Synchronized Mammalian Cell Culture: Part I—A Physical Strategy for Synchronized Cultivation Under Physiological Conditions Oscar Platas Barradas, Uwe Jandt, Max Becker, Janina Bahnemann, Ralf P€ortner, and An-Ping Zeng Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestr. 15, 21071, Hamburg, Germany DOI 10.1002/btpr.1944 Published online July 29, 2014 in Wiley Online Library (wileyonlinelibrary.com)

Conventional analysis and optimization procedures of mammalian cell culture processes mostly treat the culture as a homogeneous population. Hence, the focus is on cell physiology and metabolism, cell line development, and process control strategy. Impact on cultivations caused by potential variations in cellular properties between different subpopulations, however, has not yet been evaluated systematically. One main cause for the formation of such subpopulations is the progress of all cells through the cell cycle. The interaction of potential cell cycle specific variations in the cell behavior with large-scale process conditions can be optimally determined by means of (partially) synchronized cultivations, with subsequent population resolved model analysis. Therefore, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical selection methods, but not with frequently used chemical or whole-culture methods. Conventional nonsynchronizing methods with subsequent cell-specific, for example, flow cytometric analysis, can only resolve cell-limited effects of the cell cycle. In this work, we demonstrate countercurrent-flow centrifugal elutriation as a useful physical method to enrich mammalian cell populations within different phases of a cell cycle, which can be further cultivated for synchronized growth in bioreactors under physiological conditions. The presented combined approach contrasts with other physical selection methods especially with respect to the achievable yield, which makes it suitable for bioreactor scale cultivations. As shown with two industrial cell lines (CHO-K1 and human AGE1.HN), synchronous inocula can be obtained with overall synchrony degrees of up to 82% in the G1 phase, 53% in the S phase and 60% in the G2 =M phase, with enrichment factors (Ysync ) of 1.71, 1.79, and 4.24 respectively. Cells are able to grow with synchrony in bioreactors over several cell cycles. This strategy, combined with population-resolved model analysis and parameter extraction as described in the accompanying paper, offers new possibilities for studies of cell lines and C 2014 processes at levels of cell cycle and population under physiological conditions. V American Institute of Chemical Engineers Biotechnol. Prog., 31:165–174, 2015 Keywords: cell synchronization, synchronous growth, elutriation, dialysis bioreactor, bioreactor synchronous culture

Introduction Mammalian cell expression has become the dominant recombinant-protein production process for clinical applications because of its capacity for post-translational modification and human protein-like molecular structure assembly (Zhu, 2012). The high demand for these biopharmaceuticals has led to the development of large-scale manufacturing processes. Productivity improvements of these processes have mainly focused on cell physiology and metabolism, cell line development and process control (e.g., fed-batch) strategy (Costa et al., 2010). More recently, genomic and systems biology approaches have been applied to cell cultures (Hu and Zeng, 2012). So far, studies in both cell line and process analysis and optimization have treated the cell culture primarily as a Correspondence concerning this article should be addressed to A.-P. Zeng at [email protected]. C 2014 American Institute of Chemical Engineers V

homogeneous population and lumped process parameters or performance indicators are used. In reality, a cell culture is always composed of a heterogeneous population. In particular, mammalian cells exhibit a clearly defined cell cycle with distinct phases (G1, S, G2, and M). In cell biology and related fundamental studies, such as cell proliferation and apoptosis, cell cycle has received much attention and plays prominent roles. A lot about cell cycle dependent properties in bioprocesses is known in the case of baker’s yeast, little in the case of bacteria but hardly in the case of mammalian cells. Moreover, in bioprocess engineering and systems biology studies of mammalian cells, the heterogenity or population diversity of a culture has been seldom considered. One of the major obstacles is the lack of a suitable method to synchronize a cell culture under undisturbed physiological conditions and at a reasonable bioreactor scale for extensive study of effects of cell population variation. There have been various attempts to synchronize cell cultures, but 165

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only few physical selection methods are usable to actually produce synchronized cultures with reasonable accuracy and yield, while very frequently used chemical or whole-culture methods are not suitable to synchronize cultures (Cooper, 1998; Jandt et al., 2014).

