There are several manual methods to separate peripheral blood mononuclear cells (PBMCs) from leukapheresis products, but most of them are highly user-dependent and laborious. The Gibco™ CTS™ Rotea™ Counterflow Centrifugation System can be used to automate a variety of methods to separate PBMCs in a closed environment.
In this 12-page app note, you’ll see effective and consistent PBMC separation across all tested method compared to a manual density gradient centrifugation method.
Test methods include:
- Ficoll solution
- With ACK (ammonium-chloride-potassium) lysis buffer
- Without lysis buffer
Introduction
The Gibco
™
CTS
™
Rotea
™
Counterflow Centrifugation System
offers fast and effective cell separations based on size
and density by employing counterflow centrifugation (CFC)
technology. The CTS Rotea system provides great flexibility by
incorporating different cell processing methods from customer-
designed protocols to application-specific needs. Currently,
there are several manual methods to separate peripheral blood
mononuclear cells (PBMCs) from leukapheresis products,
but most of them are highly user-dependent and laborious.
Here we demonstrate the CTS Rotea system can be used to
separate PBMCs in a highly reproducible and effective manner,
by comparing the performance of CTS Rotea system protocols
and a manual density gradient centrifugation method (Figure 1).
The methods for the CTS Rotea system included using Ficoll
™
solution, ACK lysis buffer, or red blood cell (RBC) elutriation.
The data show effective and consistent PBMC separation
from the CTS Rotea system across all tested methods and
prove the system’s ability to successfully automate existing
manual methods. These data will serve as the basis for further
development and optimization of protocols for PBMC separation
using the CTS Rotea system.
Application note | CTS Rotea Counterflow Centrifugation System
PBMC separation (day 0)
Analysis of PBMCs and T cells with an automated cell counter and
Attune NxT Flow Cytometer
T cell activation and
culture (days 0–5)
Bead removal followed
by T cell expansion
(days 5–13)
• CTS Rotea system
with Ficoll solution
• Manual density
gradient
centrifugation with
Ficoll solution
• CTS Rotea system
with ACK lysis bu?er
• CTS Rotea system
without lysis bu?er
(RBC elutriation)
• CTS Dynabeads
CD3/CD28 beads
• CTS OpTmizer T Cell
Expansion Serum
Free Medium (SFM)
• CTS Immune Cell
Serum Replacement
• L-Glutamine
• GlutaMAX
Supplement
• IL-2 recombinant
human protein
• DynaMag-50 Magnet
• G-Rex 100M system
Figure 1. Overview of the experiments.
Materials and methods
Fresh leukopaks were used as starting material for all PBMC
separation experiments. Four different protocols were performed
for separating PBMCs: (1) CTS Rotea system with Ficoll solution,
(2) manual density gradient centrifugation with Ficoll solution,
(3) CTS Rotea system with ACK lysis buffer, and (4) CTS Rotea
system without lysis buffer. During processing with the CTS
Rotea system, DPBS was used as leukopak dilution buffer, and
DPBS with 2% HSA was used as washing buffer. Details of the
protocols and kit configurations are shown in Tables 1–4 and
Figure 2. A total of 3 billion input cells were processed per loop
in each experiment, except for the protocol using the CTS Rotea
system with Ficoll solution in which the number of cells was
reduced to 1–1.5 billion per loop to accommodate the volume of
Ficoll solution added into the chamber. This was to avoid possible
unwanted elutriation of the mononuclear cell layer during the
separation with Ficoll solution in the CTS Rotea system chamber.
For manual processing with Ficoll solution, the same number of
input cells (3 billion) was used as in the CTS Rotea system runs.
Separated PBMCs from each protocol were analyzed for recovery
(concentration) and viability using an automated cell counter.
Their cellular characteristics were profiled using an Invitrogen
™
Attune
™
NxT Flow Cytometer.
