Application Note
PN33506
Flexible Purecell™ Select System Enables Protocol
Modifications to Optimize Enriched MNC Population
for Downstream Applications
Introduction
Materials and Methods
Pall’s extensive knowledge and experience
in blood cell interaction with materials and medical
device development has resulted in a first-generation product named the Purecell Select System.
Designed for mononuclear cell (MNC) recovery
from whole blood (WB), the Purecell Select System
is easy to use and produces consistently high MNC
recovery in less than 15 minutes. Additionally, the
Pall system has a sterile fluid path with very few
points of direct user interaction.
Human WB Samples
• Units of human whole blood collected in
CPDA-1 anticoagulant are held at ambient
temperature until use.
The Purecell Select System process is simple –
the sample is transferred to the sample bag via
a sterile Luer-Lok◆ port, then as the sample flows
through the system under gravity flow, the MNC
are captured on Pall’s filter materials. After flow
has stopped, the user back-flushes the filter with
the optimized Harvest Solution to recover the
enriched MNC population. Purecell Select System
processing results in a sample with a significant
reduction in total red blood cells (RBC), platelets,
and volume.
Pall recognizes that for some applications the
standard performance is not optimal for downstream use. Simple modifications to the standard
protocol can significantly alter the cellular composition of the final product. This Application Note
is intended to provide useful information on the
performance of this system when the standard
protocol is modified. Results from WB starting
volume of 20-120 mL, Harvest Solution volumes
of 10-25 mL, the inclusion of a rinse step, and
fresh versus 24-, 48-, and 72-hour WB samples
are presented. Additionally, recommendations
for post-processing sample manipulation for
RBC removal, centrifugation, and freeze-thaw
are included.
• Experiments are performed within three hours
of blood collection, except for WB 1, 2, or 3
days after collection, as noted.
Mononuclear Cell Enrichment by Filtration
(8-15 minutes total processing time. Refer to
instructions for use for labeled diagram and
full details.)
• A syringe is used to transfer WB and approximately 10 mL of air into the harvest set. 50 mL
WB is used for all experiments unless noted.
• The WB passes through the Purecell Select
System by gravity flow.
• Cells are recovered by back-flushing (i.e.,
reversing the direction of the flow) with 24 mL
sterile Purecell Select System Harvest Solution,
unless noted otherwise in figure legend.
• Incorporation of the optional rinse is performed
in the following manner:
– Close the upper clamp to stop WB flow once
the WB sample reaches the mid-point of the
Y at Port A (sample input port).
– Attach a syringe filled with pre-measured
rinse solution [Phosphate Buffered Saline
(PBS) for these tests] to Port A and add
slowly. Be sure that there are no air bubbles
in the tubing between the blood and the
rinse solution. Any air bubbles can be
removed by squeezing the tubing to force
the bubbles upward, or by flicking or tapping
the tubing.
– Open the clamp to restart the sample flow.
• All Purecell Select System processing procedures were done on an open lab bench
using good laboratory practice, not in a
laminar flow biological safety cabinet.
Materials and Methods (continued)
Cell Counts
Generated on a Cell-Dyn◆ 1800 hematology analyzer (Abbott
Labs) following standard protocols. Triplicate measurements
are averaged for the concentrations of WBC (white blood
cells), RBC, and platelets. Percent recoveries for each
sub-population are determined by calculating the number
of cells before and after the preparation using the following
formula:
Concentration of cells after
isolation procedure x volume
CFU Assays (StemCell Technologies)
Performed according to manufacturer’s instructions.
• Processed samples are resuspended at a concentration
of 2 x 106 WBC/mL.
• 300 µL of cell suspension is added to 3 mL of complete
Methocult.
• 1.1 mL of Methocult-cell suspension (2 x 105 WBC/dish)
is added to each of two 35 mm culture dishes.
