Assessing the Functionality of PBMCs After Cryopreservation
App Note / Case Study
Last Updated: June 7, 2024
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Published: April 30, 2024
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Human peripheral blood mononuclear cells (PBMCs) are commonly used during the drug development process in T cell proliferation assays to determine whether a compound or drug will elicit a T cell response to a foreign substance.
As PBMCs continue to see an increase in use in the testing of new biological therapeutic compounds, ensuring purified cells retain functionality after cryopreservation is critical.
Download this app note to discover PBMCs that:
- Maintain function when isolated from fresh and cryopreserved leukopaks
- Are reliable, reproducible and interchangeable
- Can be used to generate experimental data with convenience
Discover the
Freshness of
Frozen PBMCs
An Assessment of
Cryopreservation
Impact on T Cell Activation
Enabling a Healthier World
Katherine M Dunnick, PhD, Kayla DeOca, PhD,
Aurita Menezes, PhD, Ly Nguyen, M.Sc,
Anshika Sharma, PhD
Understanding the impact of cryopreservation on performance of PBMCs is critical.
This study focuses on assessing functionality
of PBMCs after cryopreservation in standard
immune cell based assays. Here, we demonstrate that cryopreserved PBMCs are reliable, deliver reproducible data, and maintain
functionality. Utilizing freshly isolated PBMCs
directly from donation may not be as necessary as believed.
Introduction
Human peripheral blood mononuclear cells (PBMCs)
consist of a mixed population of specialized immune cells.
These immune cells (belonging to both lymphoid and myeloid lineages) play an important role in both the innate and
adaptive immune systems and are critical in protecting the
body from viral, bacterial, and parasitic infections. Upon
interaction with various pathogens, cytokines, or chemokines, these cells can fully mature and activate into T cells,
B cells, dendritic cells, and macrophages for example.
Isolated human PBMCs are utilized in various fields of research, including vaccine and drug development. Routinely,
these cells are isolated from fresh leukopaks and frozen
down for subsequent use in experimental testing. As
PBMCs continue to see an increase in use in the testing of
new biological therapeutic compounds, ensuring purified
cells retain functionality after cryopreservation of either
the leukopak or PBMCs is necessary. The ability to use
cryopreserved cells and be assured of the same functionality as fresh cells allows more options for cell sources,
reproducible results, and the ability to plan experiments
with convenience. Understanding the impact of cryopreservation on functionality of cell types isolated from PBMCs
will provide guidance on use of PBMCs from leukopaks.
This study focused on determining if PBMCs maintain function after cryopreservation in standard immune cell based
assays.
Materials and Methods
Isolation of PBMCs from fresh and cryopreserved
leukopaks
Apheresis material was collected from four individual donors. After collection, ½ the material was processed for
isolation and the other ½ of the material was cryopreserved following a proprietary method. Fresh leukopak material was processed equivalently across all donors within
24 hours of collection and PBMCs were isolated following
standard methods. Following PBMC isolation, cells were
cryopreserved in liquid nitrogen (LN2
) for future testing
(these cells are referred to as freshly isolated/single cryopreserved PBMCs). After one week, cryopreserved apheresis material was thawed. Following thaw, PBMCs were
isolated using standard isolation procedures. Cells were
subsequently cryopreserved in LN2
for future assessment
(these cells are referred to as dual cryopreserved PBMCs).
Assessment of PBMC yield and viability
To determine the impact of single and dual cryopreservation on PBMC viability and yield, PBMC samples were
thawed in a 37ºC water bath for 2 minutes, centrifuged
for 15 minutes at 300xg and resuspended in media for
counting. Cells were counted using trypan blue exclusion
method and yield and viability were determined.
T cell proliferation assay
To determine the impact of single and dual cryopreservation on PBMC function, PBMC samples were thawed in
a 37ºC water bath for 2 minutes, centrifuged for 15 minutes at 300xg and resuspended in media for counting.
