Optimizing gelatin hydrogels for volumetric 3D bioprinting

Over 74 million people across Europe suffer from diabetes, making it one of the most common chronic conditions on the continent, according to the World Health Organization. In order to address the disease and advance the development of innovative diabetes treatments, a multi-disciplinary group came together in 2021 through the ENLIGHT project. The aim of this project, which will wrap up this spring, is to 3D bioprint a living model of a pancreas—in other words, an organ-on-a-chip—which will enable pharmaceutical companies to better test diabetes medications. Besides this, a functional 3D bioprinted tissue can accelerate drug discovery and reduce the costs of drug development and healthcare, which could have significant effects as  the increasing expense of drug development is a major contributor to today’s large increase in healthcare costs. Farther down the line, the work done through ENLIGHT could also lead to the bioprinting of organ replacements capable of producing insulin directly within the body.

Among the partners spearheading the ENLIGHT project are UMC Utrecht, École Polytechnique Fédérale de Lausanne, ETH Zürich, the University of Naples Federico II, AstraZeneca, Fondazione Giannino Bassetti, bioprinting company Readily 3D and gelatin specialist Rousselot. We had the opportunity recently to talk with Jos Olijve, Rousselot’s Senior Project Manager, who updated us on the ENLIGHT project’s achievements and how Rousselot’s contribution to the project could actually have wide reaching impacts for the biomedical and bioprinting segments.

Technologies come together

“ENLIGHT is a multi-disciplinary project that involves biomaterials and a specific technology called volumetric bioprinting,” Jos Olijve tells us. “Using these technologies, the goal is to make a construct containing cells for a functional pancreas. This bioprinted pancreas tissue could then be used to improve and accelerate drug development, to eliminate animal testing and, if it is functional in the body, the bioprinted pancreas could also be used as a cell therapy product for producing insulin in the body. But the primary objective of the project is to make an organ-on-a-chip system for drug testing, which pharma companies can use to test their drugs.”

Volumetric bioprinting has played a central role in the innovative biomedical project, offering solutions to challenges associated with more conventional dispensing or extrusion-based bioprinting technologies. The volumetric bioprinting technology used in ENLIGHT comes from Readily 3D, a Swiss company that has developed a tomographic bioprinting process that creates centimeter-scale structures in mere seconds by exposing a three-dimensional light image into a vat of photosensitive hydrogel. The speed of the technology has been vital to the ENLIGHT mission, as it allows for the matrix material (hydrogel) to crosslink without applying shears and stresses to the cells suspended within it. This gives the cells a higher chance of remaining viable and maintaining their reproductive capacity.

However, the volumetric bioprinting technology itself is only part of the equation. Rousselot has played a vital role on the materials side developing a GelMA hydrogel that both mimics organic pancreatic tissue and is usable with the volumetric 3D bioprinting process.

As Olijve explains: “The biomaterial used is a methacrylamide modified gelatin, GelMA. After the addition of a photoinitiator, the material can be exposed to a selective wavelength of light, which creates a photoreactive effect that crosslinks the gelatin and makes it stable at temperatures of 37°C, which is useful for tissue engineering, organ-on-a-chip drug testing and for in-body applications.”

Gelatin is derived from collagen and has proven to be an important material for 3D bioprinting and tissue engineering applications. In the body, collagen is an essential component of the extracellular matrix, providing structure, supporting cell adhesion and regeneration processes and controlling cell fate. Derived from collagen hydrolysis, gelatin is a biomaterial that can be bioprinted as a hydrogel to provide a viable structure for cell survival and growth outside the body. GelMA, for its part, has been particularly advantageous for bioprinting applications as the addition of a methacrylate group results in a photo crosslinking ability which creates a gelatin-based structure that remains stable at body temperature.

While GelMA is broadly used across biomedical and bioprinting research, Rousselot has set a new standard in the segment by developing the first GelMA gelatins made under GMP conditions, meaning it offers batch-to-batch consistency, ultra-low impurity levels and tunable mechanical properties. Within the context of ENLIGHT, Rousselot’s expertise and high-quality gelatin products have been crucial.

