High-Throughput Verification of CAR-T Cell Function
App Note / Case Study
Published: May 16, 2025

Credit: iStock
Efficient and precise validation methods are essential to streamline cell therapy development. Conventional bulk assays often miss the functional heterogeneity of engineered T cells, delaying insights into cytotoxic performance.
This app note highlights a novel picodroplet-based platform that provides rapid, high-throughput assessment of cell product fitness, offering a valuable tool for both QC release and early-stage research.
Download this app note to explore:
- A streamlined method for functional validation of engineered T cells
- How single-cell analysis reveals hidden variability in CAR-T responses
- A practical workflow for rapid cytotoxicity screening in microfluidic droplets
High Throughput CAR-T
Cell Function Verification
in Microfluidic Picodroplets
Introduction
Over the last few years, there has been an increasing interest in cell therapy as a strategy to harness
the immune system to fight tumors. Technologies include reprogramming of T cells collected
from patients to produce personalized medicines. T-cell therapy capitalizes on the body’s immune
systems ability to recognize and kill tumor cells. This ability can be lost or overwhelmed as a cancer
develops. By extracting and manipulating a patient’s T-cells, the ability to attack cancer cells can
potentially be restored. Rapid, accurate assays to qualify the potency of the engineered T-cells
against the target tumor cell are required to support the effective and efficient development
of therapeutics.
This study presents a new picodroplet-based approach for the functional validation of CAR-T
cells. The easy-to-use and robust method combines the benefits of Sphere Bio’s high-throughput
picodroplet technology with a granzyme B assay for studying cell-mediated cytotoxicity (Figure 1).
Engineered T cells and target cells are co-encapsulated in picolitre-volume aqueous droplets
(picodroplets) in an oil emulsion, along with granzyme B substrate. Granzyme B release by the T cell,
a readout of the cells ability to recognize and kill the cancer cell, can be captured to determine the
level of potential anti-cancer activity. In doing so, this method enables rapid cell product efficacy
readouts to streamline the QC release of cell product.
Application Note 10
spherebio.com
p2 Document number: SF - 004801 - AN | spherebio.com
Figure 1. Workflow depicting high throughput CAR-T cell function verification
in microfluidic picodroplets.
About Picodroplet Technology
Picodroplet-based technology has emerged as an attractive microfluidic-based technique
for single cell functional analysis, offering several advantages over conventional tools.
Picodroplets provide a unique microenvironment in which to perform high throughput
analysis of cell secretion and cell-cell interaction studies at a single cell level.
Miniaturized picolitre volumes facilitates rapid mixing and minimized sample dilution,
increasing detection sensitivity while reducing sample requirement and reaction time.
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Figure 2. Principle of Granzyme B detection assay. A) Granzyme B substrate peptide labelled with 5-FAM
(green star) and a QXL®-520 fluorophore quencher (grey star). No fluorescence is detected on excitation
due to Fluorescence Resonance Energy Transfer (FRET) from 5-FAM to QXL®-520 (left side). On cleavage
with Granzyme B the quenching molecule is removed, and fluorescence is emitted from 5-FAM (right side).
B) Application of Granzyme B activity assay to CAR-T cell/target cell interaction. Left: CAR-T cell and nontarget cell, no CAR mediated signal, no Granzyme B release, 5-FAM fluorescence quenched. Right: CAR-T cell
interaction with target cell results in CAR mediated signal, Granzyme B release, substrate peptide cleavage,
release of the quencher and fluorescence is then detected.
About Granzyme B detection in Picodroplets
The release of Granzyme B in picodroplets is detected with a commercially available
Granzyme B assay kit (SensoLyte® Granzyme B Activity Assay Kit, Anaspec, Fremont,
CA) adapted for use in picodroplets. The assay is based on the cleavage of a Granzyme
B substrate peptide, labelled with a 5-FAM fluorophore and a QXL®-520 fluorophore
quencher. In the intact, uncleaved, state, no fluorescence is emitted upon excitation
of the fluorophore - due to the quenching effect of the nearby QXL®-520 molecule
(Figure 2A). In the presence of Granzyme B the peptide substrate is cleaved, releasing
the quenching molecule, and resulting in a fluorescent signal (Figure 2A and 2B).
