3.3

Expansion of

hMSCs in Planar

Culture

1. One vial of frozen hMSCs (generally contains 1
10⁶ cells) is

recovered by immediately thawing in a 37 ^C water bath for

30 s until a small piece of ice remains. Spray the vial with 70%

ethanol and open the vial in biological safety cabinet. Transfer

the cell suspension carefully into at least ten times of volume of

hMSC-CCM (for instance, 1 mL cell suspension in 10 mL

CCM) in centrifuge tube. Gently pipette the mixture and

then centrifuge at 400
g for 5 min.

2. After centrifugation, carefully remove the supernatant and do

not disturb the cell pellet. Resuspend the cell pellet with

1–3 mL hMSC-CCM carefully and distribute the cell suspen-

sion into 150 mm diameter petri dish at 1200–1500 cells/cm².

The culture is maintained in a standard incubator (37 ^C, 5%

CO2).

3. First medium change is performed after 12 h to remove debris

and unattached cells. Then medium is changed every 2 days

with fresh hMSC-CCM.

4. For passaging hMSCs, culture medium is removed, and the

cells are washed with sterile PBS twice. Then the hMSCs are

treated with 0.25% trypsin solution at 37 ^C for 5–7 min.

Trypsin is neutralized by adding hMSC-CCM and detached

cells are collected in a 15 mL centrifuge tube. The petri dish is

washed with fresh hMSC-CCM once to collect residue cells for

maximized yield.

5. Spin down the hMSCs at 500
g for 5 min and remove the

supernatant. Resuspend the cell pellet with hMSC-CCM and

determine the cell number by hemocytometer. hMSCs can be

expanded on tissue culture surface or on microcarriers in

PBS-VW bioreactors for EV collection (see Note 9).

3.4

Expansion of

hMSCs in PBS-VW

Bioreactors

1. hMSCs in planar cultures are harvested and resuspended with

hMSC-CCM containing EV-free FBS. Then hMSCs are mixed

with sterile Cytodex-1 microcarriers (0.25–0.5 g) at the density

of 1000–1500 cells/cm². The mixture is added to the PBS-VW

bioreactors and the volume is brought to 60 mL with hMSC-

CCM containing EV-free FBS (see Note 10).

2. On day 0, intermittent agitation is used after cells and micro-

carriers are transferred into the bioreactor. The agitation base is

set to 25 rpm for 5 min and off for 15 min for a total of 12 cycles

(roughly 4 h). Then the agitation speed is set to 25 rpm for the

rest of the culture period. The total volume is remained at

60 mL for seeding phase. After seeding, the total volume is

brought to 100 mL with fresh hMSC-CCM containing

EV-free FBS. The culture is maintained in a standard incubator

(37 ^C, 5% CO2).

Human Stem Cell-derived Extracellular Vesicles in Bioreactors

199