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Automatic Programmable Bioreactor with pH Monitoring System for Tissue Engineering Application

Automatic Programmable Bioreactor with pH Monitoring System for Tissue Engineering Application bioengineering Article Automatic Programmable Bioreactor with pH Monitoring System for Tissue Engineering Application 1 1 1 1 1 Suruk Udomsom , Apiwat Budwong , Chanyanut Wongsa , Pakorn Sangngam , Phornsawat Baipaywad , 1 1,2 1,3 1,4, Chawan Manaspon , Sansanee Auephanwiriyakul , Nipon Theera-Umpon and Pathinan Paengnakorn * Biomedical Engineering Institute, Chiang Mai University, Chiang Mai 50200, Thailand; suruk_u@cmu.ac.th (S.U.); apiwat_bouthwong@cmu.ac.th (A.B.); chanyanut_w@cmu.ac.th (C.W.); pakorn.sangngam@cmu.ac.th (P.S.); phornsawat.b@cmu.ac.th (P.B.); chawan.m@cmu.ac.th (C.M.); sansanee.a@cmu.ac.th (S.A.); nipon.t@cmu.ac.th (N.T.-U.) Department of Computational Engineering, Faculty of Engineering, Chiang Mai University, Chiang Mai 50200, Thailand Department of Electrical Engineering, Faculty of Engineering, Chiang Mai University, Chiang Mai 50200, Thailand Center of Excellence for Innovation in Analytical Science and Technology, Chiang Mai University, Chiang Mai 50200, Thailand * Correspondence: pathinan.p@cmu.ac.th Abstract: Tissue engineering technology has been advanced and applied to various applications in the past few years. The presence of a bioreactor is one key factor to the successful development of advanced tissue engineering products. In this work, we developed a programmable bioreactor with a controlling program that allowed each component to be automatically operated. Moreover, we Citation: Udomsom, S.; Budwong, developed a new pH sensor for non-contact and real-time pH monitoring. We demonstrated that A.; Wongsa, C.; Sangngam, P.; the prototype bioreactor could facilitate automatic cell culture of L929 cells. It showed that the cell Baipaywad, P.; Manaspon, C.; viability was greater than 80% and cell proliferation was enhanced compared to that of the control Auephanwiriyakul, S.; Theera-Umpon, obtained by a conventional cell culture procedure. This result suggests the possibility of a system N.; Paengnakorn, P. Automatic that could be potentially useful for medical and industrial applications, including cultured meat, Programmable Bioreactor with pH Monitoring System for Tissue drug testing, etc. Engineering Application. Bioengineering 2022, 9, 187. https:// Keywords: bioreactor; tissue engineering; automation; sensor doi.org/10.3390/bioengineering9050187 Academic Editors: Francesca Raganati and 1. Introduction Alessandra Procentese Tissue engineering has been developed with the aim of increasing the opportunity of Received: 31 March 2022 substituted tissue production for restoring or regenerating tissue and organ function. In Accepted: 22 April 2022 recent years, development in tissue engineering technology has grown rapidly, and it has Published: 25 April 2022 been widely applied to various fields apart from the medical area, such as organs-on-chip, Publisher’s Note: MDPI stays neutral bioelectronic devices, cultured meat, etc. [1,2]. The production of tissue engineering mainly with regard to jurisdictional claims in relies on the cell culture process. Therefore, quality control of cell culture conditions is published maps and institutional affil- essential for tissue engineering research [3]. The critical factors for mammalian cell culture iations. include temperature, pH, O and CO concentrations, humidity, nutrition and growth 2 2 factor content, etc. [4–6]. In a laboratory, the cell culture process is time-consuming and laborious. Therefore, in order to increase cell production capacity and reduce human error, an automatic and programmable bioreactor for tissue engineering equipped with cell Copyright: © 2022 by the authors. condition monitoring could reduce time and workload for researchers. Also, the monitoring Licensee MDPI, Basel, Switzerland. system could provide useful data for profiling cell growth and enhancing the productivity This article is an open access article of the process [7,8]. distributed under the terms and Nowadays, automation of analytical systems has been utilized in many areas, such conditions of the Creative Commons as in clinical, pharmaceutical, and biomedical applications [9–11]. The automatic system Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ has played an important role in both qualitative and quantitative analysis. Various ana- 4.0/). lytical techniques have been employed (for example, electrochemical, optical, and mass Bioengineering 2022, 9, 187. https://doi.org/10.3390/bioengineering9050187 https://www.mdpi.com/journal/bioengineering Bioengineering 2022, 9, 187 2 of 12 spectroscopic methods). The robotic automation could offer high precision and high system throughput while reducing analysis costs. A study of integrated measurement tools or sensors for cell metabolism monitoring through cell culture procedures has been widely of interest for a decade. There are several types of sensors integrated into general cell cultivation, bioreactors, and labs on a chip, such as oxygen sensors, pH sensors, and glucose-lactate biosensors [12]. The role of a cell culture monitoring tool indicates the viability of the cells, the consumption of nutrients, the nutrient yield in fermentation media, and the effect of drugs [13–15]. There have been several lab-on-chip developments for cell culture and other biomedi- cal applications. In organ-on-chip, the microfluidic platforms allowed co-culture of different cells to mimic the organ systems for drug testing [16]. Many studies involved real-time monitoring of cell activity and gene expression [17–19]. They provided a useful platform for cellular investigation. However, the number of cells on the lab-on-chip is limited and the re- sults from microscopic experiments could be different, so scale-up could be challenging [20]. Moreover, there are limitations concerning the complications and availability of fabrication technology for general research laboratories. In this work, an automatic programmable bioreactor with a pH monitoring system was developed. All system components were made in-house with computer-aided design (CAD) and 3D-printing techniques. The bioreactor consists of cell culture chamber, syringe pump, selector, pH sensor, and a controlling program. The developed system was customizable for different cell types, and the program was customizable for different culture conditions. Two types of 3D printing techniques were used. The main 3D printing technology used is liquid crystal display (LCD)-based stereolithography (SLA) that is based on pho- topolymerization of monomer and oligomer resin layer by layer [21]. This 3D printing technique has the advantage of high-resolution printing, but the size of printing object is limited by the LCD size, so it was chosen for making small and detailed components such as selector and pH sensor case. On the other hand, fused deposition modeling (FDM) 3D printing was employed for large and less-detailed components such as a syringe pump. The obtained data from the pH monitoring system could also be tracked for inspec- tion. The developed system was demonstrated with cell culture of the L929 cell line in comparison with the conventional cell culture procedure. 2. Materials and Methods 2.1. Design and Construction of Bioreactor The bioreactor consists of cell culture chamber, syringe pump, selector, pH sensor and controlling program as shown in Figure 1. The hardware parts were designed using a CAD program (Fusion 360, Autodesk Incorporation, San Rafael, CA, USA). The details of each component were described here. 2.1.1. Cell Culture Chamber A cell culture chamber was designed to accommodate one standard cell culture well plate size of 128 mm  86 mm (length  width). For a prototype, a 6-well plate was used as a model, as shown in Figure 2. The main body and lid of the chamber were made of transparent acrylic with a thickness of 5 mm that was sterilized with ethanol and UV radiation before use. Each well was covered with a piece of polydimethylsiloxane (PDMS; Dow Inc., Midland, MI, USA) and a flow channel was connected to a selector outside of the chamber via a luer connector. The cell culture chamber is also equipped with 6 micro-stirrers that can be used for suspension cell culture. Bioengineering 2022, 9, x FOR PEER REVIEW 3 of 13 Bioengineering 2022, 9, 187 3 of 12 (a) (b) Figure 1. A bioreactor with a pH monitoring system: (a) a schematic diagram showing components Figure 1. A bioreactor with a pH monitoring system: (a) a schematic diagram showing components of the bioreactor; (b) a photo of the bioreactor inside an incubator. of the bioreactor; (b) a photo of the bioreactor inside an incubator. 2.1.2. Syringe Pump 2.1.1. Cell Culture Chamber A syringe pump consists of a stepper motor and a syringe holder. A stepper motor A cell culture chamber was designed to accommodate one standard cell culture well (NEMA17, Shenzhen, China) was employed, as shown in Figure 3. A syringe holder plate size of 128 mm × 86 mm (length × width). For a prototype, a 6-well plate was used was constructed using fused deposition modeling (FDM) 3D printing (in-house) with as a model, as shown in Figure 2. The main body and lid of the chamber were made of acrylonitrile butadiene styrene (ABS) filament (eeSun, Shenzhen, China). The syringe transparent acrylic with a thickness of 5 mm that was sterilized with ethanol and UV ra- pump was designed to house a disposable sterile 10-mL syringe (Nipro (Thailand) Co Ltd., diation before use. Each well was covered with a piece of polydimethylsiloxane (PDMS; Ayutthaya, Thailand) that connected with a female luer adaptor and a silicone tubing (inner Dow Inc., Midland, MI, USA) and a flow channel was connected to a selector outside of diameter of 2 mm, Runze Fluid, Nanjing, China). The syringe pump was placed in a clear the chamber via a luer connector. The cell culture chamber is also equipped with 6 micro- acrylic box and sterilized with ethanol and UV radiation before use. stirrers that can be used for suspension cell culture. Bioengineering 2022, 9, 187 4 of 12 Bioengineering 2022, 9, x FOR PEER REVIEW 4 of 13 (a) (b) (c) Figure 2. Cell culture chamber: (a) a CAD file of a cell culture chamber design with a well plate Bioengineering 2022, 9, x FOR PEER REVIEW 5 of 13 Figure 2. Cell culture chamber: (a) a CAD file of a cell culture chamber design with a well plate holder; (b) a CAD file showing 6-well plate with PDMS lids and 6 micro-stirrers; (c) a side view of holder; (b) a CAD file showing 6-well plate with PDMS lids and 6 micro-stirrers; (c) a side view of a a cell culture on a 6-well plate inside a cell culture chamber. cell culture on a 6-well plate inside a cell culture chamber. 