3

Methods

3.1

Rehydration,

Conditioning, and

Sterilization of

Microcarriers

1. Weigh 300 mg of Cytodex 3 microcarrier beads and transfer to

50 mL centrifuge Falcon tube.

2. Add 30 mL of Ca²⁺ and Mg²⁺ free DPBS to the 50 mL Falcon

tube containing microcarriers, suspend them carefully and

incubated at room temperature. After 4 h of microcarriers

incubation in DPBS, slowly pipet out the supernatant. Next,

add 20 mL of fresh Ca²⁺ and Mg²⁺ free DPBS into

microcarrier-containing Falcon tube, and vortex shortly. After

ca. 5 min, pipet out old DPBS and add 30 mL of fresh Ca²⁺ and

Mg²⁺ free DPBS.

3. Autoclave the microcarriers at 121 ^C with 1 bar overpressure

for 25 min.

4. Wait until the Falcon tube with microcarriers cool down and

carefully pipet out the DPBS under the biological safety cabi-

net. Next, add 30 mL of sterile DMEM culture medium,

vortex shortly, and leave for incubation at room temperature

to accomplish microcarriers conditioning. After 30 min, slowly

pipet out supernatant and add 30 mL of sterile fresh DMEM

culture medium. Suspend the microcarriers by vortexing and

then transfer them to the Cellbag container according to steril-

ity regime (see Note 1).

3.2

Preparation of

CP5 Chondrocyte

Inoculum

1. Remove CP5 cell-containing culture flask from the incubator

and carry out microscope observation. If confluence of CP5

cells in the culture flask is between 70% and 90%, the chondro-

cytes are ready for the next steps.

2. Transfer the culture flask to the biological safety cabinet. Care-

fully pipet out the culture medium from culture flask and flush

still adhered CP5 cells twice with 5 mL of fresh Ca²⁺ and Mg²⁺

free DPBS. Next, detach CP5 cells by addition of 3 mL of

trypsin–EDTA and leave shortly at room temperature to

accomplish trypsinization. After ca. 5 min, add 5 mL of

DMEM culture medium and suspend cells by gently shaking

culture flask.

3. Immediately transfer suspended cells into new centrifuge Fal-

con tube and separate cells from medium by centrifugation

(3800  g, 5 min). Then carefully decant supernatant and

resuspend cells in 5 mL of fresh DMEM culture medium.

4. Count the number of cells using the methodology described in

Subheading 3.4.2. Dilute cells to reach cell density of

6  10⁵ cell mL
¹.

5. Sterilely transfer 30 mL of diluted cells into microcarrier-

containing

Cellbag

container

prepared

according

to

Microcarrier-Supported Culture of Chondrocytes in Disposable Bioreactor

151