distilled water (in the case of blank sample) and 1 mL

BioMaxima-glucose reagent, and immediately vortex it.

2. Next, incubate all samples for 20 min at 25 ^C.

3. Measure absorbance of all samples using UV-VIS spectropho-

tometer at 500 nm.

4. Calculate the values of specific glucose consumption rate

according to following equation:

r^∗

glc=cell ^{¼ Cglc1
Cglc2}

X∙Δt

g h
¹ cell
¹



where r^*

glc/cell ^{is the specific glucose consumption rate, C}glc1^,

Cglc2 is the glucose concentration in the culture medium, and

Δt is the time interval between two consecutive measurements

of Cglc.

3.4.5

Activity of Lactate

Dehydrogenase

1. Mix 1 mL of BioMaxima-LDH reagent and 20 μL of filtered

culture medium harvested daily from cultures (in the case of

test samples) or 20 μL of double-distilled water (in the case of

blank sample) in directly in the disposable UV-Vis measuring

cuvette, and vortex it shortly.

2. Next, as soon as it only possible, read the absorbance in 1-min

intervals using UV-Vis spectrophotometer at 340 nm.

3. Calculate the values of activity of lactate dehydrogenase accord-

ing to the following equation:

aLDH ¼ 267:2  ΔA μkat L
¹



where aLDH is the activity of lactate dehydrogenase, and ΔA is

the absorbance change per minute.

3.4.6

DO and pH Level

Measurement

1. DO and pH level can be automatically measured by miniatur-

ized spot-like sensors built-in inside the bottom of Cellbag

container. The correctness of both values is verified and

certified by the manufacturer of the culture bag according to

setting the blank data for both sensors, which is printed indi-

vidually on each applied Cellbag container, fixed to WAVE

25 system.

4

Notes

1. The most suitable way to transfer suspension of conditioned

microcarriers from Falcon centrifuge tube into Cellbag con-

tainer is manually pipetting them via Screwcap port.

2. Small volume (i.e., ca. 60 mL) of cell–microcarrier suspension

introduced into Cellbag container provides proper conditions

for contact between CP5 cells and beads of microcarrier which

154

Kamil Wierzchowski and Maciej Pilarek