Immediately following the separation, 100 million cells from
each protocol were activated using Gibco
™
CTS
™
Dynabeads
™
CD3/CD28 beads in complete medium (Gibco
™
CTS
™
OpTmizer
™
T Cell Expansion SFM supplemented with Gibco
™
CTS
™
Immune Cell Serum Replacement, Gibco
™
L-Glutamine, Gibco
™
GlutaMAX
™
Supplement, and interleukin 2 (IL-2)). The cells
were then cultured for five days in a T-225 flask at 37°C (initial
seeding density: 1.34 million/mL). Complete medium was added
on day 2. On day 5 post–T cell activation, the CTS Dynabeads
CD3/CD28 magnetic beads were removed using an Invitrogen
™
DynaMag
™
-50 Magnet, and T cells were expanded for an
additional 8 days. A total of 100 million activated T cells were
cultured in a G-Rex
™
100M system with 250 mL of complete
medium.
The CTS Rotea system: automation and consistency in
PBMC separation—a comparison of protocols
Cell therapy solutions
2
Table 1. Protocol to separate PBMCs using the CTS Rotea system and Ficoll solution.
Step Description Flow path
Centrifuge
force (x g)
Flow rate
(mL/min) Step type Trigger(s)
1 Pre-prime B to A 0 100
Normal
Input bubble sensor
2 Lubricate rotary coupling B to A 0 100 15 mL
3 Prime chamber and line A B to A 10 100 15 mL
4 Add leukopak dilution volume B to A 10 100 1.5x leukopak dilution (mL)
5 Prime bubble trap and line B A to B 10 100 15 mL
6 Prime line D A to D 10 50 5 mL
7 Prime line E A to E 10 50 5 mL
8 Prime line F B to F 10 50 5 mL
9 Dilute leukopak A to C 10 100 1x leukopak dilution (mL)
10 Prime pause loop J to K 10 25
Pause
3 mL
11 Ramp speed for loading J to K 2,000 25 10 seconds
12 Load leukopak C to A 2,000 35 Normal
1x leukopak draw volume (mL),
input bubble sensor, pause
13 Pause J to K 2,000 25 Pause 10 seconds
14 Wash bubble trap B to A 2,000 25 Normal 10 mL
15 Pause J to K 2,000 7 Pause 20 seconds
16
Add Ficoll solution + elutriate
white blood cells (WBCs)
D to G 2,000 7 Normal 40 mL
17 Pause for RBC harvest J to K 2,000 7 Pause 10 seconds
18 Harvest RBCs B to F 2,000 100 Harvest 50 mL
19 Pause—take count J to K 2,000 35 Pause
20 Dilute WBCs B to G 2,000 100 Normal 1x intermediate dilution (mL)
21 Pause J to K 2,000 40 Pause 10 seconds
22 Establish WBC bed E to G 2,500 35
Normal
100 mL
23 Load intermediate bag E to A 2,500 40
1x intermediate draw volume (mL),
input bubble sensor, pause
24 Pause for wash J to K 2,500 20 Pause 20 seconds
25 Wash WBCs B to A 2,500 20 Normal 6 seconds
26 Pause—stable loop J to K 2,500 20 Pause 20 seconds
27 Wash B to A 2,500 20 Normal 50 mL
28 Pause before harvest J to K 2,500 20 Pause 10 seconds
29 Harvest WBCs B to H 2,500 30 Harvest 35 mL
30 Ramp down to end J to K 500 5 Pause 10 seconds
On day 2 following the initial seeding (day 7), 250 mL of the medium was added to the
culture as a supplement. On day 9, an additional 450 mL of fresh complete medium
was added to the G-Rex 100M system. At the end of the 8-day culture (day 13), T cell
subpopulations were profiled using an Attune NxT Flow Cytometer. Growth and viability
were measured using an automated cell counter. Multicolor flow cytometry panels are
listed in Figure 3.
3
Table 2. Protocol to separate PBMCs using manual density gradient centrifugation and Ficoll solution (protocol does
not involve the CTS Rotea system).
