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(a) Magenta Matrigel-embedded cell triangles spaced at 18 and 38 μm. (b) Mouse mammary fat pad
used to print a pattern of DNA spots and rendered as single cells fully embedded in Matrigel.
(c) Cell pattern as attached to (green) glass template embedded in Matrigel (magenta).
(d) Heat map illustrating differences in global cell position in two dimensions.
Image Credit: Nature Methods

3-D printed organoids - first step to building organs

Scientists have developed a technique to build tiny models of human tissues — called organoids — that turn human cells into a biological equivalent of LEGO bricks. These 'organoids' are useful in cancer research and drug screening.

A University of California San Francisco (UCSF) led team has created organoids in a dish to study how particular tissue structures reflect normal growth — or go awry in cancer. Organoids can also be used for therapeutic drug screening and to help teach researchers how to eventually grow whole human organs.

The new technique — called DNA Programmed Assembly of Cells (DPAC) is reported in the journal Nature Methods, August 31, 2015. It allows researchers to create arrays of thousands of custom-designed organoids, such as models of human mammary glands, which contain several hundred cells each yet can be built in a matter of hours.

There are few limits to the tissues this technology can mimic, explains Zev Gartner PhD, senior author and an Associate Professor of Pharmaceutical Chemistry at UCSF.

"We can take any cell type we want and program just where it goes. That way, we can precisely control what cell is touching another cell at the earliest stages of development. Cells initially follow our programmed spatial cues to interact, move around, and in time develop into tissues."

"One potential application within the next couple of years is to take samples from a cancer patient's mammary glands and build a model of their tissue and use that for personalized drug screening. Another future application is using the rules of tissue growth we are learning with these models, and one day grow complete organs."

Zev Gartner PhD, senior author, Associate Professor of Pharmaceutical Chemistry, UCSF.

Our bodies are made of more than 10 trillion cells of hundreds of different kinds, each playing a unique role in keeping us alive and healthy. The way these cells organize themselves structurally in different organ systems helps coordinate amazingly diverse behaviors and functions that keep our whole biological machine running smoothly. But in diseases such as breast cancer, the breakdown of this order leads to the rapid growth and spread of tumors.

Gartner: "Cells aren't lonely little automatons. They communicate through networks to make group decisions. As in any complex organization, you really need to get the group's structure right to be successful, as many failed corporations have discovered. In the context of human tissues, when organization fails, it sets the stage for cancer."

Studying how the cells of the mammary gland self-organize, and then break down with disease, has been a challenge. A living organism is often too complex to reveal a specific cause for a particular cell outcome. On the other hand, cells in a dish also lack the critical element of 3-D structure.

"This technique lets us produce simple components of tissue in a dish that we can easily study and manipulate. It lets us ask questions about complex human tissues without needing to do experiments on humans."

Michael Todhunter PhD, study leader with Noel Jee PhD, when both were graduate students in the Gartner research group.

To specify the 3-D structure of their organoids, Gartner's team made use of a familiar molecule: DNA. Researchers incubated cells from tiny snippets of single-stranded DNA engineered to slip into a cells' outer membrane. The scientists cover a single cell with these snipets of DNA, much like the fibrous hairs covering a tennis ball. The DNA strands act as a sort of molecular Velcro, in addition to being a bar code that specifies where each cell goes within the organoid. When two separate cells, covered with complementary DNA come into contact, they stick fast to one another. If the DNA sequences don't match, the cells float on by each other. Cells can be incubated with several sets of DNA bar codes to attract multiple partners.

To turn these cellular LEGOs into arrays of organoids that can be used for research, Gartner's team lays down the cells in layers, with multiple sets of cells designed to stick to particular partners. Not only does this process allow cells to build up complex tissue components like the mammary gland, but also lends itself to experiments. By being able to specifically add in a single cell with a known cancer mutation to different parts of the organoid, researchers can observe that cells' effect.

To demonstrate the precision of the technique and its ability to generalize to many different human tissue types, the research team created several proof-of-principle organoids mimicking human tissues such as branching blood vessels and mammary glands.

In one experiment, the researchers created several groups of mammary epithelial cells to watch for an affect from low levels of the cancer gene RasG12V. They found normal cells grow faster in an organoid with low levels of RasG12V, but required more than one mutant cell to kick-start the abnormally fast growth. They also observed how cells with low expression of RasG12V, when placed at the tip of a tube filled with normal cells, branched and grew and pulled normal cells behind them — just as buds grow from the tip of a tree branch.

Gartner plans to use the DPAC organoid technique to investigate mammary gland cellular structure breakdown in tumor metastasis. He also hopes to learn from building simple models of various tissue types, how to ultimately build functional organs and neural circuits.

"Building functional models of complex cellular networks, such as found in the brain, is probably one of the highest challenges to which we aspire. DNA Programmed Assembly of Cells now makes a lofty goal like that seem achievable."

Michael Todhunter PhD

Abstract
Reconstituting tissues from their cellular building blocks facilitates the modeling of morphogenesis, homeostasis and disease in vitro. Here we describe DNA-programmed assembly of cells (DPAC), a method to reconstitute the multicellular organization of organoid-like tissues having programmed size, shape, composition and spatial heterogeneity. DPAC uses dissociated cells that are chemically functionalized with degradable oligonucleotide 'Velcro', allowing rapid, specific and reversible cell adhesion to other surfaces coated with complementary DNA sequences. DNA-patterned substrates function as removable and adhesive templates, and layer-by-layer DNA-programmed assembly builds arrays of tissues into the third dimension above the template. DNase releases completed arrays of organoid-like microtissues from the template concomitant with full embedding in a variety of extracellular matrix (ECM) gels. DPAC positions subpopulations of cells with single-cell spatial resolution and generates cultures several centimeters long. We used DPAC to explore the impact of ECM composition, heterotypic cell-cell interactions and patterns of signaling heterogeneity on collective cell behaviors.

Other researchers on the paper include postdoctoral fellow Alex Hughes, PhD, staff researcher Maxwell Coyle, former graduate students Alec Cerchiari, PhD, and Justin Farlow, PhD, and Tejal Desai, PhD, professor of bioengineering, all of UCSF; James Garbe, PhD, a staff scientist at UCSF and the Lawrence Berkeley National Laboratory; and Mark LaBarge PhD, a staff scientist at the Lawrence Berkeley National Laboratory.

Funders of the work include the Department of Defense Breast Cancer Research Program, the National Institutes of Health, the Sidney Kimmel Foundation, and the UCSF Program in Breakthrough Biomedical Research.

UC San Francisco (UCSF) is a leading university dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care. It includes top-ranked graduate schools of dentistry, medicine, nursing and pharmacy, a graduate division with nationally renowned programs in basic, biomedical, translational and population sciences, as well as a preeminent biomedical research enterprise and two top-ranked hospitals, UCSF Medical Center and UCSF Benioff Children's Hospital San Francisco.

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