Theoretical and Technical Backgrounds of Cell Synchronization Cell synchronization aims at bringing the majority of cells of a culture into a certain rhythm of going through the different phases of the cell cycle. During the cell cycle, the cell’s genetic material, organelles, and macromolecules double their quantity to create two genetically identical daughter cells. In the S phase (Synthesis), the chromosomes replicate. In the M phase (Mitosis), chromosome material is distributed to the new formed nuclei and cytoplasm is divided (Cytokinesis), resulting in the formation of two daughter cells. Two gap phases accomplish for the time needed by the cell for growth, and organelle and protein division: the first one between the mitosis and the S phase (G1), and the second one between the S phase and the mitosis (G2). If environmental growth conditions are not appropriate, cells in the G1 phase may not move into the S phase, but enter a quiescent state, the G0 phase. Only at appropriate growth conditions, cells can reach a point at the end of the G1 phase (commitment point or Start), at which they commit into DNA synthesis and division, even if growth and division signals cease to exist. After a synchronization attempt, cell populations should obey several criteria to be considered as truly synchronized (see, e.g., Cooper and Shedden (2003) or Jandt et al. (2014a) for a condensed version). Important criteria include a minimal increase in cell number during the interdivision time as well as a short fraction of time for division, compared with the whole duration of the cell cycle. Moreover, it is a characteristic of synchronous cells to have narrow DNA and size distributions, together with a population’s doubling time which is equal to the average one of cells with asynchronous growth. In literature, synchronization attempts can be divided in two basic groups: Physical selection methods and chemical whole-culture methods. Synchronization can be performed either chemically or physically. Chemical methods—or whole-culture methods—are believed to result mostly in a reversible blocking of cells at a certain growth phase by means of deprivation of a medium component or addition of chemicals. Periodical nutrient deprivation by a change of feast and famine substrate supply is usually referred to as phased cultivation (Dawson, 1972; Fritsch et al., 2005). Colcemid or Nocodazole have been used for trying to block cells in the M phase (Boxberger, 2007; Knehr et al., 1995; Lindl and Gstraunthaler, 2008); Aphidicolin, Hydroxyurea, Mimosine, and Thymidine excess, for trying to block cells in the S phase (Knehr et al., 1995; Matherly, 1989); serum or amino acid starvation, DMSO, and Lovastatin for trying to block cells in the G1/G0 phase (Boxberger, 2007; Fiore et al., 2002). Previous synchronization attempts (Cooper, 2002a; Enninga et al., 1984; Fiore et al., 2002; Keyomarsi et al., 1991; Knehr et al., 1995; Krishan et al., 1976; Moore et al., 1997) have mostly focused on arresting cell growth at a determined cell cycle phase and studying the consequences of growth arrest. Temperature reduction methods have been

applied frequently (Boxberger, 2007; Enninga et al., 1984; Lindl and Gstraunthaler, 2008; Moore et al., 1997). In literature, it was discussed and practically shown (Cooper, 1998, 2003) that whole-culture, or chemical, methods are not able to synchronize cells, including lovastatin (Cooper, 2002b), (double) thymidine block (Cooper et al., 2008), serum starvation (Cooper and Gonzalez-Hernandez, 2009), and nocodazole (Cooper, 2006). Furthermore, the chemical blockage of cellular processes, however, is normally not reversible in a short period of time and thus disturbs the cell physiology. For a chemical which acts during a specific phase of the cell cycle, the maximum theoretical reaction time for the blockage would account for the whole duration of the cell cycle. The time needed for washing off the chemicals out of the culture, intra and extracellularly, is another disadvantage of chemical blockages. Physical methods—or selection methods—are based on physical properties of the cells like size or density. Most physical methods focus on the synchronization of only a fraction of cells, separating them from the rest of the population. They comprise mitotic shake-off (Zwanenburg, 1983), gradient centrifugation (Holley, 1988; Rola-Pleszczynski and Churchill, 1978), membrane elution, (Cooper, 2002a; Helmstetter and Cummings, 1963; Helmstetter et al., 2003), fluorescence-activated cell sorting (FACS) (Jorgensen and Tyers, 2004; Rieseberg et al., 2001), magnetic-activated cell sorting (MACS) (Miltenyi et al., 1990), centrifugal elutriation (Banfalvi, 2008), microfluidic sorting (reviewed in Autebert et al. (2012)), and more. In most cases, the duration of a process for physical synchronization can be significantly reduced compared with most chemical methods. This might be translated into a reduction in the metabolic stress caused to the cells during synchronization. The selection of a physical method relies on its capacity to yield high numbers of synchronous cells with a high synchrony degree and a low cell damage. To obtain scalable results for production relevant processes at reactor scales of, for example,  1 L, the number of viable synchronized cells must reach a magnitude of 5 3 108 cells L21 in a time scale that is short compared with the cell cycle duration, that is, in maximum  1 h. Moreover, synchronization of cells in different phases of the cell cycle from one and the same culture source (the so-called “simultaneous synchronization”) is another desirable feature to study the dynamics of cells obtained from a common parental population. In this study, we set out to establish a suitable method to synchronize mammalian cell cultures for further cultivation under physiological and bioreactor conditions. A previous evaluation of synchronization methods in our laboratory considered methods that should be able to achieve acceptable synchrony yields, high numbers of synchronous cells in a short period of time, and cause as little as possible perturbation to the cells (Helmstetter et al., 2003; Macdonald and Miller, 1970; Moore et al., 1997). The results of this evaluation are shortly summarized in Table 1. The immobilization of cells onto different substrates was implemented successfully with the goal of harvesting cells by the time of division following the principle shown by (Helmstetter et al., 2003). However, although cells adhered to most of the tested substrates (some polylysine treated), cell agglomeration took place during adherent growth and synchrony could not be verified in the newborn cells.

