Gene Delivery

An Optimized Workflow for Generating Anti-CD19 CAR T Cells
by mRNA Electroporation

Michael Kirmiz, Kristin Lind, Elizabeth Dreskin, Chris Brampton
Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547

Abstract

Chimeric antigen receptor (CAR) T-cell therapy is a revolutionary and promising approach for cancer
treatment. However, its potential is limited by technical challenges and safety risks associated withviral transduction–based CAR T-cell generation workflows. Nonviral methods, such as electroporation,
represent a potentially safer and less challenging approach for generating CAR T cells. Here, we describe
the generation of anti-CD19 CAR T cells using the Gene Pulser™ Xcell Electroporation System in an
mRNA electroporation–based CAR T-cell workflow optimized for efficiency and viability. Our workflow
and optimized conditions resulted in CAR transfection efficiencies as high as ~70 and ~90% in primaryand immortalized human T cells, respectively, at 6 hr after electroporation, and a viability of ~60% inprimary human T cells. These data highlight the utility of the Gene Pulser Xcell Electroporation System forgenerating CAR T cells without the use of viral transduction.
Introduction
CAR-expressing T cells are a promising therapeutic strategy
for addressing cancers in humans, as demonstrated by theirsuccess in the treatment of various human B cell lymphomas andleukemias. In this approach, patient or donor T cells are harvested,
genetically reprogrammed to express a CAR that targets anantigen selectively expressed on the surface of cancer cells, andinfused into a patient for therapeutic use. The binding of a CAR T
cell to its ligand results in major histocompatibility complex (MHC)–
independent activation and mobilization of CAR T cells against the
antigen-expressing cancer cells. CAR T-cell therapy is associatedwith numerous successful therapeutic outcomes in patients withspecific B-cell lymphomas and/or leukemias (Sterner and Sterner2021). The best known and most clinically validated CAR T cells
recognize CD19, a cell surface antigen found on normal andmalignant B cells. CD19-directed CAR T cells are shown to target
different leukemias and lymphomas, leading to long-term disease
remission. Given these significant results, much biomedical andclinical research is currently focused on workflows leading tosuccessful production of viable CAR T cells for therapeutic use.

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To date, all U.S. Food and Drug Administration–approved CAR T
cell therapies have relied on viral transduction to achieve
expression of a CAR in patient T cells (National Cancer Institute,2022). This method is used primarily because of its relatively highrate of gene transfer, scalability, and the well-studied techniques
associated with viral transduction. However, this approachcomes with several potential health risks, including insertionalmutagenesis with oncogenic potential and other associated side
effects (Vannucci et al. 2013; Zhang et al. 2017; Stock et al. 2019;
Sterner and Sterner 2021).
Transiently expressed CARs produced by mRNA delivery can
avoid the potential risks associated with viral transduction
because transfected CAR mRNA cannot be integrated into thehost genome and its expression diminishes with time due tomRNA turnover (Foster et al. 2019). In a pivotal study, Krug et al.
(2014) successfully implemented mRNA transfections in a good
manufacturing practice (GMP)–compliant CAR T-cell workflow.
In their study, mRNA transcripts encoding CARs were delivered
via electroporation into human T cells, resulting in highly functional
CAR T cells. More recently, the same group implemented
this mRNA electroporation workflow to produce CAR T cells
at clinical scale using the Gene Pulser Xcell Electroporation
System, resulting in cytolytic CAR T cells that targeted melanomas(Wiesinger et al. 2019). While primary human T cells are relatively

An Optimized Workflow for Generating Anti-CD19 CAR T Cells by mRNA Electroporation

