Overview and flow circuit of the designed bioreactor system
Figure 8 shows an overview of the bioreactor system and the perfusion circuit. The bioreactor vessel was made of stainless-steel and placed inside the incubator (37℃) during experiments. The bioreactor was equipped with sensors for respectively measuring pH, dissolved oxygen and temperature. The above parameters were controlled by BioBundles of Applikon™ (Holland)  and recorded by a computer automatically. The culture medium (pink part in Fig. 8A) was driven by a peristaltic pump and flowed in the clockwise direction between the culture chamber (the right side of bioreactor) and the control chamber (the left side of bioreactor), as shown in Fig. 8C. These two chambers were disconnected at the bottom. And the sensors were placed in the control chamber, for we expected the cell culture area in the culture chamber to be as large as possible. Dissolved oxygen and pH were adjusted by N2 and CO2, respectively. The cell suspension bottle was connected to the outlet at the bottom of the bioreactor via a four-way valve, and the medium storage bottle was also connected to the perfusion circuit via this valve. A real-time camera system was equipped on top of the bioreactor vessel, and the image data were recorded automatically by the computer.
Multi-layer parallel plate bioreactors have been previously used to develop an artificial liver  and to culture heart cells . In this study, we used a similar configuration that provided a stable fluid environment for cell culture. Circular parallel plates and the drainage column were installed in the center of culture chamber. We designed six circumferential homogeneously distributed diversion holes in each layer. Streamline diagram of the circulation are shown in Fig. 8C. During self-circulation process the medium firstly enters the control chamber on the left side of the bioreactor from the inlet and then flows upwards. In ascension process, bubbles in the medium gradually rupture to prevent damage to the cells in the culture chamber. Subsequently, the medium enters the culture chamber on the right side. In this chamber, the medium flows in the space between two adjacent layers, primarily in the radial direction, into the central drainage column through the diversion holes, and finally out of the bioreactor. This flow field design provides laminar flow in the bioreactor, preventing turbulence from damaging the cell growth.
In this study, 7 layers of plates were used for the experiment. To create different laminar velocities, the diameters of the diversion holes of the layers were 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 and 1.4 mm, in ascending order from bottom to top, and the diameters of the other diversion holes were 1.0 mm. The other parameters are listed in Table 2.
The parameters represent
The dimension in this study (mm)
diameter of the central drainage column
diameter of the parallel circular plate
diameter of the culture chamber
thickness of parallel plate
height of the interstitial space between adjacent layers
The inner diameter, outer diameter and thickness of the silicon plates were 15 mm, 100 mm and 1 mm, respectively. To enable quick and easy assembly and disassembly, we designed a 3D printing scaffold, as shown in Fig. 9C. The silicon plates were placed in scaffold layer by layer. During cell culture, the scaffold and plates were fixed to the central drainage column. The silicon plates were reflective, which enabled diffuse reflection imaging, as shown in Fig. 9D. And silicon plates could be sterilized without degeneration in high pressure and temperature about 120℃.
Computational Fluid Dynamics (CFD) Modeling And Simulation
The geometric model of the bioreactor was created using SolidWorks™ (2018, USA). Meshing and flow field calculations were processed using commercial CFD software (ANSYS™ 15.0, USA). The simulation was performed with the following settings: non-structural tetrahedral meshing; 300 K isothermal; an incompressible, Newtonian fluid with a dynamic viscosity of 0.0025 kg/(m·s) and a density of 1007.4 kg/m3; and 9.8 m/s2 acceleration due to gravity. The inlet velocity was set at 0.000138 m/ s (i.e. a flow rate of 1.828 mL/s), and the outlet of the central drainage column was set as the outflow. A laminar flow model was used in the calculation and energy transfer was ignored. The fluid shear stress (FSS) and fluid velocity were simulated with the CFX unit of ANSYS.