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Table 1. Apparent Synchrony Degree (%sync ) and Cell Yield (no. of cells) Achieved by Previous Experiments Using Physical (top) and Whole Culture Methods (bottom) Method Synchronization Principle %sync (%) No. of Cells Immobilization Centrifugation Elutriation Temperature reduction

adhesion strength size and shape size and shape growth control points

1  106 2  106 3  107 whole culture

65% G1 75% G1 95%; G1 ; 75% G2 =M 80% G1 =S

Part of these results has been published in Platas Barradas et al. BMC Proc 2011, 5, 49, BioMed Central Ltd.

Furthermore, cell centrifugation in media with higher densities using sucrose or ficoll led to a slight increase in the percentage of G1 cells in the collected subpopulations (75%). However, the low synchrony yield and the cell number were not satisfactory to pursue this method. The reduction in temperature during culture was followed by accumulation of cells in the G1 and early S phases up to 80%. However, the temperature shifts lasted over 90 h, which is at least two times the duration of the cell cycle of industrial cell lines. Furthermore, cell death was observed after resumption of the cultivation temperature. Centrifugal elutriation allowed during initial experimentation not only for higher synchrony yields, but also for the collection of higher numbers of cells in a short period of time among the evaluated selection methods. Countercurrent centrifugal elutriation In this work, centrifugal elutriation has been successfully used for the synchronization of the industrial cell lines CHO-K1 and AGE1.HNAAT. The former cell line was chosen due to its widely established relevance in the field of production of biologics. The latter was used in this work as a model human industrial cell line, which has shown the capacity of producing a complex and highly fucosylated protein with the same biological activity compared with that isolated from human serum (Blanchard et al., 2011). This makes the AGE1.HNAAT cell line potentially interesting for research and industrial applications. Both cell lines have been synchronized in the G1, S, and G2/M phases of the cell cycle and cultivated with synchrony in benchtop bioreactors. Synchrony has been assessed by following the viable cell density as well as the distribution of the cell cycle phases and the change of cell size during culture. This synchronization method is demonstrated as a fundamsental for population dependent bioprocess and systems biology studies of mammalian cell cultures at bioreactor scales. In an accompanying article (Jandt et al., submitted for publication), we show how the data generated from deliberate synchronization of cell culture can be used for a more sophisticated and population modeling of cell cultures. In centrifugal elutriation, separation takes place in a rotor with a built-in funnel-shaped elutriation chamber (Beckman Coulter, 2012). Two opposite forces act on the cells in the chamber: the centrifugal force (driving it away from the axis of rotation) and the fluid velocity (driving it toward the axis of rotation, in counterflow). While the rotor is spinning in the centrifuge, a suspension of cells is pumped at a determined flow rate from outside the centrifuge into the rotor to the narrow end of the elutriation chamber. Cells move within the chamber according to their sedimentation rates and to the point where equilibrium between the two opposite forces takes place (elutriation boundary). After increasing the flow velocity in the chamber by means of the pump rate, small

cells with low sedimentation rates move faster toward the axis of rotation. After crossing the elutriation boundary, cells are dragged by the increasing flow velocity due to the narrowing chamber walls. These cells are washed out of the chamber into a collection vessel. By increasing the flow rate in gradual steps, successive fractions of increasingly larger or denser cells can be washed out of the rotor and collected (Beckman Coulter, 2012). An easy method for determining the percentage of synchronous cells within a cell cycle phase, or so called Synchrony Degree, is the use of the percentage values %sync obtained from flow cytometry for the different cell cycle phases. This simple method allows for a parallel and rapid assessment of synchrony in cell subpopulations prior to their further processing (e.g., inoculation of bioreactors). According to %sync , a cell subpopulation with %G1 5100% will contain all of its cells with a G1 DNA content. This value, however, does not consider the possible age difference of the cells within the specific cell cycle phase. The importance of regarding this age difference will be explained by the following example: if we think of the possible age difference between two single G1 cells, the maximum value this difference will account for is the whole duration of the G1 phase (e.g., in AGE1.HN cells it is 24 h). This would also be the time needed by the whole population for complete doubling during growth, even if they are regarded as with 100% synchrony. On the other hand, a cell population with %G2 =M 5 100% would display shorter age differences, as the G2 =M phase is in general at least three times shorter than the G1 phase. Therefore, whenever DNA content is the only measurement used for assessment of synchrony, a cell subpopulation with a high percentage of cells in G1 might not grow with a higher synchrony than a counterpart with a lower percent of synchrony in the S or G2/M phases. To avoid a biased understanding of results, the percentile duration of the cell cycle phase can be introduced to the calculation of synchrony in cell subpopulations, which will be instead expressed by relating the synchrony degree for a regarded phase, %sync , to the percentage of cells in the same cell cycle phase within the parental asynchronous population, %par : Ysync 5

%sync %par

(1)

The resulting dimensionless coefficient represents the enrichment factor of a specific subpopulation.