+Granzyme B
A
B
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Granzyme B detection in Picodroplets
To demonstrate the general feasibility
of the assay, we initially generated
two populations of picodroplets of
~450 pL volume with our Pico-Capture®
instrument. One population contained
only the fluorogenic Granzyme B
substrate peptide (negative control),
while the other co-encapsulated
recombinant Granzyme B together
with the substrate peptide.
Figure 3 shows micrographs of the
two populations in brightfield (left)
and green fluorescence (right) after 2h
incubation at 37ºC. Only picodroplets
containing both Granzyme B and the
substrate peptide (bottom) show clearly
detectable fluorescence after a 2h
incubation, while picodroplets with
substrate peptide only (top) exhibit only
very low to no background fluorescence.
Granzyme B detection assay
with T cells and target cells
In the next step we co-encapsulated
polyclonal human donor T cells
genetically modified to express a CAR
directed against Prostate-Specific
Membrane Antigen (PSMA), with
target cells expressing PSMA (PC3-
LN3-PSMA) and the fluorogenic
Granzyme B substrate peptide.
We used an in-house custom-made
biochip with two separate aqueous inlets
to keep CAR T and target cells apart until
immediately prior to encapsulation. Cell
concentrations were adjusted so that
only ~20% of picodroplets contained
a CAR T cell, while >75% received at
least one target cell (according to a
Poisson distribution). Note, 150,000 out
of 1 million generated picodroplets will
contain one CAR-T cell and at least one
target cell. Consequently, ~15% (0.75 x
0.2) of picodroplets contained at least one
CAR-T and one target cell. As a control we
co-encapsulated CAR T cells with PC3-LN3
cells lacking PSMA expression, together
with the fluorogenic peptide substrate
under otherwise identical conditions.
Figure 3. Detection of Granzyme B activity in picodroplets using
a fluorogenic 5-FAM/ QXL®-520 substrate peptide. Brightfield (left
side) and Fluorescence micrographs (right side) 2h post generation.
Scale bar: 100 μm.
Substrate peptide only
Substrate peptide +
Granzyme B
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Picodroplets were collected in an
Eppendorf tube and imaged after
2, 4, and 24 hours of incubation
under a fluorescence microscope to
detect Granzyme B activity. Figure
4 shows fluorescent micrographs of
picodroplets containing non-expressing
control cells (top) and PC3-LN3-PSMA
target cells (bottom), imaged after
2, 4, and 24 hours (left to right).
Picodroplets containing PC3-LN3 control
cells showed very little Granzyme B
activity after 2-4h. In picodroplets with
PC3-LN3-PSMA cells, multiple fluorescent
picodroplets are indicative of Granzyme
B released by CAR-T cells in response to
encountering one or more target cell(s).
Only after prolonged incubation (24h)
is increased fluorescence also observed
in the negative control, possibly due to
unspecific stimulation or Granzyme B
leaking from dead or apoptotic CAR-T cells.
Figure 4. Granzyme B
assay in picodroplets
with co-encapsulated
CAR-T and target cells
(bottom) or control
cells (top). Fluorescence
micrographs of
picodroplets after 2, 4,
and 24 h (left to right).
Scale bar: 100 µm.
CAR-T + control cells
CAR-T + target cells
2h 4h 24h
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Granzyme B detection using Cyto-Mine®
After establishing that Granzyme B release
by CAR-T cells can be detected with the
5-FAM/QXL®-520 fluorogenic peptide
substrate we proceeded to establish
detection and sorting of Granzyme B
positive picodroplets using Sphere Bio’s
Cyto-Mine® Single Cell Analysis System.
We co-encapsulated CAR-T cells with PC3-
LN3-PSMA target cells and the 5-FAM/
QXL®-520 peptide substrate in 450 pL sized
picodroplets as described above with
Sphere Bio’s Pico-Capture® instrument. The
emulsion was collected and loaded into
a Cyto-Cartridge® for injection, analysis
and sorting on the Cyto-Mine® platform.
The fluorescence signal was detected
immediately after all picodroplets had
been injected into Cyto-Mine® (t=0), as
well as after an additional incubation of
1 and 2h at 37ºC inside the instrument.
