Frequently Asked Questions

Technical FAQs

Why is 3D culture in hydrogel better for cell biology research than conventional 2D culture?

A hydrogel matrix presents a more realistic microenvironment to cells than a plastic surface. Cells behave differently in a 3D matrix. Studies have shown that 3D cell models better represent human tissues[1] and more accurately replicate biological processes and drug responses[2].

  1. Picollet-D’hahan (2016) Trends Biotechnol. 34, 757; Yamada (2007) Cell 130, 601; Knight (2015) J. Anat. 746;
  2. Breslin (2013) Drug Discov. Today 18, 240; Mogilner (2011) Trends Biotechnol. 21, 692

How is RASTRUM™ different to other bioprinters?

RASTRUM is not a typical bioprinter. It is a platform designed from the ground-up for the simple creation of 3D cell culture models. To enable this, there are two major aspects unique to RASTRUM. 

Firstly, RASTRUM is based on proprietary Digital Bioprinting technology. This includes hardware, software and printable biomaterials that together enable a robust drop-on-demand bioprinting approach, as opposed to the common extrusion-based bioprinter (see “What are the key differences between drop-on-demand and extrusion bioprinting?”). 

Secondly, RASTRUM is designed for a cell biologist, not a tissue engineer. Many bioprinters require a user to design 3D structures, tweak printing parameters, and optimise printing protocols depending on the hydrogel and desired outcome. This is great if the core focus of your research is bioprinting, but not so great if all you are after is better 3D cell models. RASTRUM delivers a platform where hydrogels, printable structures and printing parameters are pre-validated, enabling a simple and efficient workflow for the creation of 3D cell models, with no prior bioprinting knowledge required.

What are the key differences between drop-on-demand and extrusion bioprinting? What are the advantages of drop-on-demand printing for a cell biologist?

RASTRUM’s Digital Bioprinting technology is a drop-on-demand bioprinting approach. This is akin to inkjet printing, but instead of depositing pixels of colour onto a page, RASTRUM deposits hydrogel components and cell suspensions onto the surface of a well plate and builds these components layer-by-layer to form a 3D structure. 

Extrusion bioprinting is akin to squeezing toothpaste out of a tube, where pre-formed hydrogel with or without cells is forced out of a needle that is in contact with the surface under the application of pressure.

One of the advantages of drop-on-demand printing is higher cell viability, as the pressure required to eject a droplet from the RASTRUM print head is lower than that required to extrude pre-formed hydrogel from a needle, thus lowering shear stresses on cells.

Extrusion bioprinting requires contact of a needle with the surface of the well plate, whereas the RASTRUM print head moves seamlessly across a well plate and builds 3D cell models using fly-by droplet deposition, enabling a step-change in printing speed and throughput.

Additionally, with RASTRUM it is possible to print multiple cell and matrix components in a single pass of the printhead. This multiplexing capability makes it easier and more efficient to build complex 3D cell models.

RASTRUM™ Specifications

What is the printing resolution?

The smallest droplet volume is 15 nL and the typical droplet volume ranges from 20-25 nL. The size of the printed gel structure depends on the specific properties of the bioink. The most common structure yields a basic structural building block of approximately 250 µm in diameter and  50 µm in height.

What is the maximum printing frequency?

The printer is capable of depositing 1,000 droplets per second onto the surface.

Does the printer feature inbuilt temperature regulation?

No. Our hydrogels form at room temperature, and cells are within the system for a short amount of time (<30 mins), so temperature control is not required.

Can RASTRUM™ print large cells?

RASTRUM has successfully printed large cells such as hepatocytes and cardiomyocytes. For cells that do not easily dissociate into single cells (e.g. cells from patient-derived tissues), we recommend filtering suspensions through a 40 µm cell strainer prior to use. For further information about specific cell types, please contact Sales & Support.

RASTRUM™ Hydrogels

What are RASTRUM™ Hydrogels?

RASTRUM Hydrogels are a printable, synthetic hydrogel system, comprising a large library of matrix environments by tuning combinations of:

  • A PEG-based backbone, which can be tailored to tune mechanical properties of the hydrogel
  • Tethered adhesion peptide sequences
  • MMP-sensitive crosslinks
  • Full-length proteins

How do RASTRUM™ Hydrogels gelate?

Gelation occurs instantly upon the combination of two droplets deposited from independent print nozzles - one drop of RASTRUM Activator and one drop of RASTRUM Bioink.

RASTRUM Activator is an aqueous peptide-based cross-linking solution which interacts with the RASTRUM Bioink to form a hydrogel. The user resuspends cells in Activator solution prior to loading into RASTRUM, so when Bioink and Activator combine to form hydrogel, cells are encapsulated within.

This two component system is key to the formation of controlled 3D hydrogels using a drop-on-demand approach. In this way, we do not need to rely on temperature- or UV light-induced gelation.

Can RASTRUM™ print any hydrogels?

RASTRUM will only operate with validated RASTRUM Hydrogels. Our hydrogel components exhibit optimal biochemical properties for drop-on-demand dispensing and printing parameters are optimised by our engineers to build a robust 3D structure. RASTRUM takes this optimisation effort away from the user so that you can focus on the biology.

What is the benefit of RASTRUM™ Hydrogel over natural hydrogels (i.e. Matrigel)?

