Assembling cells into artificial 3D microtissues, including a tiny gland

Berkeley 05 March 2009As synthetic biologists cram more and more genes into microbes to make genetically engineered organisms produce ever more complex drugs and chemicals, two University of California (UC) chemists have gone a step further. They have assembled different types of genetically engineered cells into synthetic microtissues that can perform functions such as secreting and responding to hormones, promising more complex biological capabilities than a single cell alone could produce.

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Scientists at the United States Department of Energy's Lawrence Berkeley National Laboratory can now control how cells connect with one another in vitro and assemble themselves into three-dimensional, multi-cellular microtissues. The researchers demonstrated their method by constructing a tailor-made artificial cell-signaling system, analogous to natural cell systems that communicate via growth factors.

Artificial tissues are presently used in medicine for a range of applications such as skin grafts, bone marrow transplants, or blood substitutes, as well as in basic medical and biological research. Tissue engineers try to improve upon or repair natural tissues by manipulating living cells from one or more donors, sometimes in combination with synthetic materials. Unfortunately, in this "top down" approach, the cells assemble themselves randomly, losing the 3D organisation that is key to many tissue functions.

"Our method allows the assembly of multi-cellular structures from the 'bottom up'", stated Carolyn Bertozzi, principal investigator in the research, who directs DOE's Molecular Foundry nanoscience research facility at Berkeley Lab and is a member of the Lab's Materials Sciences and Physical Biosciences Divisions. "This is like another level of hierarchical complexity for synthetic biology", stated co-author Carolyn Bertozzi, who is also UC Berkeley professor of chemistry and of molecular and cell biology. "People used to think of the cell as the fundamental unit. But the truth is that there are collections of cells that can do things that no individual cell could ever be programmed to do. We are trying to achieve the properties of organs now, though not yet organisms."

While the synthetic tissues today comprise only a handful of cells, they could eventually be scaled up to make artificial organs that could help scientists understand the interactions among cells in the body and might some day substitute for human organs.

"We are really taking this into the third dimension now, which for me is particularly exciting", stated first author Zev J. Gartner, a former UC Berkeley post-doctoral fellow who recently joined the UC San Francisco faculty as an assistant professor of pharmaceutical chemistry. "We are not simply linking cells together, we are linking them together in 3D arrangements, which introduces a whole new level of cellular behaviour which you would never see in 2D environments."

Zev J. Gartner and Carolyn Bertozzi, the T.Z. and Irmgard Chu Distinguished Professor at UC Berkeley and a Howard Hughes Medical Institute investigator, report on their assembly of three-dimensional microtissues in the on-line early edition of the journal Proceedings of the National Academy of Sciences.

One type of cell that needs other cells to make it work properly is the stem cell, Carolyn Bertozzi noted. Theoretically, using Zev J. Gartner and Carolyn Bertozzi's chemical technique, it should be possible to assemble stem cells with their helper cells into a functioning tissue that would make stem cells easier to study outside the body. "In principal, we might be able to build a stem cell niche from scratch using our techniques, and then study those very well defined structures in controlled environments", Carolyn Bertozzi stated.

Carolyn Bertozzi noted that most of the body's organs are a collection of many cell types that need to be in actual physical contact to operate properly. The pancreas, for example, is a collection of specialized cells, including insulin-secreting beta cells, that "sense glucose from the environment and respond by producing insulin. A complex feedback regulatory loop goes into all of this, and you need more than one cell type to achieve such regulation".

"If you really want to understand the way these cells behave in an organism, especially a human, you would like to recapitulate that environment as closely as possible in vitro", Zev J. Gartner stated. "We are trying to do that, with the aim that the rules we learn may help us control them better."

Zev J. Gartner and Carolyn Bertozzi assembled three types of cultured cells into onion-like layers by using two established technologies: DNA hybridization and Staudinger chemistry. DNA hybridization is like a "programmable glue", she stated, that can stick cells together because of the highly precise nature of binding between complementary DNA strands: One strand of the DNA helix binds only to its complementary strand and nothing else. By putting a short DNA strand on the surface of one cell and its complementary strand on another cell, the researchers assure that the two lock together exclusively.

A 3D reconstruction using deconvolution fluorescence microscopy of a single multi-cellular structure encapsulated in agarose gel. Cells are stained different colours according to the oligonucleotide sequence attached to their surfaces. Photo: Courtesy of Bertozzi lab, UC Berkeley.

The researchers induced the cells to express artificial sugars bearing special chemical groups. Lengths of synthetic DNA, introduced into the cell-growth medium, were equipped to recognize these synthetic sugars on the cell surfaces and chemically bind to them. The researchers coated cell surfaces from one group with strands of single-stranded DNA only 20 bases long, and the surfaces of another group with the complementary DNA strand. When a cell from one group meets its counterpart, the single strands recognize each other and form double-stranded DNA, which binds the cells together.

To get these specific DNA strands onto the cells, they used chemical reactions that do not interfere with cellular chemistry but nevertheless stick desired chemicals onto the cell surface. The technique for adding unusual but benign chemicals to cells was developed by Carolyn Bertozzi more than a decade ago based on a chemical reaction called the Staudinger ligation. After proving that they could assemble cells into microtissues, Zev J. Gartner and Carolyn Bertozzi constructed a minute gland - analogous to a lymph node, for example - such that one cell type secreted interleukin-3 and thereby kept a second cell type alive.

