While traditional 2D cell culture usually consists of growing cells on a planar surface (e.g. on plastic or glass as a monolayer), 3D cell culture enables cells to grow into 3D structures within a scaffold or matrix. This has many benefits, particularly for complex biology, or when modelling certain biological scenarios (e.g. tumour microenvironment, migration and metastasis). While a number of options for 3D cell culture are currently available (e.g. Matrigel), the Rastrum platform utilises specialised hydrogels which are in a ‘printable’ form, which are pre-validated for individual cell types and biology. This approach enables a simple, efficient and robust workflow for the development and creation of 3D cell models, with little prior bioprinting knowledge required.

Rastrum’s drop-on-demand bioprinting technology is similar to that of an inkjet printer. The Rastrum can deposit tiny droplets of a PEG (polyethylene glycol)-based bioink and cells mixed with matrix ‘activator’ onto the surface of a microplate layer-by-layer, which combine to instantly form a hydrogel at room temperature to form a desired 3D cell structure. The Rastrum is capable of depositing up to 1000 droplets per second onto the surface of the plate, with a typical droplet volume of 20-25nL, and the small hydrogel plug structure volume of 300nL. The print head contains independently addressable nozzles, which are capable of printing up to four different cell types within a single well and/or across the same plate.

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The simplest model that can currently be printed is the 3D Small Plug Cell model in a 96 well plate. This starts with the printing of a base layer of inert hydrogel matrix to prevent cells attaching to the bottom of the well. Next, cells are encapsulated in a small plug of biofunctional hydrogel matrix on top of the inert base layer.

Rastrum^™ PEG Synthetic-Based Hydrogel Matrix

Choosing a suitable extracellular matrix which enables viability and/or growth of cellular structures is critical to the success of any 3D cell culture model. Matrix-embedded 3D cell models are considered to be more physiologically relevant than 2D cultures, because they enable inclusion of the key elements of the extracellular matrix (ECM), which can better recapitulate the in vivo context. Generating a 3D environment which enables the optimal cellular/structural characteristics (e.g. morphology, metabolism, migration, invasion etc) requires time and development in a process called ‘tuning’, where ECM stiffness and the presence of proteins within the ECM can heavily influence the biology in both in vitro and in vivo scenarios. The characteristics of the Rastrum PEG backbone define the stiffness of the resulting hydrogel matrix. The PEG backbone can also be functionalised with addition of adhesion peptides, full-length proteins and MMP-sensitive sites.

The advantage of Rastrum^™ hydrogels over other hydrogel systems include:

• Tunable 3D cell culture environment for each cell type and research question through:
  • Modifying the stiffness to provide a physiologically relevant environment
  • The addition of adhesion peptides that mediate integrin binding
  • The addition of full-length proteins enriched in natural extracellular matrices (ECM)
  • The addition of MMP-sensitive sites to enable cleavage of the matrix by cellsecreted
    proteases.
• Precise batch control and consistency
• Highly permeable to antibodies, growth factors and small molecules
• Dissolvable to enable cell extraction (if required)

Current Applications

Fundamental Biology

• Create complex 3D cell models with multiple cell and/or matrix components
• Probe specific cell-cell or cell-matrix interactions in a controlled and quantitative
  way
• Open your experimental design to rapid testing of multiple variables in 3D

Drug Discovery

• Unprecedented reproducibility well-to-well, day-to-day and site-to-site
• Better quantitative 3D analysis due to controlled structure-readout correlation

Personalised Screening

• Work confidently with low numbers of patient-derived cells without loss
• Rapidly screen an optimal matrix environment for an individual tumour
• Efficient patient-to-3D workflow for rapid screening capability

Current Models

• Tumour/cellular spheroids
• 3D migration/invasion assays
• Cancer co-culture
• Immuno-oncology models
• Neural co-culture

Compatible Cell Types

• Tumour cells
• Stromal cells
• Neuronal cell types
• Hepatocytes
• Cardiomyocytes
• Induced Pluripotent Stem Cells (iPSCs)

Available Matrix

Inventia has developed a library of hydrogel formulations and 3D structures. The user can select a program that uses optimised printing parameters to build the 3D cell model using a hydrogel formulation matched to a cell type of interest as below. The currently available range of off-the-shelf matrix formulations include:

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Each kit contains a Rastrum cartridge, which acts as a reservoir for the reagents within the printer, an inert activator and bioink that are required for the printing of the inert base, and the biofunctional bioink and activator which encapsulate the cells. Current matrices under development include human dermal fibroblast matrix and human liver cancer matrix.

We recommend that clients purchase a manual matrix panel kit initially to test which condition may suit their cell type the best. The test kit includes four different matrices as outlined below (Conditions A-D). Currently, the panel kit is designed for clients to manually encapsulate cells in four unique matrix formulations. Once you have found one condition that suits your 3D model the best, you can purchase the single matrix kit to be used with the Rastrum 3D Bioprinter.