Tracer liquid imaging velocity (TLIV) measurements
This technique was performed by adding tinted droplets to the fluid, recording these flowing droplets by camera, and calculating the speed of the droplet centroid, which represents the fluid velocity. Figure 10 shows the experimental setup of the TLIV measurement, which included a camera, a glass bioreactor with multi-layer plates, a tracer liquid injector, peristaltic pumps and a reservoir (not shown in Fig. 10). For better lighting, a glass bioreactor of the same size was used in the measurement. In the case of constant liquid flow, the velocity distribution in the bioreactor is independent of fluid viscosity , so pure water was used instead of the medium for better imaging. In this experiment, water was pumped from the reservoir to the bioreactor at the same speed as in the CFD simulation and flowed along the path shown in Fig. 8C. The tracer droplet was injected to the outer edge between two plates, and flowed toward the diversion holes in the water and the camera recorded trajectory images of the tracer droplet.
The relative error between the velocities obtained from CFD and TLIV was calculated as follows:
Online Image System
To monitor the cell culture in the bioreactor, an online camera system was designed. The camera system includes an industrial lens with 0.7-4.5x magnification (T168, SunTime™, Taiwan), a CMOS image camera with 10 million pixels, 4–40 frames per second and 1/2.3 imaging area (D1000E, SunTime™, Taiwan), and LED ring lights, as shown in Fig. 9. The light radiated from the LED, passed through the observation window in the bioreactor, and then reached the plate and diffusely reflected. The schematic diagram of the optical path is also shown in Fig. 9A. The observation window was designed as follows: a waterproof cabin was located on the top of the bioreactor, and an optically transparent glass was mounted at the bottom of the cabin (as shown in Fig. 9B). Except the inlets and outlets, the bioreactor was isolated from the external environment by a sealed design.
The Vero cells and hUCMSCs were purchased from Sciencecell™ (Carlsbad, CA, USA) and cultured in T75 cell culture flasks (Corning™, USA) containing α-MEM medium (HyClone™, Logan, Utah) which was supplemented respectively with 10% fetal bovine serum (FBS) from Gibco™ (USA) and Excell™ (USA), and a 1% penicillin-streptomycin (PS) solution (Gibco™, USA) in 5% CO2 at 37 ºC. The sixth passage of Vero cells and the fifth passage of hUCMSCs were used in corresponding experiments.
Preparation Before Experiments
The flow chart of the cell culture experiment is shown in Fig. 11.
To be modified, silicon plates were added to 20 mg/mL poly-l-lysine and kept soaked for about 1 h. The cleaned bioreactor system was connected by silicone tubes according to the flow circuit. The modified silicon plates with the drainage column were installed inside the bioreactor, which constructed a contact bioreactor system. The gas tightness of bioreactor system was tested, which made sure the whole system was completely isolated from the external environment. Subsequently the prepared bioreactor system was entirely sterilized by high pressure steam in autoclave and then was exposed to ultraviolet for about 30 min.
Cell Seeding And Circulation
900 mL culture medium with 1 × 107 cells were prepared in the cell suspension bottle connected to the outlet of the bioreactor with silicone tube, while this outlet for circulation was also used as inlet in cell seeding. After cell seeding the bioreactor kept static for about 24 h in incubator to facilitate cell adherence on silicon plates. For the culture and control chamber were disconnected at the bottom, the cell suspension solution was kept in the culture chamber during seeding and adherence processes, with the purpose to enhance the chance of cell attachment on silicon plates. During these processes, there was no medium existing in the control chamber, which resulted in that the pH, dissolved oxygen sensors and the corresponding controllers did not work.
After cell adherence, 600 mL fresh medium was added into the culture chamber via the same outlet. Subsequently to initiate the self-circulation the medium was transferred from the culture chamber to the control chamber, which formed a self-circulating system realizing the control of medium parameters and renewal of medium ingredients. The circulation provided constant and stable flow shear stresses for cell growth on seven parallel silicon plates, which was same with the results of CFD simulations.