2.1.2. Syringe Pump A syringe pump consists of a stepper motor and a syringe holder. A stepper motor (NEMA17, Shenzhen, China) was employed, as shown in Figure 3. A syringe holder was constructed using fused deposition modeling (FDM) 3D printing (in-house) with acrylo- nitrile butadiene styrene (ABS) filament (eeSun, Shenzhen, China). The syringe pump was designed to house a disposable sterile 10-mL syringe (Nipro (Thailand) Co Ltd., Ayut- thaya, Thailand) that connected with a female luer adaptor and a silicone tubing (inner diameter of 2 mm, Runze Fluid, Nanjing, China). The syringe pump was placed in a clear acrylic box and sterilized with ethanol and UV radiation before use. (a) (b) Figure 3. Syringe pump: (a) a CAD file of syringe pump design; (b) a photo of syringe pump with a Figure 3. Syringe pump: (a) a CAD file of syringe pump design; (b) a photo of syringe pump with a 10-mL syringe in an acrylic box. 10-mL syringe in an acrylic box. 2.1.3. Selector A selector acts as a media flow switch between a syringe pump, a cell culture well, a media reservoir, and sensors. It has 3 servo motors (MG996R, Shenzhen, China) that rotate to open and close the desired channel as illustrated in Figure 4. The rotating parts and flow channels were constructed using LCD-based 3D printing (ANYCUBIC Photon Mono X, Hong Kong Anycubic Technology, Hong Kong, China) with acrylic-based resin (eResin PLA biophotopolymer resin, eSun, Shenzhen, China). The outlet was fitted with PTFE flangeless fittings and TPFE tubing (inner diameter of 1 mm, Runze fluid, Nanjing, China). The selector was placed in a clear acrylic box and sterilized with ethanol and UV radiation before use. (a) (b) Figure 4. Selector: (a) a CAD file of a selector design; (b) a photo of a selector with 3D-printed chan- nels. 2.1.4. pH Monitoring System An on-line pH monitoring system was based on colorimetric measurement of cell culture media containing phenol red indicator. As shown in Figure 5, a pH monitoring system consists of an LED light source, an ambient light sensor (TEMT 6000, Vishay Inter- technology, Malvern, PA, USA), and a tubing holder that allows for non-contact measure- ment. The tubing holder is designed to fit a PTFE tube and was constructed using LCD- based 3D printing (ANYCUBIC Photon Mono X, Hong Kong Anycubic Technology, Bioengineering 2022, 9, x FOR PEER REVIEW 5 of 13 (a) (b) Bioengineering 2022, 9, 187 5 of 12 Figure 3. Syringe pump: (a) a CAD file of syringe pump design; (b) a photo of syringe pump with a 10-mL syringe in an acrylic box. 2.1.3. Selector 2.1.3. Selector A selector acts as a media flow switch between a syringe pump, a cell culture well, a A selector acts as a media flow switch between a syringe pump, a cell culture well, media reservoir, and sensors. It has 3 servo motors (MG996R, Shenzhen, China) that rotate a media reservoir, and sensors. It has 3 servo motors (MG996R, Shenzhen, China) that to open and close the desired channel as illustrated in Figure 4. The rotating parts and rotate to open and close the desired channel as illustrated in Figure 4. The rotating parts flow channels were constructed using LCD-based 3D printing (ANYCUBIC Photon Mono and flow channels were constructed using LCD-based 3D printing (ANYCUBIC Photon X, Hong Kong Anycubic Technology, Hong Kong, China) with acrylic-based resin (eResin Mono X, Hong Kong Anycubic Technology, Hong Kong, China) with acrylic-based resin PLA biophotopolymer resin, eSun, Shenzhen, China). The outlet was fitted with PTFE (eResin PLA biophotopolymer resin, eSun, Shenzhen, China). The outlet was fitted with flangeless fittings and TPFE tubing (inner diameter of 1 mm, Runze fluid, Nanjing, China). PTFE flangeless fittings and TPFE tubing (inner diameter of 1 mm, Runze fluid, Nanjing, The selector was placed in a clear acrylic box and sterilized with ethanol and UV radiation China). The selector was placed in a clear acrylic box and sterilized with ethanol and UV before use. radiation before use. (a) (b) Figure 4. Selector: (a) a CAD file of a selector design; (b) a photo of a selector with 3D-printed chan- Figure 4. Selector: (a) a CAD file of a selector design; (b) a photo of a selector with 3D-printed channels. nels. 2.1.4. pH Monitoring System 2.1.4. pH Monitoring System An on-line pH monitoring system was based on colorimetric measurement of cell cul- ture media containing phenol red indicator. As shown in Figure 5, a pH monitoring system An on-line pH monitoring system was based on colorimetric measurement of cell Bioengineering 2022, 9, x FOR PEER REVIEW 6 of 13 consists of an LED light source, an ambient light sensor (TEMT 6000, Vishay Intertechnol- culture media containing phenol red indicator. As shown in Figure 5, a pH monitoring ogy, Malvern, PA, USA), and a tubing holder that allows for non-contact measurement. system consists of an LED light source, an ambient light sensor (TEMT 6000, Vishay Inter- The tubing holder is designed to fit a PTFE tube and was constructed using LCD-based technology, Malvern, PA, USA), and a tubing holder that allows for non-contact measure- 3D printing (ANYCUBIC Photon Mono X, Hong Kong Anycubic Technology, China) with ment. The tubing holder is designed to fit a PTFE tube and was constructed using LCD- China) with opaque acrylic-based resin (eResin PLA biophotopolymer resin, eSun, Shen- opaque acrylic-based resin (eResin PLA biophotopolymer resin, eSun, Shenzhen, China). based 3D printing (ANYCUBIC Photon Mono X, Hong Kong Anycubic Technology, zhen, China). (a) (b) Figure 5. On-line pH monitoring system. (a) a CAD file of a pH sensor; (b) a photo of a pH sensor Figure 5. On-line pH monitoring system. (a) a CAD file of a pH sensor; (b) a photo of a pH sensor with green LED. with green LED. 2.1.5. Controlling Program The bioreactor is controlled by simple software as shown in Figure 6 and Table 1. We developed software for controlling syringe pump, which can set the volume of culture media fed into and out of the culture chamber. For the selector, the software can control the selector to select the channel of each culture well, fresh media, and waste. Also, this software can control the stirrer of each well plate for mixing solution in each well. The pH monitoring system readings are recorded and a graph was plotted on the graphic user interface (GUI). In addition, we can write the preset script of the culture condition in ad- vance and this software can control the bioreactor automatically. The GUI of this software is shown in Figure 7. Figure 6. Schematic diagram showing workflow of controlling program. Bioengineering 2022, 9, x FOR PEER REVIEW 6 of 13 China) with opaque acrylic-based resin (eResin PLA biophotopolymer resin, eSun, Shen- zhen, China). (a) (b) Figure 5. On-line pH monitoring system. (a) a CAD file of a pH sensor; (b) a photo of a pH sensor Bioengineering 2022, 9, 187 6 of 12 with green LED. 2.1.5. Controlling Program 2.1.5. Controlling Program The bioreactor is controlled by simple software as shown in Figure 6 and Table 1. We The bioreactor is controlled by simple software as shown in Figure 6 and Table 1. We developed software for controlling syringe pump, which can set the volume of culture developed software for controlling syringe pump, which can set the volume of culture media fed into and out of the culture chamber. For the selector, the software can control media fed into and out of the culture chamber. For the selector, the software can control the selector to select the channel of each culture well, fresh media, and waste. Also, this the selector to select the channel of each culture well, fresh media, and waste. Also, this software can control the stirrer of each well plate for mixing solution in each well. The pH software can control the stirrer of each well plate for mixing solution in each well. The monitoring system readings are recorded and a graph was plotted on the graphic user pH monitoring system readings are recorded and a graph was plotted on the graphic interface (GUI). In addition, we can write the preset script of the culture condition in ad- user interface (GUI). In addition, we can write the preset script of the culture condition vance and this software can control the bioreactor automatically. The GUI of this software in advance and this software can control the bioreactor automatically. The GUI of this software is shown in Figure 7. is shown in Figure 7. Figure 6. Schematic diagram showing workflow of controlling program. Figure 6. Schematic diagram showing workflow of controlling program. Table 1. List of commands for controlling bioreactor. Command Value Description Loop number Set number of scripts to repeat Vol number Set volume of syringe pump to fill or drain (mL) Goto number Jump to a line number that set of the script Wait hh:mm:ss Set delay time Dir F or B Set syringe pump direction (Forward or Backward) Selector channel Ch Select selector channel (A or B or C or else) Stirrer channel Stir ON/OFF selected stirrer (A or B or C or else) Start Starting syringe pump Msgbox Text Displays the specified text in the message box Bioengineering 2022, 9, x FOR PEER REVIEW 7 of 13 Table 1. List of commands for controlling bioreactor. Command Value Description Loop number Set number of scripts to repeat Vol number Set volume of syringe pump to fill or drain (mL) Goto number Jump to a line number that set of the script Wait hh:mm:ss Set delay time Dir F or B Set syringe pump direction (Forward or Backward) Selector channel Ch Select selector channel (A or B or C or else) Stirrer channel Stir ON/OFF selected stirrer (A or B or C or else) Bioengineering 2022, 9, 187 7 of 12 Start Starting syringe pump Msgbox Text Displays the specified text in the message box Figure 7. A screenshot of user interface of controlling program. Figure 7. A screenshot of user interface of controlling program. 