Prepare Precursor Materials

Primary Human T Cells

CAR mRNA

Monitor Cell Growth

Assay T-Cell Viability

Electroporate T Cells

ZOE Cell Imager

TC20 Cell Counter

Gene Pulser Xcell Electroporation System

Assay Expression and CAR T-Cell Viability

ZE5 Cell Analyzer

ReadiDrop 7-AAD

StarBright Antibodies

ZOE Cell Imager

Recover CAR T Cells

TC20 Cell Counter

CAR T Cells

Fig. 1. CAR T-cell workflow. First, precursor materials are prepared: primary human T cells are obtained by leukapheresis and negative selection, and CAR mRNA is analyzed
for quality, size, and concentration using a 2100 Bioanalyzer Instrument (Agilent Technologies; not shown). T-cell growth is checked using the ZOE Fluorescent Cell Imager,
and viability is confirmed using the TC20 Automated Cell Counter. Then, the T cells are electroporated with CAR mRNA using the Gene Pulser Xcell Electroporation System.
Transfection efficiency is measured with the ZOE Cell Imager using GFP. The viability of CAR T cells recovered from electroporation is then tested using Trypan Blue and theTC20 Cell Counter as well as ReadiDrop 7-AAD and the ZE5 Cell Analyzer. CAR expression is also analyzed using StarBright Antibodies with the ZE5 Cell Analyzer. CAR T cells
can then be prepared for infusion in a patient. 7-AAD, 7-Aminoactinomycin D; CAR, chimeric antigen receptor; GFP, green fluorescent protein.

difficult to transfect, Wiesinger and colleagues reported a hightransfection efficiency (~90%) while concurrently preserving
cellular health and viability (~70%). Here, we describe theoptimization of an mRNA transfection–based CAR T-cell workflowthat includes the development of a CAR, optimization of anmRNA electroporation protocol using green fluorescent protein(GFP), electroporation of primary and immortalized human T
cells with CAR mRNA, confirmation of GFP and CAR expression
by live cell imaging and flow cytometry, and pre- and postelectroporation viability analysis by automated cell counting andflow cytometry (Figure 1).

Human Cell Culture, Cell Counting, and Imaging

Jurkat T cells (American Type Culture Collection, Clone E61) were cultured in RPMI 1640 (Thermo Fisher Scientific Inc.,#21875034) containing 10% Fetal Bovine Serum (Hyclone,#SH30071.03) and 1% Penicillin-Streptomycin (Thermo Fisher
Scientific, #15140122) using standard cell culture procedures.
Cells were grown in suspension and passaged every 2–4 days
and kept below a density of 3 x 106 cells/ml in culture media.
Frozen human CD3+ T-cells (AllCells, product LP, CR, CD3+, NS,
25M) were thawed and expanded for 48 hr at a concentrationof 1 x 106 cells/ml in ImmunoCult-XF T Cell Expansion Media(STEMCELL Technologies Inc., #10981) supplemented with Human
Recombinant Interleuken-2 (IL-2) (STEMCELL Technologies,#78036) at a concentration of 10 ng/ml and ImmunoCult Human
CD3/CD28 T Cell Activator (STEMCELL Technologies, #10991)
at a concentration of 25 μl beads/ml. Cells in suspension wereroutinely counted using a TC20 Automated Cell Counter (Bio-Rad™
Laboratories, Inc., #1450102) and Trypan Blue (Bio-Rad, #1450021)
at a 1:2 dilution. Cells were routinely examined using a ZOE
Fluorescent Cell Imager (Bio-Rad, #1450031).

Materials and Methods
Generation of an Anti-CD19 CAR and mRNA

The anti-CD19 CAR used in this study was designed to containextracellular granulocyte-macrophage colony–stimulating
factor (GM-CSF) leader sequence, anti-CD19 VH and VL
chains, transmembrane and cytoplasmic CD28 domains, anda cytoplasmic CD3z domain. See Figure 2A for a schematic of
the anti-CD19 CAR used in this study. DNA encoding this CAR
was synthesized and cloned into pTwist-CMV (Twist Bioscience
Corporation). We purchased a large-scale custom in vitro mRNA
synthesis kit that incorporates the 5' Cap 1 structure (Aldevron).
This mRNA was modified to include a more substantial poly(A)tail using E. coli Poly(A) Polymerase (NEB, catalog #M0276S) andpurified using the Monarch RNA Cleanup Kit (NEB, #T2030S).
Concentration, size, and purity of mRNA was estimated usinga 2100 Bioanalyzer Instrument (Agilent Technologies, Inc.,#G2939BA) in combination with an RNA 6000 Nano Kit (Agilent
Technologies, #5067-1511). DasherGFP mRNA (Aldevron,#3870-0200) containing a 5' Cap 1 was used to track mRNAdelivery and expression as a control. RNA was aliquoted andstored at –80°C to minimize freeze-thaw cycles.
© 2022 Bio-Rad Laboratories, Inc.