• Culture dishes are transferred to a tissue culture incubator
at 37 ºC, 5% CO2, and 95% humidity.
x 100
Concentration of cells before isolation
procedure x WB volume
Three-Part Differential
Based on WBC counts from Cell-Dyn analyzer and lymphocyte,
monocyte, and granulocyte percentages from forward versus
side scatter plots on a BD FACSCalibur◆ flow cytometer (BD
Biosciences) unless noted in figure legend. MNC numbers are
a sum of lymphocytes and monocytes. The formula used is as
follows:
(Concentration of WBC x volume x percent of population) / 100
Viability Determination
Percent viability of WBC in the starting material and after processing is determined by dye exclusion using propidium
iodide (PI).
• 50 µL sample, pre- and post-filtration, is incubated in 1 mL
1x H-lyse buffer (R&D Systems) for 18 minutes at RT.
• 10 or 2 µL of PI (1 mg/mL, Molecular Probes) is added
(except no stain controls) and incubated an additional
2 minutes at RT.
• The cells are pelleted by centrifugation at 500 x g for
5 minutes, then resuspended in 0.5 mL PBS for analysis.
• Percent viability is determined by calculating the percentage
of unstained versus total cells as determined by flow
cytometry (BD FACSCalibur).
RBC Depletion for Colony Forming Unit (CFU) Assays
RBC depletion prior to cell culture is recommended. NH4Cl
solution (StemCell Technologies) is used with slight modifications to manufacturer’s protocol.
• 5 mL of Purecell Select System processed cells are added
to 20 mL of ice-cold NH4Cl solution (final ratio is 4:1 lysis
buffer:sample).
• The mixture is incubated on ice until lysis is complete
(~10-15 minutes).
• After complete lysis, 25 mL of PBS with 2% fetal bovine
serum (FBS) is added. Then WBC cells are centrifuged
at 500 x g for 10 minutes.
• The cell pellet is resuspended in 25 mL of PBS with 2%
FBS and centrifuged again at 500 x g for 10 minutes.
• The final cell pellet is resuspended in Iscove’s Medium with
2% FBS.
• The CFUs are enumerated after 14-16 days in culture.
Enumeration of CFU
The CFU is counted using an inverted microscope (Olympus
1x51). Cultures are evaluated for the presence of erythroid,
myeloid, and multi-potential CFU as described in the technical
manual, Human Colony-Forming Cell Assays Using Methocult
(Stem Cell Technologies).
• Erythroid CFC include Burst-Forming Units-Erythoid
(BFU-E) and Colony-Forming Unit-Erythroid (CFU-E).
• The Myeloid CFC include Colony-Forming Unit-Granulocyte
(CFU-G), Colony-Forming Unit-Macrophage (CFU-M), and
Colony-Forming Unit-Granulocyte, Macrophages (CFU-GM).
• Multi-potential CFC include Colony-Forming Units
with mixed populations of erythroid and myeloid cells
(CFU-GEMM).
Results
WB Volume Testing – Purecell Select System Results
Using 20-120 mL of WB
The bar graph in Figure 1 shows total number of MNC and
granulocytes (left and right, respectively) and percent recovery
from the Purecell Select System processing of 20, 40, 80,
and 120 mL of human WB. As expected, the number of MNC
and granulocytes in the Purecell Select System sample
increases with increasing volume of WB (blue bars). There is
a slight decrease in the percent recovery of MNC as the WB
volume increases, as seen by the increasing gap between cell
number in the WB sample (orange bars) and the Purecell Select
System sample (blue bars) at higher volumes. Interestingly,
there is a dramatic decrease in granulocyte recovery as the WB
volume increases. This results in a relatively greater enrichment of MNC as compared to granulocytes with increasing
volume. When the volume is increased to 150 mL, there is
a significant reduction in percent MNC recovery (data not
shown). Thus, 120 mL is the upper limit recommended for
this filter harvest set design.