Step Description Flow path
Centrifuge
force (x g)
Flow rate
(mL/min) Step type Notes
1
Add Ficoll solution to 50 mL
conical tubes
– – – – 10 mL Ficoll solution/tube
2 Add PBS – – – – 15 mL PBS/tube
3 Add leukopak – – – – 15 mL leukopak/tube
4 Centrifuge tubes – 500 – – 30 min at 22°C, brake set to “off”
5 Aspirate 2/3 of the top layer – – – –
6 Collect lymphocyte layer – – – –
7
Centrifuge, wash, and discard
the supernatant
– 100 – – 10 min at 22°C
8
Centrifuge, wash, and discard
the supernatant
– 100 – – 10 min at 22°C
9 Resuspend cell pellet in a medium – – – –
Table 3. Protocol to separate PBMCs using the CTS Rotea system and ACK lysis buffer.
Step Description Flow path
Centrifuge
force (x g)
Flow rate
(mL/min) Step type Trigger(s)
1 Pre-prime B to A 0 100 Normal Input bubble sensor
2 Lubricate rotary coupling B to A 0 100 Normal 15 mL
3 Prime chamber and line A B to A 10 100 Normal 15 mL
4 Add priming volume B to A 10 100 Normal 1.5x leukopak dilution (mL)
5 Prime bubble trap and line B A to B 10 100 Normal 15 mL
6 Prime line D and dilute leukopak A to D 10 100 Normal 1x leukopak dilution (mL)
7 Prime line F A to F 10 100 Normal 5 mL
8 Prime pause loop J to K 10 25 Pause 3 mL
9 Pressure prime A to E 10 0
Pressure
prime
10 Mix leukopak “>>” to advance J to K 2,300 32 Pause
11 Load leukopak and remove platelets D to A 2,300 32 Normal
1x leukopak aliquot (mL), input
bubble sensor, pause
12 Pause J to K 2,300 32 Pause 10 seconds
13 Wash cells to remove platelets B to A 2,300 32 Normal 32 mL
14 Pause before lysis J to K 2,300 16 Pause 10 seconds
15 Lyse RBCs F to A 2,300 16 Normal 16 mL
16 Pause J to K 2,300 16 Pause 10 seconds
17 Lysis 2 F to A 2,10 0 34 Normal 2 minutes
18 Lysis 3 F to A 2,10 0 34 Normal 6 minutes
19 Pause after lysis J to K 2,10 0 34 Pause 10 seconds
20 Wash to remove RBCs and lysis buffer B to A 2,10 0 34 Normal 50 mL
21 Concentrate PBMCs J to K 2,600 20 Pause 10 seconds
22 Harvest PBMCs B to H 2,600 100 Harvest 1x harvest volume
23 Ramp to stop K to J 300 30 Pause 5 seconds
4
Table 4. Protocol to separate PBMCs using the CTS Rotea system without lysis buffer.*
Step Description Flow path
Centrifuge
force (x g)
Flow rate
(mL/min) Step type Trigger(s)
1 Pre-prime B to A 0 100 Normal Input bubble sensor
2 Lubricate rotary coupling B to A 0 100 Normal 15 mL
3 Prime chamber and line A B to A 10 100 Normal 15 mL
4 Add priming volume B to A 10 100 Normal 1.5x leukopak dilution (mL)
5 Prime bubble trap and line B A to B 10 50 Normal 15 mL
6 Dilute leukopak A to D 10 100 Normal 1x leukopak dilution (mL)
7 Prime line F A to F 10 100 Normal 5 mL
8 Prime pause loop J to K 10 25 Pause 3 mL
9 Pressure prime A to E 10 0
Pressure
prime
10 Mix leukopak “>>” to advance J to K 2,300 32 Pause
11 Load leukopak and remove platelets D to A 2,300 32 Normal
1x leukopak aliquot (mL), input
bubble sensor, pause
12 Pause J to K 2,300 32 Pause 10 seconds
13 Wash cells to remove platelets B to A 2,300 32 Normal 50 mL
14 Pause J to K 2,300 25 Pause 10 seconds
15 Ramp speeds to elutriate RBCs J to K 1,200 30 Pause 30 seconds
16 Elutriate RBCs B to A 1,200 38 Normal 150 mL
17 Concentrate lymphocytes J to K 2,600 20 Pause 15 seconds
18 Harvest lymphocytes B to H 2,600 50 Harvest 1x harvest volume
19 Ramp to stop K to J 10 30 Pause 5 seconds
* This protocol separates PBMCs using the CTS Rotea system without the use of any density gradient medium or lysis buffer. It solely depends on the capability of the CTS Rotea system to fractionate cells based
on size and density.
