Subheading 3.1 (see Note 2). Incubate inoculated system at

37 ^C for ca. 4 h, that is, CP5 cells must completely attach to

the outer surface of beads of the conditioned microcarriers.

After this time, add 300 mL of DMEM culture medium to

obtain final volume of the culture system inside Cellbag con-

tainer (see Note 3).

3.3

Maintaining CP5

Cells in Bioreactor

System

1. Install Cellbag container which contains microcarriers inocu-

lated with CP5 in the rocker of WAVE 25 system.

2. Set the operating parameters (see Table 1) in control unit of

WAVE 25 system (see Note 4).

3. Next, stabilize the temperature of culture system at level of

37 ^C, as well as DO concentration at the value at 100%

saturation of the aqueous phase (for gas mixture composed of

21% O2, 5% CO2, and 74% N2).

4. Maintain oscillatory wave-agitated culture for 7 days, with daily

monitoring and harvesting 5 mL samples of culture medium

(see Note 5). To fully quantitatively characterize the culture

parameters for each day of bioprocess, determination the values

of cells density, viability of living cells, metabolic activity of

cells, and specific glucose consumption rate (see Subheadings

3.4.2–3.4.4) as quantitative characteristics of biomass, as well

as determination the values of DO, pH level and activity of

lactate dehydrogenase (see Subheadings 3.4.5 and 3.4.6) as

quantitative characteristics of culture medium, should be

performed.

3.4

Analytical

Methods

3.4.1

Preparation of

Samples for Analysis

1. Use 3 mL of each of collected samples, in a form of suspension

of cell-occupied microcarriers, to analyze metabolic activity of

cells.

2. For other 2 mL of each of collected samples, separate cell-

occupied microcarriers from the culture medium by carefully

pipetting out the culture medium. Filter culture medium

through 0.22 μm syringe filter into new and sterile 2 mL

Eppendorf tube. Sample of filtered culture medium will be

used for analysis of specific glucose consumption rate and

level of activity of lactate dehydrogenase.

3. Rinse the separated microcarriers occupied by CP5 cells with

2 mL of fresh Ca²⁺ and Mg²⁺ free DPBS for 5 min twice.

Carefully pipet out DPBS, then add 1 mL of trypsin–EDTA,

and gently vortex once a minute. After 5 min incubation at

room temperature, add 1 mL of fresh culture medium (see

Note 6). Vortex Falcon tube and immediately carefully pipet

out cell-containing liquid above microcarriers (see Note 7).

Cell-containing liquid phase will be used for counting cells, as

well as for determination of living cells viability.

152

Kamil Wierzchowski and Maciej Pilarek