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UNICAEN: New Bioprinted Tumor Models Help Researchers in France Study Its Biology

Studying cancer biology is among the top priorities for researchers around the world. From consortiums to universities, pharma companies, newcomers in the drug development industry, and research institutions, current research to understand how tumors develop is crucial to progress against the disease. At the University of Caen Normandie (UNICAEN), in France, two teams of more than 30 researchers, clinicians, and doctoral students are developing a new 3D bioprinted tumor model that will provide a novel alternative tool for studying tumor biology and response to anti-cancer treatments.

Many of them are part of CERVOxy, one of the scientific teams of the Imaging and Therapeutic Strategies of Cerebral and Tumoral Pathologies (ISTCT) unit, which was created in early 2012 by the French National Centre for Scientific Research (CNRS), Commission for Atomic Energy and Alternative Energies (CEA) and the UNICAEN, and hosted at the GIP CYCERON imaging platform in Caen, France. CERVOxy’s scientific team focuses on hypoxia and its role in glioblastoma (a fast-growing brain tumor) and brain metastases.

All of these topics are developed in different axes to study tumorigenic or tumor-forming processes, to develop new therapeutic strategies. For example, they are researching how to use hadrontherapy (protons and carbon ions) to treat brain tumors. Moreover, the effects of these therapies on healthy brain tissue are also being evaluated using in vitro and in vivo methods, which is why they have started to develop new models based on bioprinting technology.

3DPrint.com spoke to Nolwenn Pasquet, a post-doctoral fellow from the University of Caen and one of the researchers at CERVOxy focused on studying the effects of radiotherapy and hadrontherapy on the brain healthy tissue in the context of a glioblastoma. Along with her colleagues, Pasquet is using Cellink’s INKREDIBLE+ to perform a great deal of the work.

“Despite recent improvements, treatment of glioblastoma is still challenging and the physiopathology of these tumors is so complex that the use of 2D in vitro models fails to recapitulate the in vivo situation,” indicated Pasquet. “Moreover, there is a lack of relevant models to mimic interactions between the cells, for example, it is not possible for the 2D models to reflect the tumor microenvironment such as the hypoxic gradient and the presence of surrounding cerebral and inflammatory cells. In this context, new 3D brain models obtained by bioprinting are very attractive for glioblastoma studies.”

For this study, Pasquet and fellow researchers used a murine glioblastoma cell line to develop a novel 3D bioprinted glioblastoma model. These cells were then embedded into specific bioinks from Cellink to mimic the extracellular matrix, and followed by bioprinting of the models, which was performed by the INKREDIBLE+ bioprinter, provided to CERVOxy by the LARIA team, part of the François Jacob Institute of Biology, and a collaborator in the development of the model.

According to Pasquet, in further experiments, it will be possible to observe the crosstalk between glioblastoma cells and surrounding cells (astrocytes, inflammatory cells, and more) by combining these cells in the same 3D model and analyzing cell progression, invasiveness, and interactions between them.

“In terms of preliminary results, we observed after bioprinting that glioblastoma cells have a homogeneous distribution until six days and then start to form cell clusters at the periphery of the model at 14 days of cell culture,” explained Pasquet. “Interestingly, these models recapitulate one of the most important features of glioblastomas: hypoxia. Indeed, 14 days after biobrinting we observed a hypoxic gradient in our model with hypoxic cells in the core of the model not observed in the periphery or at six days.”

Pasquet indicated that they also performed x-ray irradiation on these models. X-ray radiotherapy as a complement to surgery and chemotherapy is part of the standard protocol for the treatment of brain tumors. As in medical radiography, it involves delivering photons in different doses, except in this case it is to destroy cancer cells. Through these 3D bioprinted models, the researchers wished to evaluate the response and sensitivity of the cells to irradiation and thanks to specific markers, they were able to evaluate the proliferation of the cells which gives them indications on the evolution of the tumor in its environment.

