Bioprinting multi-cellular liver models

The liver plays a central role for drug metabolism and toxicity. Understanding liver function is very important for pharmacology and toxicology research, especially in the context of drug-induced liver toxicity, the leading cause of acute liver failure, and for increased knowledge of liver diseases.

Cell-based tissue models are important components in the path towards increased understanding of tissue and organ function. To achieve biological relevance, cell-based tissue models need to mimic the cellular architecture found in organs.

With Biopixlar, it is possible to create complex multi-cellular tissue models in 3D with single-cell precision. Here, we show how the unique single-cell printing technology of Biopixlar can be used to create detailed, multicellular liver models that improve hepatocyte functionality.

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Two scientists using the Biopixlar

The multicellular in vitro liver model is created by assembling two separate cell layers on top of each other, directly in culture media. The two layers are held together with a connecting cell-adhesive layer, as shown in Figure 1. Figure 2 shows the assembled structure with the arrangement of cells in the two different layers.

Bottom layer The bottom layer consists of a larger uniform patch of 3T3-J2 fibroblasts acting as an underlying support structure.

Top layer The second layer is the functional part of the model, built from a central patch of hepatocytes (HepG2) with a surrounding fibroblast structure.

Creating detailed bioprinted in vitro models has many important benefits compared to conventional cell culture techniques:

  • Biological relevance – Complex tissue models can be constructed with improved physiological response compared to monoculture systems.
  • Simplicity – Users have direct control of cell placement through Fluicell’s easy-to-use interface.
  • Viability – Printing occurs directly in culture media with minimal mechanical stress on the cells.
liver model assembly

Figure 1. Liver model assembly

 

liver model schematic

Figure 2. Assembled liver model

Step 1: Print supporting fibroblast layer

The liver model is constructed by first assembling a support layer consisting of  fibroblasts (3T3-J2).

To prepare for the printing of the hepatocyte layer, cell-adhesive agent is printed on top the fibroblasts. This allows adhesion of the second layer of cells.

Step 2: Print hepatocyte structure

Hepatocytes (HepG2) are printed on top of the support layer in a 0.5 x 0.5 mm square, which is depicted in the image below.

Newly printed hepatocytes. The shadow in the righ hand side of the pictures is the Biopixlar printhead, which is held out of focus.

During printing, cells are ejected from the microfluidic printhead in a controlled fashion and are deposited onto the support layer. The desired pattern is obtained by moving the substrate while cells are being ejected.

Cell-adhesive agent is added on top of the fibroblast support around the hepatocyte square, to allow attachment of a second layer of fibroblasts.

Step 3: Print surrounding fibroblast layer

Following this, a layer of fibroblast is printed around the hepatocyte square, shown in detail in the second video. The fibroblasts help to promote the metabolic capacity of the hepatocytes.

The image below show the complete liver model with hepatocytes printed on top of and surrounded by a layer of fibroblasts. The colored regions in the right panel highlight the structure of the top layer of cells, with hepatocytes in red and fibroblasts in blue.

Bright field images of liver model showing the bottom fibroblast layer together with upper fibroblast and hepatocyte layer. Right images highlight the structure of hepatocytes and fybroblasts in the top layer.

Creating detailed structures with complex architectures on the cellular level is both fast and simple with Biopixlar. Both printhead positioning and printing speed can be controlled directly by the user, providing a wide range of possibilities in model design.

Printing video 1

The video below shows the printing of the central patch of hepatocytes with Biopixlar in real-time.

Printing video 2

Fibroblasts are ejected around the hepatocytes and attach to the underlying support structure.

The multi-cellular liver model is constructed by arranging hepatocytes (HepG2) and fibroblast (3T3-J2) in two layers. The fibroblasts provide a supporting layer beneath and around the hepatocytes.

To facilitate distinction between the different cell types, the cells are fluorescently labeled. The image below shows the composition of the liver model directly after printing in bright field and fluorescence imaging. In the fluorescence image, the hepatocytes appear in red and the fibroblasts in blue.

Bright field and fluorescence image of the liver model. In the fluorescence image (right), hepatocytes are red and fibroblasts blue.

Albumin production, which is one of the important functions of hepatocytes, was used to probe the functionality of the liver model created using Biopixlar. The results presented below show a significant increase in albumin production for the 3D Biopixlar model after 7 days incubation, when comparing to our bioprinted 2D and monoculture tissues.

 

liver model albumin production

Hepatocyte albumin production for different liver models relative to monoculture after 7 days incubation.

The results presented here show that liver models created using the single-cell printing capacity of Biopixlar have greatly improved hepatocyte albumin production compared to the monoculture system, resulting in increased biological relevance. Furthermore, it is easy to further increase model complexity with Biopixlar, either by building additional cell structures or by adding additional cell types.