2.2. Bioreactor Setup 2.2. Bioreactor Setup Befor Before e using usingthe the developed developed bior biore eactor actor, , a a syringe syringepump pumpwas wascalibrated calibrated by by weighting weighting water flew out at different flow rates using a four-digit balance. A pH monitoring system water flew out at different flow rates using a four-digit balance. A pH monitoring system was was calibrated calibrated using using cultur cultue re media media with withdif differen ferenttpH pH values, values, which which wer were e pr pred edetermined etermined with with a a l laboratory aboratory p pH H me meter te.r. A A set setof of PDMS PDMS lids, lids, containers containers for for fr fr esh eshmedia media and and waste, waste, and and silicone silicone tub tubin ing g wer were e sterilized sterilized using using a annautoclave autoclave beforeh beforehand. and. All All components componentsexcept except aa micr microc ocontr ontro oller ller and and aa notebook notebook with with a a contr control olling ling pr program ogram wer were e sts erilized terilized with with ethanol ethanol and and then thenUV UVradiation radiation for for15 15 min minin ina a biosafety biosafety cabinet cabinet befor before e use. use. A A 6-well 6-well plate plate containing containing cell cell c cultur ultur ee was wasplaced placed in in aa chamber chamber and and a a m media edia rreservoir eservoir and waste container were connected to a selector in a biosafety cabinet. A bioreactor with and waste container were connected to a selector in a biosafety cabinet. A bioreactor with cell cell cu cultur lture e was was then then placed placed i inn an an incub incubator ator . . 2.3. Bioreactor Performance Test The performance of the developed bioreactor was tested by measuring the cell viability and cell proliferation rate of cells cultured in the bioreactor compared with that of a conventional cell culture procedure. A mouse fibroblast cell line (L929: NCTC clone 929, JCRB cell bank, Osaka, Japan) was used as a model for this test. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and 1% antibiotic- antimycotic (100X, Thermo Fisher Scientific, USA). 2.3.1. Cell Viability Testing The L929 cells were seeded in a 6-well plate at 1  10 cells per well. The well plate was then placed in the bioreactor and was incubated at 37 C with 5% CO and a humidified atmosphere for 48 h. The control group was place in the same incubator. The cell viability was measured using an MTT assay [22]. Bioengineering 2022, 9, x FOR PEER REVIEW 8 of 13 2.3. Bioreactor Performance Test The performance of the developed bioreactor was tested by measuring the cell via- bility and cell proliferation rate of cells cultured in the bioreactor compared with that of a conventional cell culture procedure. A mouse fibroblast cell line (L929: NCTC clone 929, JCRB cell bank, Osaka, Japan) was used as a model for this test. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and 1% antibiotic- antimycotic (100X, Thermo Fisher Scientific, USA). 2.3.1. Cell Viability Testing The L929 cells were seeded in a 6-well plate at 1 × 10 cells per well. The well plate was then placed in the bioreactor and was incubated at 37 °C with 5% CO2 and a humidi- fied atmosphere for 48 h. The control group was place in the same incubator. The cell viability was measured using an MTT assay [22]. 2.3.2. Cell Proliferation Assay The L929 cells were seeded in a 6-well plate at 7 × 10 cells per well with 5 mL of complete media and then cultured automatically in the bioreactor for 72 h using a pre-set program where 2 mL of cultured media was disposed of and refilled every 6 h. At 24 h and 72 h, the cells were trypsinized using 0.25% trypsin-EDTA solution (Thermo Fisher Bioengineering 2022, 9, 187 8 of 12 Scientific, USA) and the number of cells was counted with an automated cell counter (Countess II, Thermo Fisher Scientific, USA). The obtained proliferation rate was com- 2.3.2. Cell Proliferation Assay pared to that obtained from conventional cell culture with the same starting cell numbers. The L929 cells were seeded in a 6-well plate at 7  10 cells per well with 5 mL of complete media and then cultured automatically in the bioreactor for 72 h using a pre-set program where 2 mL of cultured media was disposed of and refilled every 6 h. At 24 h 2.4. pH Monitoring System Test and 72 h, the cells were trypsinized using 0.25% trypsin-EDTA solution (Thermo Fisher Scientific, ThUSA) e pH and mo the ninumber toring of system cells was wa counted s test with ed an duri automated ng thecell inc counter ubation of L929 cells for 48 h. (Countess II, Thermo Fisher Scientific, USA). The obtained proliferation rate was compared The program was set to refresh 2 mL of cell culture media every 6 h. to that obtained from conventional cell culture with the same starting cell numbers. 2.4. pH Monitoring System Test 3. Results The pH monitoring system was tested during the incubation of L929 cells for 48 h. 3.1. Syringe Pump Calibration The program was set to refresh 2 mL of cell culture media every 6 h. A calibration of the syringe pump with a 10 mL syringe was performed. The different 3. Results 3.1. Syringe Pump Calibration flow rates were set for one minute and the flow out water was collected and precisely A calibration of the syringe pump with a 10 mL syringe was performed. The different weighted. In Figure 8, the calibration plot showed that the mass of flow out water is cor- flow rates were set for one minute and the flow out water was collected and precisely related with the set flow rate. This result indicates that the flow rate of the calibrated sy- weighted. In Figure 8, the calibration plot showed that the mass of flow out water is correlated with the set flow rate. This result indicates that the flow rate of the calibrated ringe pump was accurate. syringe pump was accurate. Figure 8. A calibration graph of an in-house syringe pump, n = 3. Figure 8. A calibration graph of an in-house syringe pump, n = 3. 3.2. pH Calibration The pH monitoring system was calibrated with DMEM media, which is used for the culture of the L929 cell model. The media contains a phenol red indicator that changes color in the range of 6.8–8.2 from yellow to pink, respectively, as shown in Figure 9a. An increase in the pH of the media caused an increase in the red color in- tensity, which absorbed more green light and will be detected by the sensor. Figure 9b shows a calibration plot obtained from DMEM at a pH range between 6.90 and 7.83; green intensity = 57.7 (pH) + 1032, n = 3. Bioengineering 2022, 9, x FOR PEER REVIEW 9 of 13 3.2. pH Calibration The pH monitoring system was calibrated with DMEM media, which is used for the culture of the L929 cell model. The media contains a phenol red indicator that changes color in the range of 6.8–8.2 from yellow to pink, respectively, as shown in Figure 9a. An increase in the pH of the media caused an increase in the red color intensity, which ab- sorbed more green light and will be detected by the sensor. Figure 9b shows a calibration Bioengineering 2022, 9, 187 9 of 12 plot obtained from DMEM at a pH range between 6.90 and 7.83; green intensity = −57.7 (pH) + 1032, n = 3. (a) (b) Figure 9. Optical pH sensor: (a) effect of pH on green intensity measured by flowing DMEM media Figure 9. Optical pH sensor: (a) effect of pH on green intensity measured by flowing DMEM media at various pH (shown in inset) thought a sensor; (b) a calibration plot between pH of DMEM media and green intensity, n = 3. at various pH (shown in inset) thought a sensor; (b) a calibration plot between pH of DMEM media and green intensity, n = 3. 