Researcher at the CERVOxy lab (Image: CERVOxy)

“For now, we are starting with this new methodology and it is necessary to further characterize the model well and to know its limitations in order to reach a conclusion on the results obtained. For example, it is difficult to rule out the fact that cells interact with each other in this model and real-time microscopy experiments would allow us to verify it. This is an important point and is part of the reason why we decided to develop this type of model in order to recreate the microenvironment that these cells have within the patient’s brain tissue. These results are positive and encourage us to continue our research in this direction.”

The project is led by the laboratory, which is a French National Center for Scientific Research (CNRS) unit –a public-funded institution that covers all scientific disciplines. It is financed by several sponsors, notably the ARCHADE center for hadrontherapy in Caen; HABIONOR European project, co-funded by the Normandy County Council, the French State in the framework of the interregional development Contract “Vallée de la Seine”, and Région Normandie for the Normandy Network for Therapeutic Innovation in Oncology (ONCOTHERA) project.

Pasquet suggested that without bioprinting technology, the information obtained would not have been the same. She explained that “this technology is in full development,” and that “we’ve only been using it for a short period of time, just over a year, and there’s an important characterization step depending on what you want to study before you can do tests.”

The expert concluded that she “believes that there is a relevant distinction to be made about the models used in bioprinting, and many are going to be used to reproduce a fully functional organ in the fields of medicine and tissue engineering. The interest in 3D bioprinting is to create complex cellular structures through a process of superimposition of successive layers, and it is this aspect that is of particular interest to us in order to have a new and more complex study model for our research.”

The CERVOxy research team (Image: CERVOxy)

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New Method: Immersion Bioprinting of Tumor Organoids Will Increase the Throughput of 3D Drug Screening

Drug testing and screening for cancer drug discovery can take years and the 2D cell cultures and animal models used to estimate their efficacy before reaching human trials are often not representative of the human body, which is why researchers are turning to bioprinting technologies to increase the success rate during human trials by providing human-specific preclinical data. In 2018 there were 17 million new cases of cancer worldwide, and the disease is expected to affect 27.5 million people each year by 2040, this high incidence level makes tackling the disease enough of a reason for researchers to consider new technologies that could accelerate drug discoveries and screenings. Although still in its lab phase, a new development that uses immersion bioprinting of human organoids could change 3D drug screening.

Researchers from Cornell University, Wake Forest School of Medicine, Virginia Polytechnic Institute and State University and The Ohio State University have published an article in Micromachines, demonstrating an immersion printing technique to bioprint tissue organoids in 96-well plates to increase the throughput of 3D drug screening. Using a hydrogel bioink comprised of hyaluronic acid (HA) and collagen they were able to bioprint it into a viscous gelatin bath, which blocks the bioink from interacting with the well walls and provides support to maintain a spherical form.

According to the article, the use of bioengineered human cell-based organoids may not only increase the probability of success during human trials, but they could also be deployed for personalized medicine diagnostics to optimize therapies in diseases such as cancer. However, they suggest that one limitation in employing organoids in drug screening has been the difficulty in creating large numbers of homogeneous organoids in form factors compatible with high throughput screening, so bioprinting can be used to scale up the deposition of such organoids and tissue constructs.

The team of scientists employed two commercially available bioprinters to evaluate the compatibility of the collagen-HA hydrogel and the HyStem-HP hydrogel: Cellink‘s INKREDIBLE bioprinter and Allevi‘s Allevi2 bioprinter. This method was validated using several cancerous cell lines and then applied to patient-derived glioblastoma (GBM) –a fast-growing brain tumor– and sarcoma (or malignant tumor) biospecimens for drug screening.

For the initial analysis of hydrogel biocompatibility, researchers used two common cell lines: human liver cancer and human colorectal cancer.

While carrying out patient-derived tumor biospecimen processing, they obtained two glioblastomas and one sarcoma biospecimen from three surgically treated patients in adherence to the guidelines of the Wake Forest Baptist Medical Center IRB protocols. These biospecimens were processed into cell suspensions, successfully yielding millions of viable cells from each sample. The cells were then combined with the collagen–HA bioink for deployment in immersion bioprinting. After bioprinting, the GBM and sarcoma patient-derived tumor organoids (PTOs) were maintained for seven days in the incubator, after which a chemotherapy screening study was initiated.

