Name: Design and Validation of a Volumetric-extrusion Bioprinter for Bioprinting of Soluble Basement Membrane Extract for Translational Research (Video) | JoVE Journal
Uploaded: 2025-03-28T14:20:00.000+00:00
Duration: 8 min 27 s
Description: Learn a volumetric-extrusion bioprinting method for cancer research using soluble basement membrane extracts. This protocol enables precise 3D construct fabrication with high cell viability and reliable shape fidelity.

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This work was partially funded by the Accelerator Award n&#176; A26815 entitled: &#34;Single-cell cancer evolution in the clinic&#34; funded through a partnership between Cancer Research UK and Fondazione AIRC (n&#176; 22790) and by the European Union - Next Generation EU, Mission 4, Component 1, CUP D53D23003310006.

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The authors have no conflicts of interest to declare.

In translational research, SBMes are particularly valuable in a variety of research fields, from tissue engineering to drug testing2,3. In particular, they have become fundamental in cancer research, as they replicate mechanical and biochemical features of the tumor microenvironment, enabling the development of models that better reflect tumor growth, metastasis, and treatment responses. SBMe is the most widely used biological matrix, especially for 3D organoids<...

In translational research, creating 3D in vitro models that closely replicate the in vivo environment is essential for advancing scientific understanding and therapeutic development1. A key component in building them is the use of extracellular matrix (ECM) analogs, such as Soluble Basement Membrane Extracts (SBMes), which better reflect the complexities of natural tissues. SBMes are particularly valuable in a variety of research fields, from tissue engineering to drug testing2,3. In particular, they have become fundamental in cancer research, as they replicate mechanical and biochemical features of the tumor microenvironment, enabling the development of models that better reflect tumor growth, metastasis, and treatment responses.
Indeed, despite considerable advancements over the past few decades, many therapies continue to fail, leading to frequent treatment failure in clinical settings. This failure is often attributed to drug resistance mechanisms caused by high levels of cell heterogeneity4,5. Effective in vitro models, capable of accurately recapitulating the complexities of human tumors, are crucial to better understanding cancer evolution and anticipating this phenomenon.
Traditional 2D cell cultures have been foundational in cancer research but can lead to misleading information about drug efficacy and tumor behavior, as they fail to capture the 3D structure and microenvironment of actual tumors6,7,8. In contrast, 3D in vitro models have emerged as promising alternatives, offering a more accurate representation of tumor microenvironment and biological behavior by mimicking 3D complexity9,10.
In cancer research, organoids are widely used as 3D models for studying disease and drug efficacy, since they provide more accurate and relevant models of human tissues and organs. The use of SBMe, derived from Engelbreth-Holm-Swarm mouse sarcoma cells, has been pivotal in advancing cancer research11,12. It provides a gel-like substrate in which cells can grow, proliferate, and self-organize into organoid structures, ensuring proper development and growth13,14.
However, SBMe poses a number of challenges due to its unique and complex characteristics. As a non-Newtonian fluid, SBMe exhibits shear-thinning properties, meaning its viscosity decreases with increasing shear stress, making its handling and application inconsistent under variable forces. Additionally, its thixotropic behavior adds to the complexity, as it can recover its viscosity over time when shear stress is removed15,16. These properties, together with low mechanical strength (with storage modulus values ranging from 10 Pa to 5,000 Pa depending on protein concentration) and thermosensitive behavior, worsen these challenges, requiring meticulous control during preparation and use as well as bioprinting it pure.
Standard pneumatic-driven bioprinting systems fail to properly control SBMe dispensing as the application of pressure causes uncontrollable behavior after its ejection from the syringe. It is affected by a phenomenon similar to the &#34;spurt&#34; effect&#34; described for polymeric melts, in which the flow rate abruptly increases above a certain critical pressure value17,18,19. As reported in our previous work16, the volumetric-control dispensing strategy allows uncoupling SBMe extrusion from the pressure generated in the dispenser, which is dependent on the rheological properties of the matrix, working conditions, and nozzle geometry. In recent years, several attempts have been made in the market to exploit the same principle, with Corning&#39;s Matribot leading the way. It allows the handling and deposition of SBMe and other temperature-sensitive bioinks thanks to controlled temperature conditions. However, it might be expensive, limiting accessibility for smaller labs.
In our prior paper, we have presented a low-cost custom alternative-a volumetric-driven bioprinting system16. This version was based on a modified entry-level 3D printer with a custom 3D printed extruder. Despite its effectiveness in bioprinting, it presented some limitations in printing repeatability. Being derived from an entry-level 3D printer, it had some problems with the Z-movements and auto-homing. To address these needs, we developed an advanced version of our custom low-cost volumetric dispensing system and a specific protocol for bioprinting structures using SBMe.
Here, we provide protocols for the construction and use of this system. It is composed of a custom volumetric-controlled extruder, based on our previous study16, a bioprinter case, and a control system. Production and assembly are made for each module. When assembled and programmed with a specific gcode file, following the protocol description for matrix handling, the system is capable of bioprinting pure or diluted constructs using the SBMe with good shape fidelity in both single and multiple layers.