3.3. Cell Viability—MTT Assay 3.3. Cell Viability—MTT Assay The cell viability of L929 cells was measured after incubation in the developed biore- actor for 48 h. Figure 10a shows that the cell viability of the L929 cell in the bioreactor was The cell viability of L929 cells was measured after incubation in the developed biore- greater than 80% when compared to the control obtained through conventional cell cul- actor for 48 h. Figure 10a shows that the cell viability of the L929 cell in the bioreactor was ture. In addition, no morphologic change was observed, as shown in Figure 10b. These greater than 80% when compared to the control obtained through conventional cell culture. results suggest that the developed bioreactor including setup conditions and sterilization In addition, no morphologic change was observed, as shown in Figure 10b. These results process did not cause a cytotoxic effect on the cells. Bioengineering 2022, 9, x FOR Psuggest EER REVIEW that the developed bioreactor including setup conditions and sterilization 10 of 13 process did not cause a cytotoxic effect on the cells. (a) (b) Figure 10. Cell viability of L929 incubated in the developed bioreactor: (a) percent cell viability ob- Figure 10. Cell viability of L929 incubated in the developed bioreactor: (a) percent cell viability tained from MTT assay with respected to control group (n = 6); (b) a microscopic image of cells after obtained incubati from on iMTT n the bi assay oreactor with for 48 respected h. to control group (n = 6); (b) a microscopic image of cells after incubation in the bioreactor for 48 h. 3.4. Cell Proliferation Assay 3.4. Cell Proliferation Assay An automatic cell culture in the developed bioreactor was demonstrated. Figure 11 shows cell proliferation from L929 cells cultured in the bioreactor with the program to An automatic cell culture in the developed bioreactor was demonstrated. Figure 11 refresh the media every 6 h automatically compared to that cultured parallelly in a con- shows cell proliferation from L929 cells cultured in the bioreactor with the program to ventional manner (no media changed). It was found that the number of cells in the biore- refresh the media every 6 h automatically compared to that cultured parallelly in a conven- actor increased significantly at 72 h in comparison with the number of cells in the control tional manner (no media changed). It was found that the number of cells in the bioreactor plate. The results suggested that the automation of regular partial media change could help enhance the cell growth in the bioreactor. Figure 11. Cell proliferation of L929 incubated in the developed bioreactor. The number of cells was counted at 24 and 72 h after incubation and the data were analyzed by two-way ANOVA and post- hoc using R program. * Statistical significance was set at p < 0.05, n = 3. Bioengineering 2022, 9, x FOR PEER REVIEW 10 of 13 (a) (b) Figure 10. Cell viability of L929 incubated in the developed bioreactor: (a) percent cell viability ob- tained from MTT assay with respected to control group (n = 6); (b) a microscopic image of cells after incubation in the bioreactor for 48 h. 3.4. Cell Proliferation Assay An automatic cell culture in the developed bioreactor was demonstrated. Figure 11 Bioengineering 2022, 9, 187 10 of 12 shows cell proliferation from L929 cells cultured in the bioreactor with the program to refresh the media every 6 h automatically compared to that cultured parallelly in a con- ventional manner (no media changed). It was found that the number of cells in the biore- actor increased significantly at 72 h in comparison with the number of cells in the control increased significantly at 72 h in comparison with the number of cells in the control plate. plate. The results suggested that the automation of regular partial media change could The results suggested that the automation of regular partial media change could help help enhance the cell growth in the bioreactor. enhance the cell growth in the bioreactor. Figure Figure 11. 11. Cell Cell prolife proliferation ration of L929 of L929 incubate incubated d in the de in vel the oped developed bioreactor.bior The eactor number . The of celnumber ls was of cells counted at 24 and 72 h after incubation and the data were analyzed by two-way ANOVA and post- was counted at 24 and 72 h after incubation and the data were analyzed by two-way ANOVA and hoc using R program. * Statistical significance was set at p < 0.05, n = 3. post-hoc using R program. * Statistical significance was set at p < 0.05, n = 3. Bioengineering 2022, 9, x FOR PEER REVIEW 11 of 13 3.5. pH Monitoring Results The pH measurement of the cultured media was displayed in real-time and recorded 3.5. pH Monitoring Results as .CSV file for further analysis. The pH was measured by the pH sensor located on the The pH measurement of the cultured media was displayed in real-time and recorded channel before the waste container, indicating the pH of the used media. In general, acidic as .CSV file for further analysis. The pH was measured by the pH sensor located on the conditions could inhibit cell growth. A change in pH could reflect the cell condition, such channel before the waste container, indicating the pH of the used media. In general, acidic as a lower pH suggesting cell death or bacterial contamination. conditions could inhibit cell growth. A change in pH could reflect the cell condition, such as a lower Figur pH s e ugg 12 e shows sting cell de a plot ath or of bact the erial pH conchange tamination. with time during a 48-h cell culture of Figure 12 shows a plot of the pH change with time during a 48-h cell culture of L929 L929 cells in the developed bioreactor. It was found that a program set for regular media cells in the developed bioreactor. It was found that a program set for regular media change change was able to maintain the pH of the cell culture within 7.4  0.2, which is suitable was able to maintain the pH of the cell culture within 7.4 ± 0.2, which is suitable for fibro- for fibroblast cell line [23]. This could be related to the higher cell proliferation rate in the blast cell line [23]. This could be related to the higher cell proliferation rate in the bioreac- bioreactor observed in Figure 11. tor observed in Figure 11. Figure 12. pH changes during L929 cell incubation in the developed bioreactor. Figure 12. pH changes during L929 cell incubation in the developed bioreactor. 4. Discussion In this work, we developed an automatic programmable bioreactor with a pH moni- toring system that was suitable for tissue engineering cell culture. This bioreactor’s com- ponents can be controlled via programable script to the desired cell culture condition, and this system can operate automatically. This system helps to reduce time and labor com- pared to manual cell culture. For pH sensor, we applied a colorimetry technique to monitor the pH of the cell cul- ture on-line. This sensor was compact and easy to use. In addition, non-contact measure- ment has an advantage in real-time monitoring, and there is no contamination to cell cul- ture. The data were collected and can be analyzed for cell culture condition scripts next time. In future work, we plan to use machine learning methods, for example, the deep learning method [24,25], to optimize and find the suitable parameters in controlling bio- reactors in order to enhance the productivity of the cell culture in the bioreactor. Furthermore, this prototype can be applied to culture cells for a long period to study cell differentiation and tissue formation, which could be potentially useful for medical and industrial applications, including cultured meat, drug testing, etc. For example, this system could be used to study muscle cell differentiation and determine optimal condi- tions for cultured meat production. For drug testing, the bioreactor could be programmed Bioengineering 2022, 9, 187 11 of 12 4. Discussion In this work, we developed an automatic programmable bioreactor with a pH mon- itoring system that was suitable for tissue engineering cell culture. This bioreactor ’s components can be controlled via programable script to the desired cell culture condition, and this system can operate automatically. This system helps to reduce time and labor compared to manual cell culture. For pH sensor, we applied a colorimetry technique to monitor the pH of the cell culture on-line. This sensor was compact and easy to use. In addition, non-contact measurement has an advantage in real-time monitoring, and there is no contamination to cell culture. The data were collected and can be analyzed for cell culture condition scripts next time. In future work, we plan to use machine learning methods, for example, the deep learning method [24,25], to optimize and find the suitable parameters in controlling bioreactors in order to enhance the productivity of the cell culture in the bioreactor. Furthermore, this prototype can be applied to culture cells for a long period to study cell differentiation and tissue formation, which could be potentially useful for medical and industrial applications, including cultured meat, drug testing, etc. For example, this system could be used to study muscle cell differentiation and determine optimal conditions for cultured meat production. For drug testing, the bioreactor could be programmed to vary the dose or frequency of the treatment. In the future, an additional analytical system could be added to the bioreactor to detect disease-specific biomarkers or to monitor other biomolecules such as glucose and lactate dehydrogenase, which would indicate cells’ activity. Author Contributions: Conceptualization, S.U., S.A. and N.T.-U.; methodology, S.U., A.B., C.W., P.S., P.B., C.M. and P.P.; software, A.B.; validation, S.U., C.M. and P.P.; formal analysis, S.U., P.B. and P.P.; investigation, P.P.; resources, N.T.-U.; data curation, S.U., A.B., C.W., C.M., P.B. and P.P.; writing— original draft preparation, S.U., P.B. and P.P.; writing—review and editing, S.U., P.P., S.A. and N.T.-U.; visualization, S.U., A.B., P.S., P.B. and P.P.; supervision, S.A. and N.T.-U.; project administration, N.T.-U.; funding acquisition, S.A. and N.T.-U. All authors have read and agreed to the published version of the manuscript. Funding: This research project is supported by TSRI (71717). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors acknowledge the Biomedical Engineering Institute and Chiang Mai University for facility and financial support. We are thankful to Sarawut Kumphune for his advice on cell culture technique. We would like to thank Patipat Kamdenlek and Sirapat Boonsirijarungradh for their assistance in sample preparation. Conflicts of Interest: The authors declare no conflict of interest. References 1. Chandra, P.K.; Soker, S.; Atala, A. Chapter 1—Tissue engineering: Current status and future perspectives. In Principles of Tissue Engineering; Lanza, R., Langer, R., Vacanti, J.P., Atala, A., Eds.; Academic Press: London, UK, 2020; pp. 1–35. ISBN 978-0-12-818422-6. 2. Khademhosseini, A.; Langer, R. A decade of progress in tissue engineering. Nat. Protoc. 2016, 11, 1775–1781. [CrossRef] [PubMed] 3. O’Mara, P.; Farrell, A.; Bones, J.; Twomey, K. Staying alive! Sensors used for monitoring cell health in bioreactors. Talanta 2018, 176, 130–139. [CrossRef] 4. Kropp, C.; Kempf, H.; Halloin, C.; Robles-Diaz, D.; Franke, A.; Scheper, T.; Kinast, K.; Knorpp, T.; Joos, T.O.; Haverich, A.; et al. Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors. Stem Cells Transl. Med. 2016, 5, 1289–1301. [CrossRef] 5. Abbasalizadeh, S.; Larijani, M.R.; Samadian, A.; Baharvand, H. Bioprocess Development for Mass Production of Size-Controlled Human Pluripotent Stem Cell Aggregates in Stirred Suspension Bioreactor. Tissue Eng. Part C Methods 2012, 18, 831–851. [CrossRef] [PubMed] Bioengineering 2022, 9, 187 12 of 12 6. Gaydhane, M.K.; Mahanta, U.; Sharma, C.S.; Khandelwal, M.; Ramakrishna, S. Cultured meat: State of the art and future. Biomanufacturing Rev. 2018, 3, 1. [CrossRef] 7. Choi, J.; Mathew, S.; Oerter, S.; Appelt-Menzel, A.; Hansmann, J.; Schmitz, T. 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Automatic Programmable Bioreactor with pH Monitoring System for Tissue Engineering Application

