Cost effective open source microvalve bioprinting of therapeutic cells
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1
Newcastle University International Singapore, Faculty of Science, Agriculture and Engineering, Singapore
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2
Republic Polytechnic, School of Applied Sciences, Singapore
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3
University of Alberta, Department of Mechanical Engineering, Canada
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4
Newcastle University, Newcastle Upon Tyne, School of Mechanical and Systems Engineering, United Kingdom
Three Dimensional (3D) printers are being used to underpin major innovations in manufacturing, engineering, education and medicine. Advances in the technology have also enabled cells and biocompatible materials to be incorporated into bioprinting. Biofabrication involves the dispensation of cells onto a stage or printed structure (Hendriks et al. 2015). This process is being researched for the ability to create reliable models for the study of the development of cells and tissues. Biofabricated structures have increasing complexity and are being constructed with more similarity to native tissues and organs (Costa 2015). Biofabrication technologies currently include inkjet bioprinting, laser assisted forward transfer, valve based bioprinting (Hendriks et al. 2015). Bioprinting can be useful in dispensing therapeutic cells in order to increase efficiency and improve productivity. Dispensing cells using manual pipetting is labour-intensive and error prone. The aim of this project is to automate this high throughput cell dispensing with the use of a modified, custom-designed, microvalve-based cell printer. The project consists of three phases; Phase I is the design and fabrication of the 3D printer, along with development of the dispensing mechanism, Phase II consists of improvement, in terms of accuracy, efficiency and total time taken for the dispensing process, and Phase III includes experiments conducted with human colorectal carcinoma cells (HCT116) obtained from ATCC (Atcc 2012) for cell viability and consistency testing. Phase I of the project involved the research, design and development of an open source, cost effective, and reliable system, with the objective of getting accurate results as compared to a manual dispensing method. The system when assembled was made up of multiple components that that can be programmed to function systematically. Due to the given operational requirements, certain considerations have to be taken into account when designing the system. (1) Any experiment concerning biomaterials should be done under the biosafety cabinet to protect the user, the cells and the environment. (2) Optimization of the time taken to dispense cells, to prevent clogging in the nozzle. (3) Very small amounts (in the range of µl) of liquid is being dispensed by the system, therefore it has to be accurately designed to ensure efficiency.
The system was required to dispense a 96 well plate in specific volumes therefore, an extensive program was developed for the function of the system. A program to control the system was written on Arduino software (Arduino.cc n.d.) using Arduino Mega which controls three components: movements on X, Y and Z axes, the injection and aspiration of the liquid by the syringe pump and the ejection of cells in media from the microvalve. The main structure was obtained from a modifiable 3D printer from RepRapPro Ormerod 3 (RepRapPro n.d.) and the syringe pump module supplying pressure to the microvalve was made with 3D printed parts and a lead screw. Different versions of the extruder system was tested before selecting the microvalve technology; the first extruder system included a syringe with needle gages of 18G and 23G. However, this system included safety risks such as the exposure of the needle. The system then moved on to version 2, which is the microvalve module. The microvalve used was the VHS Nanolitre Dispense Valve and the 062 MINSTAC Straight Tube Dispensing Nozzle from Lee Products (Lee Products Ltd n.d.), both of which were attached to the system in Phase III. A magnetic agitator module purchased from Thermo Fisher Scientific (ThermoFisher n.d.) was added to prevent the cells from aggregating in the nozzle. A program written in Arduino will send commands to the electronic components and allow the movement of the 3D printer, syringe pump and dispensing from the valve. The final system and the specific modules is shown in Figure 1 below.
Fig. 1 Final version of the system.
Experiments were conducted in three sets and comparisons were made with manually pipetted cells vs bioprinted cells (with and without cell agitation). For the negative control, cells were treated with hydrogen peroxide and this showed 18.83% cell viability as compared to the positive control with 86.63% viability. Cell viability percentages for manual method was from 36.92% - 53.49%, whereas results for bioprinted and bioprinted plus mixer method was from 61.53% - 66.06% and 68.54% - 75.31%, respectively. Moreover, the coefficient of variance showed unitless values of 23.9 - 34.7, 15.9 - 17.5 and 8.3 - 9 for the manual dispensing, bioprinted and bioprinted plus mixer, respectively. The higher variance of the manual results can be attributed to human error and pipetting inaccuracies. The entire process of dispensing cells into a 96-well plate lasted about 3 minutes whereas manual method lasted around 20 minutes. Since the bioprinted plus mixer method gave the best results, further experimentation will be done with other types of cells with therapeutic value, for example mesenchymal stem cells.
This work demonstrates that valve-based printing is able to dispense cells with higher viability, lower variability, reduced time taken, and a higher efficiency than a manual pipetting method.
Keywords:
Bioprinting,
Therapeutic cells,
microvalve technology,
Cells,
Technology
Conference:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting, Seberang Jaya, Penang, Malaysia, 17 Nov - 18 Nov, 2016.
Presentation Type:
Oral
Topic:
Biomaterials and Tissue Regeneration
Citation:
Okubo
N,
Quershi
AJ,
Derebail
S,
Dalgarno
K and
Goh
KL
(2016). Cost effective open source microvalve bioprinting of therapeutic cells.
Front. Bioeng. Biotechnol.
Conference Abstract:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting.
doi: 10.3389/conf.FBIOE.2016.02.00003
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Received:
08 Dec 2016;
Published Online:
19 Dec 2016.
*
Correspondence:
Dr. Nami Okubo, Newcastle University International Singapore, Faculty of Science, Agriculture and Engineering, Singapore, Singapore, 569830, Singapore, nokubo7@gmail.com