Several organs and various tissue types have been successfully printed since 3D bioprinting techniques were developed in the early 2000s. Among those are mouse ovaries, which were printed and implanted in mice by researchers from Northwestern University. The ovaries were functional, and mice could conceive and gave birth to offspring.
The researchers, from Shah’s lab at Northwestern, bio-printed layers of gelatin to form small 3D microporous hydrogel scaffolds of varying pore geometry and seeded mouse follicles containing precursor ovaries into the scaffolds.
The synthetic, follicle-seeded ovaries were implanted into female mice who had been surgically sterilized (their natural ovaries removed). These synthetic ovaries became highly vascularized in a few days, and after mating, the female mice bearing 3D-printed ovaries birthed healthy pups that were confirmed to have resulted from an egg ovulated from the synthetic ovary. The litters, however, were smaller than average (1-2 vs. 6-8 pups).
This study opened up the possibility of restoring reproductive health and fertility to cancer patients who have been sterilized by chemotherapy treatment and represented an important advance in the applications of tissue bioengineering to the reproductive system.
The results were published in Nature Communications; you can read the article here.
During the past two decades, we have witnessed significant scientific and technical advances in the fields of drug discovery and translational medicine along with advances in predictive in vitro model systems. As of now, microfabrication techniques and tissue engineering have enabled the development of a wide range of 3D cell culture technologies, including multicellular spheroids, organoids, scaffolds, hydrogels, organs-on-chips, and 3D bioprinting, each with its own advantages and disadvantages. 3D culture models have been penetrating into the early drug discovery process, starting from disease modeling to target identification and validation, screening, lead selection, efficacy, and safety assessment.
This year’s TEDD Annual Meeting brings together experts from diverse fields with a shared interest in advanced 3D models. The idea is to help to foster collaborations between 3D cell culture developers, and experts in advanced analysis methods: microscopy, sensors, data modelling, and high-throughput screening. Several companies will exhibit during our famously long lunch break at the Greenhouse, where we have the opportunity to interact. Join us for this meeting to celebrate another fruitful collaboration year with the new perspectives ahead.
Louisa Dean, MSc is a young researcher in the field of Cell Matrix Biology and Regenerative Medicine at the University of Manchester. In her recent project, she investigated novel 3D bioactive biomaterials to evaluate their suitability as substrates for wound-healing acceleration in ex vivo human skin tissue assays. In the following interview, she is sharing her experiences working with BiogelxTM-Collagen.
1. What research does your lab focus on?
Our research focuses on developing new therapies for the management of large-surface and chronic skin wounds, which remain an unresolved clinical challenge – posing a serious burden to patients and public health systems. Hence, with a clear need for effective treatment of these kind of wounds, our project aim was to design and produce novel 3D bioactive biomaterials and evaluate their suitability as substrates for wound-healing acceleration in ex vivo human skin tissue assays. Specifically, we investigated the suitability of synthetic collagen proteins as agents to promote cell spreading in both 2D and 3D (hydrogel substrate) adhesion assays.
2. Why is it so important to use 3D models?
As the critical focus of this research is tissue regeneration, a 3D substrate is needed to act as a porous scaffold for cells to colonize, ideally mimicking the extracellular matrix (ECM) that surrounds cells in their natural context – replicating an in vivo environment.
3. What are the features you think are key for a 3D matrix?
Personally, I think two fundamental properties for 3D substrates are biocompatibility and reproducibility. Another important feature is that the substrate is adaptable to various applications (e.g. mimic various tissues), with mechanical properties that are tuneable – without concerns for how changing the crosslinking chemistry of the matrix would influence the functionalization of the gels with cell-adhesive components.
4. Why did you choose BiogelxTM-Collagen?
Ease of use, easily adjustable range of stiffnesses to produce gels that suitably mimic the desired cell environment for our research application and functionality of gels to incorporate specific components (in this case a collagen biomimetic peptide sequence) into the 3D matrix.
5. How easy was it to use the gel? How easy was to repeat your research results?
Once you are familiar with preparing pre-gel solutions at a desired stiffness from the hydrogel powder, and decide the best gel format for your application, I found that my results (i.e. the level of attachment and spreading by cells) were quite consistent across my experiments.