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bioengineering Article Automatic Programmable Bioreactor with pH Monitoring System for Tissue Engineering Application 1 1 1 1 1 Suruk Udomsom , Apiwat Budwong , Chanyanut Wongsa , Pakorn Sangngam , Phornsawat Baipaywad , 1 1,2 1,3 1,4, Chawan Manaspon , Sansanee Auephanwiriyakul , Nipon Theera-Umpon and Pathinan Paengnakorn * Biomedical Engineering Institute, Chiang Mai University, Chiang Mai 50200, Thailand; suruk_u@cmu.ac.th (S.U.); apiwat_bouthwong@cmu.ac.th (A.B.); chanyanut_w@cmu.ac.th (C.W.); pakorn.sangngam@cmu.ac.th (P.S.); phornsawat.b@cmu.ac.th (P.B.); chawan.m@cmu.ac.th (C.M.); sansanee.a@cmu.ac.th (S.A.); nipon.t@cmu.ac.th (N.T.-U.) Department of Computational Engineering, Faculty of Engineering, Chiang Mai University, Chiang Mai 50200, Thailand Department of Electrical Engineering, Faculty of Engineering, Chiang Mai University, Chiang Mai 50200, Thailand Center of Excellence for Innovation in Analytical Science and Technology, Chiang Mai University, Chiang Mai 50200, Thailand * Correspondence: pathinan.p@cmu.ac.th Abstract: Tissue engineering technology has been advanced and applied to various applications in the past few years. The presence of a bioreactor is one key factor to the successful development of advanced tissue engineering products. In this work, we developed a programmable bioreactor with a controlling program that allowed each component to be automatically operated. Moreover, we Citation: Udomsom, S.; Budwong, developed a new pH sensor for non-contact and real-time pH monitoring. We demonstrated that A.; Wongsa, C.; Sangngam, P.; the prototype bioreactor could facilitate automatic cell culture of L929 cells. It showed that the cell Baipaywad, P.; Manaspon, C.; viability was greater than 80% and cell proliferation was enhanced compared to that of the control Auephanwiriyakul, S.; Theera-Umpon, obtained by a conventional cell culture procedure. This result suggests the possibility of a system N.; Paengnakorn, P. Automatic that could be potentially useful for medical and industrial applications, including cultured meat, Programmable Bioreactor with pH Monitoring System for Tissue drug testing, etc. Engineering Application. Bioengineering 2022, 9, 187. https:// Keywords: bioreactor; tissue engineering; automation; sensor doi.org/10.3390/bioengineering9050187 Academic Editors: Francesca Raganati and 1. Introduction Alessandra Procentese Tissue engineering has been developed with the aim of increasing the opportunity of Received: 31 March 2022 substituted tissue production for restoring or regenerating tissue and organ function. In Accepted: 22 April 2022 recent years, development in tissue engineering technology has grown rapidly, and it has Published: 25 April 2022 been widely applied to various fields apart from the medical area, such as organs-on-chip, Publisher’s Note: MDPI stays neutral bioelectronic devices, cultured meat, etc. [1,2]. The production of tissue engineering mainly with regard to jurisdictional claims in relies on the cell culture process. Therefore, quality control of cell culture conditions is published maps and institutional affil- essential for tissue engineering research [3]. The critical factors for mammalian cell culture iations. include temperature, pH, O and CO concentrations, humidity, nutrition and growth 2 2 factor content, etc. [4–6]. In a laboratory, the cell culture process is time-consuming and laborious. Therefore, in order to increase cell production capacity and reduce human error, an automatic and programmable bioreactor for tissue engineering equipped with cell Copyright: © 2022 by the authors. condition monitoring could reduce time and workload for researchers. Also, the monitoring Licensee MDPI, Basel, Switzerland. system could provide useful data for profiling cell growth and enhancing the productivity This article is an open access article of the process [7,8]. distributed under the terms and Nowadays, automation of analytical systems has been utilized in many areas, such conditions of the Creative Commons as in clinical, pharmaceutical, and biomedical applications [9–11]. The automatic system Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ has played an important role in both qualitative and quantitative analysis. Various ana- 4.0/). lytical techniques have been employed (for example, electrochemical, optical, and mass Bioengineering 2022, 9, 187. https://doi.org/10.3390/bioengineering9050187 https://www.mdpi.com/journal/bioengineering Bioengineering 2022, 9, 187 2 of 12 spectroscopic methods). The robotic automation could offer high precision and high system throughput while reducing analysis costs. A study of integrated measurement tools or sensors for cell metabolism monitoring through cell culture procedures has been widely of interest for a decade. There are several types of sensors integrated into general cell cultivation, bioreactors, and labs on a chip, such as oxygen sensors, pH sensors, and glucose-lactate biosensors [12]. The role of a cell culture monitoring tool indicates the viability of the cells, the consumption of nutrients, the nutrient yield in fermentation media, and the effect of drugs [13–15]. There have been several lab-on-chip developments for cell culture and other biomedi- cal applications. In organ-on-chip, the microfluidic platforms allowed co-culture of different cells to mimic the organ systems for drug testing [16]. Many studies involved real-time monitoring of cell activity and gene expression [17–19]. They provided a useful platform for cellular investigation. However, the number of cells on the lab-on-chip is limited and the re- sults from microscopic experiments could be different, so scale-up could be challenging [20]. Moreover, there are limitations concerning the complications and availability of fabrication technology for general research laboratories. In this work, an automatic programmable bioreactor with a pH monitoring system was developed. All system components were made in-house with computer-aided design (CAD) and 3D-printing techniques. The bioreactor consists of cell culture chamber, syringe pump, selector, pH sensor, and a controlling program. The developed system was customizable for different cell types, and the program was customizable for different culture conditions. Two types of 3D printing techniques were used. The main 3D printing technology used is liquid crystal display (LCD)-based stereolithography (SLA) that is based on pho- topolymerization of monomer and oligomer resin layer by layer [21]. This 3D printing technique has the advantage of high-resolution printing, but the size of printing object is limited by the LCD size, so it was chosen for making small and detailed components such as selector and pH sensor case. On the other hand, fused deposition modeling (FDM) 3D printing was employed for large and less-detailed components such as a syringe pump. The obtained data from the pH monitoring system could also be tracked for inspec- tion. The developed system was demonstrated with cell culture of the L929 cell line in comparison with the conventional cell culture procedure. 2. Materials and Methods 2.1. Design and Construction of Bioreactor The bioreactor consists of cell culture chamber, syringe pump, selector, pH sensor and controlling program as shown in Figure 1. The hardware parts were designed using a CAD program (Fusion 360, Autodesk Incorporation, San Rafael, CA, USA). The details of each component were described here. 2.1.1. Cell Culture Chamber A cell culture chamber was designed to accommodate one standard cell culture well plate size of 128 mm  86 mm (length  width). For a prototype, a 6-well plate was used as a model, as shown in Figure 2. The main body and lid of the chamber were made of transparent acrylic with a thickness of 5 mm that was sterilized with ethanol and UV radiation before use. Each well was covered with a piece of polydimethylsiloxane (PDMS; Dow Inc., Midland, MI, USA) and a flow channel was connected to a selector outside of the chamber via a luer connector. The cell culture chamber is also equipped with 6 micro-stirrers that can be used for suspension cell culture. Bioengineering 2022, 9, x FOR PEER REVIEW 3 of 13 Bioengineering 2022, 9, 187 3 of 12 (a) (b) Figure 1. A bioreactor with a pH monitoring system: (a) a schematic diagram showing components Figure 1. A bioreactor with a pH monitoring system: (a) a schematic diagram showing components of the bioreactor; (b) a photo of the bioreactor inside an incubator. of the bioreactor; (b) a photo of the bioreactor inside an incubator. 