Monitoring Muscle Stem Cell Cultures with Impedance Spectroscopy

Yaiza Yuste

, Juan A. Serrano

, Alberto Olmo

, Andrés Maldonado-Jacobi

, Pablo Pérez

Gloria Huertas

, Sheila Pereira

, Fernando de la Portilla

and

Alberto Yúfera

Instituto de Microelectrónica de Sevilla (IMSE), Universidad de Sevilla, Av Americo Vespuccio, S/N, Sevilla, Spain

Instituto de Biomedicina de Sevilla (IBIS), Campus Hospital Universitario Virgen del Rocío, Sevilla, Spain

Keywords: Impedance Spectroscopy, Oscillation-Based Test (OBT), Skeletal Muscle, Myoblast, Stem Cell

Differentiation.

Abstract: The aim of this work is to present a new circuit for the real-time monitoring the processes of cellular growth

and differentiation of skeletal myoblast cell cultures. An impedance spectroscopy Oscillation-Based

technique is proposed for the test circuit, converting the biological system into a voltage oscillator, and

avoiding the use of very high performance circuitry or equipment. This technique proved to be successful in

the monitoring of cell cultures growth levels and could be useful for determining the degree of

differentiation achieved, of practical implications in tissue engineering.

1 INTRODUCTION

Impedance spectroscopy is currently used for real

time monitoring of different biological processes,

such as cell toxicity, cell invasion or inflammation

(Giaever and Keese, 1984; Daza et al., 2013; Pérez

et al., 2017). Impedance spectroscopy has the

advantage of being a non-invasive technique

(current intensity can be kept at minimum levels)

and is relatively non-expensive (only one sample or

petri plate is required for a performance curve).

Different circuits have been used for these

applications, using different topologies and

electrodes depending on the application.

The use of impedance spectroscopy in the

monitoring of the growth and differentiation of stem

cells is recently being studied for different tissue

engineering applications. Human mesenchymal stem

cells (hMSCs) development has been studied with

impedance spectroscopy in different works (Eun et

al., 2011; Hildebrandt et al., 2010). The impedance

spectra of osteogenic treated hMSCs reported a

significant increase of the magnitude of impedance

compared to controls cultivated in normal growth

medium (Hildebrandt et al., 2010). In this work, it is

concluded that impedance spectroscopy is an

appropriate method for non-invasive

characterization of osteogenic differentiation of

hMSCs, which is relevant for quality control of cell-

based implants and cell-based test systems for drug

development (Hildebrandt et al., 2010).

Other interesting and recently used stem cell

lines in tissue engineering are adipose stem cells

(Nordberg et al., 2017) or myoblasts (Liao and

Zhou, 2009). Skeletal muscle tissue engineering

holds great promise for regenerative medicine.

However, ex vivo cultivation methods typically

result in a low differentiation efficiency of stem cells

as well as graft that resemble the native tissues

morphologically, but lack contractile function.

In this work, a new circuit is proposed to apply

the impedance spectroscopy technique in myoblast

assays, to see whether this technique is useful in the

study of growth and differentiation of these cells

into muscular structures, in a similar way as it was

studied for others (Hildebrandt et al., 2010). The

circuit is based on the Oscillation-Based Test

technique, with amplitude and frequency values

obtained dependant on the cell culture

bioimpedance.

In section 2, the description of this circuit is

presented, together with the description of the initial

experiments performed.

Yuste, Y., Serrano, J., Olmo, A., Maldonado-Jacobi, A., Pérez, P., Huertas, G., Pereira, S., Portilla, F. and Yúfera, A.

Monitoring Muscle Stem Cell Cultures with Impedance Spectroscopy.

DOI: 10.5220/0006712300960099

In Proceedings of the 11th International Joint Conference on Biomedical Engineer ing Systems and Technologies (BIOSTEC 2018) - Volume 1: BIODEVICES, pages 96-99

ISBN: 978-989-758-277-6

2 MATERIALS AND METHODS

2.1 Impedance Spectroscopy

Monitoring System

For cell culture assays, commercial electrodes

8W10E PET, from Applied Biophysics (Applied

Biophysics) were employed. The multi-well is

composed of eight separated wells; each one

contains ten circular biocompatible gold

microelectrodes of 250 µm diameter in parallel, and

a surrounding reference electrode.