Following counts, cells were resuspended at 10x106/mL
and incubated with 5 µM CellTrace CFSE Cell Proliferation
dye (Invitrogen, C34554) for 20 minutes. Cells were then
incubated with 5x media volume to stop the CFSE reaction
and remove unbound dye for 10 minutes. Cells were then
centrifuged 300xg for 10 minutes and resuspended in
X-Vivo® 15 Serum-free Hematopoietic Cell Medium (Lonza,
04-418Q) at 10x106/mL. After 5 minutes, cells were added
to 24- or 48-well plates at 1x106/mL. Cells were then combined with either X-Vivo® 15 Media alone, X-Vivo® 15 Media +
5 ng/mL IL-2 (R&D Systems, 202-IL-500), or X-Vivo® 15 Media
+ 5 ng/mL IL-2 + 50 μL CD3/CD28 Dynabeads (ThermoFisher, 11131D). IL-2 was added to the media to maintain T cell
health and allow for proliferation and growth after activation, it is not expected to result in T cell proliferation alone.
CD3/CD28 Dynabeads were selected to provide prolonged
T cell activation in culture without the need for media
changes or replacement. Cells were incubated for
4 days at 37ºC, 5% CO2
to allow for T cell proliferation.
After 4 days, cells were collected, stained with Live/Dead
fixable violet dead cell dye (ThermoFisher, I34964) for 20
minutes and analyzed via FACS on the BD Canto II.
Results
PBMCs isolated from both fresh leukopaks and
cryopreserved leukopaks retain high viability
PBMCs were isolated from fresh (within 24 hours of
donation) or cryopreserved leukopaks. Following isolation, viability was measured via trypan blue and cells were
subsequently cryopreserved. Cells were thawed after at
least two weeks in cryopreservation and viability was again
measured via trypan blue. Results indicate PBMCs isolated
from fresh leukopaks had higher viability when measured
pre-cryopreservation as compared with post-cryopreservation and when compared to the PBMCs isolated from
cryopreserved leukopaks at both time points (Figure 1);
however, both single and dual cryopreserved cells maintained viability above 90% and 85% respectively.
T cell activation as measured by cytokine production
in the presence of IL-2
To determine the impact of single and dual cryopreservation on T cell generation of cytokines, PBMCs were
thawed in a 37ºC water bath for 2 minutes, centrifuged
for 15 minutes at 300xg and counted. Following counts,
cells were resuspended at 1x106/well in 24-well plates and
incubated with either X-Vivo® 15 media alone, X-Vivo® 15
media + 5 ng/mL IL-2 (R&D Systems, 202-IL-500), or X-Vivo®
15 Media + 5 ng/mL IL-2 + 50 µL CD3/CD28 Dynabeads
(ThermoFisher, 11131D) for four days. On days 0, 2, and 4,
media was collected from each well and frozen for future
cytokine analysis. After collection of all timepoints, samples
were thawed, centrifuged at 10,000xg for 5 minutes at 4ºC
and assessed for IL-1β, IL-12, IL-4, IFNγ, TNFα, IL-10, and IL-6
cytokines using a 32x8 Simple Plex Cartridge (Protein Simple, SPCKE-PS-003426) and the ELLA automated cytokine
instrument.
T cell activation without additional IL-2
To determine PBMC functionality as measured by cytokine
production in the absence of IL-2, PBMCs were thawed in
a 37ºC water bath for 2min, centrifuged for 15 minutes at
300xg and counted. Following counts, cells were resuspended at 1x106/well in 24-well plates and incubated with
either X-Vivo® 15 Media alone, or X-Vivo® 15 Media + 50 µL
CD3/CD28 Dynabeads (ThermoFisher, 11131D) for four days.
On days 0, 2, and 4, media was collected from each well
and frozen for cytokine analysis. After collection of all timepoints, samples were thawed, centrifuged at 10,000xg for
5 minutes at 4ºC and assessed for IL-2 and IFNγ cytokines
using a 32x2 Simple Plex Cartridge (Protein Simple, SPCKC-PS-006011) and the ELLA automated cytokine instrument.
