quantity of UCMSCs, multilayer culture flasks with automatic

culture systems are introduced, but this still requires large produc-

tion footprint for incubators. Thus, three-dimension (3D) dynamic

cell culture process based on microcarriers and perfusion of

medium in automated bioreactor is developed [6].

Here we illustrate a novel culture system developed by Beijing

CytoNiche Biotechnology Co., Ltd. for large-scale hUCMSC

expansion, with their proprietary dissolvable 3D TableTrix^® micro-

carrier tablets in a low-speed stirred tank 3D FloTrix^® vivaSPIN

bioreactor. The use of these microcarriers provides anchorage for

MSCs during the suspension culture, and the porous nature of

these microcarriers dramatically increases surface-to-volume ratio

of the whole culture system [7]. In comparison to traditional

microcarriers, 3D TableTrix^® is fully dissolvable using the compli-

mentary 3D FloTrix^® digest solution, which simplifies cell harvest-

ing from microcarriers as no additional separation of microcarriers

from cells is required. This gentle harvesting protocol also ensures

cell quality. More than 10⁹ cells can be produced in 4 days using

one bioreactor and cells retained their MSC phenotypic markers,

i.e., >95% expression of CD73, CD90, and CD105, and < 2% for

the myeloid lineage cell marker CD14 [8]; B-cell marker CD19 [9],

hematopoietic cell markers CD34, CD45; and HLA-DR [10].

2

Materials

2.1

Cell Culture

Reagents

1. General MSC culture reagents (i.e., hUCMSCs complete cul-

ture medium, phosphate-buffered solution (PBS)) and con-

sumables (T75 culture flasks) are required.

2. hUCMSCs: 2.5
10⁸ cryopreserved hUCMSCs are needed

for this culture protocol. Thaw 10 tubes of 2.5
10⁷/tubes

cryopreserved hUCMSCs seed cells (passage 4–5) in a 37 ^C

water bath, dilute to 50 mL by slowly adding warm complete

hUCMSC culture medium. Centrifuge at 179
g for 5 min.

Discard supernatant and resuspend with 50 mL warm complete

hUCMSC culture medium (see Note 1).

2.2

3D TableTrix^®

Microcarrier Tablets

Microcarrier suspension: 3D TableTrix^® microcarrier tablets (F01,

Beijing CytoNiche Biotechnology Co., Ltd.) are sterile products

and are provided as weight-defined tablets (Fig. 1a). Handle these

materials under sterile condition. Disperse 500 tablets (equivalent

to 10 g, 4,893  288 microcarriers/mg, offering at least

9000 cm²/g surface area) with 500 mL hUCMSC complete

medium (20 mg/mL) and gently agitate by shaking the bottle to

fully disperse them into individual microcarriers (Fig. 1b). This

should take about a minute. After dispersion, most microcarriers

are about 200 μm (D10–D90 size distribution between 125 and

114

Huanye Xu et al.