AtlaZ as a High-Throughput System for Advanced
Functional Cell Analysis to Develop Immunotherapies
Marta Lemme1, Martin Ziller1, Max Engelstädter1, Matthias Gossmann2, Peter Linder2, Bettina Lickiss2, Elena Dragicevic1, Johannes Stepper1, Michael George1, Niels
Fertig1, Sonja Stoelzle-Feix1
1Nanion Technologies, GmbH, 2innoVitro GmbH, Artilleriestr. 2, 52428 Jülich, Germany
Contact: sonja@nanion.de1

AtlaZ for sophisticated high-throughput quantitative live-cell analysis

Within classical treatment types for cancer, advancing therapies such as immunotherapy have emerged lately.
Identifying T cells that kill cancer cells in vivo and monitoring CAR-T cell activity in vitro is critical to thedevelopment of successful cell therapies.
We here developed an in vitro system allowing for killing assays within immune-therapeutic efforts, and to searchfor pharmaceutical drugs or for cytotoxic effects of substances. The assay technology, Electric Cell-substrateImpedance Sensing (ECIS), offers possibilities to study the response of living cells to a stimulus in a label-free, timeresolved, and non-invasive manner. The impedance of planar gold-film electrodes that are used as growth
substrate for cells reveals changes in e.g. electrode coverage or cell behavior. Real-time data provide insights
regarding kinetics of cell responses. Advanced information content is obtained by using multi- frequency
impedance readouts (0.1 kHz – 100 kHz): high frequency impedance is sensitive to differences in cell-confluency,
making it useful for measuring proliferation or cytotoxicity, whereas low frequency impedance data reveal
barrier integrity and allow to quantify cell adhesion.
We used the A549 epithelial lung adenocarcinoma cell line that was derived from a primary lung cancer. Effector cells co-cultured in the killing assay were
purified human cytotoxic T- lymphocytes. We found that after 27 h the cytolysis of A549 cells gradually increases and reaches a maximum of 37%, 48%, 59% and
57% in the presence of the target to effector cell ratio 1:2, 1:1, 2:1 and 3:1, respectively. Respective Kill Time 50 values are shown. CAR T-cells directed to thereceptor EGFR on the target cells A549 and SKOV3 were evaluated as a next step. Furthermore, we investigated H9C2 cardiac-like cells. We found that e.g.
Erlotinib is, as expected, cardio-safe, whereas compounds with a different mechanism of action show toxic effects on the cells. For example, Vincristine which isinteracting with tubulin proteins showed a concentration- and time-dependent effect on H9C2 cells.
Our aim with the newly developed 6 x 96-well platform, AtlaZ, is to elevate live cell analysis to a next level. To our knowledge, there is no platform available toquantify in vitro cell behavior like barrier function, proliferation, cytotoxicity, and more in such a time-resolved manner with this throughput.

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Accelerating your research..unique richness of information

Immune-cell mediated killing assay
Real-time cell analysis

Automated graphing of resultsACB
untreated
1:2
1:1
2:1
3:1

Untreated

Control A549 fourdays post plating

1:2

n.a.

1:1

Cytolysis (%)

Cell Signal

1000

100500

2:1

3:1

50

00

Time (h)30

BA549 cells

2530

SKOV3 cells

4
Cell Signal

Suspended cell

H9C2 differentiated towards cardiac phenotype
ControlVincristine (300pM )Vincristine (3nM )Vincristine (30nM )Vincristine (300nM )1000800

400DSKOV3 cells CAR T (1:1)

A549 cells

SKOV3 cells
0

n=2

CAR T (1:1)
n=2

CAR T (1:3)
n=2

CAR T (3:1)
n=2Cell Signal

CAR T (3:1)
n=2

100

1000
800
600
4000

A The Cell Signal value offers information on cell adherence, proliferation or cell death. B CAR T cells added in different ratios
to A549 or SKOV3 cells at t = 24 h after plating. C Kill Time 50 values calculated from measurements as shown in B (+/- SD, n = 2 wells each.
UTD = untransduced control cells). D The ratios 1:1 and 3:1 did induce a 84% and 100% cytolysis of A549 cells, and a 75% and 100% cytolysis
in SKOV3 cells, respectively. The ratio 1:3 induced a 10% (A549) or 20% (SKOV3) cytolysis.

550

1001501.0

***

***

0.5Control
Doxorubicin (100nM )Doxorubicin (300nM )Doxorubicin (1µM )Doxorubicin (3µM )1.51.0********0.50.0Time (h)Vincristine, acting on myofilaments and Doxorubicin inducing apoptosis
showed a time- and concentration dependent effect resulting in cell
death/reduced adherence of H9C2 cells.

Conclusions• AtlaZ is a quantitative live-cell analysis system and allows for cellular research on cell adhesion and proliferation, cytotoxicity, GPCR, morphology and barrier function, label-free and in real-time.
• Recordings can be performed in up to 6 x 96-well plates simultaneously or independently.
• Electrical impedance spectroscopy in combination with the throughput allows for a so far unmet quantity and richness of information which can be gained from cells, through the potential toaccess multiple kinetic and phenotypic information from in vitro cell cultures.

**0.0150

Time (h)Control
Doxorubicin (100nM )Doxorubicin (300nM )Compound addition
Doxorubicin (1µM )Doxorubicin (3µM )1200

CAR T (1:3)
n=250

Control
Vincristine (300pM)
Vincristine (3nM)
Vincristine (30nM)
Vincristine (300nM)

1.5

Compound addition

600

A549 cells

40

Cardiotoxicity

1200

C

35Time (h)A Effector cells were added at t = 24 h after plating of target A549 cancer cells. Increasing E:T cell ratios induced a ratiodependent reduction of the viability of A549 cells, represented as a reducing Cell Signal. B Cytolysis in percent (%) as
calculated from the data seen in Fig. 2. C Kill time 50 shows that 50% of A549 cells were killed after approx. 13h (E:T = 1:1)
or 6 hrs (E:T = 2:1 and 3:1).

CAR T killing assay

A040

Normalized cell signal

Cancer cells in vitro: access kinetic
and phenotypic information

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Normalized cell signal

Many cell effects can be recorded in parallel in the same well. The Cell Signal isdominated by the paracellular cell layer resistance or capacitive currents acrossthe cell membranes – depending on the readout frequency.

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