2.5

Other Reagents

1. Sodium hydroxide solution: Dissolve 20 g sodium hydroxide

(NaOH) in 5 L deionized (DI) water to prepare 0.1 M NaOH

solution.

2. Calcein-AM staining solution: Prepare Calcein-AM working

solution according to manufacturer’s instructions. Generally,

dilute 2 μL of 1 mM calcein AM stock solution in 1 mL PBS,

vortex the resulting solution to ensure thorough mixing. Keep

from light and use within 24 h.

3

Methods

The process is simply summarized in a flowchart in Fig. 2.

3.1

Setting up 3D

FloTrix^® vivaSPIN

Bioreactor on Day 1

1. Calibrate pH sensor (see Note 2).

2. Dismantle all stainless-steel tubes and two 3-blade impellers.

Rinse with ultrapure water thoroughly and sonicate for 60 min.

3. Wash the 5 L culture vessel with mild detergent. Rinse with

ultrapure water thoroughly for three times. Fill up the vessel

with 5 L 0.1 M NaOH solution and soak all stainless-steel tubes

and impellers for 12 h (see Note 3). Discard NaOH solution

safely. Rinse the vessel, tubes, and impellers with ultrapure

water for another three times.

Fig. 2 Flowchart of the protocol, with light blue boxes indicating preparation work involved and dark blue

boxes indicating the cell culture process

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