mRNA Transfections via Electroporation

Prior to and following electroporation, cell density and viability
were determined using Trypan Blue staining in conjunction
with a TC20 Automated Cell Counter. Cells were pelleted bycentrifugation at 750 x g for 5–10 minutes and resuspended in
Gene Pulser Electroporation Buffer (Bio-Rad, #1652677). For eachelectroporation, 2 x 106 Jurkat or primary human T cells were
electroporated using the Gene Pulser Xcell Electroporation System(Bio-Rad, #1652660) in 200 µL of Gene Pulser Electroporation
Buffer containing mRNA. In brief, cells were vortexed gently,
loaded into 0.2 cm gap cuvettes (Bio-Rad, #1652086), andelectroporated using the following conditions, which are based2

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An Optimized Workflow for Generating Anti-CD19 CAR T Cells by mRNA Electroporation

B. Synthesized anti-CD19 CAR mRNA

A. Anti-CD19 CAR

bp
VL

VH

GM-CSF

Ladder

CAR mRNA

bp

4,000

4,000

2,000

2,000

1,000

1,000

500

500

200

200

25

25

Anti-CD19 scFv

SpacerExtracellularTM

Intracellular

CD28

PM

Cyto
CD3z

Fig. 2. Anti-CD19 CAR design and mRNA synthesis. A, schematic of anti-CD19 CAR employed in this study. The anti-CD19 CAR is expressed at the plasma membranewith an extracellular GM-CSF leader sequence, an anti-CD19 scFv domain (containing VH and VL domains), transmembrane and cytoplasmic CD28 domains, and an
intracellular CD3z domain; B, Synthesized anti-CD19 CAR mRNA size fractioned using a 2100 Bio-Analyzer Instrument (Agilent Technologies). CAR, chimeric antigen receptor;
cyto, cytoplasmic; GM-CSF, granulocyte-macrophage colony–stimulating factor; PM, plasma membrane; scFv, single-chain variable fragment; TM, transmembrane; VH, heavy
chain variable domain; VL, light chain variable domain.

on our prior study (Wong et al., 2020): square wave, 200 V, 2 ms
pulse length, 1 pulse. Following electroporation, cells were cultured
in supplemented media until further analysis. Cell viability andcount were assayed (as described at the beginning of this section)
immediately prior to cell imaging, staining, and flow cytometry.

Table 1. Reagents for flow cytometry.

Cell Imaging, Staining, and Flow Cytometry

Brightfield and GFP fluorescence micrographs were collectedusing a ZOE Cell Imager with identical acquisition settings acrossall time points. To analyze electroporation efficiency and viability byflow cytometry, cells were harvested by centrifugation at 750 x g
for 5–10 minutes and resuspended in an ice-cold staining buffer
containing 1% Bovine Serum Albumin Solution (MilliporeSigma,#A7284) in Dulbecco’s Phosphate Buffered Saline (Thermo Fisher
Scientific, #14190144). For each sample, 1 x 106 cells were stainedwith 100 μl of an antibody cocktail, prepared using the samestaining buffer, for 30 min at room temperature. Following staining,cells were pelleted by centrifugation, washed in 1 ml of staining
buffer twice and resuspended in 0.5 ml of staining buffer. 20 μl of
ReadiDrop 7-AAD (Bio-Rad, #1351102) was added immediately
prior to flow cytometry analysis. Flow cytometry reagents andtheir dilutions are described in Table 1. Flow cytometry analysis
was performed using a ZE5 Cell Analyzer (Bio-Rad, #12004279)
configured with five lasers (355/405/488/561/640 nm) andcontrolled by Everest Software. Laser power and detector
photomultiplier tube (PMT) voltage were optimized and kept
identical for all experimental samples and compensation controls.
Experimental data were either analyzed within Everest Softwareand exported to Microsoft Excel Software for presentation, or
separately exported as Flow Cytometry Standard (FCS) files foroffline analysis using FCS Express Software (De Novo Software).