Figure 5 shows scatter plots of picodroplet
size (y-axis) versus green fluorescence
(x-axis) obtained at the indicated time
points. At the earliest time point, a clear
fluorescent signal was detectable in ~1.25%
of picodroplets. This positive population
increased over time up to 2.95% of
picodroplets after 2h. As only 15% of
picodroplets contain both a CAR-T cell and
at least one target cell, this corresponds
to an actual positive rate of ~20%.
We then proceeded to sort 5,000 positive
picodroplets to visually confirm the
presence of a fluorescent signal in these
picodroplets. After sorting, we carefully
removed the Cyto-Cartridge® from
the instrument and placed it under a
fluorescence microscope to image the
picodroplets in the dispensing chamber.
Figure 6 shows a brightfield (left)
and fluorescent micrograph (right) of
picodroplets in the dispensing chamber
of the Cyto-Cartridge®. A clear enrichment
of fluorescent picodroplets is evident.
Analysis with ImageJ software using a
Hough Circle Transform algorithm resulted
in a total count of 1,112 picodroplets.
170 of these picodroplets were deemed
to contain little to no fluorescence (i.e.,
false positive, based on near background
signal and a dark picodroplet border);
consequently, 942 picodroplets (~85%)
were true positives. This corresponds
to a 28-fold enrichment compared
to the non-sorted population with
2.95% positives after 2h incubation.
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Figure 5. Detection of Granzyme B activity in picodroplets with
Cyto-Mine®. Screenshots of Cyto-Mine® software during detection/
sorting, showing scatterplot of picodroplet size (Intra picodroplet, ms)
against Fluorescence signal (Green Average, V) at t=0 (A), 1h (B) and 2h
(C). The middle and bottom panel also show polygon gates used for
determining percentage of positive picodroplets and for sorting.
Figure 6. Brightfield (A) and fluorescent micrograph (B) of sorted
picodroplets in dispensing chamber. Scale bar 100 μm.
Figure 7. Cyto-Mine® - Sphere Bio’s automated and fully integrated
single cell analysis system.
A
B
Intra Picodroplet
Green Average
0.2
Scatterplot
0.8
0.6
0.4
0.0
0.5 1.0 1.5 2.0 2.5 3.0
1.0
1.2
1.4
A
C Intra Picodroplet
Green Average
0.4
0.8
0.6
0.2
0.5 1.0 1.5 2.0 2.5 3.0
1.0
1.2
1.4
Scatterplot
B Scatterplot Intra Picodroplet
Green Average
0.4
0.8
0.6
0.2
0.5 1.0 1.5 2.0 2.5 3.0
1.0
1.2
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Conclusions
This study demonstrated that researchers can use picodroplet technologies to
monitor CAR-T activation at relevant early timepoints after co-culture. This has
been accomplished by adapting established assays to the picodroplet format.
Current strategies rely on screening in bulk cultures to establish CAR-T cell
product profiles. Readouts of cytotoxicity take time and are not currently
included in release criteria for cell products. This information is gathered
after the fact or not at all. The ability to run a short assay, to give a readout of
potential efficacy fitness of a cell product could be of great value to cell therapy
development. Picodroplet technology enables screening at the single cell level
in a high throughput and rapid process. Coupling this utility to granzyme B
release by co-encapsulated CAR-T and target cells offers the potential to release
highly functional products with the best chance of impact for patients.
In addition to rapid release analysis for a cell product, this co-encapsulation
approach could be utilized by cell therapy developers to interrogate other
readouts of cellular fitness and cytotoxicity. This could include other proteomic
markers of cytotoxicity or direct cytotoxicity of the target cells. Bulk T cells
cultures display a variety of phenotypic and metabolic subsets. Investigating
the cytotoxic efficacy at a single T cell level has the potential to inform on each
of these subsets individually and guide T cell engineering towards more potent
subsets. An additional role for co-encapsulation in picodroplets could be rapid
screening of candidate CAR/TCR gene modified cells for drug development.
The work presented here was kindly supported by MedCity Ltd, as part of a Collaborate
to Innovate partnership with King’s College London. Grant Ref. C2N-AT.028.
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Notes
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Notes
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Notes
Sphere Bio Ltd is an ISO 9001:2015 accredited company.
The trademarks used herein are the property of Sphere
Bio Ltd or their respective owners.
For research and development purposes only.
Product specifications subject to change without notice.
Content ©Sphere Bio Ltd.
contact@spherebio.com
spherebio.com
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