6. What cell types do you use in your research? Please share your experience in growing cells in our gel(s).
This project began using HT1080 cells, a fibrosarcoma cell line, then from this we moved onto primary HFF cells, human foreskin fibroblasts – chosen for high levels of integrin expression and adherence properties. Each cell type behaved differently across various hydrogel stiffnesses and types (unfuctionalised vs. GFOGER collagen), but both responded well to the gel environment and demonstrated good attachment during culture.
7. What are the next steps in your research?
At the moment we have some promising preliminary results using collagen-functionalised hydrogels (which incorporates the GFOGER peptide into the matrix) with our primary fibroblast cells. So, the plan for the future is to investigate a panel of collagen proteins for these hydrogels – with the aim of applying these functionalised gels to ex vivowound closure assays using human skin.
Despite the successful studies and reported outstanding research efforts, the goal of 3D bioprinted organs has yet to be accomplished and there are several challenges which must be overcome.
1. Bioprinter technology
Bioprinter technology needs to increase resolution and speed and should be compatible with a wide spectrum of biocompatible materials. Higher resolution will enable better interaction and control in the 3D microenvironment. Speeding up could build the opportunity to reach a commercially acceptable level and allow the process to be scaled up.
Biomaterials are undoubtedly the primary limitation of this technology. Today, we are limited to several biocompatible synthetic and natural bio-inks. Synthetic materials provide good mechanical strength and can mimic naturally-derived materials, which are particularly adept at promoting cell attachment, proliferation, and differentiation.
3. Choice of the cell source
The choice of cell source also determines the success of the printed construct. Stem cells have the ability to differentiate into multiple different cell types and can build different tissues. Hence, their differentiation and interaction with the scaffold material are essential.
4. Vasculature of the printed construct
Another fundamental issue is the vasculature of the printed construct. In vivo 3D tissue is constantly fed by oxygen and nutrients. If the tissue is constructed using a bioprinter, then it also needs a vascular system. Diffusion by itself will only work up to 150-micrometer thickness. Beyond this thickness, the tissue will not develop properly.
These challenges will need to be addressed down this novel technology becomes fully operational and effective.
Stem cells have the ability to differentiate into specialized cells and can self-renew; dividing to give rise to new stem cells. Therapeutic uses for stem cells that are currently under investigation include tissue engineering and regenerative medicine, cell therapy for a range of diseases such as cancer, and also for drug discovery in which new pharmaceuticals are evaluated. During differentiation into a particular cell type, the stem cells undergo morphological changes in size and shape, as well as functional changes in metabolic activity and response to outside stimuli.
In the development from a zygote into a multicellular organism, the cells of the organism undergo differentiation into many complex and complementary tissue types. Beginning with the totipotent and pluripotent stem cells which give rise to all of the specialized tissues in the body, the DNA sequence of the cells does not change. Rather, the epigenetic profile of the cells changes to modify gene expression. The DNA molecule is chemically modified and packaged in such a way as to make particular genes more or less accessible for expression, effectively turning genes on and off. A stem cell used by a researcher has a particular epigenetic profile that is concurrent with pluripotency or multipotency. Likewise, a mature and specialized cell will have a different profile involving the up-regulation of specific heart, liver, etc. genes and down-regulation of genes that maintain stemness. Differentiation of a stem cell is characterized by a shift in levels of gene expression.
There are many factors that cause a stem cell to differentiate. For in vitro culture conditions, researchers can induce differentiation by introducing growth factors and/or inhibitory factors to the culture media, by co-culturing the stem cells with differentiated cells, and through mechanical signals from a three-dimensional culture environment. Cellular differentiation is a result of cellular signal transduction. A signaling molecule that is either produced by a neighboring cell or exogenously introduced binds to a specific receptor on the stem cell. Receptor-binding induces a conformational change that triggers a cascade of signaling, typically based around activation of enzymes which phosphorylate proteins and transcription factors. The activated transcription factors bind to the DNA of the cell at particular regions and influence gene expression. The elasticity of the extracellular matrix also influences stem cell differentiation. Different tissue types range from soft to stiff, and stem cells recognize these signals as part of the differentiation process. The cells sense the mechanical properties of the ECM at focal adhesions that transmit the mechanical force required to deform the matrix to the interacting cell.
In order to induce stem cell differentiation is a lab, researchers must provide the cells with the correct biochemical and mechanical signals. The cell must have all of the precise and balanced inputs that they would receive in the body during the differentiation process. These complex systems of signal transduction pathways and relationships are under investigation so that researchers can effectively translate the culture, maintenance and differentiation of stem cells into safe and operational therapies.