Schematic of the printing process using 2 bioinks in two commercially available bioprinters: Cellink Inkredible and Allevi 2 (Image: Cornell University/Wake Forest)

The researchers claim that while their PTOs have been useful for disease modeling, mechanistic study, and drug development, they have also used these models in a diagnostic sense to influence therapy, which might just be the ultimate goal of their work.

This 3D bioprinting approach called immersion bioprinting is an efficient way to surpass the limitations that have plagued tumor organoid systems. The experts, in this case, suggest that there have been few advances in regard to approaches to the printing process itself, or generation of novel, more user-friendly bioinks. Indicating that “unfortunately, many bioprinting studies are somewhat repetitive, falling back on traditional biomaterials and their crosslinking approaches, which were never developed to be bioprinted or to accurately represent the complexities of the native ECM (extracellular matrix).”

Results of the published study suggests that the realization of this technology that can fabricate PTOs in a consistent and high-throughput fashion will provide a valuable ex vivo/ in vitro tool that can be deployed for many subsequent studies, including target discovery, mechanistic investigation of tumor biology, drug development, and personalized drug screens to aid in treatment selection in the clinic.

Clinical oncology is faced with some critical challenges during this decade, from inefficient trial design to integrating new technologies in diagnostics and drug trails. However, advances in new methodologies, from hardware design to improved bioinks developed specifically for bioprinting, are opening up new opportunities for bioprinting-based applications. This new study, in particular, suggests that with advances in bioprinting hardware, software, functional ECM-derived bioinks, and modifications to printing protocols, bioprinting can be harnessed not only to print larger tissue constructs, but also large numbers of micro-scaled tissue and tumor models for applications such as drug development, diagnostics, and personalized medicine.

Employing bioprinted patient-derived tumor organoids in a clinical precision medicine setting (Image: Cornell University/Wake Forest)

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CELLINK in France: Expanding Their Portfolio in 2020

Seeking to strengthen their presence in Europe, 3D bioprinter provider and pioneer bioink company CELLINK, opened their new offices in Lyon, France, last October. Begining new partnerships and collaborations with universities, hospitals, pharmaceutical companies and more, is a big part of the core mission of CELLINK, as they combine their technology with research innovation everywhere. The city of Lyon offers a booming scene for bioprinting, with companies heavily focusing on microtumors and taking advantage of an established network that has collected more than 2000 tumors from patients in collaboration with 11 major cancer hospitals in the country via the IMODI initiative–a French consortium to develop new experimental models of cancer–which preserves and archives all tissue and cell samples developed by the consortium partners during the project.

3DPrint.com spoke to CELLINK’s Sales Director for France and Southern Europe, Edouard Zorn, who envisions a greater expansion this year, new partnerships and research collaborations: “We hope to really expand our portfolio this year in France.”

“Cellink is amazing at building bridges between a product and researchers from different backgrounds and cultures. The company has a team-oriented mindset, looking to work with people from different nationalities,” sad Zorn, a biotechnology engineer with vast experience in scientific sales management.

Zorn and his five-people work team at the Lyon offices have their agenda full, with 30 customers in France, bioprinter installations, and training sessions for new CELLINK users, Zorn can’t help but highlight how fast the field of bioprinting is moving in Europe. “There are many researchers focusing on skin and cancer, that’s really big here, but lately I have also been in contact with companies working on biopharma, vaccines and some even trying to replace animal testing assays,” he stated. 

Since January 2018, CELLINK has been working with another major player in Lyon, CTI Biotech, using bioprinting to develop microtumors. CTI Biotech uses CELLINK technology exclusively for their work and now have three bioprinters at their lab, which is also located in Lyon. CELLINK and CTI Biotech had even signed a deal to 3D print customized cancer cells, with CELLINK assisting CTI in the production of patient-specific cancer tumor replicas, which will be 3D printed by combining CTI’s bioink with a sample of patients’ own cancer cells, promising to deliver personalized treatments for cancer on a custom, patient-by-patient basis.