© 2022 Bio-Rad Laboratories, Inc.

Reagent

Ordering Information

Dilution

Rat Anti-Mouse CD8 Alpha:
StarBright Violet 440

Bio-Rad, #MCA609SBV440

1:5

Rat Anti-Mouse CD25:StarBright
UltraViolet 400

Bio-Rad, #MCA1260SBUV400

1:5

FITC-Labeled Monoclonal Anti-FMC63
scFv Antibody, Mouse IgG1 (anti-CD19
CAR antibody)

Acro Biosystems,#FM3-FY45

1:50

FITC-Labeled Human CD19 (20-291)
Protein, Fc Tag

Acro Biosystems,#CD9-HF251

1:10

ReadiDrop 7-AAD

Bio-Rad, #1351102

1:25

7-AAD, 7-aminoactinomycin D; CAR, chimeric antigen receptor; Fc, fragment
crystallizable region; FITC, fluorescein isothiocyanate; IgG1, immunoglobulin G1.

Results
Construction and Expression of an Anti-CD19 CAR

A schematic of the topology of the anti-CD19 CAR used in thisstudy is provided in Figure 2A. The presence of anti-CD19 VH and
VL chains imparts CD19-binding specificity. Plasmid DNA encoding
this CAR was the basis for synthesis of mRNA transcripts, whichwere used for the electroporation experiments performed in thisstudy. An example of synthesized mRNA, size-fractioned using a
Bioanalyzer Instrument, is shown in Figure 2B. The prominent bandcorresponds to the correct molecular weight of the anti-CD19 CAR
mRNA that has been 5'-capped and 3'-polyadenylated. A relatively
pure mRNA sample is important for achieving optimal cellularviability and transfection efficiency. We routinely confirmed thepurity and quantity of the GFP and anti-CD19 CAR mRNAs using
the Bioanalyzer Instrument.

3

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An Optimized Workflow for Generating Anti-CD19 CAR T Cells by mRNA Electroporation

the ZOE Fluorescent Cell Imager to monitor GFP expression
following transfection of GFP mRNA. Samples were evaluatedevery hour using identical acquisition settings. GFP expression
was lowest immediately after electroporation and peaked at
6 hr. Representative images acquired at 1, 2, 3, and 6 hr after
electroporation are shown in Figure 3.

Optimization of GFP mRNA Electroporation of Human T Cells

We previously identified optimal parameters for delivery of GFP
mRNA to human CD8+ T cells using the Gene Pulser Xcell
Electroporation System (square wave, 200 V pulse, 2 ms pulse
length, 1 pulse, 0.2 cm cuvette) (Wong et al. 2020). To verify thatthese conditions were sufficient for use with the CD3+ primary
human T cells and mRNA constructs used in this study, we utilized
1 hr

2 hr

3 hr

6 hr

0 μg/mlmRNA

60 μg/mlmRNA

Fig. 3. GFP expression in primary human T cells after electroporation. Merged brightfield and GFP ZOE Imager micrographs of primary human T cells mock-transfected ortransfected with 60 μg/ml GFP mRNA and imaged at 1, 2, 3, and 6 hr after electroporation. Scale bar represents 100 μm. GFP, green fluorescent protein.
B. anti-CD19 CAR

