2. Thoroughly clean the lid of the bioreactor with 70% ethanol

prior to inverting it to the vented orientation.

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

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

culture.

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

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

6. Replace the bioreactor lid and incubate it on a magnetic stirrer

set to 100 rpm for a further 7–14 days in a 37 ^C, 5% CO2

humidified incubator. Perform a full media change on day 7 if

perfusing for 14 days.

3.3

Processing and

Analysis of the

Perfused Models

To investigate the function and morphology of in vitro tissue

equivalents, a wide range of analytical techniques can be used.

The figures shown in this chapter were generated through histo-

logical and immunofluorescent techniques for visual analysis of

gross morphology and expression of individual proteins, or

through the use of an MTT assay to measure cellular metabolism.

3.3.1

Processing

Samples for Paraffin Wax

Embedding

1. Carefully unclip the Alvetex^® insert using blunt forceps and

place the membrane into a 12-well plate containing 2 mL

of PBS.

2. Aspirate the PBS and wash a further two times to remove

residual culture medium.

3. Fix in 4% paraformaldehyde at room temperature for 2 h, or

overnight at 4 ^C.

4. Wash the fixed models three times in PBS to remove residual

paraformaldehyde.

5. Dehydrate the samples through a gradient of ethanol concen-

trations (30%, 50%, 70%, 80%, 90%, 95%) for 15 min each

before finally in 100% ethanol for 30 min.

6. Transfer the dehydrated samples into tissue processing cas-

settes and submerge in Histo-Clear II for 30 min.

7. Move the samples into a 1:1 mixture of Histo-Clear II and

molten paraffin wax, and incubate at 65 ^C for 30 min.

8. Transfer the samples to pure molten paraffin wax and incubate

at 65 ^C for 1 h.

9. Cut the tissue models in half across their diameter using a

surgical scalpel and embed in molten wax with the flat edge

orientated to the base of the plastic embedding mold.

10. Allow embedded models to completely set overnight at room

temperature prior to sectioning and downstream analysis.

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

253