Materials and Methods Cell lines and culture media Two suspendable cell lines were used in this study: the human cell line AGE1.HNAAT (ProBioGen AG, Berlin, Germany) and a Chinese Hamster Ovary cell line, CHO-K1,

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Figure 1. Centrifugal elutriation process: injection of cells after bubble trap and cell separation. Modified from Banfalvi, Nat Protoc., 2008, 3, 663–673; Dorin, Developing Elutriation Protocols. Technical Information on High Speed Centrifugation. 1994, Beckman Instruments Inc.

(kindly provided by T. Noll, Bielefeld University, Bielefeld, Germany). Both cell lines grow in chemically defined media (AGE1.HNAAT: 42-Max-UB, CHO-K1: TC-42, TeutoCell AG, Germany), which were supplemented with L-glutamine at final concentrations of 5 and 4 mM, respectively. AGE1.HNAAT cells agglomerate immediately in the presence of Ca21 and Mg21. For this reason, only Ca21 and Mg21free phosphate-buffered saline (PBS) was used in this work. After dilution of the components, the pH was brought to 7.3 6 0.1 with HCl or NaOH. PBS was always filtered with a pore size of 0.22 lm.

Preculture AGE1.HNAAT cells were precultured in unbaffled 100 and 500 mL glass Erlenmeyer shake flasks (Schott Duran GmbH, Germany) with working volumes of 50 and 200 mL, respectively. Preculture of CHO-K1 cells was carried out in 250 mL plastic shake flasks (Corning, Inc., USA) with 100 mL working volume. These preculture steps were carried out on an orbital shaker at 225 rpm with a shaking diameter of 10 mm (GFL3005, Omnilab, Germany). The temperature in the incubator’s atmosphere was controlled at 36:860:2 C (Heraeus Heracell, Germany). As additional temperature reference measurement, an incubator thermometer (VWR, Germany) was placed onto the shaker’s surface. After inoculation of a shake flask, the CO2 partial pressure in the incubator’s atmosphere was set at an initial value of 5%. The pH value of the cultures was measured externally (CG 822, Schott AG, Mainz, Germany) during sampling. A scheduled reduction in the CO2 partial pressure was carried out routinely according to the measured pH value in culture: CO2 5 5% for pH > 7:3; 3% for 7:1 < pH  7:3, and 0% for pH  7:1. Exponential growth was always observed at a pH range of 7.0—7.4. This schedule was easy to follow and allowed for exponential growth at all preculture steps and

for longer exponential growth during shake flask experiments. Cell counting and viability measurements were performed manually on a Neubauer Haemocytometer (Laboroptik, Friedrichsdorf, Germany) using the trypan blue exclusion method. Lactate concentration was measured enzymatically (YSI 7100, Yellow Springs Instruments, USA).

Countercurrent centrifugal elutriation An Avanti Centrifuge with JE 5.0 Rotor (Beckman Coulter, USA), kindly provided by the working group of Prof. A. Lohse at University Medical Center HamburgEppendorf, Germany, was used for elutriation experiments. Equipment operation, settings and troubleshooting have been well described in the literature (Banfalvi, 2008; Beckman Coulter, 2012; Dorin, 1994). A schematic set-up of the elutriation system is shown in Figure 1. An 8-roller-head pump (medorex, e.K., Germany) and thin silicon tubing (di 5 2 mm, Carl Roth, Germany) were used for elutriation to reduce pulsation in the separation chamber. Furthermore, the change of the pump rate was done slowly at 2–3 mL min22 to avoid cross-mixing of different-size cells at the elutriation boundary as well as a premature cell washout out of the elutriation chamber. The elutriation path was desinfected with a 2% sodium hypochlorite solution for 2 h followed by 70% ethanol for 30 min. Afterward, a bottle containing sterile PBS was connected to the inlet tubing. The rotation speed for elutriation was set constant for all experiments at 1800 rpm. For each elutriation, 3–5 3 108 cells were centrifuged (125 g, 5 min), resuspended in 3–5 mL PBS and drawn from the centrifuge tube into a sterile syringe. The pump was turned off and the cell suspension was injected into the system. A slight modification was carried out to Banfalvi’s (2008) method by changing the injection point of the cells to

