Fusion touch

The modular 3D printed microfluidic system was set up with two micromixers, a spiral microfluidic device, and a zig-zag concentrator connecting in series with Tygon tubes. Cell harvesting was conducted in a biosafety cabinet to prevent any contamination. The upper mixer has two inlets, one was connected to the bioreactor through a peristaltic pump (Shenchen, China), and the other one was connected to a syringe pump (Fusion Touch, Chemyx Inc) for the TrypLE injection. Before cell harvesting, the whole setup was sterilised by 70% ethanol and UV irradiation. The same number of cell-attached MCs was harvested by the conventional membrane filtration method as a control. Briefly, the MCs were allowed to settle for 10 min, and then the culture media was carefully removed by a serological pipette. 40 mL of TrypLE was added to the bioreactor and incubated for 20 min. The microcarrier-cell suspension was gently pipetted before filtration. Lastly, the suspension was filtered by Steriflip Nylon Net filters (Millipore Steriflip filtration 100 μm, Merck, Australia), and the filtrated cells were collected. The recovery rate of cells and microcarriers were calculated by: $R=N_{\mathrm{target}\mathrm{outlet}}/(N_{\mathrm{target}\mathrm{outlet}}+N_{\mathrm{outher}\mathrm{outlet}})$ , N is the number of particles counted with haemocytometer. Counting was repeated 3 times.

The experimental setup is illustrated in Figure 1A, and the experiments were perfomed under room temperature. The cells and DMSO solutions were loaded into 3 mL syringes (BD plastic) and connected to the device with Tygon tubings (John Morris, Sydney, Australia). Two syringe pumps (Fusion Touch, Chemyx Inc., Stafford, TX, USA) were used to hold these two syringes separately, and the flow rate ratio was fixed to 9 to 1 for cells and DMSO syringes. The cells were collected from the outlet by a cryopreservation tube and placed in Mr. Frosty (Sigma Aldrich, Sydney, Australia) inside the −80 °C freezer immediately. This allowed the temperature of the sample to decrease in a controlled rate. Five control groups were made by adding and pipetting 100 µL of DMSO in 900 µL cells in fresh culture media. One group was put into Mr. Frosty (Thermo Scientific, Waltham, MA, USA) immediately after mixing, two groups were left at room temperature (RT) for 30 min and 60 min, and the last two groups were left in a 4 °C fridge for 30 min and 60 min before being put into Mr. Frosty. This was to mimic the cell damage caused by long-term incubation with DMSO. All groups of cells were left in the freezer for one week before thawing for cell characterisations.

Plasma was separated from whole blood by the sedimentation of the red blood cells (RBCs) in the trenches of the microfluidic devices. Before using, the devices were washed three times with IPA and twice with PBS. A syringe pump (Chemyx, Fusion Touch, version 1.7.6 c) was used to control the flow, using either positive or negative pressure, in the 1–15 µL min⁻¹ range at a constant rate. In addition, 250 µL syringes (Hamilton, Reno, NV, USA) were used to introduce the sample into the devices. PTFE tubing (0.22 in ID × 0.042 in OD, Cole-Parmer, Vernon Hills, IL, USA) and capillary tubing (polymicro flexible fused silica capillary tubing, ID 50 µm, OD 375 µm, Lisle, IL, USA) were fixed with UV-curable resin (DecorRom, Shenzhen, China) to connect the microfluidic devices to the syringe pump. The entire process is summarized in Figure 1.