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Programmed Assembly of 3-dimensional Microtissues with Defined Cellular Connectivity

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Multicellular organs comprise differentiated cell types with discrete yet interdependent functions. The cells' spatial arrangements and interconnectivities are often difficult or impossible to emulate in vitro. A research team from University of California and Lawrence Berkeley National Laboratory reported the design of microtissues with defined cell composition and stoichiometry with a novel strategy.

In vitro cell-based assays are crucial in the process of drug discovery, which provide key information on the efficacy and toxicity of some new compounds. Thus 3-dimensional tissue constructs receive more and more attention in order to increase the predictability of these assays.

However, reconstituting functional cell–cell contacts ex vivo and in 3 dimensions remains an important challenge in tissue engineering, whether for the purpose of building materials for in vivo tissue repair or for constructing realistic in vitro tissue models.

The team writing this article has envisioned a bottom-up approach to the construction of microtissues with defined connectivities by stepwise formation of contacts between individual cells. As their previous research has found a method for functionalizing cell surfaces with oligonucleotides for the purpose of cell patterning on complementary DNA-coated surfaces, they confirmed in this project that hybridization between DNA-coated cells can direct the assembly of cell–cell contacts.

Several main results are demonstrated in this article:

1. Activating Mutual Reactivity Between 2 Populations of Cells

Under certain conditions, the researchers observed individual cell clusters that, under higher magnification, demonstrated a common architecture in which the microenvironment of the limiting cell type was defined by neighbours of the cell type added in excess. These clusters formed sequence-specifically, in excellent yield, and without compromising the cell membrane.

2. Characterizing and Controlling the Kinetics of Microtissue Assembly

The researchers then designed several experiments, which showed that at least 3 independent variables could control reactivity between cells during the assembly process: cell density, cell surface DNA density, and oligonucleotide sequence complexity.

3. Purification of Assembled Microtissues and Iterative Synthesis

Some experiments were conducted to prove the feasibility of synthesizing microtissues by the iterative stepwise formation of contacts between individual cells.

4. DNA-Mediated Intercellular Contacts Are Reversible

They investigated 2 strategies to reverse DNA-bound cellular assemblies, using oligonucleotides with a 10-base region of complementarity and DNase that can rapidly degrade DNA duplexes and found that assembled structures could be templated, encapsulated within a suitable 3-dimensional matrix, and the cell surface duplexes removed without disrupting the topology of microtissues.

5. Synthesizing a Functional Paracrine Signaling Network in 3 Dimensions

Then they constructed a system comprising functionally distinct cell types wherein one produces a signal that is critical for the other's growth and survival to prove the successful synthesis of microtissues with emergent functional properties that depend on those of the underlying cellular building blocks, as in living tissues.

This research assembled 3D microtissues by building connectivities among cells by using duplex DNA as a bonding agent, which can be conducted under typical cell culture conditions without any genetic manipulation, producing products portable to any environment for fundamental studies or tissue engineering. This strategy for 3-dimensional microtissues development can provide a new way of analyzing cellular behavior in vitro as a function of overall tissue architecture, and can also serve as access to study fundamental units of tissue function such as building blocks for artificial organs and high-throughput screening platforms, the stem cell niche, and in vitro models of human disease.

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