Engineering Tissue-Scale Properties with Synthetic Cells: Forging One from Many

Abstract

In metazoans, living cells achieve capabilities beyond individual cell functionality by assembling into multicellular tissue structures. These higher-order structures represent dynamic, heterogeneous, and responsive systems that have evolved to regenerate and coordinate their actions over large distances. Recent advances in constructing micrometer-sized vesicles, or synthetic cells, now point to a future where construction of synthetic tissue can be pursued, a boon to pressing material needs in biomedical implants, drug delivery systems, adhesives, filters, and storage devices, among others. To fully realize the potential of synthetic tissue, inspiration has been and will continue to be drawn from new molecular findings on its natural counterpart. In this review, we describe advances in introducing tissue-scale features into synthetic cell assemblies. Beyond mere complexation, synthetic cells have been fashioned with a variety of natural and engineered molecular components that serve as initial steps toward morphological control and patterning, intercellular communication, replication, and responsiveness in synthetic tissue. Particular attention has been paid to the dynamics, spatial constraints, and mechanical strengths of interactions that drive the synthesis of this next-generation material, describing how multiple synthetic cells can act as one.

Keywords: adhesion; cell mechanics; intercellular communication; regeneration; synthetic cells; tissue.

Figure 1.

Synthetic tissue is inspired by the organization and dynamics of living tissue and seeks to emulate key features and functions of its living counterpart. (A) Tissue of the small intestine (left) contains (i) different cell types patterned within the villi and crypts and (ii) noncellular structures, including the extracellular matrix (ECM) and the mucus layer, that shape and contribute to the tissue and its function. Signaling-dependent differentiation and migration maintain the functional capacity of the organ for absorption under fluid flow. Synthetic tissue (right) requires the precise combination of distinct synthetic cell building blocks and substrata materials to approach the capabilities and resiliency of living tissue. (B) Six desired functionalities of synthetic cells for the construction and maintenance of synthetic tissues include adhesion between synthetic cells, synthetic cell–substrata contacts, synthetic tissue mechanics, regeneration, intercellular communication, and synthetic cell patterning. These functionalities either depend on or must operate in the presence of synthetic cell neighbors.

Figure 2.

Linkage chemistries for adhering synthetic cells to each other and to the extracellular matrix (ECM). (A) Non-native adhesions, nonspecific interactions, and native proteins have been used to interface synthetic cells into multicellular assemblies (left). This large repertoire of interactions enables engineering of the strength and dynamics of synthetic tissue assemblies. Examples and characteristics of each type of adhesion are summarized in a table (right). (B) Reconstitution of integrin heterodimers, which engage the ECM, has been achieved via electroformation (top) (reprinted from ref , with permission from Elsevier) and membrane fusion (bottom) (reprinted from ref , Copyright 2017 Nature Publishing Group). Both methods lead to synthetic cells contacting the substrata to build composite materials, analogous to living tissue.

Figure 3.

To engineer defined mechanical responses in synthetic tissue, incorporating cytoskeletal proteins, their resulting networks, and connections to the membrane remains critical and has been an intense area of focus. Successful examples include building internal structures by ligating multiple cytoskeletal elements together (top left) (from ref , CC BY 4.0), generating artificial cell cortices (top right) (from ref , CC BY 4.0), generating synthetic cell movement with photosensitive proteins (bottom left) (reprinted with permission from ref , Copyright (2018) American Chemical Society), and inducing membrane deformations with molecular motors (bottom right) (reproduced with permission from ref , Copyright (1999) National Academy of Sciences, U.S.A.). Placement of anisotropic mechanical properties within synthetic tissue can give rise to convoluted morphologies, often found in living tissue, and may provide access to collective phenomena, like collective cell migration.

Figure 4.

Hypothetical path for regenerating damaged synthetic tissue. Damaged cells within synthetic tissue must be recognized, extruded, and replaced to maintain the function and integrity of synthetic tissue. After receiving damage-specific signals, neighboring cells respond by initiating cell replication and tissue-wide compression. The neighboring cells extrude the damaged cell while simultaneously dividing to fill the gap left by the damaged cell. Ultimately, the damaged cell is extruded, and the synthetic tissue is repaired.

Figure 5.

Recent methods of synthetic cell replication and communication. (A) For replication within synthetic tissue, synthetic cells must undergo similar steps of cell division to those of living cells under the constraint of surrounding neighbors and their cumulative pressure. First, DNA must be replicated, which has been accomplished with encapsulating machinery from the phi29 replication complex. Double-stranded binding protein (DSB) and single-stranded binding protein (SSB) stabilize DNA while terminal protein (TP) and phi29 DNA polymerase (DNAP) initiate and catalyze DNA replication, respectively (from ref 140). DNA can then be segregated. A DNA droplet with photolabile sites formed through liquid–liquid phase separation undergoes segregation in response to UV light (from ref 150). Next, small unilamellar vesicles (SUVs) are fused with a parent GUV to mimic membrane growth (from ref 156). Finally, the membrane must deform and split, creating two separate synthetic cells, which has been partially demonstrated with encapsulating Min proteins in osmotically deflated vesicles (from ref 151). (B) Two general forms of communication in synthetic tissue. Contact-based communication is governed by direct cell–cell channels or through fusion of synthetic cells and mainly occurs between proximal cells, while diffusion-mediated communication relies on release of soluble signaling molecules via membrane pores, which can reach more distant neighbors.

Figure 6.

Synthetic cells can be patterned spatially to yield assemblies capable of information transfer and responsiveness. (A) Two general methods of assembling synthetic tissue have been shown in the literature. One, clustered assembly (left) relies on external forces and/or chemical cross-linkers to group cells together. While this method can organize many cells together (10–100), single-cell spatial patterning is difficult to achieve with this approach. Sequential assembly (right), on the other hand, occurs either through optical tweezing or 3D printing of single synthetic cells. Optical tweezing is capable of only patterning up to ~10 cells at once, a limitation for building large systems. (B) Transfer of information and responsiveness are defining features of living tissue. In synthetic tissue, transfer of information (left) has been implemented by endowing synthetic cells with the ability to transmit signals over long distances, requiring both spatially defined processing and transport machinery (from ref , Copyright 2013 The American Association for the Advancement of Science). Responsiveness to external stimuli (right) has also been achieved, allowing synthetic tissue to adapt to various environmental conditions, mechanical states, and to external user-defined inputs, such as light pulses and temperature changes. (C) In the future, we envision synthetic tissue–living tissue interfaces, where biocompatible synthetic cells are engineered to signal to neighboring living cells directly through hybrid junctions.