AIP Conference Proceedings 2344, 050017 (2021); https://doi.org/10.1063/5.0047233 2344, 050017
© 2021 Author(s).
A low cost, off-the-shelf bioreactor as enabling technology for physiological modeling
Cite as: AIP Conference Proceedings 2344, 050017 (2021); https://doi.org/10.1063/5.0047233 Published Online: 23 March 2021
Muhammad Satrio Utomo, Muhammad Hanif Nadhif, Ghulsan Fahmi El Bayani, and Yudan Whulanza
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A Low Cost, Off-Whe-Shelf Bioreactor as Enabling Technology for Physiological Modeling
Muhammad Satrio Utomo
1, Muhammad Hanif Nadhif
2,3, Ghulsan Fahmi El Bayani
4, Yudan Whulanza
5,6,a)1Research Center for Metallurgy and Materials, Indonesian Institute of Sciences, PUSPIPTEK Area, Building 470 South Tangerang, Banten 15314 Indonesia
2Department of Medical Physics, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, DKI Jakarta 10430, Indonesia
3Medical Technology Cluster, Indonesia Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, DKI Jakarta 10430, Indonesia
4Department of Physiology, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, DKI Jakarta 10430, Indonesia
5Department of Mechanical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI Depok, West Java 16424 Indonesia
6Research Center for Biomedical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI Depok, West Java 16424 Indonesia
a)Corresponding author: [email protected]
Abstract. Nowadays, tissue engineering has become a primary option for clinical treatment involving tissue damage or organ failure. One important enabling technological aspect for tissue engineering to produce successful outcomes is bioreactor where cells could be grown under certain conditions mimicking real physiological conditions and be prepared for in vivo integration before implantation to the patients. Physical stimulation by mechanical and electrical means could improve the development of engineered tissue to mimic the actual tissue. Mechanical stimulation could improve cellular function by improving the integrity and organization of the engineered tissue while electrical stimulation can improve the conductivity and contractility of tissue construction. The electric field would stimulate cellular calcium activity which could stimulate cell integration and gap junction formation. Thus, it is necessary to develop a bioreactor that is capable to provide a well-controlled environment and proper combination of mechanical and electrical stimulation to optimize the process of tissue engineering. Here we build a bioreactor that is capable to stimulate the engineered tissue mechanically and electrically to improve the tissue's contractile performance and functional maturity through an isovolumic contraction. The mechanical stimulation is generated by harmonic inflation and deflation of a balloon while the electrical stimulation is generated from a pair of carbon electrodes. The mechanical and electrical stimulations could function independently to each other. The bioreactor was successfully constructed and passed the functional test and ready for actual application for tissue engineering.
INTRODUCTION
The lack of sufficient stimuli when using Petri dishes is one of the reasons researchers developed bioreactors.
Using a bioreactor, tissue culture can be performed with a dynamic supply of medium and growth factor [1], which is unable to perform in Petri dishes. The mechanisms of dynamic supply for the cultured tissues can also be varied, which include using a closed loop [2], an open loop [3,4], and a mixing system [5]. A closed-loop system usually utilized a peristaltic pump to maintain the cycle of medium and waste in the loop [6]. Meanwhile, an open-loop system is more flexible in terms of pump selection: a peristaltic pump with a positive [6] or negative pressure [7]
and a syringe pump [3,4]. A mixing system, on the other hand, required a motor during the operation [8].
Apart from the dynamic flow, the cultured tissues in a bioreactor can be stimulated either mechanically or electrically. Stimulating the cultured cells and tissues may emerge with various types: uniaxial [9], biaxial [10], and multiaxial [11]. Prior studies presented that mechanical stimulation improved cellular functionalities, especially cell growth and proliferation [9–11], regardless of the types of stress. However, some reports showed that the stress working along in more axes generated a better functionality of the engineered tissues [10].
It was also reported that the effects of mechanical stimuli were profound in muscular tissues. Reports from Heher et al. [12] and Shen et al. [13] showed the improvement of the cultured skeletal and heart muscle cells, respectively, due to the mechanical stimuli applied to the tissues. The improvement was quantitatively determined by measuring the mechanical properties and beating the performance of the cultured cells [14].
