Feasibility study of a biocompatible pneumatic dispensing system using mouse 3T3-J2 fibroblasts
This paper presents results for dispensing living cells using a pneumatic dispensing system to verify the feasibility of using this system to fabricate biomaterials. Living cells (i.e., mouse 3T3-J2 fibroblast) were dispensed with different dispensing pressures in order to evaluate the effect of dispensing process on cell viability and proliferation. Based on the results of a live-dead assay, more than 80% of cell viability has been confirmed which was reasonably similar to that in the control group. Furthermore, measurement of cell metabolic activity after dispensing confirmed that the dispensed cell proliferated at a rate comparable to that of the control group. These results demonstrate that the pneumatic dispensing system is a promising tool for fabrication of biomaterials.
KeywordsPneumatic dispensing Living cell Viability Proliferation
Technologies to manipulate living cells are widely employed in biological and tissue engineering applications. For example, depositing living cells at a specific location is useful to help understand how cell behavior such as proliferation, differentiation, and migration is related to local environment (e.g., extracellular matrix and neighboring cells) [1, 2, 3, 4]. Also, in tissue engineering, which seeks to repair injured or ill organs, it is necessary to seed living cells at a precise position in three-dimensional (3D) scaffolds to achieve successful production of artificial tissues or organs [5, 6, 7].
To construct 3D cellular structures, many cell patterning technologies have been developed, including micro-contact printing using soft-lithography , and microfluidic technology . These technologies can produce a high resolution pattern, but they are inconvenient to form 3D structures and expensive to change patterns .
Recently, dispensing (e.g., inkjet printing) technology has attracted much attention for use as a tool to fabricate structures consisting of various cell types [11, 12, 13, 14, 15]. Because the dispensing technology can make a precise cell pattern directly by dispensing small droplets of biological materials containing living cells, it has advantages of simplicity and flexibility for fabrication of complex cellular structures. The dispensing technology is flexible to pattern various designs by simply controlling the position to dispense living cells in droplets from a nozzle. Furthermore, because the dispensing technology can be easily integrated with computer-assisted manufacturing systems, it can use solid free-form fabrication to precisely form complex 3D cellular structures [16, 17]. Also, it can form cellular structures that consist of different cell types by using and controlling multiple nozzles, each with a corresponding reservoir .
For tissue engineering applications, considerable research has been devoted to extending the capability of dispensing technology to create artificial tissues or organs. Because living cells are easily damaged by heat and mechanical stress , the biocompatibility of the dispensing system must be assessed by evaluating the effect of the dispensing process on cell viability and proliferation. Living cells have been dispensed using various printing systems such as thermal inkjets , piezoelectric inkjets [12, 13, 14, 15], electrostatics , lasers , and electrohydrodynamic jets .
This paper presents results for dispensing living cells using a pneumatic dispensing system to verify the feasibility of using it as a biocompatible fabrication tool. The pneumatic dispensing system uses a simple yet effective mechanism with a backflow stopper and a flexible membrane, so it is easy to control droplet volumes and to eject highly viscous liquid [20, 21]. To assess the biocompatibility of the dispensing system, experiments were conducted using mouse 3T3-J2 fibroblast cells at various operation conditions, then cell viability and proliferation were evaluated using a live-dead assay and a cell counting kit assay, respectively.
Materials and methods
Pneumatic cell dispensing system
Summary of design parameters
The flexible membrane is deflected by the applied pressure, and thus draws in or dispenses liquid. The membrane is either pulled (negative pressure) or pushed (positive pressure) depending on the programmed electric signal. The applied pressure is normally negative; a pulsed signal switches a solenoid valve to provide positive pressure (during duration time) to dispense the liquid. The liquid is drawn into the chamber when the membrane is pulled (during delay time) and dispensed when it is pushed.
Preparation of cell suspension
A cell suspension was prepared using mouse fibroblasts (3T3-J2 cells) obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithersburg, MD) supplemented with 10% bovine calf serum (Gibco), 1% mixture of penicillin and streptomycin (Gibco). Cells were cultured at 37 °C in a humidified incubator in an atmosphere containing 10% CO2 . To apply living cells to the dispensing system, cells in culture flasks were trypsinized, then cell pellets were collected, and resuspended in a phosphate-buffered saline (PBS, Gibco) solution. The suspension contained 500,000 cells mL−1, as quantified using a hemocytometer.
Cell viability assay
Cell proliferation assay
Results and discussion
To test the biocompatibility of our dispensing system, cell viability and proliferation were analyzed qualitatively using live-dead and CCK-8 assays. A statistical significance was determined using analysis of variance (ANOVA) on MINITAB version 14.2 (Minitab Inc., State College, PA, USA). A P value less than 0.05 was considered statistically significant.
The effect of our dispensing system on cell viability and proliferation was assessed at various applied pressures. Based on the results of a live-dead assay, more than 80% of cell viability has been confirmed which is reasonably compatible to the control group. Cell metabolic activity measurements confirmed that the dispensed cells were proliferating at a rate comparable to that of the control. These results confirm the feasibility of using our pneumatic dispensing system to dispense living cells for fabrication of biomaterials.
LS and KH performed the experiments, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
Ethics approval and consent to participate
This work was supported by Dong-eui University (Grant No. 201702730001) and the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MSIT) (Nos. 2017R1C1B1008045 and 2017R1C1B5076710).
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- 1.Devillard R, Pagès E, Correa MM, Kériquel V, Rémy M, Kalisky J, Ali M, Guillotin B, Guillemot F (2014) Cell Patterning by laser-assisted bioprinting. Methods Cell Biol 119:159–174. https://doi.org/10.1016/B978-0-12-416742-1.00009-3 CrossRefGoogle Scholar
- 3.Xu T, Gregory CA, Molnar P, Cui X, Jalota S, Bhaduri SB, Boland T (2006) Viability and electrophysiology of neural cell structures generated by the inkjet printing method. Biomaterials 27:3580–3588Google Scholar
- 18.Xiong R, Christensen K, Fu J, Markwald RR, Huang Y (2016) Freeform laser and inkjet printing of biological constructs. 2016 International symposium on flexible automation, Cleveland, Ohio, USA, 1–3 August 2016Google Scholar
- 19.Liaudanskaya V, Gasperini L, Maniglio D, Motta A, Migliaresi C (2015) Assessing the impact of electrohydrodynamic jetting on encapsulated cell viability, proliferation, and ability to self-assemble in three-dimensional structures. Tissue Eng Part C 21(6):631–638. https://doi.org/10.1089/ten.tec.2014.0228 CrossRefGoogle Scholar
- 23.Liberski AR, Zhang R, Bradley M (2009) In situ nanoliter-scale polymer fabrication for flexible cell patterning. JALA 14:285–293Google Scholar
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