Synthetic poly(ethylene glycol)-based hydrogel platform for in vitro cell studies in 3 dimensions

The behavior of cells cultured in two-dimensions (2D) greatly differs from that in three-dimensions (3D), and often does not reflect the actual in vivo situation. Consequently, 3D model systems are becoming more widely used as a tool for in vitro cell research. Hydrogels are polymer networks that contain large amounts of water and are potentially biocompatible. Therefore, hydrogels that mimic natural extracellular matrix (ECM) are being developed as 3D platforms for in vitro cell studies. Poly(ethylene glycol)-based (PEG) hydrogels are artificial gels that allow control over biophysical and biochemical parameters. Moreover, PEG hydrogels are known to be generally not cytotoxic which makes them widely used in a variety of biomedical applications. This thesis has two major parts. The first part is the synthesis and characterization of PEG-based hydrogels via utilizing Michael-type addition reaction of unsaturated double bonds to thiols. Characterization included observation of elastic properties using atomic force microscopy (AFM) and performing swelling study from which the theoretical mesh sizes of hydrogels were calculated. The actual mesh sizes were checked via beads incorporation into the hydrogels’ network. Moreover, the ranges, within which gels are reproducibly forming, were obtained for a variety of hydrogels and the gelation kinetics was checked using the oscillatory rheology. The second part of this thesis deals with the cell encapsulation into the hydrogel network and observation of cell viability. LS174T cell line was encapsulated into the PEG vinyl sulfone hydrogel with cleavable matrix metalloproteinase peptide (cMMP). The results of the hydrogel development and characterization part have shown that it is possible to controllably modify mechanical properties of PEG hydrogels. Change of the PEG concentration and incorporation of linear PEGs in between the tetra-functional ones has proven to have a impact on the mechanical properties of the gels. The results of cell encapsulation part have shown that it is possible to encapsulate LS174T cell line in the kind of hydrogels that was developed and evaluated in this project. However, it remains a matter of future work to optimize the setting and meet the challenges regarding the reproducibility of these experiments and viability of encapsulated cells.

Light-controlled self-assembly and self-sorting of cell-like compartments

The organization of cells within tissues allows them to work together. Tight spatiotemporal control over cell-cell interactions is essential for individual cells to self-assemble and function as tissues. In addition, during many biological processes, such as embryogenesis and cancer development, cell-cell interactions undergo dynamic changes to alter their function. Analogously, in the context of bottom-up synthetic biology it is of interest to dynamically control the interactions between minimal synthetic cells and assemble them into precise multi-compartment prototissues with high spatiotemporal resolution. The aim of the first part of this thesis was to reversibly self-assemble different types of micrometer-sized colloids, which were used as synthetic cell-mimics, with high spatiotemporal resolution using visible light. Light provides a dynamic, non-invasive, and biocompatible control with high spatiotemporal precision. In order to control the self-assembly of cell-mimics, I functionalized them with photoswitchable proteins that specifically interact with each other under blue or red light. For this purpose I used several combinations of photoswitchable proteins that are dimerizing under blue light: heterodimerizing iLID and Nano proteins, nMagHigh and pMagHigh proteins, and homodimerizing VVDHigh protein. For the red light switchable proteins I used both the heterodimerization of phytochrome B (PhyB) and phytochrome interaction partner (PIF6) proteins and the homodimerization of Cph1 protein. All of these light dependent protein interactions enabled controlling the self-assembly of cell-mimics with light. Additionally, blue light dependent protein interactions are reversible in the absence of light with red light dependent interactions reversing under far-red light illumination. Consequently, the self-assembly of cell-mimics mediated by these protein interactions was also reversible. Additionally, the high specificity and the independent response of these protein interactions to blue or red light offers the potential to self-assemble a specific population of cell-mimics in the presence of others on demand. In multicellular organisms, cells do not just self-assemble but they also self-sort into precise arrangements in order to work together. As part of this thesis, I also mimicked the self-sorting behavior with synthetic compartments inspired by the observations in nature. Self-sorting is defined by the ability to distinguish between self and non-self, and happens in different modes depending on the interactions between the particles. One mode is social self-sorting, which leads to the separation of colloids into independent colloidal families and requires heterophilic and orthogonal interactions. In this part of the thesis, I used heterodimerization of two blue light switchable protein pairs, iLID/Nano and nMagHigh/pMagHigh, for the social self-sorting between four different populations of colloids within one mixture. Each protein pair specifically and orthogonally brings together two different subpopulations of colloids providing tight and reversible control over their self-sorting into two distinct families using blue light. On the other hand, asocial sorting is another mode of self-sorting, which requires homophilic interactions to bring together compartments of the same type into isolated aggregates. This could potentially be achieved by combining homodimerization of VVDHigh and Cph1 proteins under blue and red light respectively. Eventually, all the versatile and orthogonal light-switchable proteins and different dimerization modes have the potential to be incorporated together in different combinations to achieve the desired self-sorting outcome in complex prototissues. In the second part of the thesis, I addressed the spatiotemporally controlled formation of protein patterns on synthetic cell-mimics. Protein patterns and gradients on cell membranes are important during many biological processes to locally trigger events in multicellular structures with high spatiotemporal precision. To create and control protein patterns on synthetic membranes such as giant unilamellar vesicles (GUVs) with light, I used the blue light switchable heterodimerization of iLID and Nano proteins. For this purpose, the GUVs were functionalized with iLID. This allowed for the blue light mediated, reversible recruitment of a fluorescent protein (mOrange) fused to Nano using blue light with high spatiotemporal resolution. Further, this approach allowed scaling the size of protein patterns from the level of a single GUV to the level of a tissue-like GUV carpet. Hence, these photoswitchable proteins offer a versatile, reversible, dynamic, and non-invasive method to photopattern proteins with high spatiotemporal control that operates under mild conditions. Overall, photoswitchable proteins are important building blocks in the bottom-up synthetic biology toolbox. Incorporating them onto minimal synthetic cells can be used to self-assemble and self-sort different types of cell-mimics and to generate protein patterns, thus mimicking complex processes that occur in nature. Most importantly, these protein interactions provide high spatiotemporal precision and specificity to control these biomimetic processes. Ultimately, this concept can be transferred to assemble prototissues using various types of cell-mimics that host different functionalities, which would allow for controlling, how different synthetic cells work together in a prototissue

