromagnets, are illustrated
by straight and curved lines, respectively. When the magnetic field is varied at a high frequency, the
magnetic fields of the two types of microparticles can align and they aggregate, whereas they
dissociate when the magnetic field direction changes with a high frequency. See the Materials and
Methods section for a more detailed explanation. (B) Illustration of the MagCell-based cell sorting
principles, depicting the Mixing mode (step 1), the capture of cellbound superparamagnetic
microparticles by ferromagnetic microparticles (Capture mode, step 2), bead immobilization prior to
washing out unbound cells by flushing in wash buffer (Separation mode, step 3), other mixing modes
during each wash cycle (Release mode, step 4), and finally the bead separation and elution of the
superparamagnetic bead-bound cells (Recovery mode, step 5). See the Materials and Methods
section for a detailed description of the operating procedures related to these operation modes. (C)
Procedures for cell labeling and capture using a biotinylated antibody that recognizes the protein of
interest expressed and displayed by a fraction of the cells, the association of the biotin moiety with
streptavidin-coated superparamagnetic microparticles, and their capture ferromagnetic microbeads.
(D) Following Magcell sorting and elution, as detailed in part B, fast-secreting cells dissociate first
from the superparamagnetic microbeads into the culture supernatant, upon the
completion of the secretion process, whereas those that display the protein of interest but
do not secrete it efficiently are pelleted and eliminated. Not drawn to scale.
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Fig. 3. GFP- or BFP-labeled reference cells mediating various IgG display and secretion levels.
CHO-M-derived cell clones displaying various amounts of cell surface IgG, but with variable levels of
IgG secretion rates were generated and selected by FACS as reference cell populations.
BFP labeled median displayer BS2 cells, high displayer BLC cells and very high displayer BHB cells
are compared to the GFP labeled F206 very high producer cell clone. The IgG displayed at the cell
surface was labeled with an APC conjugated anti-IgG antibody, prior to flow cytometry analysis (A).
The IgG titer produced by parallel cultures of the indicated cell clones (B), or their secretion rate
(specific productivity) expressed in picogram of protein secreted per cell and per day (C), were
determined by ELISA assays of the IgG released into the cell culture medium.
Fig. 4. MagCell automated separation of high (F206) from medium (BS2), high (BLC) and
very high (BHB) IgG displayer cells with superparamagnetic and ferromagnetic
microparticles. The cells preparation and MagCell operation conditions for sorting cells using a mix
of superparamagnetic and ferromagnetic microparticles were as described in the Material and
Methods. A mix of GFP-labeled F206 with BFP-labeled BS2 (A), BLC (B) or BHB cells, with the
indicated ratios (INPUT), were sorted, and the proportion of recovered cells from each clone was
quantified post-sorting (OUTPUT) by fluorescence microscopy.
Fig 5. MagCell sorting enriches highly-secreting cells from polyclonal populations. Polyclonal
populations of cells expressing the therapeutic IgG were sorted using MagCell as described in
Material and Methods. Captured cells eluted from the magnetic microparticles at Day 1 and Day 4
post-sorting, as well as an aliquot of input cells which served as a control, were placed in culture
without antibiotic selection for 14 days, prior to assessing IgG display at the cell surface by FACS
and IgG secretion in the cell supernatant by ELISA assays. (A) IgG surface display of the unsorted
cell population, and the cells recovered at Day 1 and Day 4 post-sorting. (B) The IgG secretion rate
(specific productivity) was assessed in the supernatant of cultures of the cells eluted at
Day 1 or Day 4 post sorting.
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Fig 6. Analysis of MagCell-enriched cell clones. Polyclonal cell populations expressing a
therapeutic IgG antibody were sorted using MagCell, and captured cells eluted from the magnetic
microparticles at Day 1 and Day 4 post-sorting, as well as an aliquot of input cells as control, were
cultivated in semi-solid medium containing anti-human IgG-FITC conjugated antibody for 10 days,
as described in the Material and Methods. After 10 days of culture, IgG-secreting colonies growing
in semi-solid medium were analyzed using the Clonepix FL Imaging software and colonies were
ranked according to the intensity of their secretion halo (A and B). The 10 clones yielding the largest
secretion halos obtained from the cells released at Day 1 or Day 4 post-sorting, or from
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unsorted control cells, were picked using the ClonePix FL device. The growth and production
performance of each clone was evaluated in a Fed-Batch cultivation process, and their specific
productivities were analyzed (C). The IgG secretion titers of the clones obtained from the cells
released at Day 1 following MagCell sorting were determined in fed-batch culture production
conditions, and the titers of secreted IgG were plotted according to the duration of the
culture expressed as days (d).
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