3.4.2

Cell Density and

Viability

1. Mix 50 μL of detached-cell suspension and 50 μL of 0.4%

trypan blue aqueous solution, and immediately vortex it.

2. Next, incubate cell–dye mixture for 3 min at 37 ^C.

3. Count live (i.e., unstained) and dead (i.e., blue-stained) cells

under

reverse

microscope

equipped

with

the

hemocytometer grid.

4. Calculate the value of cell density according to the following

equation:

X ¼ ^x

k ^{ d  5  105 cells mL
1}



where X is total concentration of cells (i.e., value summarizing

both stained and unstained cells), x is the total number of cells

counted in the grid of the hemocytometer, k is the number of

grid squares with cells, and d is the dilution of the sample

containing cells.

5. Calculate the value of cells viability as follows.

Z ¼ ^z

x

%

ð

Þ

where Z is the viability of living cells, and z is the number of

living (i.e., unstained) cells.

3.4.3

Metabolic Activity

of Cells Adhered to

Microcarriers

1. Mix 0.1 mL of PrestoBlue™reagent and 0.9 mL of suspension

of cell-occupied microcarriers (in the case of test samples) or

0.9 mL of suspension of freshly sterilized conditioned micro-

carriers (in the case of reference (i.e., blank) samples), and

immediately vortex it shortly.

2. Next, incubate all samples for 2 h at 37 ^C.

3. After incubation measure the absorbance of all samples, that is,

both test and reference ones, using UV-VIS spectrophotome-

ter at 570 nm versus referenced wavelength equaled to 600 nm.

4. Calculate the values of metabolic activity of cells according to

the following equation:

AW ¼ A570
A570REF

ð

Þ
A600
A600REF

ð

Þ

½ 

am ¼ 37, 04  AW

μkat L
¹



where AW is specific absorbance of the test sample, A570 is

absorbance of the test sample at 570 nm, A570REF is absorbance

of the reference at 570 nm, A600 is absorbance of the test

sample at 600 nm, A600REF is absorbance of the reference

sample at 600 nm, and am is metabolic activity of cells.

3.4.4

Specific Glucose

Consumption Rate

1. Mix 20 μL of filtered culture medium collected daily from the

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

Microcarrier-Supported Culture of Chondrocytes in Disposable Bioreactor

153