30,000Mean fluorescence intensity

Mean fluorescence intensity

A. GFP
25,00020,00015,00010,000
5,000
0
0

20

40

60
80
100
mRNA concentration, mg/ml

120

140

1,200
1,000
800
600
400
200

160

0
0

20

40

60
80
100
mRNA concentration, mg/ml

120

140

160

Fig. 4. Optimization of mRNA electroporation in Jurkat T cells. A, measurements of GFP expression intensity from GFP mRNA dose-response experiment; B, measurementsof anti-CD19 CAR immunolabeling intensity from anti-CD19 CAR mRNA dose-response experiment. Expression was assayed 6 hr ( ) and 24 hr ( ) after electroporation.
CAR, chimeric antigen receptor; GFP, green fluorescent protein.
Table 2. Transfection efficiency in Jurkat T cells.
Time after
Electroporation, hr

624

CAR-Positive Cells, %mRNA, µg/ml

GFP-Positive
Cells, %Anti-CD19 CAR

0

0.00

0.00

0.04

20

97.19

88.23

83.14

CD19-FITC

60

98.13

89.37

86.85

150

98.05

84.25

79.28

0

0.02

0.01

0.07

20

97.30

34.83

14.20

60

97.36

73.60

63.50

150

97.96

71.73

65.18

CAR, chimeric antigen receptor; GFP, green fluorescent protein; FITC, fluorescein isothiocyanate.

© 2022 Bio-Rad Laboratories, Inc.

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An Optimized Workflow for Generating Anti-CD19 CAR T Cells by mRNA Electroporation

We optimized the concentration of mRNA used in electroporations,as well as the length of time required after electroporation, for
optimal protein expression in human T cells. We carried out adose-response experiment in human leukemic Jurkat T cells to
examine the impact of increasing concentrations of GFP-encodingmRNA (20, 60, or 150 μg/ml) on GFP expression assayed at
6 and 24 hr after electroporation. We observed a dose-dependent
response in GFP expression (reported as mean GFP fluorescenceintensity, as measured by flow cytometry) at both 6 and 24 hr
after electroporation (Figure 4A, Table 2). Interestingly, all tested
concentrations of GFP mRNA resulted in greater than 97%
positivity rate for GFP expression at both time points, suggestingthat we had achieved optimal GFP transfection efficiency under
our experimental conditions.

Generation of Primary Human Anti-CD19 CAR T Cells by mRNA Electroporation

Using the optimized conditions described above, we next examinedthe expression of anti-CD19 CAR in primary human T cells following
electroporation-based mRNA transfections. As T-cell activation
improves transfection efficiency (Zhang et al. 2018), we activatedand expanded primary CD3+ primary human T cells with IL-2 andanti-CD3/CD28 beads prior to electroporation. At 6 and 24 hr after
electroporation, samples were analyzed for surface expression ofanti-CD19 CAR using the same approach used in Jurkat T cells(that is, staining with an anti-CD19 CAR antibody and recombinant
CD19 protein). We additionally identified CD8+ primary T cells
using CD8 immunolabeling. In these experiments, cytotoxic
CD8+ T cells demonstrated similar frequencies of anti-CD19 CAR
expression, at both 6 and 24 hr after electroporation, relative tothe global T-cell population (Figures 5 and 6). Similar to Jurkat T
cells, CAR expression was highest in primary T cells at 6 hr after
electroporation. These results demonstrate the success of thisworkflow in generating CAR T cells expressing functional anti-CD19
CAR at the cell surface.

Optimization of Anti-CD19 CAR mRNA Electroporation of Human T Cells

Following the same approach we applied earlier, we performed
a dose-response experiment examining the impact of increasing
amounts of anti-CD19 CAR-encoding mRNA (20, 60, or 150 μg/ml)
on anti-CD19 CAR expression in Jurkat T cells. In these experiments,
we employed parallel confirmatory detection methods in conjunction
with analysis by flow cytometry (Table 2):■■