Mechanical stimuli can also be generated with a dielectric elastic elastomer (DEA) [11]. The DEA is an elastomer membrane embedded with stimulating electrodes. The charge passing the electrodes generated a bulging mechanism of the membrane. However, the mentioned mechanism is uncommon. Commonly, as the name implies, a stimulating electrode is intended to provide electrical stimulation to the seeded (exciting) cells [15]. Results of the simulation include the enhancement of neural stem cell (NSC) differentiation [16] and more accurate cardiac organoid models [17].
Unfortunately, the realization of mechanical and electrical stimulations is not straightforward. In this study, we aim to build a low-cost, off-the-shelf bioreactor that is designed to stimulate the engineered tissue mechanically and electrically. Using simplified fabrication techniques, the tissue's contractile performance and functional maturity through the isovolumic contraction is expected to improve.
METHODS
A set of Arduino Uno microcontroller with L9110 motor driver was prepared to control the mechanical and electrical stimulations. A USB 2.0 to USB type-B cable functioned as a connection to the computer. A glass container with a sealed lid, silicone tube, and a 12 VDC vacuum pump were utilized to drive air for inflating and deflating a segment of latex balloon for the mechanical stimulation. Silver wires and two graphite electrodes were mounted for the electrical stimulation. Moreover, two latex O-rings and a silicone sealant was used to seal any joints, while a paper filter and a Teflon spacer was set for the exhaust. Figure 1 shows the required materials to build the proposed bioreactor.
Several steps were required to build the pneumatic-driven mechanical stimulator. First, the silicone tube was cut into a 10 cm-long segment, which one end was closed. Second, two holes were opened across each other at the wall of the silicone tube. Next, the latex balloon was cut into a 6 cm-long segment, which the silicone tube was put inside until the holes were covered. To tighten the joint and to prevent air leakage, latex O-rings were used.
The assembly of the bioreactor started by cutting holes on the container lid to put the silicone tube, graphite electrodes, and exhaust. The hole for the silicone tube was located in the center, while the holes for graphite electrodes were located on the side across each other with the same radial distance from the center. To prevent contamination, the exhaust was closed with a filter paper. Subsequently, the silicone tube and graphite electrodes were mounted in the holes using the silicone sealant. Last, the graphite electrodes were connected with wires to the microcontroller and the silicone tube to the DC vacuum pump.
FIGURE 1. Off-the-shelf materials to build the proposed bioreactor
RESULTS $1' DISCUSSION
In the current research, there is no specific design activity to build the bioreactor. The bioreactor was based on items that are available off-the-shelf in an engineering lab or workshop setting. As the main, Fig. 2 shows the schematic wiring diagram connecting the Arduino microcontroller to the DC air pump and graphite electrodes. To operate the bioreactor, the Arduino microcontroller must be connected to a power source, which in the current experiment was a USB port from a computer. The signals for the mechanical and electrical stimulations could be modified through codes in the computer. In the future, it is possible to build a function generator module and use an independent power source so that the system does not have to always rely on a computer. The current bioreactor also does not have an active system yet to regulate the fluid media. The media is changed manually to minimize the perfusion effect on the system. For further development, it is possible to use a micropump to enable the active circulation of fluid media while minimizing the perfusion effect. Moreover, by utilizing technology such as finger- actuated micropump, we could eliminate the dependence on electrical power source [18].
For comparison, a low-power consumption bioreactor capable to operate with a pair of 9 V batteries has been reported [19]. The bioreactor was capable to deliver electrical stimulation and perfusion through biodegradable tubes for 3D engineered cardiac tissue. The electrical stimulation was monophasic square wave pulses of 2 ms duration, 3 Hz, and 3 V/cm for 5 days and perfusion was done in a closed-loop system at 0.5 ml/min using a micropump.
microfluidic-based bioreactor using PDMS, electrical stimulator based on an Arduino Uno microprocessor, digital AD5206 potentiometer chips for amplitude control, and a TLV 4110 operational amplifier capable delivering electrical stimulation from 0 V to 5 V. The perfusion system consisted of a Bartels mp6 piezo micropump, a microcontroller, a digital potentiometer, and a pump controller chip to control the flow rate. It allowed the perfusion rate from 0.25 ml/min to 1 ml/min with the energy consumption at 200 mW - 800 mW.