The proposed circuit for bioimpedance

measurements is based on the well-known technique

of Oscillation-Based Test (Huertas et al., 2015). It

avoids the use of very high performance circuitry or

equipment, as well as accurate current/voltage

generators, Instrumentation Amplifiers (IA) and

exact precise demodulation circuits, converting the

bioimpedance in a voltage oscillator, whose

oscillation parameters (fosc, aosc) are dependent of

the biological sample under test. This circuit

performance is fully described in (Huertas et al.,

2015; Pérez et al., 2017). Simplified circuit diagram

is illustrated in Fig. 1C. Cell cultures are

incorporated to circuit analysis through the

electrode-cell impedance value Zcell-electrode. This

circuit works as a voltage oscillator, being

characterized by its oscillation parameters:

frequency of oscillation (fosc) and amplitude of

oscillation (aosc) at the output voltage signal (Vout).

The aim, in this application, is to characterize cell

growth and differentiation level through these

parameters. For the proposed circuit, has been

observed that the amplitude of oscillation is more

sensible to changes in bioimpedance (Huertas et al.,

2015). A limited maximum current intensity of 10

µA was applied to the cell cultures.

2.2 Cell Cultivation and Experiments

Performed

Rat skeletal myoblasts were obtained from Rattus

Norvegicus L6 cell line (ATCC® CRL-1458™) and

were cultured at 37˚C in a CO

incubator at 5% on

the Instituto de Biomedicina de Sevilla (IBIS). The

growth medium used was Minimum Essential

Medium α (12571-063, Gibco) supplemented with

10% fetal bovine serum (F7524, Sigma) and 1%

penicillin-streptomycin (15140-122, Gibco).

After the cells reached 85-90% of confluence,

they were sub-cultured using trypsin-EDTA at

0.05% (25300-062, Gibco) and seeded 10

cells in

the appropriate wells of the multi-well used (wells 2,

3, 4, 6, 7, 8) with growth medium. When the specific

wells reached 70% of confluence, after rinse with

phosphate buffered saline (L0615, Linus), the

medium was changed to differentiation medium,

MEMα supplemented with 2% horse serum (S0910,

Biowest) and 17.8mM NaHCO

(S6297, Sigma-

Aldrich). Microscope images were taken with the

Olympus IX-71 inverted phase microscope.

Figure 1: Impedance Spectroscopy monitoring system. A)

8W10E PET cultureware from Applied Biophysics (Pérez

et al., 2016) with 8 wells of 0.8 cm

. Detail of each well:

cells are measured on top of the 10 circular gold electrodes

in parallel, with 250 m diameter. The ground electrode

can be seen on the right. B) Simplified block diagram

proposed for measurements, composed by a bioimpedance

converter, comparator and bandpass filter. Oscillation

parameters: amplitude (a

osc

) and frequency (f

osc

) can be

measured over the signal V

out

. C) Communication circuit

used.

All cell cultures (wells 2, 3, 4, 6, 7, 8) were held

in growth medium for control the first days, starting

form 10.000 cells. The differentiation in myotubes

(wells 6, 7, 8) was initialized by treatment with

differentiation medium, whereas the rest were held

in growth medium for control (wells 2, 3, 4). Cell

culture growth medium and differentiation medium

were measured by the impedance system (wells 1,

Monitoring Muscle Stem Cell Cultures with Impedance Spectroscopy

5), in order to differentiate any possible effect of the

medium used. The medium was replaced every 2–3

days. Of the two group of cells, one well was left

without measuring impedance (wells 4, 8), as a

control, in order to detect any possible effect of

current intensity on stem cells. Table 1 summarizes

the wells used in the 8W10E PET cultureware.

Temperature and humidity values were also

monitored during all the experiment. In each

medium change, cells were seen under the

microscope, and photographs of all wells were

taken. Two experiments were performed, in order to

compare results.

3 RESULTS AND DISCUSSION

Similar results were obtained in both experiments.