Synthetic poly(ethylene glycol)-based hydrogel platform for in vitro cell studies in 3 dimensions

The behavior of cells cultured in two-dimensions (2D) greatly differs from that in three-dimensions (3D), and often does not reflect the actual in vivo situation. Consequently, 3D model systems are becoming more widely used as a tool for in vitro cell research. Hydrogels are polymer networks that contain large amounts of water and are potentially biocompatible. Therefore, hydrogels that mimic natural extracellular matrix (ECM) are being developed as 3D platforms for in vitro cell studies. Poly(ethylene glycol)-based (PEG) hydrogels are artificial gels that allow control over biophysical and biochemical parameters. Moreover, PEG hydrogels are known to be generally not cytotoxic which makes them widely used in a variety of biomedical applications. This thesis has two major parts. The first part is the synthesis and characterization of PEG-based hydrogels via utilizing Michael-type addition reaction of unsaturated double bonds to thiols. Characterization included observation of elastic properties using atomic force microscopy (AFM) and performing swelling study from which the theoretical mesh sizes of hydrogels were calculated. The actual mesh sizes were checked via beads incorporation into the hydrogels’ network. Moreover, the ranges, within which gels are reproducibly forming, were obtained for a variety of hydrogels and the gelation kinetics was checked using the oscillatory rheology. The second part of this thesis deals with the cell encapsulation into the hydrogel network and observation of cell viability. LS174T cell line was encapsulated into the PEG vinyl sulfone hydrogel with cleavable matrix metalloproteinase peptide (cMMP). The results of the hydrogel development and characterization part have shown that it is possible to controllably modify mechanical properties of PEG hydrogels. Change of the PEG concentration and incorporation of linear PEGs in between the tetra-functional ones has proven to have a impact on the mechanical properties of the gels. The results of cell encapsulation part have shown that it is possible to encapsulate LS174T cell line in the kind of hydrogels that was developed and evaluated in this project. However, it remains a matter of future work to optimize the setting and meet the challenges regarding the reproducibility of these experiments and viability of encapsulated cells.

Reversible Social Self-Sorting of Colloidal Cell-Mimics with Blue Light Switchable Proteins

Toward the bottom-up assembly of synthetic cells from molecular building blocks, it is an ongoing challenge to assemble micrometer sized compartments that host different processes into precise multicompartmental assemblies, also called prototissues. The difficulty lies in controlling interactions between different compartments dynamically both in space and time, as these interactions determine how they organize with respect to each other and how they work together. In this study, we have been able to control the self-assembly and social self-sorting of four different types of colloids, which we use as a model for synthetic cells, into two separate families with visible light. For this purpose we used two photoswitchable protein pairs (iLID/Nano and nHagHigh/pMagHigh) that both reversibly heterodimerize upon blue light exposure and dissociate from each other in the dark. These photoswitchable proteins provide noninvasive, dynamic, and reversible remote control under biocompatible conditions over the self-assembly process with unprecedented spatial and temporal precision. In addition, each protein pair brings together specifically two different types of colloids. The orthogonality of the two protein pairs enables social self-sorting of a four component mixture into two distinct families of colloidal aggregates with controlled arrangements. These results will ultimately pave the way for the bottom-up assembly of multicompartment synthetic prototissues of a higher complexity, enabling us to control precisely and dynamically the organization of different compartments in space and time

Mimicking adhesion in minimal synthetic cells

Cell adhesions to the extracellular matrix and to neighboring cells are fundamental to cell behavior and have also been implemented into minimal synthetic cells, which are assembled from molecular building blocks from the bottom‐up. Investigating adhesion in cell mimetic models with reduced complexity provides a better understanding of biochemical and biophysical concepts underlying the cell adhesion machinery. In return, implementing cell–matrix and cell–cell adhesions into minimal synthetic cells allows reconstructing cell functions associated with cell adhesions including cell motility, multicellular prototissues, fusion of vesicles, and the self‐sorting of different cell types. Cell adhesions have been mimicked using both the native cell receptors and reductionist mimetics providing a variety of specific, reversible, dynamic, and spatiotemporally controlled interactions. This review gives an overview of different minimal adhesion modules integrated into different minimal synthetic cells drawing inspiration from cell and colloidal science

Dynamic blue light-switchable protein patterns on giant unilamellar vesicles

The blue light-dependent interaction between the proteins iLID and Nano allows recruiting and patterning proteins on GUV membranes, which thereby capture key features of patterns observed in nature. This photoswitchable protein interaction provides non-invasive, reversible and dynamic control over protein patterns of different sizes with high specificity and spatiotemporal resolution