(b)

Autoclave the bioreactor for 20 min at 121 ^C using a stan-

dard clean cycle.

(c)

Store the bioreactor in a clean area until required (see Note 1).

3.1.5

Moving HepG2

Models into the Perfusion

System

1. Take an

autoclaved

bioreactor and

place

in

a laminar

flood hood.

2. Add 100 mL of prewarmed complete DMEM to the system.

3. Aspirate the medium from the HepG2 models after 7 days of

culture.

4. Using sterile forceps, lift the Alvetex^® insert from the plate and

place it into the holder in the bioreactor system (see Note 2).

5. Place a sterile 60 mm petri dish lid over the vessel and incubate

it on a magnetic stirrer set to 100 rpm for a further 7 days in a

37 ^C, 5% CO2 humidified incubator (see Note 3).

3.2

Creation of

Perfused Intestinal

Models

For the intestinal models an Alvetex^® Scaffold 12-well insert is

used. These require lower quantities of cells compared to 6-well

inserts while the use of Alvetex^® Scaffold instead of Strata creates a

more beneficial environment for the growth of the fibroblast com-

partment. The production of this model requires more user input

compared to the previous HepG2 model, as shown by the diagram-

matical summary of the protocol in Fig. 9.

3.2.1

Revival of Cells

1. Prepare a bottle of complete media by supplementing DMEM

with 10% FBS, 1% NEAA, and 2 mM L-glutamine. Additional

antimicrobial agents (100 U/mL penicillin and 100 μg/mL

streptomycin) is also recommended.

2. Cryopreserved Caco-2 cells should be revived and cultured in

complete DMEM as per the manufacturer’s instructions.

3.2.2

Preparation of

Alvetex^® Scaffold

The Alvetex^® Scaffold must be treated with ethanol before use, to

render the scaffold hydrophilic.

(a)

Remove the Alvetex^® Scaffold inserts using sterile forceps and

place into a sterile petri dish or beaker.

Fig. 9 Protocol for setting up intestinal bioreactor cultures in in static and perfusion. HepG2 cells are initially

seeded into Alvetex inserts in a 6-well plate and cultured for a week to allow the cells to adhere and migrate

across the membrane. The samples are then moved into bioreactors and placed on the stirrer unit with the

perfused samples on a lane set to 100 rpm and the static samples placed on a lane which is turned off. The

samples are then cultured for a further 7–14 days before fixation

Applying Stirred Perfusion to 3D Tissue Equivalents to Mimic the Dynamic In. . .

251