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a place of the silicon tubing closer to the separation chamber. This was done to avoid long residence times of the cells inside of the bubble trap. The pump was turned on at the preset lowest calculated pump rate for 1 min, after this it was increased again to elute the first cell fraction. An amount 100–150 mL of cell-containing buffer was collected at the system outlet in sterile plastic tubes before the pump rate was increased to the next value. The pump rate was varied in a range of 4–6 mL min21 for elutriation of a certain subpopulation. This value depends on the desired cell number to be collected and on the expected synchrony degree. The smaller the increase in the pump rate, the higher the synchrony of the fraction, but the lower the cell number collected in the tubes. The time for collection of a whole subpopulation consisting of 2–3 plastic tubes at the same pump rate value varied between 3–8 min depending on the pump rate. The whole elutriation process lasted around 30 min, which is very short compared with the cell cycle time ( 15 h for CHO-K1 and  32 h for AGE1.HN). Cell count on the microscope, cell size measurement and cell cycle analysis were performed for analysis of the elutriated subpopulations. For inoculation of bioreactors repeated elutriation cycles from a common parental population were performed. After inoculation, off-line sampling during synchronous culture was performed at close time intervals between 3 and 6 h depending on the cell line. Cell culture Synchronous Bioreactor Culture of CHO-K1 Cells in Batch Mode. Two experiments were performed in a benchtop bioreactor operating in batch mode for culture of synchronous CHO-K1 cells. Neither medium change nor feed were needed for these experiments, as CHO-K1 cells have shown the capacity of almost five population doublings in batch culture. Cells were elutriated in the G1 and S phases, respectively, and further cultivated in a 1-L benchtop bioreactor Vario1000 (medorex e.K., Germany) with a working volume of 250 mL and an initial cell density of 0.4 3 106 cells mL21. This bioreactor allows for controlled culture conditions despite of its low working volume. The pH was controlled at 7.20 (CO2/0.5 M Na2CO3), whereas the dissolved oxygen concentration (DO) was set to 30% air saturation. The stirring rate was kept constant at 300 rpm. Synchronous Culture of AGE1.HNAAT Cells in a Dialysis Bioreactor. In contrast to the CHO-K1 cell line, AGE1.HN cells have shown no more than two doublings in batch culture, which limits the assessment of the number of synchronous divisions as well as the number of representative samples during experimentation. To provide the cells with an unperturbed environment during growth with synchrony and to allow for more synchronous divisions of the cells, a dialysis bioreactor was used (M€arkl et al., 1989; P€ortner and M€arkl, 1998). This bioreactor consists of an inner chamber (culture chamber) and an outer chamber (dialysis chamber), both displayed in Figure 2. The culture chamber is built-in inside the dialysis chamber and is separated from the medium by a transparent nonporous dialysis membrane made of regenerated cellulose (Cuprophan, cut-off 5 10 kDa). Toxic metabolites of low molecular weight can be removed from the culture chamber through the dialysis membrane. Cells and high-molecular-weight products are retained inside the culture chamber. The use of a dialysis process allows for a lon-

Figure 2.

Dialysis bioreactor (Bioengineering AG, Switzerland). The culture chamber is built-in inside the dialysis chamber and is separated from the medium by a transparent nonporous dialysis membrane which is at the same time the culture chamber’s wall. Toxic metabolites of low molecular weight can be removed from the culture chamber through the dialysis membrane. Cells and high-molecular-weight products are retained inside the culture chamber. Modified from (P€ ortner and M€arkl, Applied microbiology and biotechnology, 1998, 403–414).

ger exponential cell growth as it prevents the culture from reaching substrate limitation in a relatively short time period, as it happens commonly in batch culture. Similarly, dialysis culture was employed to study the number of synchronous divisions of the cultured subpopulation. This bioreactor has allowed AGE1.HN cells for up to five doublings in batch mode (Jandt et al., 2012). For inoculation, elutriation was repeated so as to generate subpopulations in the G2/M phase, which were pooled before cultivation. The initial volume in the culture chamber was 800 mL, whereas the dialysis chamber was operated with 3.7 L of culture medium. During the cultivation, the pH was controlled at 7.15 (CO2 / 0.5 M Na2CO3), whereas the DO was set to 25% air saturation. The DO control in the culture chamber was done by means of a cascade controller actuating in the outer chamber (N5300  1300 rpm $ Air 55  240 mL  min21 $ Air/O2 ratio 5 0–100%). Analysis of the cell cycle The procedure for cell fixation and propidium iodide staining was adapted from Riccardi and Nicoletti (2006): 1 – 2 3 106 cells were centrifuged (125 g, 5 min) and resuspended in 1 mL cold PBS. The suspension was centrifuged again and the cell pellet was thoroughly resuspended in 288 lL PBS.