“The aim of our collaboration is to give researchers an advantage in treating specific cancer types, and in the long term, take a serious step forward in the fight to cure cancer. CTI is moving really fast to develop models and commercialize them, and they choose our machines for their versatility, intuitiveness, and easily modifiable parameters. CTI Biotech is one of the customers we most grow with, and I believe it was a very good decision for both companies to work together,” explained Zorn. 

So far, they have already commercialized CELLINK skin for drug and cosmetic testing, which they have also been working to improve, and Zorn thinks that soon they will be working on introducing some human cells to the skin, as well as perhaps vascularizing the tissue.

Edouard Zorn (far right) and CELLINK bioprinters at the CTI Biotech labs (Credit: CELLINK)

The CELLINK office in Lyon is selling their machines to central Europe, working along the french-speaking part of Switzerland and Belgium, as well as in Spanish and Portuguese markets. 

Edouard Zorn at CELLINK France (Credit: CELLINK)

“We work with a lot of universities in France. For example, at the Medicine University of Montpellier, Xavier Garric, uses the INKREDIBLE+ bioprinter to teach master students how to design and print implantable medical devices and scaffolds for tissue engineering; and Alexandra Fuchs from the Hôpital St Louis employs a BIO X for tissue engineering.”

At the University of Grenoble, Vincent Haguet is generating skin, cornea and pancreas organoids for the modeling of organogenesis (organ formation) and pathogenesis (disease development), with a BIO X. Among these applications, organoids are used to screen and test new drugs. Also wielding the power of the BIO X is Anthony Treizèbre, from the University of Lille, for the bioprinting of Tumor-On-Chip and Blood-Vessels-on-Chip for the development of multicellular microfluidic biomimicry-based devices for the study of metastasis. Their idea is to reproduce blood vessels using human umbilical vein endothelial cells (HUVEC) and modulating the surrounding extracellular matrix.

The University of Nantes‘ Pierre Weiss also works with BIO X to print calcium phosphate-based personalized medical devices for maxillo-facial bone regeneration, as well as enzyme-based hydrogel formulation for the complex systems in bone regeneration.

Zorn believes that “there is a big demand from patients that expect the medical and bioengineering field to adapt treatments to patients. There is a lot of expectation for personalized medicine, especially with regard to microtumors for drug testing. Moreover, lately, we have seen researchers focusing heavily on immunotherapy, so I see a great future in that regard and consider that CTI Biotech is trying to position itself in that field.”

Fortunately, he suggests that there is collaboration in Europe. The European Union (EU) is financing joint collaboration projects with the objective to develop medical devices and applications with therapeutic solutions, and CELLINK wants to be a part of that.  Zorn emphasized the importance of the Silk Fusion Project, which unites scientists in the development of a technology that uses silk, a natural biocompatible and sustainable material, to produce a bioink and 3D print platelet production instrumentation, attempting to solve the limited supply of human platelets. Other projects which have CELLINK as a collaborator seek to solve problems for joint articulation, bone, and even bioprinting parts of the tendon and cartilage.

“We need people who understand cell biology, chemistry, hardware, electronics, and software, as well as a good comprehension and understanding the needs inherent to each country’s culture, that is the way in which we expand,” concluded Zorn.

Edouard Zorn at the new CELLINK offices in France (Credit: CELLINK)

The French branch of the company joins the other six worldwide offices of CELLINK, in Boston, Gothenburg, Freiburg, Blacksburg, Kyoto, and Stuttgart. Zorn hopes that the sales force along with the experienced network of professionals around the world working for CELLINK will result in a stronger presence of the company in central Europe as well as more joint efforts that could bring the future of bioprinting technology closer to our present.