How to build a microtissue: At the bottom, cells bearing complementary single strands of DNA on their surfaces react with each other to form stable cell-cell contacts. At centre are Jurkat cells stained red or green, labeled with different, complementary DNA sequences, and combined at a ratio of 50 (red) to 1 (green). At top, the two cell types are shown joined in a 3D multi-cellular structure. Photo: Courtesy of Carolyn Bertozzi, Lawrence Berkeley National Laboratory.

"What we did is build a little miniaturized, stripped-down system that operates on the same principle and looks like a miniaturized lymph node, an arrangement where two cells communicate with each another and one requires a signal from the other", she stated. "The critical thing is that the two cells have to have a cell junction. If you just mix the cells randomly without connection, the system doesn't have the same properties."

She expects that eventually, clusters could be built on clusters to make artificial organs that someday may be implanted into humans. "Our method allows the assembly of multi-cellular structures from the bottom up. In other words, we can control the neighbours of each individual cell in a mixed population", she stated. "By this method, it may be possible to assemble tissues with more sophisticated properties."

One interesting aspect of the technique is that DNA hybridization seems to be temporary, like a suture. Eventually, the cells may substitute their own cell-cell adhesion molecules for the DNA, creating a well-knit and seemingly normal, biological system.

Carolyn Bertozzi and Zev J. Gartner discovered three variables that determine how cells from different groups react with one another. One is the ratio of the two kinds of cells: if both cell populations are equal, every cell finds a single partner and no complex assemblies form. But if there are, say, 50 times more cells from one group than from the other, numerous cells from the larger group will cluster around each cell from the smaller group.

Another variable is the complexity of the synthetic DNA sequence. The researchers can specify the complexity - for example, from a simple repeat of two bases such as cytosine and adenine (CACACA...) on one strand, which binds to a complementary repeating sequence of thymine and guanine (TGTGTG...) on the other, up to sequences whose base order varies over the full length of the 20-base strand of synthetic DNA. The more complex the sequences they display, the longer it takes the cells to bind together.

A third variable is the density of the DNA on the cell surface. By controlling how many artificial sugars the cells express, the researchers can control the DNA surface density. The greater the density, the faster the cells bind to one another.

Carolyn Bertozzi noted that more variables for controlling cell assemblies are possible. "For example, it might be possible to cluster DNA strands on specific cellular structures. Thus, distribution of DNA on the cell surface might be yet another parameter we can exploit to guide cell-cell interactions."

After cell types labeled with red and green dye markers are joined (bottom), the resulting 3D structures are purified to eliminate unreacted cells (centre). More cells can then be added to form even more complex structures (top). There is no theoretical limit to the number of different cell types that can be assembled; microtissues with three or four different kinds of cells should be feasible. Photo: Courtesy of Carolyn Bertozzi, Lawrence Berkeley National Laboratory.

By controlling these variables to assemble small cellular structures, then separating the desired structures from unwanted ones and unreacted cells and assembling more cells on the purified collection - then repeating the steps again - the researchers can synthesize large, complex microtissues in much the same way a synthetic organic chemist assembles a complex molecule.

Carolyn Bertozzi and Zev J. Gartner applied these methods to build a signaling network where one kind of cell controls the growth of a second kind of cell. They maintained the survival and replication of hematopoietic progenitor cells - a kind of stem cell for blood cells, which depend on the presence of the growth factor interleukin-3, by combining them in microtissues with CHO cells - Chinese hamster ovary cells - that were engineered to secrete interleukin-3.

When the two cell types were randomly mixed, the stem cells didn't grow. But structured microtissues built from the two cell types stimulated their own growth, forming a simple artificial signaling network that behaved much like the natural networks that control immune-cell expansion or tumour proliferation.

"Since DNA has essentially an unlimited capacity for information storage, there is no theoretical limit on the number of different cell types we can assemble in a structure", stated Carolyn Bertozzi. The key is to give each cell type its own unique DNA "bar code", enabling its programmed interaction with any other specified cell type. "In practice, I think structures with three or four cell types are quite feasible. Such structures would be relevant to many biological organs."

Structured microtissues have numerous research applications, explained Carolyn Bertozzi, particularly "in probing how the local cellular environment affects the behaviour of a particular cell. Also, we can study how systems of cells work together to produce complex organ functions. Examples include how T cells and B cells work together in the lymph nodes to mount an immune response against foreign antigens."

Practical challenges remain, such as scaling up the production of tailored microtissues to quantities needed for biomedical applications. Beyond that, Carolyn Bertozzi hopes to refine the present method of modifying cell-surface DNA. "As it stands, the need for unnatural sugar biosynthesis limits the kinds of cells that we can use in microtissue construction", she stated. "There are other ways in which DNA can be conjugated to cells, independent of their sugar metabolic pathways, and we intend to explore those avenues."

The research was funded by the Office of Science, United States Department of Energy as well as the Howard Hughes Medical Institute. Zev J. Gartner was supported by a fellowship from the Jane Coffin Childs Memorial Fund. The paper is titled: "Programmed assembly of 3-dimensional microtissues with defined cellular connectivity", and is written by Zev J. Gartner and Carolyn R. Bertozzi. It appeared in PNAS Early Edition, week of March 2, 2009.

Berkeley Lab is a United States Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California.


Sources: DOE/Lawrence Berkeley National Laboratory and University of California - Berkeley

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