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 semiautomated 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
•
•
•
•
3. Else
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
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
the Cell Lab Quanta SC program selected when running in automation mode.
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 13) .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 FCPCLQSC (flow
cytometer) setup with
the ASVA used for
sampling the
bioreactor.
.
Figure.2 Automated flow cytometric viable cell count versus manual trypan blue based
hemocytometer counts of CHO320 cell line in a stirred flask bioreactor.
G1/G0 Phase
S Phase
80
Percentage S phase of cell cycle
Automated obtained G1/G0 data
Manually obtained G1/G0 data
80
60
40
20
0
Automated obtained S phase data
Manually obtained S phase data
60
40
20
0
0
20
40
60
80
100
120
140
160
0
20
40
Time (hours)
60
80
100
120
Time (hours)
G2/M Phase
50
Percentage G2/M phase of cell cycle
Percentage G1/G0 phase of cell cycle
100
Automated obtained G2/M data
Manually obtained G2/M data
40
30
20
10
0
0
20
40
60
80
100
120
140
160
Time (hours)
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.
140
160
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 1. FCP command script for automated cell number and viability measurement program
Table. 2 FCP command script for automated cell cycle measurement program
Table 3. FCP command script for automated Apoptosis measurement program
Table 4. FCP command script for ASVA addition by replacing the washing and incubation steps non
ASVA commands.