Results (continued)
Figure 1
Purecell Select System Total Cell Number and Recoveries
from 20-120 mL of WB
Figure 2
Percent Recovery of Major Cell Populations with 10, 15, 20,
or 25 mL of Harvest Solution
80
400
400
71%
71%
300
300
77%
77%
200
200
100
100
00
99%
99%
20
20
85%
85%
79%
79%
40
40
80
80
Mononuclear Cells
Cells
Mononuclear
WB
WB
120
120
20
20
55%
55%
40
40
38%
38%
80
80
31
31%
Percent Recovery
6
Total
TotalCell
CellNumber
Number(x
(x10
106))
500
500
120
120
60
40
20
0
Granulocytes
Granulocytes
MNC
Purecell Select
Select System
System
Purecell
Total number and percentage of mononuclear cells and granulocytes in
WB or Purecell Select System from indicated volume of WB (20, 40, 80,
120 mL). Samples processed in duplicate. 20 mL volume from 3 donors;
all others from 5 donors. Recovery is calculated based on WBC and
three-part differential from Cell-Dyn 1800 hematology instrument. Error
bars show one standard deviation (SD) across donors.
Harvest Solution Volume Testing – Purecell Select
System Results Using 10-25 mL of Harvest Solution
(Figure 2 and Table 1)
The bar graph in Figure 2 shows the percent recovery of
MNC, lymphocytes, monocytes, and granulocytes from
the Purecell Select System processing when 10, 15, 20, and
25 mL of Harvest Solution are used for the back-flush step.
As expected, increasing the volume of Harvest Solution results
in higher recovery of the major populations. The difference in
recovery is ~6-10% of the starting cell number when comparing
10 mL to 25 mL. Although cell recovery is lower with less
Harvest Solution, the concentrations of MNC and granulocytes are significantly higher when lower volumes of Harvest
Solution are used (Table 1). MNC concentration drops from a
4.4 fold increase to a 1.6 fold increase relative to WB with 10
versus 25 mL Harvest Solution, respectively. Thus, there is a
trade-off between MNC recovery and final cell concentration.
This can be countered by processing a higher volume of WB.
The relative ratio of MNC compared to granulocytes changes
somewhat with the volume of Harvest Solution.
Lymph
Mono
Gran
15 mL
10 mL
RBC
Plts
25 mL
20 mL
Standard protocol used except that the volume of Harvest Solution for
cell recovery is varied, as indicated. Percent recovery is calculated as
described in Materials and Methods. Duplicates are averaged. Means
+/- 1 SD across 3 donors are reported.
Table 1
Effect on Final MNC and Granulocyte Concentration
of 10, 15, 20, or 25 mL of Harvest Solution
Average
Fold Increase
(x 106 Cells/mL)
(x 106 Cells/mL)
MNC
Gran
MNC
Gran
Average Final
Volume (mL)
WB
2.9
3.9
10 mL
12.5
6.8
4.4
1.8
7.6
15 mL
7.6
4.9
2.7
1.3
12.5
20 mL
5.7
3.8
2.0
1.0
17.6
25 mL
4.5
3.2
1.6
0.8
22.5
Data generation (3 donors) and analysis as described in Figure 2 legend.
Optional Rinse Step to Further Reduce RBC
and Platelets
For greater reduction in the number of RBC and platelets, an
additional rinse step is incorporated into the processing protocol. Specifically, WB flow is stopped once the level of WB has
reached the sample input valve. PBS is then added to the
system through the sample input valve. The flow is resumed
and the processing is continued as per the standard method
(see Materials and Methods). The recovery of the major cell
types is shown in Figure 3a. The standard Purecell Select
System method results in 86% depletion of RBC. When an
additional 6 or 10 mL PBS rinse is incorporated, RBC depletion
is increased to 94% and 97%, respectively. The rinse step
also results in a 5-8% further reduction in platelets. There is
a 5-20% decrease in recovery of lymphocytes, monocytes,
and granulocytes with the added rinse step. Lymphocytes,
which typically give the highest recovery, show the greatest
drop in recovery with this step. The viability of the processed
samples is similar or slightly improved with the additional rinse
step (data not shown).
