3D printing is arguably one of the most discussed trends of the moment, not only by geeks and nerds. 3D printing can be used for rapid prototyping and manufacturing, is really convenient for concept design, and can even have a great cultural value reconstructing fossils in paleontology and replicating ancient artifacts in archeology. But probably the most important application of 3D printing resides in the medical field.
Today, most common 3D printing manufacturing processes are:
• Fused Deposition Modeling (FDM), by the extrusion of thermoplastics like PLA (highly bio-compliant and used in scaffolds), or ABS (the same material of the LEGO® bricks);
• Stereolithography (SLA), by exposing a photopolymerizable resin to an UV laser light;
• Laminated object manufacturing (LOM), by gluing together layers of paper, plastic, or metal;
• Selective laser sintering (SLS), by fusing a powdery substa
nce (plastic, metal, ceramic, or glass) via a heating process into a solid form.
But latest 3D printing developments are starting to create living tissue by using living cells as ink. Such “bioprinters” could drive a revolution in many areas of medicine, by allowing the creation of tissues, and even entire organs from a culture of human cells.
Susmita Bose has been developing artificial bone-like materials with her husband, Amit Bandyopadhyay, since the late 1990s, and together they managed to successfully grow real bone tissue on artificial scaffolds. These autologous bone cells would enable doctors to repair defects or injuries without using any synthetic material and so without any issues concerning transplant rejection.
Robert Langer and others pioneered methods for encouraging certain types of organ cells to grow on polymer scaffolding.
In 2008 Makoto Nakamura created a working bioprinter that can print out biotubing similar to a human blood vessel. His idea was to print thousands of cells per second, and stack them in a three-dimensional structure.
A team of scientists at the University of Missouri, Columbia, suggested that bioprinting might be easier if cells are free to sort and fuse themselves, given that they have an innate regenerative capability to self-assemble and self-organize. In March 2008, this research group, lead by Professor Gabor Forgacs, managed to bioprint functional blood vessels and cardiac tissue using cells obtained from a chicken. Unlike other tissue engineering methods, which make use of polymers, they used only cardiac and endothelial cells, temporaly supported by a collagen scaffold during printing.
Here comes Organovo
Forgacs founded Organovo in 2007, and since the very beginning his team has worked with a company called Invetech to create a commercial bioprinter called NovoGen™ MMX. This printer includes two robotically controlled print heads: one for placing human cells, the other for placing a hydrogel, scaffold, or support matrix. According to Invetech, one of the most complex challenges in the development of the bio-printer was to perfect a means to consistently position the cell dispensing capillary tip attached to the print head within microns. Invetech developed a computer-controlled, laser-based calibration system to achieve the required repeatability.
Organovo cultures cells from a patient and use them to form bio-ink spheroids, each one containing up to 80,000 cells. The Organovo’s bioprinting process, which was selected as one of the “Best Inventions of 2010” by TIME Magazine, is divided in these phases:
1. a tissue design is established;
2. the protocols required to generate the multi-cellular building blocks (bio-ink) are developed;
3. the bio-ink droplets are dispensed from a bioprinter, using a layer-by-layer approach. Bio-inert hydrogel can be used for support and fillers to create channels or spaces within tissues to copy features of real tissue.
Dr. Sharon Presnell, CTO and Executive Vice President of Research and Development, stated:
We’ve combined three key features that set our 3D tissues apart from 2D cell-culture models. First, the tissues are not a monolayer of cells; our tissues are approximately 20 cell layers thick. Second, the multi-cellular tissues closely reproduce the distinct cellular patterns found in native tissue. Finally, our tissues are highly cellular, comprised of cells and the proteins those cells produce, without dependence on biomaterials or scaffold for three-dimensionality. They actually look and feel like living tissues.
To create its output, the NovoGen bioprinter first lays down a single layer of a water-based bio-paper made from collagen, gelatin or other hydrogels. Bioink spheroids are then injected into this water-based material. Another layer of supportive biopaper and bioink spheroids is then added, and then another, until the desired output.
Here comes the particularity of the process, and the main difference of bio-printing in respect to traditional 3D printing processes: the bioink spheroids slowly fuse together. As this occurs, the biopaper dissolves away, leaving a final bioprinted tissue. The key point is that the bioink spheroids fuse together into living tissue, because of the cells capability of rearranging themselves. So, when printing a blood vessel, the endothelial cells migrate to the inside, the smooth muscle cells move to the middle, and the fibroblasts migrate to the outside.
According to the Organovo Co-Founder and CEO Keith Murphy, going from tissues that are 1 millimeter thick to 5 millimeters thick will be harder than going from 5-millimeter tissue to building an organ.
The two biggest challenges are thickness and functionality. At this point, tissue that can be reliably made with cell cultures is still limited to thin tissue and small amounts of tissue that do not contain any blood vessels. These are simple tissues made from only one type of cell which, for the most part, lack structure. Several organizations have been able to use printers to layer stem cells or other types of cells. Much harder has been creating vascular structures that allows the tissue to be viable longer and allows for the transport of materials into and out of the tissue, enabling more complex functions, such as the production of proteins.
On April 2013, Organovo confirmed its success during a presentation at the annual Experimental Biology conference of the American Society of Pharmacology and Experimental Therapeutics held in Boston. The company showed a three-dimensional structure of an actual liver tissue. This bioprinted 3D liver tissue was composed by both hepatocytes and stellate cells, as well as blood vessels, and exhibited several key features that remained stable over 135 hours:
1. tissue-like cellular density;
2. controlled spatial positioning of specific cell types in x, y, and z axes;
3. multi-layered architecture reaching up to 500 microns thickness, with tissues comprised of up to 20 cell layers.
These 3D liver cells were able to synthetize cholesterol, produce proteins such as albumin (with a production 5-9 times greater than matched 2D controls), fibrinogen, and transferrin, and express key liver enzymes as CYP 1A2 and CYP 3A4.
Organovo’s activities are manily two: build tissues for research on drugs, and tissues for therapy and transplantation.
A bioprinted tissue can offer to the researchers something they have never had before: the opportunity to test drugs on functional human tissues before ever administering the drug to a living person.
These tissues can be used to elicit a response that’s representative of in vivo biology in an in vitro environment. Researchers can even perform serial biopsies on the same tissue, allowing them to monitor changes over time.
Organovo outlined the three key design parameters which are applicable to both research and therapeutic applications on 3D human tissues:
1. Cellular inputs – Organovo can create tissues from a wide variety of cellular inputs, including primary human cells, cell lines, iPS-derived cells, or when available, patient samples, which are critical for the study of rare diseases and genetic disorders.
2. Tissue architecture – Important questions to consider are: how many cell types are needed? What is the micro architecture?
3. Assay Design / Delivery Method – For in vitro tissue models, Organovo works from the outset to define the assays that are built into the system. For therapeutic tissues, the company studies how to maximize the therapeutic effect weighting up several options such as: grafting onto an organ, implanting peritoneally or subcutaneously, or delivering therapy via extracorporeal tissues (a dialysis-like approach).
Once human trials are complete, Organovo hopes that its bioprinters will be used to produce blood vessel grafts for use in heart bypass surgery. The intention is then to develop a wider range of tissue-on-demand and organs-on-demand technologies
3D Printing and Bio-Printing—by Giacomo Debidda