2.1.2. Syringe Pump 2.1.1. Cell Culture Chamber A syringe pump consists of a stepper motor and a syringe holder. A stepper motor A cell culture chamber was designed to accommodate one standard cell culture well (NEMA17, Shenzhen, China) was employed, as shown in Figure 3. A syringe holder plate size of 128 mm × 86 mm (length × width). For a prototype, a 6-well plate was used was constructed using fused deposition modeling (FDM) 3D printing (in-house) with as a model, as shown in Figure 2. The main body and lid of the chamber were made of acrylonitrile butadiene styrene (ABS) filament (eeSun, Shenzhen, China). The syringe transparent acrylic with a thickness of 5 mm that was sterilized with ethanol and UV ra- pump was designed to house a disposable sterile 10-mL syringe (Nipro (Thailand) Co Ltd., diation before use. Each well was covered with a piece of polydimethylsiloxane (PDMS; Ayutthaya, Thailand) that connected with a female luer adaptor and a silicone tubing (inner Dow Inc., Midland, MI, USA) and a flow channel was connected to a selector outside of diameter of 2 mm, Runze Fluid, Nanjing, China). The syringe pump was placed in a clear the chamber via a luer connector. The cell culture chamber is also equipped with 6 micro- acrylic box and sterilized with ethanol and UV radiation before use. stirrers that can be used for suspension cell culture. Bioengineering 2022, 9, 187 4 of 12 Bioengineering 2022, 9, x FOR PEER REVIEW 4 of 13 (a) (b) (c) Figure 2. Cell culture chamber: (a) a CAD file of a cell culture chamber design with a well plate Bioengineering 2022, 9, x FOR PEER REVIEW 5 of 13 Figure 2. Cell culture chamber: (a) a CAD file of a cell culture chamber design with a well plate holder; (b) a CAD file showing 6-well plate with PDMS lids and 6 micro-stirrers; (c) a side view of holder; (b) a CAD file showing 6-well plate with PDMS lids and 6 micro-stirrers; (c) a side view of a a cell culture on a 6-well plate inside a cell culture chamber. cell culture on a 6-well plate inside a cell culture chamber. 2.1.2. Syringe Pump A syringe pump consists of a stepper motor and a syringe holder. A stepper motor (NEMA17, Shenzhen, China) was employed, as shown in Figure 3. A syringe holder was constructed using fused deposition modeling (FDM) 3D printing (in-house) with acrylo- nitrile butadiene styrene (ABS) filament (eeSun, Shenzhen, China). The syringe pump was designed to house a disposable sterile 10-mL syringe (Nipro (Thailand) Co Ltd., Ayut- thaya, Thailand) that connected with a female luer adaptor and a silicone tubing (inner diameter of 2 mm, Runze Fluid, Nanjing, China). The syringe pump was placed in a clear acrylic box and sterilized with ethanol and UV radiation before use. (a) (b) Figure 3. Syringe pump: (a) a CAD file of syringe pump design; (b) a photo of syringe pump with a Figure 3. Syringe pump: (a) a CAD file of syringe pump design; (b) a photo of syringe pump with a 10-mL syringe in an acrylic box. 10-mL syringe in an acrylic box. 2.1.3. Selector A selector acts as a media flow switch between a syringe pump, a cell culture well, a media reservoir, and sensors. It has 3 servo motors (MG996R, Shenzhen, China) that rotate to open and close the desired channel as illustrated in Figure 4. The rotating parts and flow channels were constructed using LCD-based 3D printing (ANYCUBIC Photon Mono X, Hong Kong Anycubic Technology, Hong Kong, China) with acrylic-based resin (eResin PLA biophotopolymer resin, eSun, Shenzhen, China). The outlet was fitted with PTFE flangeless fittings and TPFE tubing (inner diameter of 1 mm, Runze fluid, Nanjing, China). The selector was placed in a clear acrylic box and sterilized with ethanol and UV radiation before use. (a) (b) Figure 4. Selector: (a) a CAD file of a selector design; (b) a photo of a selector with 3D-printed chan- nels. 2.1.4. pH Monitoring System An on-line pH monitoring system was based on colorimetric measurement of cell culture media containing phenol red indicator. As shown in Figure 5, a pH monitoring system consists of an LED light source, an ambient light sensor (TEMT 6000, Vishay Inter- technology, Malvern, PA, USA), and a tubing holder that allows for non-contact measure- ment. The tubing holder is designed to fit a PTFE tube and was constructed using LCD- based 3D printing (ANYCUBIC Photon Mono X, Hong Kong Anycubic Technology, Bioengineering 2022, 9, x FOR PEER REVIEW 5 of 13 (a) (b) Bioengineering 2022, 9, 187 5 of 12 Figure 3. Syringe pump: (a) a CAD file of syringe pump design; (b) a photo of syringe pump with a 10-mL syringe in an acrylic box. 2.1.3. Selector 2.1.3. Selector A selector acts as a media flow switch between a syringe pump, a cell culture well, a A selector acts as a media flow switch between a syringe pump, a cell culture well, media reservoir, and sensors. It has 3 servo motors (MG996R, Shenzhen, China) that rotate a media reservoir, and sensors. It has 3 servo motors (MG996R, Shenzhen, China) that to open and close the desired channel as illustrated in Figure 4. The rotating parts and rotate to open and close the desired channel as illustrated in Figure 4. The rotating parts flow channels were constructed using LCD-based 3D printing (ANYCUBIC Photon Mono and flow channels were constructed using LCD-based 3D printing (ANYCUBIC Photon X, Hong Kong Anycubic Technology, Hong Kong, China) with acrylic-based resin (eResin Mono X, Hong Kong Anycubic Technology, Hong Kong, China) with acrylic-based resin PLA biophotopolymer resin, eSun, Shenzhen, China). The outlet was fitted with PTFE (eResin PLA biophotopolymer resin, eSun, Shenzhen, China). The outlet was fitted with flangeless fittings and TPFE tubing (inner diameter of 1 mm, Runze fluid, Nanjing, China). PTFE flangeless fittings and TPFE tubing (inner diameter of 1 mm, Runze fluid, Nanjing, The selector was placed in a clear acrylic box and sterilized with ethanol and UV radiation China). The selector was placed in a clear acrylic box and sterilized with ethanol and UV before use. radiation before use. (a) (b) Figure 4. Selector: (a) a CAD file of a selector design; (b) a photo of a selector with 3D-printed chan- Figure 4. Selector: (a) a CAD file of a selector design; (b) a photo of a selector with 3D-printed channels. nels. 2.1.4. pH Monitoring System 2.1.4. pH Monitoring System An on-line pH monitoring system was based on colorimetric measurement of cell cul- ture media containing phenol red indicator. As shown in Figure 5, a pH monitoring system An on-line pH monitoring system was based on colorimetric measurement of cell Bioengineering 2022, 9, x FOR PEER REVIEW 6 of 13 consists of an LED light source, an ambient light sensor (TEMT 6000, Vishay Intertechnol- culture media containing phenol red indicator. As shown in Figure 5, a pH monitoring ogy, Malvern, PA, USA), and a tubing holder that allows for non-contact measurement. system consists of an LED light source, an ambient light sensor (TEMT 6000, Vishay Inter- The tubing holder is designed to fit a PTFE tube and was constructed using LCD-based technology, Malvern, PA, USA), and a tubing holder that allows for non-contact measure- 3D printing (ANYCUBIC Photon Mono X, Hong Kong Anycubic Technology, China) with ment. The tubing holder is designed to fit a PTFE tube and was constructed using LCD- China) with opaque acrylic-based resin (eResin PLA biophotopolymer resin, eSun, Shen- opaque acrylic-based resin (eResin PLA biophotopolymer resin, eSun, Shenzhen, China). based 3D printing (ANYCUBIC Photon Mono X, Hong Kong Anycubic Technology, zhen, China). (a) (b) Figure 5. On-line pH monitoring system. (a) a CAD file of a pH sensor; (b) a photo of a pH sensor Figure 5. On-line pH monitoring system. (a) a CAD file of a pH sensor; (b) a photo of a pH sensor with green LED. with green LED. 2.1.5. Controlling Program The bioreactor is controlled by simple software as shown in Figure 6 and Table 1. We developed software for controlling syringe pump, which can set the volume of culture media fed into and out of the culture chamber. For the selector, the software can control the selector to select the channel of each culture well, fresh media, and waste. Also, this software can control the stirrer of each well plate for mixing solution in each well. The pH monitoring system readings are recorded and a graph was plotted on the graphic user interface (GUI). In addition, we can write the preset script of the culture condition in ad- vance and this software can control the bioreactor automatically. The GUI of this software is shown in Figure 7. Figure 6. Schematic diagram showing workflow of controlling program. Bioengineering 2022, 9, x FOR PEER REVIEW 6 of 13 China) with opaque acrylic-based resin (eResin PLA biophotopolymer resin, eSun, Shen- zhen, China). (a) (b) Figure 5. On-line pH monitoring system. (a) a CAD file of a pH sensor; (b) a photo of a pH sensor Bioengineering 2022, 9, 187 6 of 12 with green LED. 2.1.5. Controlling Program 2.1.5. Controlling Program The bioreactor is controlled by simple software as shown in Figure 6 and Table 1. We The bioreactor is controlled by simple software as shown in Figure 6 and Table 1. We developed software for controlling syringe pump, which can set the volume of culture developed software for controlling syringe pump, which can set the volume of culture media fed into and out of the culture chamber. For the selector, the software can control media fed into and out of the culture chamber. For the selector, the software can control the selector to select the channel of each culture well, fresh media, and waste. Also, this the selector to select the channel of each culture well, fresh media, and waste. Also, this software can control the stirrer of each well plate for mixing solution in each well. The pH software can control the stirrer of each well plate for mixing solution in each well. The monitoring system readings are recorded and a graph was plotted on the graphic user pH monitoring system readings are recorded and a graph was plotted on the graphic interface (GUI). In addition, we can write the preset script of the culture condition in ad- user interface (GUI). In addition, we can write the preset script of the culture condition vance and this software can control the bioreactor automatically. The GUI of this software in advance and this software can