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Table 2. Cell Cycle Phases and Cell Size Distribution of CHO-K1 Subpopulations Obtained After Elutriation S. No. Asynchronous 1 2 3 4 5 6 7 8

Pump Rate (mL min21)

G1 (%)

S (%)

G2/M (%)

No. of Cells (106)

Xp (lm)

– 22 26 30 34 38 42 46 50

48.3 83.0 92.4 72.5 34.4 13.4 8.3 6.1 6.3

29.9 10.7 6.3 23.7 51.4 57.0 42.1 24.7 13.5

21.8 6.3 1.3 3.7 14.2 29.6 49.5 69.1 80.2

380 0.81 3.40 1.95 16.98 26.32 8.52 6.30 2.64

13.24 11.41 11.86 12.07 12.65 13.16 13.83 14.03 14.21

712 lL ice cold 96% ethanol were added to the sample followed by continuous pipetting avoiding the formation of cell agglomerates. After mixing, the sample was vortexed (about 600 rpm) for 10 s. The samples were stored for maximum of 4 months at 220 C. Before measurement cells were dyed with propidium iodide (PI): The sample tube was vortexed and then centrifuged at 400 g for 5 min. The cell pellet was resuspended once in 1.5 mL PBS and centrifuged. After centrifugation, the cells were thoroughly resuspended (pipetting 1 vortexing) in 480 lL PBS. RNAse of 10 lL (20 mg/mL, Invitrogen Cat.Nr.-12091-021, Lot.1004817) was added to the cell suspension. The suspension was vortexed for 10 s. For DNAse-free RNAse, the open RNAse containing tube was submerged into hot (almost boiling) water for 5 min. The samples were left for incubation during 30 min at room temperature before adding 10 lL propidium iodide solution (2 mg mL21, Carl Roth, Germany). The samples were vortexed and kept in darkness for another 10–30 min until analysis. Samples were analyzed in a LSR II Flow Cytometer (with kind support from A. Carambia, J. Herkel, and A. Lohse at University Medical Center Hamburg-Eppendorf, Hamburg, Germany), Analysis Software: FACS Diva v6.13 (BD Biosciences, USA). The region where the cell population of interest was located was gated using an asynchronous cell population for it. This gate was not modified within an experiment unless the cell number in the sample had varied importantly due to treatment. A variation in the cell number in the sample leads to a signal displacement, which has to be corrected by redefining the region of analysis. Cell size distribution The determination of cell size distribution of a cell population was performed with a particle counter (Z2, Beckman Coulter, USA). The resulting histogram data provides a distribution of the cells with respect to their apparent circular diameter, covering at least the range of 10–21 lm (AGE1.HNAAT) and 10–19 lm (CHO-K1) at a resolution of 0.15 lm or better. For the characterization of variations in the size distribution during synchronized cultivations, the histogram data was filtered using a sliding window averaging filter (seven times the histogram bin size) to reduce noise. Then the peak of the filtered histogram distribution was extracted, yielding the corresponding representative peak cell diameter of the given population Xp. At-line microscopy Due to the significantly higher growth rate of the CHO-K1 cells compared with AGE1.HN, an in situ microscope system

(ISM) (kindly provided by T. Scheper and A. Babitzky, Institute for Technical Chemistry (TCI), Leibniz University Hannover, Hannover, Germany) was used for cell counting parallel to manual counting. The autoclavable system consists of two tubes with lenses at the tip; one tube has a smaller diameter and can be placed inside the other one. When doing this, the remaining small space between both lenses forms a chamber. At chamber’s height, two tube ports allow for culture inlet and outlet. Culture conveyance to the microscope and back into the bioreactor is done by a peristaltic pump. O-rings between all components keep sterility in the chamber during culture flow. A digital camera (XCDSX 910, Sony, Japan) with a large objective is mounted into the inner tube. The separation between lenses can be adjusted and calibrated with a motor integrated in the system, and the object can be focused by a second motor to which the camera is attached. A high-intensity light-emitting diode (LED) is used as a light source, which is set beneath the microscope lens. Images were acquired at user-defined intervals of 1 min and were further analyzed by an image analysis software. A detailed description of this system and its applications was published by Prediger et al. (2011). For automatic determination of the cell sizes, the acquired images have been analyzed using the free ImageJ software suite, using maximum entropy threshold clustering, watershed method to separate cells in clusters and automated particle analysis, including sizes of 250—600 square pixels (equal to approximately 110–270 lm2 projected cell area, or approximately 11.9–18.5 lm2 equivalent cell diameter, assuming spherical cells) and a circularity above 0.8. The resulting histogram was low-pass filtered in the time domain with a sliding window average comprising 630 min ffi 630 pictures. A two-roller pump (Watson Marlow, USA) and wide pump silicon tubing (di 5 5 mm) were used for liquid conveyance to the microscope so as to reduce the volume specific contact area during peristalsis. Here, the pump rate was set to 10 mL min21. Furthermore, possible temperature oscillations in the flow pathway to the at-line microscope were solved by setting the microscope into an incubator.

