4

Notes

1. It must be considered that this bioreactor can be configured to

operate in different modes of action, including compression,

tension and fluid flow. For each mode, the components used in

the assembly of the equipment’s chambers are different. In the

described method, the association of the compression modes

and fluid flow was chosen. The total number of mounted

chambers are suggested when sample comparison is a desired

parameter to be evaluated.

2. Medium characteristics may vary due to cell type.

3. The hermetic feature should be previously tested before auto-

claving through a manual sphygmomanometer.

4. The open/close apertures setup must allow the fluid flow

oriented as desired, that is, from top to bottom.

5. 3D porous scaffolds of materials sensitive to the autoclave

temperature can be disinfected with alcohol baths. To facilitate

the alcohol flow within the scaffold, the material can be

immersed in alcohol and subjected to vacuum. Successive

washes in saline solution, also subjected to the same vacuum,

will be necessary for the removal of alcohol.

6. Cell attachment and matrix synthesis during the preculture

period are important for a more effective mechanotransduction

and resultant tissue stiffening when under the dynamic-

mechanical environment [20]. One must also observe that

differentiated cell types must have distinct conduct as com-

pared to undifferentiated ones. Mature cells that exhibit high

matrix expression should be treated with additional care to

properly adapt the medium content.

7. According to the desired flow direction, one must choose

different inputs and outputs apertures, and previous in/out

flow tests should be performed.

8. If an expressive amount of medium is lost during air removal,

the medium must be completed to a final volume of 180 mL.

The o-ring must be completed dry with no medium residue to

avoid contamination.

9. For convenience, create a schematic map of the designed flow

orientation.

10. Cell adhesion can be affected according to the cell type and

material properties, such as roughness, porosity, and chemical

composition. Thus, in previous test trials, the ideal flow rate for

each specific purpose should be assessed.

11. The main difference in this protocol is that the system is set up

for a sinusoidal displacement approach until it detects a specific

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Flavia Pedrini et al.