0
60
40
20
100
80
Viability
Fresh Cryo
****
0
60
40
20
100
80
Viability
Single Dual
*
Figure 1.
Viability comparison immediately post-PBMC isolation from fresh or cryopreserved leukopaks (A) and post-PBMC cryopreservation of these fresh
or cryopreserved leukopaks resulting in cells being cryopreserved once or
twice P value is <0.0001 (B). Data is presented as the mean± standard error
of the mean (SEM). Populations were analyzed by two-sided t-test P value
is 0.0484.
A) B)
were combined and assessed together, dual cryopreservation did not negatively impact T cell proliferation potential,
meaning similar T cell proliferative responses were observed for both the single and dual cryopreservation samples. As expected, variations in T cell response were donor
dependent. Overall, these data support the hypothesis
that both single and dual cryopreservation samples display
similar T cell proliferative responses.
Cryopreservation does not change pro- and
anti-inflammatory cytokines secretion profile
The next question was to investigate the relative difference between single and dual cryopreserved samples in
their ability to be activated and secrete pro-inflammatory
and anti-inflammatory cytokines. To test this, PBMCs were
incubated with IL-2 and anti-CD3/CD28 DynaBeads for
7 days to allow for activation and cytokine secretion.
Following incubation, cell supernatants from days 0, 2, and
7 were assessed for pro- (Figure 3) and anti-inflammatory (Figure 4) cytokines. As expected, cultures containing
X-Vivo® 15 Media alone did not activate and induce cytokine
production for all donors and all time points assessed (Figures 3 and 4). By days 2 and 7, there was slight production
of pro-inflammatory cytokines IL-1β and TNFα in the cultures
containing IL-2 however this cytokine secretion was very
minimal (Figure 3C and E). Following the pro-inflammatory
trend, by days 2 and 7 there was slight production of the
anti-inflammatory cytokine IL-4. However, this production
was only seen in the cultures containing IL-2 and activated
with beads (Figure 3D). Both pro-inflammatory and anti-inflammatory cytokine production was most prominent in
cultures containing IL-2 and beads (Figures 3 and 4). The
overall trend demonstrates that both anti- and pro-inflammatory cytokines were produced and secreted at measurable levels. Importantly, dual cryopreserved cells did not
inhibit this cytokine release.
T cell proliferative response is donor dependent but
not altered by cryopreservation
Since both the fresh and cryopreserved leukopaks resulted
in high viability post-PBMC cryopreservation, the next step
was to assess the functional response of the cells. Cells
were stained with CFSE dye as a means to measure downstream proliferation. After treating cells for four days with
IL-2+Dynabeads, CFSE intensity was measured via FACS. To
allow visual comparison, mean fluorescence intensity (MFI)
of the dye dilution was assessed and a fold change was calculated compared to the negative control X-Vivo® 15 Media
only. Data demonstrated that IL-2 alone did not significantly
increase T cell proliferation but cultures containing IL-2 and
CD3/CD28 beads induced a strong proliferative response
resulting in an average of 0.5 fold change decrease (Figure
2A). Furthermore, when data from all four donors tested
0.0
0.5
1.0
1.5
Fold Change
Combined CFSE Donors
IL2 Single IL2 Dual IL2+Beads Single IL2+Beads Dual
0.0
0.5
1.0
1.5
Fold Change
IL2 Single IL2 Dual IL2+Beads Single IL2+Beads Dual
CFSE Fold Change
Donor 1 Donor 2 Donor 3 Donor 4
Figure 2.
Single and dual cryopreserved PBMCs were CFSE stained and cultured for
for days with X-Vivo® 15, X-Vivo® 15 + IL-2, or X-Vivo® 15 + IL-2 + Dynabeads.
Shown is the fold change decrease in CFSE staining compared to
X-Vivo® 15 only treated PBMCs. (A) Individual donors, data is represented
as mean± standard deviation (SD) (B) combined donors, data is represented
as mean±SEM.