Fig. 2 shows the signal registered for the different

cell cultures, corresponding to the amplitude of the

oscillations of the circuit (a

osc

), together with the

measurement of the temperature and humidity

values. The OBT circuit used successfully detected

the initial cell growth, in a similar way as in other

cell types (Daza et al., 2013; Pérez et al., 2017). The

amplitudes observed are useful parameters to

determine the confluence level of the cell culture or

fill factor (defined as the area occupied by cultured

cells divided by the total culture area). A practical

threshold could be set at a fill factor of 70%, as cells

should change to differentiation medium at this

moment. In Fig. 2 it can be seen the behaviour of

muscular stem cells without differentiation (wells 2,

3), following a similar behaviour to other cell

cultures (Eun et al., 2011; Hildebrandt et al., 2010),

in comparison with muscular stem cells that have

followed a differentiation process (well 7). Stem

cells cultures that changed to the differentiation

medium show an initial decrease in the amplitude

values (day 4), as growth is then limited. This initial

decrease in cell proliferation is in accordance with

other works (Eun et al., 2011). However, after a few

hours, Fig. 2 shows a typical linear increase in the

monitored amplitude, corresponding to the

differentiation process (contrasted with microscope

images), reaching final higher amplitude levels than

cell cultures that don´t differentiate, similar to

reported in (Eun et al., 2011; Hildebrandt et al.,

2010; Bagnaninchi and Drummond, 2011).

Cellular growth and differentiation was observed

with microscope images, as shown in figure 3. In

each medium change, cells were seen under the

microscope, and photographs of all wells were

taken.

Table 1: Wells used in the 8W10E PET cultureware.

Well

Culture

Measurement

of impedance

Well 1

Growth medium

Yes

Well 2

Stem cells without differentiation

Yes

Well 3

Stem cells without differentiation

Yes

Well 4

Stem cells without differentiation

Well 5

Differentiation medium

Yes

Well 6

Stem cells for differentiation

Yes

Well 7

Stem cells for differentiation

Yes

Well 8

Stem cells for differentiation

Figure 2: A) Amplitude signals monitored for stem cells

without differentiation (W2 W3) and for differentiation

(W7). Cell culture medium was also measured (W1). An

electrical error was found in W6. After an initial transient

regime, all signals corresponding to cell cultures started to

rise, corresponding to cellular growth. After differentiation

medium was used (Day 2) and after a transitory stop in the

measured amplitude corresponding to a decrease in cell

proliferation, stem cells following the differentiation

process (W7), showing a higher increase in the monitored

amplitude.

Figure 3: Microscope images of the cell cultures. In each

medium change, cells were seen under the microscope,

and photographs of all wells were taken. Left) Well 7

(stem cells for differentiation) on the eighth day from the

start of differentiation. Tubular structures corresponding to

muscle myotubes can be observed. Right) Well 2 (stem

cells without differentiation) at the eighth day. Differences

between these two different wells are significant, although

a more quantitative work should be carried out in the

future.

A good level of differentiation was observed at

the end of the differentiation process. Tubular

structures corresponding to myotubes were clearly

BIODEVICES 2018 - 11th International Conference on Biomedical Electronics and Devices

observed at the end of the differentiation process, as

shown in Fig. 3.

These results suggest the differentiation in cell

lines correspond to differences in bioimpedance

measured, although the work should be completed in

the future with more quantitative analysis.

4 CONCLUSIONS

A new oscillating circuit based on Impedance

Spectroscopy has been presented for the real-time

monitoring of the cellular growth and differentiation

processes of stem cells. The technique has been first

applied to muscle stem cells.

The circuit proved to be useful for monitoring

the processes of cell growth and estimating the fill

factor of muscular stem cell cultures. The

oscillation-based circuit proposed successfully

detected this cell growth, in a similar way as in other

cell types. A useful threshold for the fill factor of

70% has been positively tested on stem cell-cultures,

to activate them towards differentiation by changing

the medium.

Real-time monitoring of cell differentiation can

be also enabled with the proposed impedance

spectroscopy method. An initial decrease in cell

proliferation was detected at the change of medium

to differentiation medium. However, after a few

hours, a linear increase in the monitored amplitude

was recorded, corresponding to the differentiation

process, which was contrasted with microscope

images. A final higher amplitude levels in

differentiated cell cultures were detected. The

technique could be useful for determining the degree

of differentiation achieved, although more detailed

tests would be needed.

No significant differences between cell cultures

where electrical impedance was used and the control

ones. However, higher levels of intensity could be

used, which could influence the process of cellular

differentiation and facilitate the development of

cells, or even facilitate the contraction of muscular

structures, what could be of importance in the design

of new bioreactors for tissue engineering.

ACKNOWLEDGMENT

This work was supported in part by the Spanish

founded Project: Integrated Microsystems for cell

culture test (TEC2013-46242-C3-1-P): Spanish, co-

financed with FEDER program.

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Monitoring Muscle Stem Cell Cultures with Impedance Spectroscopy