**2.2 Spatiotemporal control over cell–cell adhesions using light responsive small molecules**

Light sensitive small molecules, such as nitrobenzenes and azobenzenes, have been used to control cell–cell adhesions in space and time. For example, light cleavable nitrobenzene groups can be introduced to oxyamine linkers at the cell surface. When this cell population is mixed with a second population of cells with a ketone group at the cell surface multicellular clusters formed. These cell cluster can then be broken up into single cells upon UV-light illumination since UV-light cleaves the nitrobenzyl moiety [51]. Such a photocleavable linker only allows for a single reversion of the cell–cell adhesions. To achieve cell–cell adhesions that can be switched on and off repeatedly a linker with a photoswitchable azobenzene group was developed. β-cyclodextrins can be clicked onto the surfaces of cells and when a divalent photoswitchable azobenzene (azo) linker (azo-PEG-azo) is added in the dark the cells will link together. This is because, in the dark, the trans configuration of the azobenzenes binds to the cyclodextrin moieties linking the cells together. Upon UV illumination the azobenzene switch to the cis conformation, which results in the release from the cyclodextrin and the dissociation of the cell–cell interactions. The azobenzene can then be switched back to the trans configuration with blue light illumination, thus allowing for the formation of new cell–cell adhesions [52]. These studies represent great advances in the field and allow for spatiotemporal control over cell–cell adhesions. However, the use of UV-light is damaging to DNA and therefore to cells, and secondly, the chemical modifications cannot be maintained over long periods of time. Thus, a system which utilizes biocompatible light and can be expressed over long times would be more beneficial to bottom-up tissue engineering since cell proliferation is a key component of any built tissue. For this purpose, a genetically engineered system, which allows for propagating the modification at the cell surface would be desirable.

### **2.3 Optogenetic control of cell–cell interactions**

Cell–cell adhesions can be photoregulated by expressing bioartificial lightresponsive proteins on the surfaces of cells as adhesion receptors. Numerous lightresponsive proteins from algae, plants, bacteria, and engineered proteins change their conformation upon light illumination and bind to other proteins in a light-dependent manner through non-covalent protein–protein interactions [53–56]. In these optogenetic approaches, complementary light-dependent binding partners are expressed in the surfaces of different cell types by transfecting these proteins along with a plasma membrane localization sequence and a membrane anchoring sequence. Following translation, the localization sequence ensures that the protein is exported to the