control the bioreactor automatically. The GUI of this software is shown in Figure 7. is shown in Figure 7. Figure 6. Schematic diagram showing workflow of controlling program. Figure 6. Schematic diagram showing workflow of controlling program. Table 1. List of commands for controlling bioreactor. Command Value Description Loop number Set number of scripts to repeat Vol number Set volume of syringe pump to fill or drain (mL) Goto number Jump to a line number that set of the script Wait hh:mm:ss Set delay time Dir F or B Set syringe pump direction (Forward or Backward) Selector channel Ch Select selector channel (A or B or C or else) Stirrer channel Stir ON/OFF selected stirrer (A or B or C or else) Start Starting syringe pump Msgbox Text Displays the specified text in the message box Bioengineering 2022, 9, x FOR PEER REVIEW 7 of 13 Table 1. List of commands for controlling bioreactor. Command Value Description Loop number Set number of scripts to repeat Vol number Set volume of syringe pump to fill or drain (mL) Goto number Jump to a line number that set of the script Wait hh:mm:ss Set delay time Dir F or B Set syringe pump direction (Forward or Backward) Selector channel Ch Select selector channel (A or B or C or else) Stirrer channel Stir ON/OFF selected stirrer (A or B or C or else) Bioengineering 2022, 9, 187 7 of 12 Start Starting syringe pump Msgbox Text Displays the specified text in the message box Figure 7. A screenshot of user interface of controlling program. Figure 7. A screenshot of user interface of controlling program. 2.2. Bioreactor Setup 2.2. Bioreactor Setup Befor Before e using usingthe the developed developed bior biore eactor actor, , a a syringe syringepump pumpwas wascalibrated calibrated by by weighting weighting water flew out at different flow rates using a four-digit balance. A pH monitoring system water flew out at different flow rates using a four-digit balance. A pH monitoring system was was calibrated calibrated using using cultur cultue re media media with withdif differen ferenttpH pH values, values, which which wer were e pr pred edetermined etermined with with a a l laboratory aboratory p pH H me meter te.r. A A set setof of PDMS PDMS lids, lids, containers containers for for fr fr esh eshmedia media and and waste, waste, and and silicone silicone tub tubin ing g wer were e sterilized sterilized using using a annautoclave autoclave beforeh beforehand. and. All All components componentsexcept except aa micr microc ocontr ontro oller ller and and aa notebook notebook with with a a contr control olling ling pr program ogram wer were e sts erilized terilized with with ethanol ethanol and and then thenUV UVradiation radiation for for15 15 min minin ina a biosafety biosafety cabinet cabinet befor before e use. use. A A 6-well 6-well plate plate containing containing cell cell c cultur ultur ee was wasplaced placed in in aa chamber chamber and and a a m media edia rreservoir eservoir and waste container were connected to a selector in a biosafety cabinet. A bioreactor with and waste container were connected to a selector in a biosafety cabinet. A bioreactor with cell cell cu cultur lture e was was then then placed placed i inn an an incub incubator ator . . 2.3. Bioreactor Performance Test The performance of the developed bioreactor was tested by measuring the cell viability and cell proliferation rate of cells cultured in the bioreactor compared with that of a conventional cell culture procedure. A mouse fibroblast cell line (L929: NCTC clone 929, JCRB cell bank, Osaka, Japan) was used as a model for this test. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and 1% antibiotic- antimycotic (100X, Thermo Fisher Scientific, USA). 2.3.1. Cell Viability Testing The L929 cells were seeded in a 6-well plate at 1  10 cells per well. The well plate was then placed in the bioreactor and was incubated at 37 C with 5% CO and a humidified atmosphere for 48 h. The control group was place in the same incubator. The cell viability was measured using an MTT assay [22]. Bioengineering 2022, 9, x FOR PEER REVIEW 8 of 13 2.3. Bioreactor Performance Test The performance of the developed bioreactor was tested by measuring the cell via- bility and cell proliferation rate of cells cultured in the bioreactor compared with that of a conventional cell culture procedure. A mouse fibroblast cell line (L929: NCTC clone 929, JCRB cell bank, Osaka, Japan) was used as a model for this test. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and 1% antibiotic- antimycotic (100X, Thermo Fisher Scientific, USA). 2.3.1. Cell Viability Testing The L929 cells were seeded in a 6-well plate at 1 × 10 cells per well. The well plate was then placed in the bioreactor and was incubated at 37 °C with 5% CO2 and a humidi- fied atmosphere for 48 h. The control group was place in the same incubator. The cell viability was measured using an MTT assay [22]. 2.3.2. Cell Proliferation Assay The L929 cells were seeded in a 6-well plate at 7 × 10 cells per well with 5 mL of complete media and then cultured automatically in the bioreactor for 72 h using a pre-set program where 2 mL of cultured media was disposed of and refilled every 6 h. At 24 h and 72 h, the cells were trypsinized using 0.25% trypsin-EDTA solution (Thermo Fisher Bioengineering 2022, 9, 187 8 of 12 Scientific, USA) and the number of cells was counted with an automated cell counter (Countess II, Thermo Fisher Scientific, USA). The obtained proliferation rate was com- 2.3.2. Cell Proliferation Assay pared to that obtained from conventional cell culture with the same starting cell numbers. The L929 cells were seeded in a 6-well plate at 7  10 cells per well with 5 mL of complete media and then cultured automatically in the bioreactor for 72 h using a pre-set program where 2 mL of cultured media was disposed of and refilled every 6 h. At 24 h 2.4. pH Monitoring System Test and 72 h, the cells were trypsinized using 0.25% trypsin-EDTA solution (Thermo Fisher Scientific, ThUSA) e pH and mo the ninumber toring of system cells was wa counted s test with ed an duri automated ng thecell inc counter ubation of L929 cells for 48 h. (Countess II, Thermo Fisher Scientific, USA). The obtained proliferation rate was compared The program was set to refresh 2 mL of cell culture media every 6 h. to that obtained from conventional cell culture with the same starting cell numbers. 2.4. pH Monitoring System Test 3. Results The pH monitoring system was tested during the incubation of L929 cells for 48 h. 3.1. Syringe Pump Calibration The program was set to refresh 2 mL of cell culture media every 6 h. A calibration of the syringe pump with a 10 mL syringe was performed. The different 3. Results 3.1. Syringe Pump Calibration flow rates were set for one minute and the flow out water was collected and precisely A calibration of the syringe pump with a 10 mL syringe was performed. The different weighted. In Figure 8, the calibration plot showed that the mass of flow out water is cor- flow rates were set for one minute and the flow out water was collected and precisely related with the set flow rate. This result indicates that the flow rate of the calibrated sy- weighted. In Figure 8, the calibration plot showed that the mass of flow out water is correlated with the set flow rate. This result indicates that the flow rate of the calibrated ringe pump was accurate. syringe pump was accurate. Figure 8. A calibration graph of an in-house syringe pump, n = 3. Figure 8. A calibration graph of an in-house syringe pump, n = 3. 3.2. pH Calibration The pH monitoring system was calibrated with DMEM media, which is used for the culture of the L929 cell model. The media contains a phenol red indicator that changes color in the range of 6.8–8.2 from yellow to pink, respectively, as shown in Figure 9a. An increase in the pH of the media caused an increase in the red color in- tensity, which absorbed more green light and will be detected by the sensor. Figure 9b shows a calibration plot obtained from DMEM at a pH range between 6.90 and 7.83; green intensity = 57.7 (pH) + 1032, n = 3. Bioengineering 2022, 9, x FOR PEER REVIEW 9 of 13 3.2. pH Calibration The pH monitoring system was calibrated with DMEM media, which is used for the culture of the L929 cell model. The media contains a phenol red indicator that changes color in the range of 6.8–8.2 from yellow to pink, respectively, as shown in Figure 9a. An increase in the pH of the media caused an increase in the red color intensity, which ab- sorbed more green light and will be detected by the sensor. Figure 9b shows a calibration Bioengineering 2022, 9, 187 9 of 12 plot obtained from DMEM at a pH range between 6.90 and 7.83; green intensity = −57.7 (pH) + 1032, n = 3. (a) (b) Figure 9. Optical pH sensor: (a) effect of pH on green intensity measured by flowing DMEM media Figure 9. Optical pH sensor: (a) effect of pH on green intensity measured by flowing DMEM media at various pH (shown in inset) thought a sensor; (b) a calibration plot between pH of DMEM media and green intensity, n = 3. at various pH (shown in inset) thought a sensor; (b) a calibration plot between pH of DMEM media and green intensity, n = 3. 