Results and Discussion Synchronous bioreactor batch culture of CHO-K1 cells An elutriation experiment with CHO-K1 cells was performed previous to the inoculation of the bioreactor. The results of this elutriation experiment are shown in Table 2. In a first experiment (CHO 1), the bioreactor was inoculated with cells enriched in the G1 phase by pooling the subpopulations 2 and 3 as denoted in Table 2. This corresponds to a calculated synchrony yield of 82.2% and an enrichment factor of YG1 51:71. The inoculum for the second experiment (CHO 2) considered subpopulations 5 and 6, with a

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Figure 3. Bioreactor batch culture of synchronous CHO-K1 cells enriched in the G1 phase (left) and in the S phase (right). Top: total cell density from at-line microscopy; middle: cell cycle phases distribution; bottom: peak cell diameter.

calculated synchrony degree in the S phase of 53.3% and an enrichment factor of YS 5 1.79. Figure 3 displays the results of the cultivations. A slight synchronous growth can be observed for both experiments according to the growth curves, especially during the first 24 h in the second experiment (Figure 3, right). According to the growth curve, cell division occurs every 15–16 h in both experiments. This doubling time shows an unperturbed cell growth according to the measured value for this cell line in continuous culture in our laboratory (t d 51662:6 h, (data not

shown). Although both experiments were performed with similar enrichment factors (1.71 and 1.79 respectively), the profiles of the cell cycle phases in both experiments seem hardly comparable at first sight. However, the reason for that are clearly the too long sampling time intervals during the first experiment. This shows the importance of keeping a minimum sampling period while working with synchronous cell populations. A minimum sampling period is determined by the dynamics of the culture metabolism and is introduced in Part II of this paper series (Jandt et al., submitted for

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Table 3. Cell Cycle Phases and Cell Size Distribution of AGE1.HN Subpopulations Obtained After Elutriation S. No Asynchronous 1 2 3 4 5 6 7 8

Pump Rate (mL min21)

G1 (%)

S (%)

G2/M (%)

No. of Cells (106)

Xp (lm)

– 14 18 22 26 30 35 41 47

65.4 93.6 95.0 89.8 75.9 57.7 16.3 6.4 6.4

23.6 4.8 4.1 8.1 19.1 32.5 53.3 42.2 18.0

11.0 1.6 0.9 2.1 5.0 9.8 30.4 51.4 75.6

350 12.0 36.9 37.5 29.1 22.8 25.2 17.7 12.3

14.23 13.35 13.49 13.74 14.13 14.49 14.98 15.48 15.78

Figure 5. Mitotic growth rate of AGE1.HNAAT (black), overlayed with mean (or representative) cell volume (blue).

Synchronous dialysis culture of AGE1.HNAAT cells

Figure 4. Synchronous growth of AGE1.HNAAT cells in a dialysis bioreactor and the relative phase fractions of the cell cycle during cultivation. The inoculum used for cultivation contained mostly cells in the G2/M phase.

publication). The cells enriched in the S phase (CHO 2) displayed a clear oscillation of the cell cycle phases, with minimum initial content of cells in the G1, as expected. Cells starting at the S phase left this phase shortly after inoculation and joined the G2/M phase population. The time needed for the cell population to show first observable signs of division was almost 5 h after inoculation. Very constant peak heights can be observed in the cell cycle phase distribution, where even after 50 h in culture, almost no loss of synchrony can be observed (see Figure 3).

A total of 17 elutriations were performed to reach the needed cell number for inoculation of the dialysis bioreactor at a cell density of 9 3 105 cells mL21. The results of a previous reference elutriation used for this experiment are shown in Table 3. The overall enrichment factor (YG2 =M ) calculated for the G2/M pooled cell subpopulations used for inoculation of the dialysis bioreactor was 4.24. A maximum enrichment factor of YG2 =M 56:8 was calculated for a single subpopulation in previous elutriation experiments with AGE1.HN cells (data not shown). Figure 4 shows the results of the dialysis cultivation with the synchronous cells. According to the cell cycle analysis of the first sample, the culture was started with an initial percentage value of 60% of cells in the G2/M. During a period of 120 h at least 4 synchronous divisions can be observed; first at the time of inoculation, and then after 36, 66, and 104 h in culture. The average doubling time of the cell population (considered from the beginning of the G1 phase to the end of the G2 =M phase) remained constant (td 5 40 h). This is the best practical culture indicator for no cell damage or disturbance during the elutriation process and during synchronized cell growth in the dialysis bioreactor. Cell damage would be reflected in increased cell death and lower growth rates. A very clear oscillation of the cell cycle phases can be observed in this experiment. Thus, minimum values in the G1 phase show the time points at which the cells just have divided and show a minimum DNA content. During a part of the cultivation, at-line microscopy was employed and pictures of the cells were taken every minute. At low cell densities, that is, at t < 12 h, the cell density was too low to obtain useful results from the microscopy data,

