9. Cast PDMS mixture on the mold and degas the mold with

PDMS in the vacuum pot until there is no bubble left in the

PDMS. Cast PDMS on a petri dish to create a layer of PDMS

substrate for device binding as well.

10. Leave the mold in 45
C oven until the PDMS device is cured

(see Note 13).

11. Peel off the device from the mold.

12. Punch the inlet and outlet holes with the 3 mm biopsy punch,

carefully clean the punching holes by flushing IPA through it

and dry with an air nozzle.

13. Tape the chip with scotch tape to remove dust and debris.

14. Cut the PDMS substrate out of the petri dish, use the bottom

side as substrate and leave it in the plasma machine with the

PDMS device (channel side facing up). Vacuum for 4 min and

plasma treat for 2 min in the machine (see Note 14) and bind

them together.

15. Place the silicone tubing inside the inlet and outlets of the

spiral chip.

3.4

Operation

Carry out all processes of separation/harvesting in a biosafety

cabinet to reduce pathogen contamination risks (Fig. 2).

1. Spray 70% ethanol on all equipment and devices, move them

into the biosafety cabinet and leave them under UV light for

30 min.

2. Use one syringe to inject 70% ethanol into the device for

sterilization of the system for 5 min.

3. Use another syringe to inject DPBS into the device, wash away

the 70% ethanol and perfuse for 10 min. Keep the perfused,

sterile DPBS inside the channel.

4. Detach the cells from microcarriers by removing the superna-

tant as much as possible with a serological pipette, and add

enough digestive enzymes (e.g., TrypLE express, Gibco) to

cover all microcarriers and incubate for 10–15 min in the

incubator, depending on the concentration and cell density.

5. Resuspend the cells and microcarriers for 10 times with a

serological pipette.

6. Dilute the cells to certain concentration with DPBS: up to

0.75% v/v ratio for direct-printed chip design and 1.68% v/v

ratio for PDMS chip design.

7. Load the cell-microcarrier solution into the syringe when using

a syringe pump or leave it inside the bioreactor/spinner flask or

a reservoir with a magnetic stirrer if the solution is delivered by

a peristaltic pump.

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