Protocol Guide:
Online Flow Cytometry Protocol for Monitoring
Apoptosis, Cell Cycle and Viable Cell Number in
Suspension Mammalian Cell Cultures
Mohamed Al-Rubeai and Darrin Kuystermans
School of Chemical and Bioprocess Engineering and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland
Brief Introduction
Process analytical technology (PAT) has led to process improvements using real-time or semi real-time monitoring systems. Flow cytometry can be used to accurately monitor cell culture providing vital information on cell number and viability. apoptosis, and cell cycle. Integration of flow cytometry into an automated scheme for improved process monitoring can benefit PAT in bioreactor-based biopharmaceutical productions by establishing optimum process conditions and better quality protocols.
Herein are the protocols that outline an automated process for online flow cytometry from a bioreactor system with a focus on suspension mammalian cell cultures.
1. Materials
1.1.1 Instrumentation and Software
1. For the purpose of these protocols the use of the FlowCytoPrep™ (FCP) 5000 (MSP Corporation, MN, USA) automated cell preparation instrument was used as a test bed for protocol development and execution. This instrument has been developed for programmable sample withdrawal, washing, fixing, staining, diluting, and sample injection into a flow cytometer. The FCP uses MS Windows XP
2. Cell Lab Quanta SC (CLQSC) Flow Cytometer (Beckman Coulter, Miami, USA), with MS windows XP
3. WinAutomation software (Softomotive Solutions Ltd, Athens, Greece) on CLQSC
1.1.2 Reagents and cell culture materials
1. Autoclavable or SIP Bioreactor (stirred vessel for suspension culture) 2. Automated Sampling Valve Assembly (ASVA)
3. GL45 cap with 2 port sampler (Bellco Glass, NJ, USA) 4. 0.2 µm syringe filters (Sarstedt, Ireland)
5. 0.2 µm air filters (Whatman International, Maidstone, UK, Product Number. 6784-0402) 6. 20 ml Syringe (Sarstedt, Ireland)
7. Silicon Tubing with an I.D. of 1.6 mm
8. Polytetrafluoroethylene (PTFE) tubing with an ID of 0.38 mm (VICI Valco Instrument Co. Inc)
9. Nuclear Isolation Media-4',6-diamidino-2-phenylindole (NIM-DAPI) (NPE Systems, Pembroke Pines, FL, USA)
10. NaCl (Sigma-Aldrich, UK) 11. KCl (Sigma-Aldrich, UK) 12. Na2HPO4 (Sigma-Aldrich, UK) 13. KH2PO4 (Sigma-Aldrich, UK)
14. Propidium Iodide solution at 1.0 mg/ml (Sigma-Aldrich, UK, Product Number P4864) 15. FITC Annexin V (BD pharmingen 556419)
16. Water bath (Julabo, Petersborough, UK)
17. Male and Female Quick Disconnect coupling (Colder Products Company) 18. 20L of Iso-Diluent (Beckman Coulter, UK, Catalog # 629966)
2. Methods
2.1.1 Cell culture setup for online flow cytometry.
Several bioreactor configurations can be used which have either automated and/or semi-automated sampling ports (i.e. the apPAT project, Automated Sampling Valve Assembly (ASVA)) connected to an autoclavable or steam in place bioreactor. An autoclavable bioreactor can haves a clamped 80 cm silicon tube with an MPC connector attached to the GL45 cap with a 2-port sampler before autoclave sterilization. For manual sample withdrawal from the autoclavable bioreactor, a sampling device is connected to a syringe with a 0.2 µm air filter. Each sample is withdrawn into a fresh sterile tube using the syringe.
2.1.2 FCP-CLQSC Setup
An Ethernet transmission communications protocol (TCP) must be established to share the FCP trigger folder with the CLQSC by using MS Windows map drive function as follows: Connect the CLQSC and FCP via an Ethernet cable then open My Computer from the Windows Start menu on the CLQSC. From the Tools menu, click Map Network Drive. In the Map Network Drive window, choose Y as an available drive letter from the dropdown list located next to the "Drive:" option. Click the browse button to find the “Trigger” folder by browsing available network shares.