A B C
Figure 2. Gibco
™
CTS
™
Rotea
™
Single-Use Kit configuration for PBMC separation. Configurations are shown for the following protocols: (A) CTS
Rotea system with Ficoll solution, (B) CTS Rotea system with ACK lysis buffer, and (C) CTS Rotea system without lysis buffer.
Bu?er 500 mL Ficoll solution
100 mL
RBCs 0 mL
Waste 0 mL Leukopak 70 mL
Harvest WBCs 0 mL
B D F
A EGC
H
Intermediate 0 mL
Waste 0 mL
A
DPBS + 2% HSA
2000 mL
Leukopak 300 mL ACK lysis bu?er
2000 mL
D F
PBMCs 0 mL
H
B
Waste 0 mL
A
DPBS 1000 mL Leukopak 75 mL
D
Lymphocytes 0 mL
H
B
5
Group Target Conjugate Clone
1
CD45 FITC HI30
CD56 PerCP-eFluor 710 CMSSB
CD11c APC-eFluor 780 3.9
Viability eFluor 506 –
2
CD45 FITC HI30
CD16 PE-eFluor 610
eBioCB16
(CB16)
CD14 APC 61D3
Viability eFluor 506 –
3
CD45 FITC HI30
CD3 Super Bright 702 OKT3
CD19 PE-Cyanine5 HIB19
Viability eFluor 506 –
4
CD235a PE HIR2 (GA-R2)
CD41a APC-eFluor 780 HIP8
Viability eFluor 506 –
Group Target Conjugate Clone
5
CD3 PE UCHT1
CD8 FITC RPA-T8
CD4 APC RPA-T4
Viability eFluor 506 –
6
CD8 FITC RPA-T8
CD25 PerCP-Cy5.5 BC96
FOXP3 PE PCH101
CD4 APC RPA-T4
CD127 APC-eFluor 780 eBioRDR5
Viability eFluor 506 –
7
CD62L FITC DREG-56
CD95 PE DX2
CD45RA APC HI100
Viability eFluor 506 –
8
CD62L FITC DREG-56
CCR7 PE 3D12
CD45RA PE-Cyanine5 HI100
CD45RO APC-eFluor 780 UCHL1
Viability eFluor 506 –
A B
Figure 3. Multicolor flow cytometry panel design. (A) Antibodies in groups 1–4 were designed to characterize immune cell subpopulations within
the separated PBMCs. (B) Antibodies in groups 5–8 were used for T cell immunophenotyping.
Results
Viability and recovery
For all 5 biological replicates for each CTS Rotea system protocol
and manual protocol, viability of PBMCs immediately following
separation was maintained above 97%, with slightly lower
viability observed for the manual protocol (Figure 4). Regarding
the recovery rate, the CTS Rotea system protocol with ACK lysis
buffer resulted in the highest recovery (91%), while the lowest
recovery rate was observed for the manual protocol (40%). The
manual protocol resulted in a wide recovery range, spanning
from 20% to 60% (Figure 4). Such variability can be attributed
to intended technical focus (quantity vs. purity), depending on
downstream use of the separated population. Cells with higher
purity generally have lower recovery rate and vice versa. For
this study, manually separated cells with higher purity were
characterized and compared.
Figure 4. (A) Viability and (B) recovery of separated cells for
each protocol.
0
20
40
60
80
100
CTS Rotea
system with
Ficoll solution
Manual with
Ficoll solution
CTS Rotea
system with
lysis bu?er
CTS Rotea
system without
lysis bu?er
Recovery (%)
90
92
94
96
98
100
Viability (%)
CTS Rotea
system with
Ficoll solution
Manual with
Ficoll solution
CTS Rotea
system with
lysis bu?er
CTS Rotea
system without
lysis bu?er
A
B