Building tissue from cells as the basic building block based on principles of self-assembly is a challenging and promising approach. Understanding how far principles of self-assembly and self-sorting known for colloidal particles apply to cells remains unanswered. In this study, we demonstrate that not just controlling the cell–cell interactions but also their dynamics is a crucial factor that determines the formed multicellular structure, using photoswitchable interactions between cells that are activated with blue light and reverse in the dark. Tuning dynamics of the cell–cell interactions by pulsed light activation results in multicellular architectures with different sizes and shapes. When the interactions between cells are dynamic, compact and round multicellular clusters under thermodynamic control form, while otherwise branched and loose aggregates under kinetic control assemble. These structures parallel what is known for colloidal assemblies under reaction- and diffusion-limited cluster aggregation, respectively. Similarly, dynamic interactions between cells are essential for cells to self-sort into distinct groups. Using four different cell types, which expressed two orthogonal cell–cell interaction pairs, the cells sorted into two separate assemblies. Bringing concepts of colloidal self-assembly to bottom-up tissue engineering provides a new theoretical framework and will help in the design of more predictable tissue-like structures.
Controlling cell–cell interactions is central for understanding key cellular processes and bottom‐up tissue assembly from single cells. The challenge is to control cell–cell interactions dynamically and reversibly with high spatiotemporal precision noninvasively and sustainably. In this study, cell–cell interactions are controlled with visible light using an optogenetic approach by expressing the blue light switchable proteins CRY2 or CIBN on the surfaces of cells. CRY2 and CIBN expressing cells form specific heterophilic interactions under blue light providing precise control in space and time. Further, these interactions are reversible in the dark and can be repeatedly and dynamically switched on and off. Unlike previous approaches, these genetically encoded proteins allow for long‐term expression of the interaction domains and respond to nontoxic low intensity blue light. In addition, these interactions are suitable to assemble cells into 3D multicellular architectures. Overall, this approach captures the dynamic and reversible nature of cell–cell interactions and controls them noninvasively and sustainably both in space and time. This provides a new way of studying cell–cell interactions and assembling cellular building blocks into tissues with unmatched flexibility.
The self-assembly of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. Here, we not only independently control the self-assembly of two cell types into multicellular architectures with blue and red light, but also achieve their self-sorting into distinct assemblies. This required developing two cell types that form selective and homophilic cell–cell interactions either under blue or red light using photoswitchable proteins as artificial adhesion molecules. The interactions were individually triggerable with different colors of light, reversible in the dark, and provide noninvasive and temporal control over the cell–cell adhesions. In mixtures of the two cells, each cell type self-assembled independently upon orthogonal photoactivation, and cells sorted out into separate assemblies based on specific self-recognition. These self-sorted multicellular architectures provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them.
Micropatterns of functional protein are important in biotechnology and research.
cell-cell adhesions leads to the progression of cancer [1c] and subsequent metastasis. [4] Additionally, the control of cell-cell interactions is of interest in the field of bottomup tissue-engineering, which aims to assemble cells as the basic unit into functional tissues. [5] Precise control in time and space over cell-cell interactions is required to successfully assemble appropriate multicellular architectures that resemble the in vivo structures and to control how cells work together within a tissue.In recent years, many tools have been developed to control the formation and the disassembly of cell-cell interactions in a controlled manner and thereby have given insight into the role of cell-cell adhesions. [6] For instance, chemical modifications of the plasma membrane with bioorthogonal reactive functional groups [7] and specific noncovalent interaction partners [8] including complementary DNA strands, [9] biotin-streptavidin, [10] and supramolecular binding partners [11] result in the formation of chemical bonds between neighboring cells. However, in only a few examples, it is possible to reverse these cell-cell interactions once formed. [11] To overcome general concerns related to the chemical modification of the cell membrane (e.g., degradation over time, off-target cell toxicity), it is possible to regulate the expression and the activity of genetically encoded native cell-cell adhesion molecules such as cadherins [12] or artificial surface receptors. [1d,9b,6,12,13] These adhesions allow cells to not only just be brought together but in some cases also to transduce intracellular signals. [1e,12,14] Photoregulation of cell-cell adhesions provides high spatiotemporal control, since light, as a stimulus, can be focused on the desired area and delivered at any given time. Using photocleavable nitrobenzyl [15] and switchable azobenzyl [14] chemical linkers, it has been shown possible to control cell-cell interactions with UV light. [11b,15,16] More recently, optogenetic tools for the regulation of cell-cell interactions with visible light have improved the biocompatibility, [13b,e] while also making it possible to dynamically and reversibly control cell-cell interactions between multiple cell types. [13b,e,16b,17] In these reports, photoswitchable proteins were expressed on the cell's plasma membrane as artificial adhesion molecules, which induced cell-cell interactions through the light-dependent dimerization of these proteins. [6] Depending on the photoswitchable proteins employed cell-cell adhesions between the same or differentThe regulation of cell-cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell-cell interactions with high precision are of great interest to a better understanding of their roles and building tissuelike structures. Herein, the green light-responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion rece...
Group A streptococcus (GAS) is an important Gram-positive pathogen that causes various human diseases ranging from peripheral lesions to invasive infections. The M protein is one of the main virulence factors present on the cell surface and is associated with invasive GAS infections. Compared with other M types, serotype M3 has a predominant role in lethal infections and demonstrates epidemic behaviors, including streptococcal toxic shock syndrome, bacteremia, and necrotizing fasciitis. Traditional methods for M typing are time-consuming, tedious, contradictory, and generally restricted to reference laboratories. Therefore, development of a new M-typing technique is needed. Aptamers with the ability to detect their target with a high degree of accuracy and specificity can be ideal candidates for specific M-typing of Streptococcus pyogenes. In this study DNA aptamers with a high binding affinity towards S. pyogenes serotype M3 were selected through 12 iterative rounds of the Systematic Evolution of Ligands by EXponential (SELEX) enrichment procedure using live cells as a target. We monitored the progress of the SELEX procedure by flow cytometry analysis. Of several aptamer sequences analyzed, 12L18A showed the highest binding efficiency towards S. pyogenes type M3, with an apparent dissociation constant (K) of 7.47 ± 1.72 pmol/L being the lowest. Therefore the isolated aptamer can be used in any tool, such as a biosensor, for the detection of S. pyogenes and can be used in the development of a novel M-typing system.
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