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.
Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles. Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.
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...
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