Results (continued)
Figure 3b
Effect of a Rinse Step on Hematopoietic CFU Assays
Figure 3a
Effect of a Rinse Step on Recovery of Major
Cell Populations
35
Percent
TotalRecovery
Cell Number (x 106)
80
400
71%
300
60
200
77%
85%
100
40
55%
99%
31%
38%
79%
25
20
15
10
5
0
20
20
CFU Number/2 x 105
30
500
40
80
120
20
Mononuclear Cells
WB
40
80
120
Lymph
No rinse
Myeloid
Purecell Select System
Mono
Gran
6 mL rinse
RBC
Mixed
CFU Type
0
MNC
0
Erythroid
Granulocytes
Plts
10 mL rinse
The standard protocol +/- the optional PBS rinse step is followed, as
indicated on the graph. Means +/- 1 SD across 3 donors are reported.
Data from rare cell enumeration using CFU assays for erythroid,
myeloid, and mixed colonies demonstrate the presence of
hematopoietic progenitor cells in WB samples processed
using the Purecell Select System (Figure 3b). Although the
level of RBC in the harvested sample is much lower than the
starting material, further depletion is necessary before plating
to achieve accurate colony counts. Data comparing the
Purecell Select System rinse protocol versus the standard
protocol with RBC lysis suggests that some of the erythroid
progenitor cells are lost during the RBC lysis and/or subsequent washing step. The PBS rinse step saves time while
resulting in equivalent or higher numbers of hematopoietic
progenitor cells. Thus, even though the rinse step does result
in a ~5-20% decrease in total MNC recovery, the rare cells
do not appear adversely effected when compared to the
incorporation of an RBC lysis step.
WB
RBC Lyse
Pall
RBC Lyse
Pall
6 mL Rinse
Pall
10 mL Rinse
CFU assay results from samples processed as indicated. The standard
method sample required an RBC lysis step prior to use in this assay, while
samples processed with the additional rinse step were diluted and used
directly in the assay. Duplicates are averaged. Means +/- 1 SD across
3 donors are reported.
Purecell Select System Processing of Fresh Versus
1, 2, or 3-Day Old WB
Recovery and viability data from WB pre- and post-processing
with the Purecell Select System (Table 2) indicates that fresh
and aged WB samples (from 3 donors) behave similarly. There
are no significant differences in percent recovery of the MNC,
WBC, or cell composition of the samples (data not shown)
between WB processed on the day of collection or 1-3 days
after collection when compared to pre-processing values.
There may be a slight decrease over time in total MNC and
WBC number, but donor variability is high for these counts.
The viability of major cell populations post-processing is always
equal to or slightly higher than the WB starting sample of the
same age. There is a slight downward trend in WB viability
over time, which is to be expected.
Table 2
Purecell Select System Processing of Fresh WB Compared to Aged WB – Percent Recovery and Viability
% Recovery
MNC Recovery
WBC Recovery
Cell Number x 106/50 mL WB
or Processed Sample
MNC
WBC
Viability
252.8 +/- 80.3
164.2 +/- 39.5
90.1 +/- 9.0
92.4 +/- 5.7
WB Age
Sample
Fresh
WB
Pall
74.0 +/- 5.9
66.3 +/- 6.5
128.2 +/- 30.8
94.3 +/- 20.7
WB
Pall
76.4 +/- 6.9
72.7 +/- 3.3
117.5 +/- 29.7
90.6 +/- 29.3
236.7 +/- 67.7
170.7 +/- 43.9
83.9 +/- 12.6
86.0 +/- 7.1
WB
Pall
78.2 +/- 5.2
72.8 +/- 1.4
108.2 +/- 18.2
85.3 +/- 19.9
212.7 +/- 61.1
164.8 +/- 44.0
87.2 +/- 5.9
89.9 +/- 3.9
WB
Pall
73.7 +/- 10.4
68.0+/- 2.9
114.0 +/- 22.7
84.2 +/- 23.2
223.9 +/- 63.5
151.1 +/- 37.4
82.6 +/- 3.2
82.6 +/- 4.6
Day 1
Day 2
Day 3
Comparison of MNC and total WBC recovery and viability from matched donors using fresh WB versus the same WB stored at 4 ºC for 1-3 days. Chilled
WB is equilibrated to room temperature prior to processing. Percent viability is determined with PI. Averages from 3 donors +/- 1 SD are reported.