Natural hydrogels are more susceptible to batch-to-batch variation, lack of physical/biochemical modularity, and can be difficult to handle manually. Synthetic hydrogels can overcome some of these limitations, providing a reproducible and more tunable system than natural gels. Our library of RASTRUM hydrogels are completely synthetic, and can be precisely tuned to exhibit a variety of mechanical and biofunctional properties.

What are the mechanical properties exhibited by RASTRUM™ hydrogels?

RASTRUM Hydrogels can be biochemically fine-tuned to exhibit a stiffness suitable for the mechanical support of 3D cell growth. We support hydrogels with a mechanical stiffness range (G’) between 0.5 - 3.5 kPa.

What are the biofunctional properties exhibited by RASTRUM™ hydrogels?

RASTRUM Hydrogels exhibit covalently tethered adhesion peptides - the core integrin binding sequences found in the most common extracellular matrix proteins - to the PEG backbone of RASTRUM Hydrogel, enabling cell attachment to and interaction with the matrix.

Additionally, we crosslink the hydrogel with a matrix metalloproteinase (MMP)-sensitive peptide sequence, enabling hydrogel remodelling by cell-secreted proteases.

It is also possible to include full-length proteins (such as collagen and laminin) into RASTRUM Hydrogels.

Are the hydrogels stable over time in culture?

Most RASTRUM Hydrogels are designed to be degraded and remodelled by the action of cell-secreted proteases. This means that as the cell model matures (and potentially excretes its own ECM), the printed hydrogel will soften. If it is necessary that a particular model be stable over longer culture periods, a portion of non-cleavable crosslinker can be incorporated into the hydrogel to prevent degradation and retain gel stability.

Can the hydrogel formulation (activator and bioink) be used again once it has been thawed?

Re-freezing is not recommended as this would compromise product stability. 

What is the shelf life of the bioink and activator?

The bioinks and activators are stable for approximately 6 months from the shipment date (or until the expiration date) and should always be stored at -20°C.

Applications

What cell types are compatible with RASTRUM™?

We have tested a range of cell types, including tumour cells, stromal cells, neuronal cell types, hepatocytes and cardiomyocytes. For further information, please contact Sales & Support.

How does the applied pressure during the printing process affect the cells? Can I print sensitive cell types?

Our tests have shown no negative impacts on cell viability or function. This includes testing with sensitive primary cells types and stem cells. For further information, please contact Sales & Support.

How do you maintain printer sterility?

RASTRUM has inbuilt laminar flow with dual HEPA filtration, ensuring a sterile printing environment. Like a Biological Safety Cabinet, the internal stainless steel surfaces of the printer can be cleaned with 70% ethanol. Before and after every print, the fluidics are automatically cleaned and sterilised. Additionally, we provide a RASTRUM Sterilisation Kit that involves a more thorough deep clean of the fluidics and can be used routinely.

How does the user define the structure to be printed? Can I change printing parameters for my experiments?

We have 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.

What is the minimum number of cells required?

While cell density is dependent on the cell model, it is typical to resuspend 1x10^6 cells in 200 µL of Activator solution (5x10^6 cells/mL). Optimal cell density in a 3D cell model should always be determined empirically.

Can RASTRUM™ construct 3D cell models containing multiple cell types?

Yes, the print head contains independently addressable nozzles capable of printing up to four different cell types within a single well and/or across the same plate.

How long does it take to print an entire 96-well plate?

Printing time depends on the complexity of the 3D structure. However, our simplest 3D cell models can be printed in 96 wells in ~5 minutes.

Can you control bioink/cell placement for more accurate downstream image acquisition?

Yes, printing your cells with RASTRUM has the added advantage of controlling the cell placement within the well and Z-plane, enabling consistent cell placement for downstream image analysis.

Downstream Analysis

Can you retrieve cells from the printed hydrogel?

Yes, hydrogels can be dissolved in minutes using our RASTRUM Dissociation Solution, enabling retrieval of cells for downstream analysis.

Can you stain and image cells within the hydrogel?

Yes, most standard staining and imaging protocols are compatible within the 3D hydrogels. The gels themselves are completely transparent and compatible with brightfield/phase microscopy. The region of interest is typically kept close to the base of the well plate so that working distance is rarely an issue. Staining of the cells in situ is possible with both small molecule and antibody-based dyes, which readily permeate the gel. Standard fixation and permeabilisation protocols can be used. The gels do not autofluoresce and there are no issues with non-specific background fluorescence in these gel systems.

Can you fix and section the hydrogels?

Yes, the hydrogels can be snap-frozen and cryosectioned, or embedded and sectioned as per standard methods for tissue samples.

Laboratory Requirements

What are the printer dimensions and how much does it weigh?

482 mm W x 560 mm D × 491 mm H and weighs approximately 50 kg. Further information is available in the specification sheet.

Does RASTRUM™ have any specialised laboratory requirements?

No - the platform requires 1x standard power outlet for the printer, and 1x standard power outlet for the computer (supplied). The instrument requires compressed air, but if this is not available in the lab, we provide a small external air compressor with the unit. Further information is available in the specification sheet.

Does RASTRUM™ require a specialised computer?

The RASTRUM software is supported by Windows 10 and the printer requires USB connectivity. A computer with preloaded software is provided with RASTRUM upon purchase.