A
B
IL-2 and IFNγ cytokine secretion is variable and
dependent on donor but is not altered by
cryopreservation
PBMCs were incubated with DynaBeads for 4 days in the
absence of IL-2 to ensure the added IL-2 present in the
previous experiment was not influencing the response
and production of the other cytokines. Supernatants were
harvested and assessed for IL-2 and IFNγ cytokine responses from days 0, 2, and 4. The three donors assessed
demonstrated a variable IL-2 and IFNγ cytokine production
response in dual cryopreserved cells compared to single
cryopreserved cells (Figure 5). No significant difference
was measured between IL-2 and IFNγ levels from single and
dual cryopreserved PBMCs.
Discussion
Commonly, PBMCs are used during the drug development
process in T cell proliferation assays to determine whether
a compound or drug will elicit a T cell response to a foreign
subsance. This T cell response can be measured by multiple ways in vitro, but standardly is assessed through proliferation to determine if an adverse drug reaction (ADR) will
potentially occur in vivo. If a drug compound inappropriately binds to T cell receptors, it can result in T cell stimulation and activation associated clinically with delayed-type
hypersensivity (Pichler et. al. 2015). Delayed-type hypersensitvity, as well as other ADRs are negative consequences
that are screened for in vitro; thus ensuring PBMCs
maintain the ability to respond to stimulus is important
for drug development. In addition to T cell interactions,
immune cell signaling is important for compounds to elicit
an immune response, which can also be measured through
cytokine production following incubation of PBMCs with
the compound of interest. For example, during a Th1 reIL2 Single
IL2 Dual
IL2+Beads Single
IL2+Beads Dual
X-Vivo® Single
X-Vivo® Dual
0
02
Day of Timecourse
Single Dual
7 02 7 02 7 02 7 02 7 02 7
200000
100000
150000
50000
pg/mL
IFNy
**
0
0 2
Day of Timecourse
Single Dual
7 0 2 7 0 2 7 0 27 02 7 02 7
100
40
60
80
20
pg/mL
IL-4
0
02 Day of Timecourse
Single Dual
7 02 7 02 7 02 7 0 2 7 02 7
40
20
30
10
pg/mL
IL-12
0
02 Day of Timecourse
Single Dual
7 02 7 02 7 02 7 0 2 7 02 7
400
200
300
100
pg/mL
IL-1β
0
0 2
Day of Timecourse
Single Dual
7 0 2 7 0 2 7 0 27 02 7 02 7
15000
10000
5000
pg/mL
TNFα
Figure 3.
Single and dual cryopreserved PBMCs were cultured with X-Vivo® 15,
X-Vivo® 15 + IL-2, or X-Vivo® 15 + IL-2 + Dynabeads for 4 days. Following treatment, levels of pro-inflammatory cytokines IFNγ (A) IL-12 (B), IL-1β (C), IL-4 (D),
TNFα (E) cytokine release was measured. This data is combined data from
all donors. Data is represented as Mean±SEM, data was analyzed with 2-way
ANOVA and Tukey’s post test, only statistical differences between single
and dual are shown * p<0.05, ** p<0.01, *** p<0.001, p<0.0001.
A
C
E
B
D
X-Vivo Beads Single Beads Dual ® Single X-Vivo® Dual
0
0 2
Day of Timecourse
Single Dual
7 0 2 7 0 2 7 0 2 7 0 2 7 0 2 7
1500
500
1000
pg/mL
IL-10
**
0
0 2
Day of Timecourse
Single Dual
7 0 2 7 0 2 7 0 2 7 0 2 7 0 2 7
8000
4000
2000
pg/mL
6000
IL-6
Figure 4.
Single or dual cryopreserved PBMCs were cultured for seven days with
X-Vivo® 15, X-Vivo® 15 + IL-2, or X-Vivo® 15 + IL-2 + Dynabeads. Shown are
the levels of anti-inflammatory cytokines IL-10 (A) and IL-6 (B) released
following treatment This data is combined data from all donors. Data is
represented as mean±SEM, data was analyzed with 2-way ANOVA and
Tukey’s post-test, only statistical differences between single and dual of
the same condition are shown * p<0.05, ** p<0.01, *** p<0.001, p<0.0001.