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Recommendations for Centrifugation of Purecell Select
System Processed Samples
Although isotonic, the Harvest Solution has higher density
than typical buffers. As a result, good recovery from a centrifugation step requires the use of non-standard conditions. Pellet
the cells at 300-400 x g for 5 minutes with no brake. It is
crucial to turn off the centrifuge brake! If this is not done,
the cell pellet will be highly disrupted, resulting in poor recovery.
Resuspend the cell pellet in appropriate buffer. This procedure
will result in a significant decrease in platelets (40-75% relative
to pre-centrifugation), but high recovery of all other cell types.
Cell Freeze Recommendations for Samples Processed
Using the Purecell Select System
Several factors such as cell concentration, solutions, and
serum proteins used for preparation of cell suspension,
concentration of cryopreservation agent, cooling rate, storage
conditions, and thawing methods directly affect the viability
and quality of cells after freezing and thawing. Freeze and
thaw procedures should be optimized for the MNC samples
and any post-thaw manipulation step.
RBC lysis during a freeze-thaw cycle contributes to post-thaw
debris in any RBC-containing sample. If desired, RBC may be
removed prior to cell freezing using an RBC lysis method or
by adding a rinse step to the Purecell Select System standard
processing protocol after cell capture and before cell harvest
(see note above regarding rinse step). If an RBC lysis step is
included, be aware that the number of RBC in the processed
sample will be much lower as compared to WB (< 15% of
total from 50 mL WB). We typically use half the amount of
RBC lysis solution recommended for WB samples when
performing RBC lysis of Purecell Select System processed
samples. During the post-RBC lysis wash and centrifugation,
turn off the brake (as noted above) unless sample dilution is
10-fold or greater. Post-thaw viability determination of MNC
or specific target cell population is recommended. Analysis
of thawed samples indicates that a high percentage of the
granulocytes (CD66b positive cells) die during freeze-thaw, as
assessed with PI staining (data not shown). This is consistent
with the well-known fact that granulocytes do not survive
freeze thaw cycles as well as other cell types. As a result, postthaw viability on total WBC is not indicative of MNC viability.
Conclusions
Pall’s Purecell Select System provides fast sample processing
with an easy-to-use method resulting in high MNC recovery.
In addition to use of the standard protocol, simple alterations
to the procedure change the final MNC sample composition.
The system is very robust and flexible, readily accommodating
WB volumes from 20-120 mL, Harvest Solution volumes of
10-25 mL, and an added rinse step to reduce RBC or older
WB samples (fresh to 3 days). Guidelines are provided for
post-processing sample manipulation, specifically cell freezing
and centrifugation. Matched donor samples are used for all
testing to eliminate donor-to-donor variability, which is very
high. Most experiments are done with 50 mL volumes from
split blood units and data from multiple donors is combined,
although generally with a limited number of samples. Data
from duplicates shows a high degree of processing method
reproducibility, consistent with overall high reproducibility
reported in Application Note Performance Characterization
of the Purecell Select System for Enrichment of Mononuclear
Cells from Human Whole Blood (PN 33505). Although it is
unlikely that the Purecell Select System will replace Ficoll◆
cell processing for all applications, the ability to modify the
protocol to alter final sample composition is beneficial to
users. This method flexibility increases the range of downstream applications for which the Purecell Select System
processing method is directly applicable.
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© 2008, Pall Corporation. Pall,
, and Purecell are trademarks of Pall Corporation. ® indicates
a trademark registered in the USA. ◆Ficoll is a trademark of GE Healthcare Bio-Sciences. Cell-Dyn is
a trademark of Abbott Laboratories. BD FACSCalibur is a trademark of BD Biosciences. Luer-Lok is a
trademark of Becton-Dickinson.
This product, and its use, may be covered by one or more patents including US 6,544,751 and EP 973,587.
1/09, 1.5k, GN08.2340
PN33506