Cellular printing using laser technology to create tissue and organ models

Protective gear, such as body armor for servicemen and women, seat belts and air bags in cars, and helmets for high school athletes have been developed and tested in recent years on increasingly sensitive mannequins. While such models approximate height, weight, and other general factors, they aren’t sensitive enough to reveal how diverse human tissues behave in traumatic situations. For example, in a sports collision, do the brain’s neural synapses and networks handle impact differently than the microcapillaries that supply blood to the brain? Or, what about the connection between the inner ear and brain, specifically the cell-cell interfaces in the cochlea that translate sound into neural signals to the brain? The answers to these questions might begin to explain the medical responses that servicemen and women experience as a result of post-traumatic stress, and the medical impacts of repeated concussions to athletes Conducting tests on highly sensitive models can provide engineers with important information as they strive to create safer, even more effective protective gear.

Brad Ringeisen, Ph.D., of the Naval Research Laboratory (NRL) in Washington, D.C., has developed new cellular printing technologies that may be the first steps toward creating such highly sensitive models. Other research has relied on implanting different types of cells, often stem cells, in a gel-like medium with the result that tissue would grow, but in a random and homogenous manner, not unlike scar tissue. The most successful engineered tissues have been limited to structures such as skin and the bladder because these tissues are less complex and thin. Thick, three dimensional tissues are often much more heterogeneous and require vasculature to bring in nutrients and remove waste. With cellular printing, Ringeisen is able to precisely place individual cells on a substrate so that the cells recreate normal functions and natural inter-relationships with other types of cells. For example, Ringeisen is able to place vascular endothelial cells, a type of cell that lines the interior of capillaries and facilitates blood flow, adjacent to smooth muscle cells, thus mimicking the interface between those cell types in natural veins and capillaries. Human tissue is enormously complex; for example, the vascular matrix is so dense that every 100 microns of tissue is fed with oxygen by veins, arteries, and capillaries.

Of the handful of groups working on cellular printing worldwide, only NRL has patented a laser-based process that permits the ‘pixel by pixel’ approach to recreate structured, differentiated tissues. Ringeisen’s technique lies at the intersection of chemistry and physics and employs a laser energy transfer process. Using a thin layer of quartz, which he calls a ribbon, he lays down a bioink on top of a thin layer of titanium dioxide, which acts to absorb the incident laser energy. Each laser pulse creates a bubble that forms a jet of liquid bioink containing the cells he intends to implant. That jet then directs the cell, or groups of cells, to a sheet of biopaper with a precision of tens of microns. Because the approach is both highly focused and yet an indirect means, individual cells are deposited gently, whole and intact. The laser printing process is also fast, allowing Ringeisen to deposit 40 to 50 droplets of cells per second. One group in France has now adopted this approach in a way that enables thousands of cell droplets to be deposited per second. Biopapers, which are thin gel/membrane hybrids, containing ‘printed’ cells are stacked and allowed to grow in vitro. Ringeisen has observed that by placing the cellular material so closely, the material seems to retain and replicate cellular function. In fact, vascular endothelial cells self-recognize their environment and form lumens, the ‘tubes’ of our capillaries that carry blood, along the length of the printed pattern.

Ringeisen is working with others at NRL to further develop his approach. Within the next few years, he envisions they will be able to make a model that mimics the brain and be able to conduct impact studies, testing how different cells and cell interfaces respond biologically to mechanical, acoustic and blast impacts.

Starting from the seeds of scientific research funded by the Department of Defense, Ringeisen’s chemistry innovations stand to greatly benefit many critical efforts. Fromengineering new helmets to protect U.S. servicemen and women, to medical research into new drugs and treatments for traumatic brain injury and post-traumatic stress, to improving protective gear for our young and growing athletes, Ringeisen’s research promises to deliver improved health and safety for future generations.