A NOVEL BIOREACTOR FOR CELLULAR ELECTRICAL STIMULATION: DESIGN AND DEVELOPMENT

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2 Δεκ 2013 (πριν από 3 χρόνια και 11 μήνες)

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A NOVEL BIOREACTOR F
OR CELLULAR ELECTRIC
AL
STIMULATION: DESIGN
AND DEVELOPMENT


Fabio Zomer Volpato
1,2
, Enrico Merzari
1
, Claudio Migliaresi
1
, Dietmar W. Hutmacher
2

1
BIOtech Research Center, University of Trento, Trento (TN), Italy

2
Institute

of Health and Biomedical Innovation, Queensland University of Technology, Brisbane (QLD),
Australia

E
-
mail:
fabio.zomervolpato@unitn.it

/
zomer.volpato@qut.edu.au




ABSTRACT
.

In this work we present the design and development of a bioreactor for in vitro electrical
stimulation
,

which delivers direct or alternated electrical potential difference (ΔV) to seeded scaffolds. This
manuscript presents the technical considerations regarding the development of the device and its potential in
tissue engineering, more specifically for neur
al stimulation. The equipment was built using National Instruments
(NI) CompactRIO hardware
.

Technical specifications of the hardware include a real
-
time controller,
compactRIO 9075 chassis, alternated/continuous output voltage supply (+
-
10 V and max 1 mA
per channel) and
current input for experiment feedback. Culture chambers where produced from polycarbonate with gold
electrodes. The controlling software was designed in NI Labview environment, which allows users to control the
delivery of the voltage to f
our independent channels and can stimulate

the

culture chambers independently and
simultaneously. Electrical stimulation through functional electrical stimulators (FES) or bioreactors is a fairly
simple, flexible and realistic technique to translate the bi
oelectricity present in the human body to in vitro and in
vivo two
-

or three
-
dimensional cell cultures.



Key
words:

Tissue Engineering
,
Bioreactor
,
Electrical Stimulation, Neural Cells Stimulation



1.

INTRODU
CTION



Tissue
E
ngineering
(TE)
is a multidisciplinary field that unifies cell and molecular
biology, materials science, and medical procedures. It aims to tackle the problem of tissue and
organ regeneration and replacement. Tissue regeneration strategies can be either conducted
entirely

in vivo

or assisted by an
in vitro

phase, which may provide more stable environment
for cell survival. Commonly static cultures are used during the
in vitro

phase. Nevertheless,
with advances in tissue engineering in the past decade, scientists have start
ed to apply
dynamic conditions through the use of bioreactors for
in vitro

cultures. A bioreactor can be
defined as ‘any apparatus that attempts to mimic physiological conditions in order to maintain
and encourage tissue regeneration in three
-
dimensional s
caffolds’.
Tissue Engineering has
been exploring biomimetic approaches in order to enhance tissue formation and healing.
Mimicking t
he physiological conditions can

enhance and guide cell adhesion, proliferation
and differentiation, increase extracellular m
atrix synthesis and growth factors secretion

[1
-
3]
.
Specific bioreactors designs have been developed t
o improve structure and function of
engineered tissues, such as: applying mechanical stimulus for bone and cartilage tissue

[4, 5]
,
electrical stimulus for neural and muscular tissues

[6, 7]
, pulsati
l
e
-
flow stimulus for heart
tissue
[8, 9]

and perfusion for liver tissue

[10, 11]
.
Several works have shown the benefits of
the

application of electrical stimuli

to cardiomyocytes
[12, 13]
, spiral ganglion neuron

[3]

and
cochlear neurons

[14]

cultures.

This
manuscript presents the

design and development of a bioreactor for in vitro
electrical stimulation
,

which
can

deliver direct or alternated electrical potential difference
(
ΔV) to seeded scaffolds
.
The design was tune
d

to neural stimulation; nevertheless, the
versatile platform in which it has been built allows users to simply modify and deliver a
variety of signa
ls.
Here
,

we present the technical considerations regarding the development of
the device.


2.

DESIGN AND DEVELOPMENT


2.1

Hardware design


The equipment was
designed

on a

real
-
time embedded controller

National Instruments
(NI) CompactRIO

(cRIO
-
9075)

(
Figure
1
A)

platform. The
real
-
time embedded controller

cRIO
-
9075 was connected to a NI cRIO
-
9075 chassis, containing an FPGA
(Field
Programmable Gate Array
)
Spartan
-
6

of 3 Mgate reconfigurable I / O w
ith a clock frequency
of 40 MHz.

The cRIO

system is a versatile platform that allows users to deliver and acquire a
variety of signals, in parallel, through its embedded
FPGA. The user is able to
modulate the
input and output signals, as required by each a
pplication,
simply

interchanging the I/O
modules (
Figure
1
B
-
D) that
are placed in the cRIO chassis.

The developed bioreactor with its culture chambers and support is presented in
Figure
2
.

The device was designed to
stimuli

four independent
set of samples

working in
parallel.