WinAutomation is a program that allows the CLQSC to be automated when a trigger command is received from the FCP. Below are the instructions used to control the CLQSC using the FCP:
1. Open the WinAutomation program icon and execute the visual job designer in order to enter the following script:
1. Label Main Section
2. If File Exists If File Y:\go.txt exists
• Send Keys Send the following keystrokes: S to the active window
• Wait Wait for 15 seconds
• Delete File(s) Delete the file(s) Y:\go.txt
• Go to Main Section
3. Else
• If File Exists If File Y:\rinse.txt exists
o Send Keys Send the following keystrokes: r to the active window
o Wait Wait for 15 seconds
o Delete File(s) Delete the file(s) Y:\rinse.txt
o Go to Main Section
• Else
o Wait Wait for 2 seconds
o Go to Main Section
• End If
4. End If
2. Save the job and leave the program to run in the background. Remember to always have
2.1.3 CLQSC Setup and Calibration
The Cell Lab Quanta Flow Cytometer (CLQSC) will be used for the automation of cell analysis due to its ability to provide accurate cell size or volume
information in combination with fluorescent characteristics of the cell analysis. The electronic/coulter measurement enhances the accuracy of the cell size
measurements compared to traditional forward scatter, which is an estimate of cell size via a reference bead. The optical setup uses a 488-nm laser and a mercury arc lamp in combination or individually with three photomultiplier tubes (PMTs), and a side scatter channel. The filters used for each PMT is a 465 band-pass, 525 band-pass (BP) and 670 long pass (LP). The 488nm laser provides cell viability information via PI detection using the 525 BP filter, although the 670 LP filter may also be utilized if so desired (Fig.1a) while the mercury arc lamp is used for cell cycle detection via DAPI measurement using UV light and a 357/22 nm exciter (Fig.1b). Our lab has used both filters successfully for the same protocol.
2.1.3.1 CLQSC startup
1. Open the Cell Lab Quanta program and choose startup from the menu. 2. Follow the on-screen instructions turning on the laser.
3. It is critical that you leave the laser to warm up for at least 15 minutes before continuing to the next few steps to determine if energy output is stable.
2.1.3.2 Fluorescence and volume calibration
1. Use the Flow-Check beads from Beckman Coulter to check laser alignment for FL1 FL2, and FL3 using a flow rate of an average of 150 events per second for 5000 events and verify that the CVs are below 3.
2. Recover the sample and rinse the instrument.
3. Load a protocol for cell number, viability and cell size, using the Beckman Coulter 10 µm calibration beads to calibrate the diameter set in the EV display channel using 10,000 events.
4. For ease of use, the CLQSC is set up so that the cell cycle data can also be obtained from the same protocol loaded for cell number, viability, and cell size, so that switching between CLQSC protocols is not needed. This means FL1 channel is used for cell cycle setup while FL2 is set up for the viability and cell number detection.
5. Recover the sample and rinse the instrument.
2.1.3.3 CLQSC Data Acquisition Setup
The CLQSC can be set up to run each individual protocol where the coulter counter, the FL2 PMT detector, and the 488nm laser are used for cell number, cell size and cell viability analysis, respectively, while the FL1 PMT detector and the mercury arc lamp is used for cell cycle analysis. The setups below are used for each type of analysis:
1. Head to the Current Settings window from the Main Menu and select the auto rinse to be on.
2. The critical step here is to use the auto-save option to assign a directory, of your choice, for automatically saving the analysis files after each run. An auto incrementing number will be added to each filename to sequentially add a number to each file.
3. Select to add the date to the directory path before closing the current settings screen and setting up the cell number and viability protocol on the flow cytometry.
2.1.4 FCP Setup
The FCP's main advantage in sample preparation for CLQSC data acquisition is that it uses a sample loop (SL) in combination with a stream selector to allow for cell staining and transport (Figure 1). Although there is also the possibility of using a microchamber on the system for buffer exchange, we have found that using the sample loop has the least cell loss for the current protocols designed on the system.
2.1.4.1 FCP Setup for Cell Number and Viability
An example of the results obtained from previous runs is shown in Figure 2 where the manual and automated online measurements have been compared to illustrate the accuracy of the automated online flow cytometry system setup
1. Open the FCP program icon on the instrument screen and choose protocol. 2. Enter the protocol steps as indicated in Table 1.
3. Prime the mobile phase FCP line with 70% alcohol for at least 5 prime functions to clean the system.
4. Fill a 2 liter bottle with Beckman Coulter Iso-Diluent media, label mobile phase and connect to the mobile phase line of the FCP.