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Figure 6. Example for the production of a metabolite, here lactate, during the cultivation. (a) Raw measured data. No clear oscillation can be seen directly from raw data. (b) Cell volume-specific turnover (production) rate of lactate after low-pass filtering. Oscillations can be observed and are aligned to the progress of the cell cycle, as indicated by the overlayed relative S-phase amount of cells (blue). Note that in the dialysis process, the medium volume is approximately five times larger than the cultivation volume; therefore, the apparent turnover rates are reduced by a factor of approximately 5.

while at comparably high cell densities, that is, at t > 96 h, cell clumping became serious and at-line microscopy was therefore disabled. However, in the time span between, useful cell volume data could be derived using automated image analysis methods as described in the methods section. The corresponding mean (or representative) cell volumes are plotted in Figure 5, showing oscillating behavior in synchrony with the cell cycle: The diameter peaks (biggest cells) correspond with the peak of G2 phases and are approximately 1/3 cell cycle duration before the step-wise increase in the cell numbers, which correspond to the peak mitotic growth rate. Variations in the consumption or production rates of metabolites are often difficult to determine. Subpopulationresolved modeling and significance analysis of cell cycle specific parameter adaptation like presented in part II of this paper series can help to identify cell cycle specific variations from other potential variations. Figure 6a demonstrates the increasing concentration of lactate during cultivation, a typical by-product. Variations or oscillations cannot be directly determined from that plot. However, after low-pass filtering, either with a Gaussian filter kernel or with a sliding window average filter of size Dt around time point t, and relating to the total cell volume (product of representative volume per cell Vc ðtÞ and viable cell number Xv ðtÞ), that is, c0Lac ðtÞ



cLac t1 12 Dt 2cLac t2 12 Dt  ; Vc ðtÞ Xv ðtÞ Dt

(2)

an oscillation of the resulting volume-relative production rate c0Lac ðtÞ can be seen as depicted in Figure 6b. (Note that in the dialysis process, the medium volume is approximately five times larger than the cultivation volume, therefore the apparent turnover rates are reduced by a factor of approximately 5.) This oscillation is correlated to the progress of the cell cycle, here represented by the S phase contribution (superimposed in green color), indicating a high probability of cell cycle related variation.

Conclusions In this contribution, we developed and implemented a strategy for the synchronization of mammalian production

cell lines at bioreactor scale suitable for subsequent cultivation under physiological conditions, analysis and populationresolved model description. For this purpose, chemical, or whole-culture, treatments were excluded due to their inability to produce actually synchronized cultures and their potential interference with cellular metabolism. The utilized physical selection method of countercurrent flow centrifugal elutriation showed to be superior with respect to yield compared with other physical methods. It allows for reproducible and scalable cell synchronization, yielding synchronous cell subpopulations with synchrony degrees of up to 82% in the G1 phase, 53% in the S phase and 60% in the G2 =M phase. This represents a relative increase in at least 25, 50, and 300%, respectively, to the original asynchronous population. The cell subpopulations generated after elutriation were further cultivated in a batch and a dialysis bioreactor under controlled physiological conditions and a cultivation volume of up to 1 L. In a dialysis and a benchtop bioreactor, both cultivated cell lines exhibited unperturbed synchronous growth for at least four cell divisions with clear oscillations of the cell cycle phases during growth. Distinct oscillations could be also extracted by analysis of the distribution of the cell cycle phases. The time span for sampling turned out to play a very important role in the verification of the synchrony in cell subpopulations. The quantity and reproducibility of information that can be generated by the use of this strategy makes it a valuable and necessary prerequisite for model-based analysis as described in part II of this paper series and hence research on cell cycle dependent metabolism and its population dynamics.

Acknowledgments The authors thank Volker Sandig, ProBioGen GmbH, Berlin, Germany, for supplying the AGE1.HN cell line, Thomas Noll, University of Bielefeld, for supplying the CHO-K1 cell line, the working group of Prof. A. Lohse, UKE, Hamburg, Germany, as well as Alexander Babitzky, for assistance during some of the cultivations and always fruitful discussions. The joint research projects “SysLogics” and “SysCompart” are funded by the German Federal Ministry of Education and Research (BMBF) (grants: FKZ 0315275A and FKZ

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0315555A). The research project “TransExpress” is funded by Deutsche Forschungsgemeinschaft (DFG) (grant: ZE 542/3-3).

Notations CHO = FACS = lmax = td = VsG = Xv = Xt = Xw = Ysync =

Chinese hamster ovary fluorescence activated cell sorting specific growth rate (d21) doubling time (h) settling velocity viable cell density (cells mL21) total cell density (cells mL21) mean cell diameter (lm) synchrony degree

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