Overview: BIOMIMESYS hydrogels for 3D culture 

BIOMIMESYS is a range of new patented hyaluronic acid scaffolds for 3D cell culture. Celenys scaffold is made of RGDS and galactosamine-grafted hyaluronic acid, adipic acid dihydrazide crosslinker, and extracellular matrix (ECM) proteins (collagen type I and IV). BIOMIMESYS is suitable for robotic tests due to the thickness of the scaffold – around 600 μm – with an average porosity ranging from 100 to 200 μm (see Figure 1).

Step-by-step procedure

1. Cell seeding on BIOMIMESYS hydrogel with the VIAFLO 96/384

Homogenize HepG2 cells and seed on the ready-to-use BIOMIMESYS Hepatocyte hydrogel. This step takes less than two minutes.

HepG2 cells are homogenized in an automation friendly reagent reservoir (150 ml or 300 ml) by aspiration and dispensing. This is achieved using the Pipet/mix mode of the VIAFLO 96/384. Then, cells are transferred into a 96 well plate containing the BIOMIMESYS Hepatocyte hydrogel. Selecting the right height (z-height limit: 2.9 mm) and pipetting speed (set on 2) settings on the VIAFLO 96/384 is essential to guarantee homogenous seeding of the cells on the hydrogel (see Figure 2). Cell medium (150 μl) is added by setting the z-height limit to 3.2 mm and the pipetting speed to 2.

2. Chlorpromazine treatment of HepG2 cell line

Remove culture medium and add the hepatotoxicity-inducing drug chlorpromazine.

Carefully remove 100 μl of medium and add 100 μl of the drug, using a pipetting speed of 1 to prevent harming the cells. A z-height limit of 3 mm is set to ensure spheroids are not accidently aspirated or destroyed by the pipette tip.

3. HepG2 viability assay

Determine the metabolic activity of HepG2 cells using a WST-1 assay.

Remove 100 μl of culture medium using the normal Pipet mode at speed 2 and a z-height limit of 3 mm. Add 10 μl of the WST-1 reagent using the same pipette settings described in step 2.

4. Biliary canaliculi activity of HepG2

Biliary canaliculi activity of HepG2 cells treated with chlorpromazine was examined using 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDFDA) (21884, Sigma).

Remove 100 μl of culture medium and add 200 μl of PBS to the HepG2 cells, using the normal Pipet mode with the same z-height and speed described in step 2. Wash the HepG2 cells by adding and removing 200 μl of PBS (repeat this step five times). The convenient three position stage of the VIAFLO 96/384 accommodates the waste and reagent reservoirs (positions A and AB, respectively) needed for the washing step, and a target sample plate (position B).