2.2

Processing and

Analysis of Samples

100% ethanol.

Serial dilutions of ethanol: 95%, 90%, 80%, 70%, 50%, 30%.

Histo-Clear II.

Paraffin wax.

Tissue embedding cassettes.

Tissue embedding molds.

Microtome.

Positively charged microscope slides.

Distilled water.

Mayer’s hematoxylin (Sigma).

Alkaline ethanol (3% ammonia in 70% ethanol).

Eosin.

Neonatal calf serum.

Triton X-100.

Primary antibodies.

Fluorophore-conjugated secondary antibody.

Vectashield HardSet with DAPI.

Glass coverslips.

Thiazolyl blue tetrazolium bromide powder.

Phenol-free DMEM.

Isopropanol.

Acidified isopropanol (2% hydrochloric acid in isopropanol).

96-well plate.

Plate rocker.

Absorbance microplate reader.

3

Methods

3.1

Growth of HepG2

Liver Models for Drug

Toxicity Testing

Culture of HepG2 in the perfusion system provides a simple

method to create physiologically relevant culture conditions while

maintaining ease of use and low cost. The culture of the models in

this system can be summarized in a simple 3 step protocol, as shown

in Fig. 8.

3.1.1

Revival of HepG2

Cells

1. Prepare a bottle of complete Dulbecco’s modified essential

medium (DMEM) made up of DMEM supplemented with

10% fetal bovine serum (FBS), 2 mM

L-glutamine, and

100 U/mL of penicillin–streptomycin.

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

249