Each set of samples can contain up to 4 scaffolds, as seen
in
Figure
2
, to arrive to a
total of 16 samples being stimulated contemporaneously. Two current input modules give the
current feedback of each scaffold during the experiments. The input channels were designed
to
allow 8 samples to work at high frequencies and 8 to work at lower frequencies but with a
high sensitivity of the input signal.

The culture chambers and its support were designed and manufactured in
polycarbonate. Gold electrodes were used in the chambers.

Such materials allow the
sterilization of the chambers via autoclave.

A summary of the hardware specifications is
presented in

Table
1
.



Figure
1
:

National Instruments (NI) CompactRIO, [A] cRIO 9075 chassis, [B] output N9263 module, [C] and [D] input
modules N9203 and N9208, respectively.


Table
1
:
Hardware specifications for the developed
bioreactor
.

Output voltage

± 10 V

Output current per channel

max 1 mA

Number of output channels

4 analog

Digital
-
to
-
analog converter resolution

16 bits

Input current per channel

max ± 20 mA

max ± 22 mA

Number of input channels

8

16

Samples rate

200
kS/s

500 S/s

Analog
-
to
-
digital converter resolution

16 bits

24 bits

Type of stimulation

Continuous or alternated
(square, triangular or sinusoidal
waves)

Max stimulation frequenc
y

2 kHz

5 Hz



Figure
2
:
Designed bioreactor system depicting the
controlling box,

the
culture chambers’
support

and few chambers
.


2.
2

Software design


The controlling software was programmed in NI Labview 11.0 environment.
The
software architecture is expanded over three main
levels. The FPGA programming, wher
e all
I/O signals are generated,
acquired and analyzed from the controller to the stimulation
component.
Signals are generated at high frequency (400 MHz), while it acquires and
amplifies the measurements. The FPGA works i
n a deterministic way which allows
simultaneous operations. Thus, the software permits the user to run parallel operations.

An
overview of the FPGA program can be seen in

Figure
3
.



Figure
3
:
Block diagram of the FPGA program.


The real
-
time microprocessor and the FPGA are synchronized in a deterministic way
by the second programming level, which is permanently downloaded in the controller.
This
allows

the

autonomous control of the stimulation
chambers
,

providing a high reliability, the
possibility to manage all the working parameters and finally enabling the operator access to
the front panel, directly or remotely via
i
nternet network. The measured data are then
published on the loc
al network via TCP/IP

protocol. An overview of the real
-
time program
can be seen in
Figure
4
.



Figure
4
:

Block diagram o
f the real
-
time
prog
ram
.


The third programming level is a user
-
friendly graphical interface
. Such interface is
not downloaded in the controller, instead it resides in the operator
’s

computer. This client
interface downloads the data from the real
-
time via TCP/IP protocol.

The con
trol panel allows
the user to set the desired parameters for the experiments, such as type of stimulus
(continuous or alternated), type of wave (triangular, sinusoidal or square), periods of
stimul
ation

and rest, and the data to be saved. The data is saved

in a .txt ASCII file and stored
in the controller FTP

file
. The user panel can be seen in
Figure
5
.



Figure
5
:
U
ser interface

control panel
.


2.3

Stimulation description



The applicable
electrical potential difference can range from
±
0.001
to
±
10 V in
continuous mode or with square, triangular or sinusoidal
ramps at frequencies ranging from
0.001
Hz
to 2 kHz. A number of stimulation profiles can be imposed to each independent
channel by modifying the applied voltage

and

offset, frequency of stim
ulation, wave type,
number of stimulations

per day, duration of each stimulation and

resting time between
consecutive stimulations
, as seen in
Figure
6
.


Figure
6
:
Example of a stimulation profile.




The system was developed to work inside a standard humidified incubator at 37 °C and
5% CO
2
. Once the scaffolds are positioned in the culture chambers, the single chambers can
be fixed at the support and placed in the incubator.


3.

FINAL REMARKS


Bioelectric potentials are generated by a number of different biological processes, and
are used by cells to govern
metabolism
, to conduct impulses along nerve fibres, and to
regulate muscular contraction. It results from the conversion of chemical energy
into electrical
energy. Human bioelectricity is mostly present the nervous system signalling, muscular
contraction and wound healing.

The presented
work

is part of a project that aims to apply the latest advances in Tissue
Engineering to enhance
the
repair

outc
ome after spinal cord injuries. Nevertheless, the
applicability of such system in different areas of TE has
a
high potential. The developed
bioreactor

was built in a versatile platform which allows the adaptation to different scaffold
systems as well
as additional output signals such as current.


ACKNOWNLEDGEMENTS


The research leading to these results has received funding from the European Union, Seventh
Framework Programme [
FP7/2007
-
2013
] under
grant agreement [
Marie Curie FP7


PCOFUND
-
GA
-
2008
-
226070, acronym “progetto Trentino”
].

The authors also acknowledge
BIOTOOLS s.l.r. for the cooperation on the development of the bioreactor.


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