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Researchers develop 3D printable wound dressings based on fruit

Researchers from the Aristotle University of Thessaloniki (AUTh), Greece, have developed 3D printable direct and indirect patches with wound-healing capabilities.  In an article published in the journal Pharmaceutics, 3D printable inks based on pectin, a naturally occurring substance found in berries, apples, and other fruit, which form free‐standing transparent films designed to treat shallow wounds and ulcers. “The application […]
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Interview: BIOLIFE4D is The First US Company to Bioprint a Mini-Heart (for Cardiotoxicity Testing)

After quite a few teaser pictures on their social media platforms since August, BIOLIFE4D finally announced one of the biggest milestones for the company: they successfully 3D printed a tiny heart. But how small is the mini heart? Actually, it is about one quarter the size of a human heart.

The ability to 3D bioprint a mini-heart now gives the biotech firm a roadmap to achieve their ultimate goal: bioprinting a full-scale human heart viable for transplant. It is now a matter of optimizing processes and scaling up the technology for the pioneering company headquartered in Illinois.

Ravi Birla

With the structure of a full-sized heart and four internal chambers, the mini heart is replicating partial functional metrics compared to a full-sized heart – as close as anyone has gotten to producing a fully functional heart through 3D bioprinting. The scientific milestone was accomplished at the company’s research facility at JLABS in Houston, led by Ravi Birla, Chief Science Officer of BIOLIFE4D

3DPrint.com asked Birla about their achievement to understand how functional it is and how this project could lead to a fully beating organ in the future.

“The functional performance of our mini-heart is not the same as a normal mammalian heart, though this is a future objective of the research,” explained Birla. “Our mini-heart is intended for use in drug cardiotoxicity screening, which means that the bar that it must achieve is less than the bar required for a viable transplanted organ. This is why the performance requirements for our mini-heart do not need to mimic a fully-functional animal heart at this point.”

Bioprinting at BIOLIFE4D

“As we move forward we will be optimizing our bioink as well as the bioprinting parameters which are needed for optimal functional performance,” suggeted the expert, who also previously served as the Associate Director of the Department of Stem Cell Engineering at the Texas Heart Institute in Houston. 

So how did they do it? First on their list was developing a proprietary bioink using a very specific composition of different extracellular matrix compounds that closely replicate the properties of the mammalian heart. There is still no formal name to the bioink as it was developed in-house and for now, it is currently intended for BIOLIFE4D use only.

Then, they got around creating a novel and unique bioprinting algorithm, consisting of printing parameters optimized for the whole heart. Coupling its proprietary bioink with patient-derived cardiomyocytes and its enabling bioprinting technology, BIOLIFE4D was able to bioprint a heart. Birla suggested that because of the strategic partnerships that they have developed, they have access to and utilize most of the commercially available printers which are on the market, but the mini-heart was essentially biofabricated in their labs using a CELLINK INKREDIBLE+.

“We currently used a commercial source of human cells, through the expected use of the technology in using patient derived autologous cells,” claimed Birla. “Utilizing patient specific cells is really a cornerstone to our technology.”

“Currently those lucky enough to receive a donor heart transplant are really only trading one disease for another. The donor heart will save their life, but to prevent rejection the patient needs to take a large regiment of immunosuppressant therapy which causes many significant challenges for the patient. By bioengineering the heart out of the patient’s own cells we eliminate the need for that immunosuppressant therapy which could allow for a much better quality of life for the patient,” he continued.

With this platform technology in place, BIOLIFE4D is now well-positioned to build upon it and work towards the development of a full-scale human heart. This latest milestone also positions the company as one of the top contenders at the forefront of whole heart bioengineering, a field that is rapidly advancing.

However, beyond the scientific advancements the mini-heart represents, this is also an opportunity to provide the pharmacological industry and drug discovery companies a new tool for cardiotoxicity testing of new drugs and compounds. Until now the model used for predicting the cardiotoxicity effects of a new drug or compound was essentially limited to the animal model. But BIOLIFE4D intends to ultimately provide the mini-hearts as a more reliable model of predicting cardiotoxicity, claiming that there is no better predictor of how a human heart will react than a human heart. This also represents an opportunity to reduce the number of animals used for testing purposes, something which is already banned in quite a few regions, including India, the European Union, New Zealand, Israel, and Norway.