A B
Figure 5.
Single and dual cyropreserved PBMCs were cultured with X-Vivo® 15 alone
or X-Vivo® 15 with Dynabeads for 4 days. Shown are the cytokine levels of
IL-2 (A) or IFNγ (B) released following treatment. This data is combined data
from all donors. Data is represented as mean±SEM.
5
4
3
2
1
0
0 2
Day of Timecourse
Single Dual
4 0 2 4 0 2 4 0 2 4
400000
300000
200000
100000
pg/mL
IFNy
X-Vivo Beads Single ® Single X-Vivo Beads Dual ® Dual
0 2
Day of Timecourse
Single Dual
4 0 2 4 0 2 4 0 2 4
A B IL-2
We further assesed the effects of single versus dual
cryopreservation on cytokine production. Supernatants
collected from days 0, 2, and 7 of PBMC culture with IL-2
and CD3/CD28 Dynabeads revealed detectable levels of
both the classical anti-inflammatory and pro-inflammatory
cytokine profiles (Figures 3 and 4). Cells that were cultured
in IL-2 alone however lacked superlative cytokine production, indicating that the PBMCs required sufficient stimulation via IL-2 and activation via CD3/CD28 Dynabeads. To
rule out the effect of exogenous IL-2 in the culture systems
on other cytokine production, cells were cultured without
exogenous IL-2 and IL-2 and IFNγ production was measured. Once again, the data revealed high levels of cytokine
production from cells cultured with CD3/CD28 Dynabeads
and no difference between the single or dual cryopreserved cell samples (Figure 5).
Our data indicate that ready to use Lonza PBMCs provide
a functional product to detect T cell activation and proliferative response to stimulus and produce high levels
of cytokines. Further, cryopreserving the leukopaks prior
to PBMC isolation did not impact the isolation and proliferative potential of PBMCs. Thus frozen leukopaks can
serve as another viable option from which PBMCs can be
isolated.
Both fresh and cryopreserved leukopak types performed
similarly in all assays tested, demonstrating that PBMCs
isolated from both types of starting material are reliable,
reproducible, and interchangeable. Processing samples directly from donation may not be as necessary as believed.
As donor variation and in vivo relevance to a population
becomes more important, there is need to increase the
options of tools to choose from. Researchers now have the
ability to obtain cells from donors of a certain race, medical
history, or genetic predisposition based on the needs of
the research. By providing researchers with the option to
select fresh or cryopreserved leukopaks, as well as cryopreserved PBMCs, we provide an expanded offering of
donors to meet research needs.
Knowledge that PBMCs isolated from fresh and cryopreserved leukopaks maintain function increases the confidence to use previously isolated frozen cells and generate
experimental data with convenience.
sponse, T cells secrete inflammatory cytokines to support
cell-mediated immunity (IFNγ, IL-2, TNFα) while during a
Th2 response, T cells secrete cytokines associated with a
humoral-type immune response (IL-4, IL-5, IL-10), and lastly
during a Th17 response, T cells produce pro-inflammatory cytokines to support responses against extracellular
pathogens (IL-17, IL-21, IL-22) (Oosterhout & Motta, 2005,
Brinkhoff et. al. 2018). Measuring the cytokines produced
by PBMCs after exposure to a compound allows for potential determination of the type of T cell response (Th1 vs Th2
vs Th17). If a product causes a rapid release of unwanted
pro-inflammatory cytokines, negative in vivo reactions such
as anaphylaxis can occur (US FDA, 2014). Furthermore,
ensuring dual cryopreservation does not negatively impact
the ability of PBMCs to release cytokines and therefore
appropriately respond to stimulus, is vital to ensure in vitro
relevance.