. FIGURE 2. Schematic wiring diagram of the proposed bioreactor
Figure 3 shows the realized bioreactor with silicone tubes connected to the DC air pump and graphite electrodes connected to the microcontroller by silver wires. The vent hole is covered with filter fabric and tightened using a rubber band to prevent leakage. Ideally, the container then should be filled with fluid media containing growth factors and other chemical stimulants. The cells would be placed on the outer side of the balloon.
The current bioreactor design is based on a report by Morgan and Black [20]. To control the stimulation, they connected an air pump and electrical muscle stimulator to a NI-DAQ with programming in LabView. Compared to the current setting which utilizes Arduino-based microcontroller and open source programming, this setting is more preferable to be deployed as an accessible and low-cost bioreactor for laboratories around the globe.
A similar open-source bioreactor project has been reported by Beland et al who used NI DAQ instruments and Python library to build the electrical stimulator [21]. They used Dino-Lite and Matlab to observe the cyclic stretch of mechanical stimulation, two linear stepper motors to stretch a PDMS membrane containing the cells, Arduino Mega 2560 as the microcontroller, and a pair of carbon plate electrodes for the electrical stimulator that allows up to 16 V and 250 mA delivered to the system. Cells were grown under electrical stimulation of 2 ms pulses of 4.5 V/cm at 1 Hz. The mechanical stimulation signals include static, linear, and cyclic and the relation between motor displacement and PDMS substrate is linear for both stretched and unstretched states. In the current setting, the mechanical stimulation pattern was controlled by signals sent to the DC air pump.
FIGURE 3. Realized bioreactor without fluid media
Figure 4 shows the signals to activate the mechanical and electrical stimulations. Blue and red lines represent mechanical and electrical stimulation signals. Mechanical stimulation was realized by activating the air pump to flow air at 3.2 LPM for 0.5 s. The airflow inflated the latex balloon, providing mechanical strain to the tissue.
Meanwhile, the electrical stimulation was realized by sending 5 V for 0.4 s to both graphite electrodes. The delayed combination of mechanical and electrical stimulations increases the expression of cellular proteins responsible for calcium handling and contractility compared to synchronized combination [20]. This result indicates the importance of timing between mechanical and electrical stimulations.
Lu et al reported the design and validation of a bioreactor that incorporates uniaxial cyclic stretch, electrical stimulation, and constant perfusion [22]. They stated that the materials should be biocompatible and can be autoclaved for sterilization. Their system uses a cam coupled to a stepper motor ranging from 15 - 240 rev/min to stretch the PDMS membrane for mechanical stimulation and 0.5 mm-wide and 5 cm-long carbon rods for electrical stimulation. Their result shows higher MTT signals indicating a higher proliferation of human atrial fibroblasts in the stretched group compared to non-stretched and well plate-grown for positive control. Besides, 24 hours of cyclic stretch stimulation also rearrange the cell orientation from random orientation to aligned perpendicularly to the direction of stretch. electrical stimulation was tested using rat cardiomyocytes. The electrical stimulation of 7 V/cm at 1 Hz and 2 Hz could contract the cells for approximately 10%. In the current setting, biocompatibility and sterilization criteria for materials involved are not yet considered. Possible interaction between bioreactor components and fluid media should be further investigated.
FIGURE 4. Time series diagram for mechanical and electrical stimulation signals
CONCLUSION
The design and build of the current bioreactor were intended to mimic the physiological condition of cardiac cells growth and differentiation by providing mechanical and electrical stimulations to the cardiomyocytes stem
cells. The functional test showed that the current bioreactor built from off-the-shelf materials was functioning as planned.
We hope that the built bioreactor would be further utilized using actual cells and scaffolds. The uses of off-the- shelf materials would provide the basic necessity to operate the bioreactor even though some aspects could be upgraded to make it more sterile and reliable. Upgrades for the current bioreactor should be done without eliminating the essential aspect of low-cost and simplicity.
ACKNOWLEDGMENT
This research was supported by the Universitas Indonesia Grant PIT9 in 2019 with Contract Number: NKB- 0084/UN2.R3.1/HKP.05.00/2019. The authors would like to thank the Department of Mechanical Engineering, Universitas Indonesia for providing the materials used in the current experiment.
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