5. Fill a 1L bottle with 70 % ethanol and connect this to port 8. Port 8 can be controlled by the cleaner function.
6. Fill a 1L bottle with 0.1M NaOH and connect this to port 9. Port 9 can be controlled by the cleaner function on the FCP.
7. Connect the light-protected 50 ml PI tube to port 7 using PTFE tubing kit that comes with the FCP.
2.1.4.2 FCP setup for cell cycle analysis
An example of the results obtained from previous runs is shown in Figure 3 where the manual and automated online measurements have been compared to demonstrating the accuracy of the FCP-CLQSC system
1. Open the FCP program icon on the instrument screen and choose protocol. 2. Enter the protocol steps as indicated in Table 2.
3. Prime the mobile phase FCP line with 70% alcohol for at least 5 prime functions to clean the system.
4. Fill a 2 liter bottle with Beckman Coulter Iso-Diluent media, label mobile phase and connect to the mobile phase line of the FCP.
5. Connect to port 8 a 1L bottle filled with 70% ethanol.
6. Connect to port 9 a 1L bottle filled with 0.1M NaOH.
7. Place 20 ml of NIM-DAPI in a tube connected to port 6 using PTFE tubing kit that comes with the FCP.
The critical steps here are to make sure the lines are properly primed with all the solutions fed into the system from each port. It is also important to keep the staining solutions at 4oC before each run.
2.1.4.3 FCP setup for apoptosis analysis
An example of the results obtained from previous runs is shown in Figure 4 where the manual and automated online measurements have been compared demonstarting the accuracy of the online FCP-CLQSC system
1. Open the FCP program icon on the instrument screen and choose protocol. 2. Enter the protocol steps as indicated in Table 1.
3. Prime the mobile phase FCP line with 70% alcohol for at least 5 prime functions to clean the system.
4. Fill a 2 liter bottle with Beckman Coulter Iso-Diluent media, label mobile phase and connect to the mobile phase line of the FCP.
5. Fill a 1L bottle with 70 % ethanol and connect this to port 8. Port 8 can be controlled by the cleaner function.
6. Fill a 1L bottle with 0.1M NaOH and connect this to port 9. Port 9 can be controlled by the cleaner function on the FCP.
7. Connect the light-protected 50 ml PI tube to port 7 using PTFE tubing kit that comes with the FCP.
8. Connect the light-protected 10ml diluted (50µl stock in 10ml PBS) FITC Annexin V tube to port 6 using PTFE tubing kit that comes with the FCP.
2.1.5 Automated sampling protocol without ASVA
The following protocol is used to automatically withdraw a sample from an autoclavable bioreactor culture and monitor the cell number, viability, and cell size using the Quanta SC flow cytometer.
1. Before connecting the autoclavable bioreactor to the FCP and the FCP to the CLQSC, make sure the total tubing length from the bioreactor to FCP is 17 inches (43.18 mm) and the tubing length from the FCP to the QFP is 14 inches (35.56 mm).
2. Connect 70% alcohol 200 ml bottle with a male quick disconnect fitting to sample port 1 via a silicon tube (1.6 mm ID) with a female quick disconnect fitting attached to the port. (Note: sample port 2 is used for air input, so make sure it is not blocked).
3. Connect a 0.2 µm air filter to sample port 2 after priming the port with 70% ethanol.
4. Use the clean function once to rinse the system (70% ethanol followed by filtered mobile phase).
5. Prime the FCP lines using the prime function for all connected lines.
6. Connect the mammalian cell culture flask via the autoclaved silicon tube (1.6 mm ID) attached to the stirred flask reactor using the quick disconnect coupling interface to sample port 1 under a 70% alcohol bath.
7. Prime FCP sample port 1, 3 times using the prime function.
8. The sample transfer line between the FCP and CLQSC is via a PTFE tubing connection: connect the PTFE tubing using a female luer attachment to a male luer with an integral lock ring of 1/16" semi-rigid tubing hose inserted 1.5 cm from the base of the CLQSC sample receptacle. This PTFE tubing is then connected to stainless steel injection port 2 via the PTFE tubing kit that comes with the FCP.
9. Before initiating the automated sampling, make sure the following are running on the CLQSC:
a. Mapped network drive TCP.
b. WinAutomation in background with indicated job designer script running. c. Quanta SC software in foreground with a calibrated cell size and viable
cell number protocol.