Viability Analysis of Anti-CD19 CAR T Cells

Cellular viability is a critical parameter in the execution of any
transfection workflow. Therefore, in our experiments, we examined
the impact of these anti-CD19 CAR mRNA electroporation–basedtransfections on primary human T-cell viability using two
orthogonal and corroborative methods: automated cell countingof Trypan Blue–stained cells, and flow cytometric analysis of
ReadiDrop 7-AAD–stained cells. Viability measurements fromboth methods were largely in agreement (Table 3). We found that
untransfected cells had a resting viability of 70–80%, and thatmock electroporations (without addition of mRNA) had a negligible
impact (≤2% reduction). Electroporation of anti-CD19 CAR mRNA
resulted in a viability of ~60%. We speculate that this further impact
on viability would occur with the inclusion of any protein-coding
mRNA, as the inclusion of GFP mRNA in place of anti-CD19
CAR mRNA also reduced viability (to 69%). Figure 7 and Table 3
summarize these data and illustrate the relatively low impactof using these specific electroporation conditions on primary
T-cell viability in mRNA CAR T-cell workflows. These results also
highlight the accuracy and reproducibility of viability measurements
captured via these two distinct and orthogonal approaches.

 o confirm surface expression of the anti-CD19 CAR, weT
employed anti-CD19 CAR surface immunolabeling using ananti-CD19 CAR antibody
 o confirm that the anti-CD19 CAR was retaining anti-CD19
Treactivity (i.e., that the anti-CD19 CAR was functional), we
stained cells with FITC-conjugated recombinant CD19 protein

Further, we analyzed the extent of staining at 6 and 24 hr after
electroporation. Consistent with our GFP mRNA results, we saw adose-dependent response in anti-CD19 CAR expression (reported
as mean fluorescence intensity), detected via either staining
method, at both 6 and 24 hr after electroporation (Figure 4B).
Additionally, at 6 hr after electroporation, we did not observea substantial dose-dependent response in the total number ofanti-CD19 CAR-positive cells (Table 2); all tested concentrationsof anti-CD19 CAR mRNA resulted in greater than 79% positivity
for CAR expression. This suggests that we had achieved maximalanti-CD19 CAR transfection efficiency at this time point with
our experimental conditions. This result was generally similar
at 24 hr after electroporation, although 20 μg/ml mRNA resulted
in substantially fewer CAR-positive cells (14–35%) than both
60 and 150 μg/ml mRNA (64–74% CAR-positive cells) (Table 2).
Further, all mRNA doses resulted in higher expression at 6 hr after
electroporation than at 24 hr after electroporation (Table 2). Basedon these results, we elected to transfect anti-CD19 CAR mRNA ata concentration of 150 μg/ml for the remainder of the experimentsin this study, expecting that this would result in the highest levelof anti-CD19 CAR expression.

© 2022 Bio-Rad Laboratories, Inc.

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An Optimized Workflow for Generating Anti-CD19 CAR T Cells by mRNA Electroporation

Total T Cells
No mRNA, pulse

CAR mRNA, pulse

CAR mRNA, pulse
105

104

104

104

104

103
2.25%

102
101

103
2.33%

102
101

10 0
101

102
103
anti-CD19 CAR

104

105

103
67.99%

102
101

10 0
10 0

CD25 SBUV 400

105

CD25 SBUV 400

105

CD25 SBUV 400

CD25 SBUV 400

No mRNA, no pulse
105

101

102
103
anti-CD19 CAR

104

105

66.37%

102
101

10 0
10 0

103

10 0
10 0

101

102
103
anti-CD19 CAR

104

105

10 0

101

102
103
CD19 FITC

104

105

CD8+ T Cells
No mRNA, pulse

CAR mRNA, pulse

CAR mRNA, pulse
105

104

104

104

104

103
2.43%

102
101

103
2.26%

102
101

10 0
101

102
103
anti-CD19 CAR

104

105

103
69.01%

102
101

10 0
10 0

CD25 SBUV 400

105

CD25 SBUV 400

105

CD25 SBUV 400

CD25 SBUV 400

No mRNA, no pulse
105

101

102
103
anti-CD19 CAR

104

105

69.98%

102
101

10 0
10 0

103

10 0
10 0

101

102
103
anti-CD19 CAR

104

105

10 0

101

102
103
CD19 FITC

104

105

Fig. 5. Anti-CD19 CAR expression in total and CD8+ primary human T cells 6 hr after electroporation. Flow cytometry scatter plots (CD25+ vs. anti-CD19 CAR+
or CD19 FITC) measured from total or CD8+ primary human T cells. The gate shown in each scatter plot circumscribes CAR-positive cells. CAR, chimeric antigen receptor;
FITC, fluorescein isothiocyanate; SBUV, StarBright UltraViolet.
Total T Cells
No mRNA, pulse

