allows

for

sufficient

adhesion

of

CP

chondrocytes,

as

anchorage-dependent cells, onto microcarrier surface.

3. The addition of DMEM culture medium up to reach the total

value of 300 mL liquid phase inside Cellbag container, allows

obtaining

starting

cell

concentration

level

equaled

to

6  10⁴ cell mL
¹.

4. Based on our experience, the most suitable operating para-

meters supporting the most efficient propagation of CP5 cells

biomass are as following: angle of oscillations equaled to 6⁰,

frequency of oscillations equaled to 20 min
¹, gas flow rate

equaled to 0.55 L min
¹, volume of liquid phase equaled to

300 mL, temperature equaled to 37 ^C, O2 concentration in

gas phase equaled to 21%, CO2 concentration in gas phase

equaled to 5% and rocking motion acceleration equaled to

30%. In the case of other mammalian cells, the most suitable

operating parameters will be depend strongly on resistance of

given cells to hydrodynamic shear stress. Because of that,

operating parameters must be individually designated for each

cell line.

5. The most suitable way to harvest sample of culture medium

from Cellbag container is to use syringe with Luer Lock tip and

connect it to CLAVE™sampling port, what allows collecting

sample in sterile conditions outside the biological safety

cabinet.

6. Addition of fresh DMEM medium allows to neutralize the

proteolytic effects caused by trypsin.

7. The sedimentation time for microcarriers equaled to ca. 2 min.

After microcarrier beads sediment to the bottom of Falcon

centrifuge tube, cells can separate from microcarriers to slowly

manual pipette out the cell-containing liquid phase over the

layer of beads and transfer it into 2 mL Eppendorf tube.

References

1. Shukla A, Tho¨mmes J (2010) Recent advances

in large-scale production of monoclonal anti-

bodies and related proteins. Trends Biotechnol

28:253–261.

https://doi.org/10.1016/j.

tibtech.2010.02.001

2. Kaiser S, Kraume M, Eibl D, Eibl R (2015)

Single-use bioreactors for animal and human

cells. In: Ai-Rubeai M (ed) Animal cell culture.

Cell engineering. Springer, Basel, Switzerland

3. Wierzchowski K, Grabowska I, Pilarek M

(2020) Efficient propagation of suspended

HL-60 cells in a disposable bioreactor support-

ing wave-induced agitation at various Reynolds

number.

Bioproc

Biosystems

Eng

43:1973–1985.

https://doi.org/10.1007/

s00449-020-02386-6

4. Singh V (2001) Method for culturing cells

using

wave-induced

agitation,

US

Patent

6190913

5. Wierzchowski K, Grabowska I, Pilarek M

(2019) Propagation of non-adherent HL-60

cells in batch cultures maintained in static and

wave-type agitated systems. Chem Process Eng

40:167–177. https://doi.org/10.24425/cpe.

2019.126109

6. Eibl R, Eibl D (2011) Single-use technology in

biopharmaceutical

manufacture.

Wiley,

Hoboken

Microcarrier-Supported Culture of Chondrocytes in Disposable Bioreactor

155