Tuning GelMA properties

“Our main task in ENLIGHT was to establish a 3D hydrogel culture system for pancreatic cells, mimicking the native pancreatic extracellular milieu,” Olijve says. “It was important and confirmed that the developed GelMA hydrogels have a high-cell viability and create high-resolution for volumetric bioprinting. We varied the molecular weight, the MA modification degree and the gelatin concentration in the resin formulations to obtain a GelMA hydrogel matrix. The matrix can be used to select the best GelMA and formulation conditions not only to obtain the required Enlight pancreatic mechanical and structural hydrogel properties but also select the best GelMA and formulation conditions to make other tissue hydrogel constructs.”

In other words, Rousselot was able to vary the properties of the biomaterial, such as molecular weight and concentration, to tune the mechanical properties of the gelatin to be suitable for the pancreas. This process involved testing a porcine pancreas and then matching the properties with those of a modified GelMA. “We measured the strength of the porcine pancreas tissue and then figured out what the best concentration and molecular weight for the GelMA was.

In the end, not only did Rousselot develop a suitable gelatin material for the pancreas bioprinting application, it also developed a matrix of different gelatins, enabling the bioprinting of different types of tissues. “Our aim was not only to look for the pancreas, but also to make a kind of toolbox for customers if they have a specific requirement for mechanical properties.”

“An important reason to establish the matrix or tool-box is the fact that cell fate is determined by the interplay between chemico-mechanical signals from the ECM and different cell types,” he adds. “The local availability of these signals is dictated by the shape and geometry of the tissue. Capturing this intricate shape-function relationship is the major bottleneck in the field of bioprinting. In other words, Rousselot GelMA tunability matrix can solve this bottleneck and stimulate and enhance bioprinting and tissue engineering developments.”

ENLIGHT and beyond

Since it launched in 2021 with nearly four million euros in backing from the European Innovation Fund Horizon 2020, the ENLIGHT project has progressed significantly. To date, the partners have succeeded in creating an organ-on-a-chip system, which is now being evaluated by a pharmaceutical company for drug testing. In addition to this in vitro testing, there is also an ongoing in vivo study to investigate the potential therapeutic use of the bioprinted construct in Diabetes care. As a first step, Olijve explains, the gelatin hydrogel containing cells is implanted in mice to test whether it will be stable and functional. The goal of this in vivo investigation is to see if the bioprinted tissue can be functional and stable and produce insulin in a body.

In addition to ENLIGHT, which will conclude in April 2025, Rousselot is involved in a number of other projects. As Olijve tells us, the company is working closely with the University of Utrecht in the Netherlands on other volumetric 3D bioprinting applications, like developing organoid systems. “With that, we are also looking at specific gelatin needs in relation to crosslinking and cell adaptability,” he adds. “We are looking at what is needed in the 3D bioprinting market and how we can develop and optimize our gelatin for those requirements.”

On the horizon, Rousselot sees more and more current research projects advancing to clinical studies. “For example, a group from Stanford led by Prof. Mark A. Skylar-Scott is currently bioprinting cardiac patches and heart valves which can go into patients. This is beyond R&D, and within the near future this and other applications will go to clinical testing,” Olijve adds. “From our perspective, it’s important to be able to offer a gelatin that can support these clinical translations.”

Looking ahead, Rousselot has filed a series of patents and published (with the University of Utrecht) scientific papers, which among others cover the tunability of its modified gelatins to meet the requirements of different tissues and cells. Also notable is the fact that the ENLIGHT project and Rousselot’s GelMA has been featured on the European Commission’s Innovation Radar and has been classified as “tech ready”. “For us it’s important that the EU recognizes that our gelatin is a very important innovation,” Olijve concludes. Evidently, this recognition is not only validating, it also could have important ramifications for the future of the ENLIGHT project and Rousselot’s own gelatin products, creating avenues for funding, setting the stage for a successful business and providing solutions to enhance medical development and applications for patients.