CAR mRNA, pulse

CAR mRNA, pulse
105

104

104

104

104

103
3.29%

102
101

103
4.14%

102
101

10 0
101

102
103
anti-CD19 CAR

104

105

103
51.10%

102
101

10 0
10 0

CD25 SBUV 400

105

CD25 SBUV 400

105

CD25 SBUV 400

CD25 SBUV 400

No mRNA, no pulse
105

101

102
103
anti-CD19 CAR

104

105

43.89%

102
101

10 0
10 0

103

10 0
10 0

101

102
103
anti-CD19 CAR

104

105

10 0

101

102
103
CD19 FITC

104

105

CD8+ T Cells
No mRNA, pulse

CAR mRNA, pulse

CAR mRNA, pulse
105

104

104

104

104

103
4.99%

102
101

103
5.64%

102
101

10 0
101

102
103
anti-CD19 CAR

104

105

103
53.77%

102
101

10 0
10 0

CD25 SBUV 400

105

CD25 SBUV 400

105

CD25 SBUV 400

CD25 SBUV 400

No mRNA, no pulse
105

101

102
103
anti-CD19 CAR

104

105

47.56%

102
101

10 0
10 0

103

10 0
10 0

101

102
103
anti-CD19 CAR

104

105

10 0

101

102
103
CD19 FITC

104

105

Fig. 6. Anti-CD19 CAR expression in total and CD8+ primary human T cells 24 hr after electroporation. Flow cytometry scatter plots (CD25+ vs. anti-CD19 CAR+
or CD19 FITC) measured from total or CD8+ primary human T cells. The gate shown in each scatter plot circumscribes CAR-positive cells. CAR, chimeric antigen receptor;
FITC, fluorescein isothiocyanate; SBUV, StarBright UltraViolet.
© 2022 Bio-Rad Laboratories, Inc.

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An Optimized Workflow for Generating Anti-CD19 CAR T Cells by mRNA Electroporation

No mRNA, no pulse

No mRNA, pulse
73.40%

64

128

64

0
101

102

103
7-AAD

104

105

192

128

128

64

0
10 0

62.56%

192
FSC 488/10-A

128

256
68.79%

192
FSC 488/10-A

192

CAR mRNA, pulse

256

73.26%

FSC 488/10-A

GFP mRNA, pulse

256

FSC 488/10-A

256

64

0
10 0

101

102

103
7-AAD

104

105

0
10 0

101

102

103
7-AAD

104

105

10 0

101

102

103
7-AAD

104

105

Fig. 7. Viability analysis of primary human T cells transfected with anti-CD19 CAR. Flow cytometry scatter plots (7-AAD+ vs. FSC) measured from primary human T cells.
Cells were assayed 6 hr after electroporation (or mock-electroporation). Gates circumscribe live cells (cells negative for 7-AAD). Percentages shown in each panel refer to thepercentage of viable cells. 7-AAD, 7-aminoactinomycin D; CAR, chimeric antigen receptor; FSC, forward scatter; GFP, green fluorescent protein.

indicates that either approach is sufficient to assay cell healthand viability in this CAR T-cell workflow. Our immunostaining
and flow cytometry experiments revealed robust cell surface
expression of anti-CD19 CAR in immortalized and primary human
T cells, including CD8+ primary human T cells. Importantly, in our
experiments, the binding of the anti-CD19 CAR to FITC-conjugatedrecombinant CD19 protein most likely indicates that the anti-CD19
CAR is properly folded and able to bind its ligand.

Table 3. Viability.
Viability, %Electroporation Condition

Trypan Blue

ReadiDrop 7-AAD

No mRNA, no pulse

81

73.26

No mRNA, pulse

79

73.40

GFP mRNA, pulse

69

68.79

CAR mRNA, pulse

58

62.56

7-AAD, 7-aminoactinomycin D; CAR, chimeric antigen receptor; GFP, green
fluorescent protein.