“We are already working closely with companies that provide cardiotoxicity testing services to the Pharma and drug discovery industries. All drugs, new compounds and anything else that currently undergoes cardiotoxicity testing requirements prior to entering the human market could be candidates for the mini-heart. After all, what would provide a better predictive model of how a human heart will respond than a human heart (albeit a scaled-down version)?” revealed Birla.

The mini-heart has many of the features of a human heart even though BIOLIFE4D has not been able to recreate the full functionality of a human heart yet.

“While we have bioengineered mini-hearts, and this in itself is a major accomplishment, a significant advancement in the field of whole heart engineering and moves us closer to bioprinting human hearts for transplantation, this accomplishment does not provide us with a specific time-line or a significant guidance on when the fully funcitional heart will be available.”

According to Birla, the most difficult part to 3D print a human heart at this point is the valves, due to the complex tri-leaflet geometry. But as they begin to scale up, they can anticipate that the complex vasculature that is needed to keep an organ viable could prove to be a big challenge.

Birla is convinced that “the algorithm used as a fundamental part of the mini-heart could change the way labs will bioprint organs in the future. We used very specific and highly customized printing parameters to bioprint the mini-heart which we have customized for our use in our lab and for our specific purposes. Some of the process ultimately could be leveraged for the bioengineering of other organs, but our overall process to bioengineer a human heart is unique to a heart.”  

One of the huge advantages BIOLIFE4D enjoys is that they have been able to form strategic partnerships with various major research institutions and hospitals to provide them access to some of the most state-of-the-art facilities and equipment. Nevertheless, because of the highly confidential nature of their work, most of it is done in-house at the labs and by their own researchers.  

The successful demonstration of a mini heart is the latest in a string of scientific milestones from BIOLIFE4D as it seeks to produce the world’s first 3D bioprinted human heart viable for transplant. Earlier in 2019, they successfully 3D bioprinted various individual heart components, including valves, ventricles, blood vessels, and in June of 2018 they 3D bioprinted human cardiac tissue (a cardiac patch).

The company states that their innovative 3D bioprinting process provides the ability to reprogram a patient’s own white blood cells to iPS cells, and then to differentiate those iPS cells into different types of cardiac cells needed to 3D bioprint individual cardia components and ultimately, a human heart viable for transplant.

This is crucial for a company that seeks to disrupt how heart disease and other cardiac impairments are treated, particularly by improving the transplant process so that in the future they can eliminate the need for donor organs. Heart disease is the number one cause of death of men and women in the United States each year. Heart diseases even claim more lives each year than all forms of cancer combined, yet countless individuals who need transplants are left waiting as there are not enough donors to meet demand and every 30 seconds, someone dies in the US of a heart disease-related event.

“While we have come a long way, and we are moving forward at a fast pace, we just don’t know how long it will take to achieve a full-scale heart.  We have to keep in mind that mother nature had millions of years to perfect this process inside our bodies, while we just aren’t sure exactly how long it is going to take us to perfect the process outside of the body,” concluded Birla.

At BIOLIFE4D, they know there are still challenges on the way to the full-size human heart viable for transplantation, however, this achievement signals that they are on the right path. They highlighted that their success, as well as the significant advancements they have been able to achieve already, are a result of an incredible team effort, a multi-disciplinary group of researchers working on the project, from bioengineers to life scientists. Their team consists of people with specific skill sets and areas of expertise, all working hard to bring this incredible life-saving technology to the market in the shortest time possible.

[Images: BIOLIFE4D]

The post Interview: BIOLIFE4D is The First US Company to Bioprint a Mini-Heart (for Cardiotoxicity Testing) appeared first on 3DPrint.com | The Voice of 3D Printing / Additive Manufacturing.