design and validation of a volumetric-extrusion bioprinter for bioprinting of soluble basement membrane extract for translational research

Design and Validation of a Volumetric-extrusion Bioprinter for Bioprinting of Soluble Basement Membrane Extract for Translational Research

In translational research, 3D in vitro models are essential for advancing scientific understanding and therapeutic development. To this aim, Soluble Basement Membrane Extracts (SBMes), derived from mouse sarcoma cells, play the key role of replicating mechanical and biochemical features of in vivo microenvironment. They are particularly valuable in a variety of research fields, from tissue engineering to drug testing. In particular, they have become fundamental in cancer research as traditional 2D cell cultures, widely used in the field, can provide misleading information since they do not capture the three-dimensional (3D) structure and tumor microenvironment.
In cancer research, effective 3D in vitro models are crucial for better understanding cancer evolution and anticipating challenges, such as drug resistance mechanisms. Organoids have emerged as promising 3D in vitro models, offering a more accurate representation of tumor biology. They grow in SBMes as they create an environment where cells can grow and self-organize into 3D structures. However, SBMe presents significant challenges, including low mechanical properties and complex rheological behavior, preventing its use with commercially available pneumatic-extrusion bioprinting systems. 
To address these limitations, we developed a low-cost, volumetric-controlled bioprinting system and a specific protocol for printing structures with SBMe. This system is based on a volumetric extruder and overcomes the limitations of traditional pneumatic-driven approaches. Once assembled and programmed with a specific g-code, the system can bioprint both pure and diluted SBMe to obtain both single and multi-layer constructs. This approach offers a more reliable and scalable method for producing 3D cell culture models, paving the way for more effective research into novel treatments and drug testing.

In this article, we present a protocol for bioprinting constructs made of SBMe using a low-cost, custom-made, volumetric bioprinter. We provide a detailed description and 3D printing *.STL files to build the system and we demonstrate its potential use in cancer research by printing single and multi-layer structures. Each 3D printed component has been properly cleaned from support structure or unwanted printing residues before assembling. Figure 8 shows different views of the overall assembly...

Soluble basement membrane extracts are the most widely used biological matrices in cancer research, but their complex rheological behaviour makes their bioprinting difficult with commercially available bioprinting systems. This work presents a customized bioprinting strategy to produce pure matrix constructs with good shape fidelity in both single and multiple layers.

In translational research, 3D in vitro models are essential for advancing scientific understanding and therapeutic development. To this aim, Soluble ...

design-validation-volumetric-extrusion-bioprinter-for-bioprinting

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Learn a volumetric-extrusion bioprinting method for cancer research using soluble basement membrane extracts. This protocol enables precise 3D construct fabrication with high cell viability and reliable shape fidelity.

Design and Validation of a Volumetric-extrusion Bioprinter for Bioprinting of Soluble Basement Membrane Extract for Translational Research (Video) | JoVE Journal

Design and Validation of a Volumetric-extrusion Bioprinter for Bioprinting of Soluble Basement Membrane Extract for Translational Research

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