10.Press the “run protocol” button on the FCP to initiate the automated sampling. 11.The first run is used to establish that the CLQSC has the correct PMT voltages for
each channel by running an unstained sample (temporarily remove step 14 for this to occur) followed by a stained sample to determine that the correct gating has been established for viability analysis and that the G1 peak is fixed at a specific
channel number so that a minimum of 10,000 nuclear signals are collected.
The critical steps here are to make sure the lines are properly primed, but also to make sure the tubing is the correct length indicated, as the protocol is designed for the length indicated and will need to be changed via the FCP if different lengths are to be used. Another critical step is the calibration of the G1 peak channel number, when running the
cell cycle protocol, so that distribution of cells in the G1, S, and G2/M phases can also be
analyzed.
2.1.6 Automated sampling protocol with ASVA
If the run is going to be carried out in an SIP bioreactor a pneumatic sample port
connected to the ASVA (see apPAT project for details) can be implemented to deliver a sample to an intermediary sample distribution unit such as the Groton Biosystems ARS 100 if sterility of the sample is wished to be kept before FCP operation is carried out. A secondary option is a direct ASVA-FCP connection (see Fig.1.). With the direct configuration the ASVA port is open to allow sample withdrawal and once sample withdrawal is finished ASVA will flush left over media out with steam. To allow for a clean in place process in between sample extractions the steps outlined in table 4 replace the “washing and incubation steps” described in the each of the prior protocols (Tables 1-3) .The end of an ASVA-FCP protocol includes a standard pause for ASVA cool down. This setup cleans out any left-over cells and media before the next sample withdrawal using the cold condensate/air to wash the tubing out via the ASVA waste valve, V5, (See ASVA schematics from apPAT project).
Two critical points have to be mentioned, first, is that these steps occur once the sample is analysed and the ASVA has cooled, thus it is important to experimentally determine the cooling stage time of the ASVA after a SIP operation ( which can depend on the room temperature) and include this as necessary in the protocol design on the FCP. The second critical point is that for optimal operation the ASVA can be triggered by the FCP (not discussed in this manual) instead of relying on synchronized timed operation of the instruments).
The sample preparation and flow cytometric analysis is set to 5-hour intervals. This can be altered to any time setting as long as there is at least 15 minutes between each sample run. Data analysis of the cell number, viability, apoptosis, and cell cycle characteristics can be done on the CLQSC via the semi real-time data obtained, or off-line on separate software which can provide deeper analysis of the cell cycle data.
When running these protocols for the first time it can be important to use a control to verify the first run is consistent with the manual data obtained for viable cell number, cell cycle and apoptosis.
Figure 1.
Figure 1. The FCP-CLQSC (flow
cytometer) setup with the ASVA used for sampling the bioreactor.
.
Figure.2 Automated flow cytometric viable cell count versus manual trypan blue based
G1/G0 Phase Time (hours) 0 20 40 60 80 100 120 140 160 P e rc e n ta g e G1 /G0 p h a s e o f c e ll c y c le 0 20 40 60 80 100 Automated obtained G 1/G0 data Manually obtained G 1/G0 data S Phase Time (hours) 0 20 40 60 80 100 120 140 160 P e rc e n ta g e S p h a s e o f c e ll c y c le 0 20 40 60 80
Automated obtained S phase data Manually obtained S phase data
G2/M Phase Time (hours) 0 20 40 60 80 100 120 140 160 P e rc e n ta g e G2 /M p h a s e o f c e ll c y c le 0 10 20 30 40 50 Automated obtained G 2/M data Manually obtained G 2/M data
Figure3. Automated cell cycle analysis comparative to manual cell cycle analysis of CHO320
cells. The G1/G0 phase (a), S phase (b) , and G2/M phase (c) plots show that the two techniques
give similar general trends for each phase of the cell cycle with the difference between the two not being significant (P>0.05) reinforcing the advantages of the automated data collected.
Figure 4. Online flow cytometry to measure apoptosis versus manual flow cytometry measurements via Annexin-V FITC and PI dual staining on CHO320 cultured cells grown in a stirred flask bioreactor.
Tables
Table 4. FCP command script for ASVA addition by replacing the washing and incubation steps non ASVA commands.