PBMCs isolated from cryopreserved leukopaks had a lower
viability than PBMCs isolated from fresh leukopaks. (Figure
1). While dual cryopreservation did decrease viability, the
specifications were still well above industry standards
(70% viability), therefore feasible for further downstream
cell analyses. Other research has also demonstrated that
cryopreservation minimally impacts yield and viability of
PBMCs (Adriana et al. 2009) and does not impact functionality when cells are stored correctly (Angel et al. 2016).
Further, previously published data from Lonza, indicated
the PBMCs maintained similar phenotypes regardless of
cryopreservation type, (Dunnick et. al, 2022). Other studies
also confirm this finding as single cryopreservation did
not impact cell subsets (Thyagarajan et. al. 2018) does not
impact cell subsets.
The lack of impact on the phenotypes detected implies
the cells should maintain similar levels of cytokine release
profiles and T cell proliferative activity. To test whether the
former statement was true, single and dual cryopreserved
cells were exposed to CD3/CD28 Dynabeads to stimulate,
activate, and elicit a T cell proliferative response. Following
T cell activation, cells were measured for proliferation via
CFSE dye dilution. Results indicated that dual cryopreservation of cells did not negatively impact T cell proliferation
as both singly and dually cryopreserved cells had high
levels of proliferation (Figure 2).
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References:
1. Adriana, W., Lin-Ye, S., Cynthia, W., Anne, S., Bruce, B., Raul, L., Dana, S.,
Patricia, D., Deborah, D., Eric, R., Nancy, R., Renee, J., Betty, B., Fran, K.
M., Ruth, D., Elizabeth, M., & Terence, F. (2009). Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for
Functional and Phenotypic T-Cell Characterization. Clinical and Vaccine
Immunology, 16(8), 1176–1186. www.doi.org/10.1128/CVI.00342-08
2. Angel, S., von Briesen, H., Oh, Y.-J., Baller, M. K., Zimmermann, H., &
Germann, A. (2016). Toward Optimal Cryopreservation and Storage for
Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality. Biopreservation and Biobanking, 14(6),
539–547. www.doi.org/10.1089/bio.2016.0046
3. Brinkhoff, A., Sieberichs, A., Engler, H., Dolff, S., Benson, S., Korth, J.,
Schedlowski, M., Kribben, A., Witzke, O., & Wilde, B. (2018). Pro-Inflammatory Th1 and Th17 Cells Are Suppressed During Human Experimental
Endotoxemia Whereas Anti-Inflammatory IL-10 Producing T-Cells Are
Unaffected. In Frontiers in Immunology (Vol. 9, p. 1133).
www.frontiersin.org/article/10.3389/fimmu.2018.01133
4. Dunnick, K. Ngyuen,L, Menezes, A. Sharma A. (2022), Cryopreservation
of Peripheral Blood Mononuclear Cells Does Not Alter Immune Cell
Phenotype. Lonza Tech Note
5. Pichler, W. J., Adam, J., Watkins, S., Wuillemin, N., Yun, J., & Yerly, D.
(2015). Drug Hypersensitivity: How Drugs Stimulate T Cells via Pharmacological Interaction with Immune Receptors. International Archives of
Allergy and Immunology, 168(1), 13–24. www.doi.org/10.1159/000441280
6. Thyagarajan, B., Barcelo, H., Crimmins, E., Weir, D., Minnerath, S., Vivek,
S., & Faul, J. (2018). Effect of delayed cell processing and cryopreservation on immunophenotyping in multicenter population studies. Journal
of Immunological Methods, 463, 61–70.
www.doi.org/10.1016/j.jim.2018.09.007
7. van Oosterhout, A. J. M., & Motta, A. C. (2005). Th1/Th2 paradigm: not
seeing the forest for the trees? European Respiratory Journal, 25(4), 591
LP – 593. www.doi.org/10.1183/09031936.05.00014105
8. US FDA, Guidance for Industry: Immunogenicity assessmen
for therapeutic protein products August 2014,
www.fda.gov/media/85017/download
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