The optimal electroporation conditions determined for primary
human T cells in this study, including electrical parameters,
expression time point, and mRNA concentration, should serve as a
useful guide for CAR T-cell development (Table 4). However, theseparameters may not be optimal for all experimental conditions(that is, for all cell types, CAR constructs, mRNA preparations, etc.).
We suggest conducting a set of pilot experiments to empiricallydetermine the optimal parameters for your experimental systemthat will lead to the best efficiency and viability results, as we didhere and in our previous study (Wong et al. 2020). The Bio-Rad
Gene Pulser Xcell Electroporation System is uniquely suited to thisoptimization process, as it enables flexible modification of the key
parameters of electroporation.

Conclusions
While viral transduction is the most widely used method in CAR
T-cell workflows, there are many established nonviral methods for
introducing molecules into cells, and the choice will vary depending
on application, cargo, and cell type, among other variables(Chong et al. 2021; Atsavapranee et al. 2021). We anticipate thatthe identification of a high-efficiency CAR mRNA transfection
approach, as executed here via electroporation, will catalyze thewider adoption of mRNA transfection in CAR T-cell workflows.
In this study, we describe a successful nonviral method to produce
CAR T cells using a high-efficiency and high-viability strategy
for executing mRNA electroporations. These two transfection
characteristics are vital in any CAR T-cell workflow to produce CAR
T cells at a sufficient scale for clinical and therapeutic applications.
More broadly, these attributes are critical to any experimental
workflow involving transfections. We also note that this workflow ishighly scalable and could easily be modified to produce a higher
volume of CAR T cells for direct therapeutic applications, as
previously described (Wiesinger et al. 2019).

Taken together, our study highlights the utility of the Gene Pulser
Xcell Electroporation System in executing high-efficiency mRNAtransfections of primary human T cells with low impact on cellular
health and viability.
Table 4. Optimized electroporation conditions used in this study for Jurkat
and primary human T cells.

It is generally well accepted that healthy cells are required fortransfection workflows. Additionally, appropriate handling ofprimary T cells in this workflow requires treatment with activating
reagents, routine examination of cell health and viability during cell
culture, and determination of the optimal time point to perform
electroporation.
The parallel use of automated cell counting of Trypan Blue–stained
cells and flow cytometric analysis of 7-AAD–stained cells allowed
us to determine the relatively low impact of our mRNA transfections
on cell health and viability. The concordance between these assays© 2022 Bio-Rad Laboratories, Inc.

Parameter

Item/Condition

Electroporation system

Gene Pulser Xcell Electroporation System

mRNA concentration

150 μg/ml

Pulse voltage

200 V

Wave typeSquarePulse length

2 ms

Pulse number

1 pulseCuvette size

0.2 cmElectroporation buffer

Gene Pulser Electroporation Buffer

Electroporation volume

0.2 mlCell number

2x106 cells

Expression time (following electroporation) 6 hr
7Bulletin 3350

An Optimized Workflow for Generating Anti-CD19 CAR T Cells by mRNA Electroporation

References
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critical for generation of a robust T-cell response. Hum Gene Ther 30, 168–178.
Krug C et al. (2014). A GMP-compliant protocol to expand and transfect cancer
patient T cells with mRNA encoding a tumor-specific chimeric antigen receptor.
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National Cancer Institute (2022). CAR T cells: Engineering patient’s immune cells to treat
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Vannucci L et al. (2013). Viral vectors: a look back and ahead on gene transfer
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Wiesinger M et al. (2019). Clinical-scale production of CAR-T cells for the treatment ofmelanoma patients by mRNA transfection of a CSPG4-specific CAR under full GMP
compliance. Cancers (Basel) 11, 1198.
Wong L et al. (2020). Optimizing electroporation conditions for high-efficiency mRNAtransfections of CD8+ T cells with the Gene Pulser Xcell Electroporation System.
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