3.3. Cell Viability—MTT Assay 3.3. Cell Viability—MTT Assay The cell viability of L929 cells was measured after incubation in the developed biore- actor for 48 h. Figure 10a shows that the cell viability of the L929 cell in the bioreactor was The cell viability of L929 cells was measured after incubation in the developed biore- greater than 80% when compared to the control obtained through conventional cell cul- actor for 48 h. Figure 10a shows that the cell viability of the L929 cell in the bioreactor was ture. In addition, no morphologic change was observed, as shown in Figure 10b. These greater than 80% when compared to the control obtained through conventional cell culture. results suggest that the developed bioreactor including setup conditions and sterilization In addition, no morphologic change was observed, as shown in Figure 10b. These results process did not cause a cytotoxic effect on the cells. Bioengineering 2022, 9, x FOR Psuggest EER REVIEW that the developed bioreactor including setup conditions and sterilization 10 of 13 process did not cause a cytotoxic effect on the cells. (a) (b) Figure 10. Cell viability of L929 incubated in the developed bioreactor: (a) percent cell viability ob- Figure 10. Cell viability of L929 incubated in the developed bioreactor: (a) percent cell viability tained from MTT assay with respected to control group (n = 6); (b) a microscopic image of cells after obtained incubati from on iMTT n the bi assay oreactor with for 48 respected h. to control group (n = 6); (b) a microscopic image of cells after incubation in the bioreactor for 48 h. 3.4. Cell Proliferation Assay 3.4. Cell Proliferation Assay An automatic cell culture in the developed bioreactor was demonstrated. Figure 11 shows cell proliferation from L929 cells cultured in the bioreactor with the program to An automatic cell culture in the developed bioreactor was demonstrated. Figure 11 refresh the media every 6 h automatically compared to that cultured parallelly in a con- shows cell proliferation from L929 cells cultured in the bioreactor with the program to ventional manner (no media changed). It was found that the number of cells in the biore- refresh the media every 6 h automatically compared to that cultured parallelly in a conven- actor increased significantly at 72 h in comparison with the number of cells in the control tional manner (no media changed). It was found that the number of cells in the bioreactor plate. The results suggested that the automation of regular partial media change could help enhance the cell growth in the bioreactor. Figure 11. Cell proliferation of L929 incubated in the developed bioreactor. The number of cells was counted at 24 and 72 h after incubation and the data were analyzed by two-way ANOVA and post- hoc using R program. * Statistical significance was set at p < 0.05, n = 3. Bioengineering 2022, 9, x FOR PEER REVIEW 10 of 13 (a) (b) Figure 10. Cell viability of L929 incubated in the developed bioreactor: (a) percent cell viability ob- tained from MTT assay with respected to control group (n = 6); (b) a microscopic image of cells after incubation in the bioreactor for 48 h. 3.4. Cell Proliferation Assay An automatic cell culture in the developed bioreactor was demonstrated. Figure 11 Bioengineering 2022, 9, 187 10 of 12 shows cell proliferation from L929 cells cultured in the bioreactor with the program to refresh the media every 6 h automatically compared to that cultured parallelly in a con- ventional manner (no media changed). It was found that the number of cells in the biore- actor increased significantly at 72 h in comparison with the number of cells in the control increased significantly at 72 h in comparison with the number of cells in the control plate. plate. The results suggested that the automation of regular partial media change could The results suggested that the automation of regular partial media change could help help enhance the cell growth in the bioreactor. enhance the cell growth in the bioreactor. Figure Figure 11. 11. Cell Cell prolife proliferation ration of L929 of L929 incubate incubated d in the de in vel the oped developed bioreactor.bior The eactor number . The of celnumber ls was of cells counted at 24 and 72 h after incubation and the data were analyzed by two-way ANOVA and post- was counted at 24 and 72 h after incubation and the data were analyzed by two-way ANOVA and hoc using R program. * Statistical significance was set at p < 0.05, n = 3. post-hoc using R program. * Statistical significance was set at p < 0.05, n = 3. Bioengineering 2022, 9, x FOR PEER REVIEW 11 of 13 3.5. pH Monitoring Results The pH measurement of the cultured media was displayed in real-time and recorded 3.5. pH Monitoring Results as .CSV file for further analysis. The pH was measured by the pH sensor located on the The pH measurement of the cultured media was displayed in real-time and recorded channel before the waste container, indicating the pH of the used media. In general, acidic as .CSV file for further analysis. The pH was measured by the pH sensor located on the conditions could inhibit cell growth. A change in pH could reflect the cell condition, such channel before the waste container, indicating the pH of the used media. In general, acidic as a lower pH suggesting cell death or bacterial contamination. conditions could inhibit cell growth. A change in pH could reflect the cell condition, such as a lower Figur pH s e ugg 12 e shows sting cell de a plot ath or of bact the erial pH conchange tamination. with time during a 48-h cell culture of Figure 12 shows a plot of the pH change with time during a 48-h cell culture of L929 L929 cells in the developed bioreactor. It was found that a program set for regular media cells in the developed bioreactor. It was found that a program set for regular media change change was able to maintain the pH of the cell culture within 7.4  0.2, which is suitable was able to maintain the pH of the cell culture within 7.4 ± 0.2, which is suitable for fibro- for fibroblast cell line [23]. This could be related to the higher cell proliferation rate in the blast cell line [23]. This could be related to the higher cell proliferation rate in the bioreac- bioreactor observed in Figure 11. tor observed in Figure 11. Figure 12. pH changes during L929 cell incubation in the developed bioreactor. Figure 12. pH changes during L929 cell incubation in the developed bioreactor. 4. Discussion In this work, we developed an automatic programmable bioreactor with a pH moni- toring system that was suitable for tissue engineering cell culture. This bioreactor’s com- ponents can be controlled via programable script to the desired cell culture condition, and this system can operate automatically. This system helps to reduce time and labor com- pared to manual cell culture. For pH sensor, we applied a colorimetry technique to monitor the pH of the cell cul- ture on-line. This sensor was compact and easy to use. In addition, non-contact measure- ment has an advantage in real-time monitoring, and there is no contamination to cell cul- ture. The data were collected and can be analyzed for cell culture condition scripts next time. In future work, we plan to use machine learning methods, for example, the deep learning method [24,25], to optimize and find the suitable parameters in controlling bio- reactors in order to enhance the productivity of the cell culture in the bioreactor. Furthermore, this prototype can be applied to culture cells for a long period to study cell differentiation and tissue formation, which could be potentially useful for medical and industrial applications, including cultured meat, drug testing, etc. For example, this system could be used to study muscle cell differentiation and determine optimal condi- tions for cultured meat production. For drug testing, the bioreactor could be programmed Bioengineering 2022, 9, 187 11 of 12 4. Discussion In this work, we developed an automatic programmable bioreactor with a pH mon- itoring system that was suitable for tissue engineering cell culture. This bioreactor ’s components can be controlled via programable script to the desired cell culture condition, and this system can operate automatically. This system helps to reduce time and labor compared to manual cell culture. For pH sensor, we applied a colorimetry technique to monitor the pH of the cell culture on-line. This sensor was compact and easy to use. In addition, non-contact measurement has an advantage in real-time monitoring, and there is no contamination to cell culture. The data were collected and can be analyzed for cell culture condition scripts next time. In future work, we plan to use machine learning methods, for example, the deep learning method [24,25], to optimize and find the suitable parameters in controlling bioreactors in order to enhance the productivity of the cell culture in the bioreactor. Furthermore, this prototype can be applied to culture cells for a long period to study cell differentiation and tissue formation, which could be potentially useful for medical and industrial applications, including cultured meat, drug testing, etc. For example, this system could be used to study muscle cell differentiation and determine optimal conditions for cultured meat production. For drug testing, the bioreactor could be programmed to vary the dose or frequency of the treatment. In the future, an additional analytical system could be added to the bioreactor to detect disease-specific biomarkers or to monitor other biomolecules such as glucose and lactate dehydrogenase, which would indicate cells’ activity. Author Contributions: Conceptualization, S.U., S.A. and N.T.-U.; methodology, S.U., A.B., C.W., P.S., P.B., C.M. and P.P.; software, A.B.; validation, S.U., C.M. and P.P.; formal analysis, S.U., P.B. and P.P.; investigation, P.P.; resources, N.T.-U.; data curation, S.U., A.B., C.W., C.M., P.B. and P.P.; writing— original draft preparation, S.U., P.B. and P.P.; writing—review and editing, S.U., P.P., S.A. and N.T.-U.; visualization, S.U., A.B., P.S., P.B. and P.P.; supervision, S.A. and N.T.-U.; project administration, N.T.-U.; funding acquisition, S.A. and N.T.-U. All authors have read and agreed to the published version of the manuscript. Funding: This research project is supported by TSRI (71717). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. 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Journal

Bioengineering – Multidisciplinary Digital Publishing Institute

Published: Apr 25, 2022

Keywords: bioreactor; tissue engineering; automation; sensor

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