Self-Assembled Microspheres for Cell Encapsulation and 3D Cell Culture

Self-Assembled Microspheres for Cell Encapsulation and 3D Cell Culture


We outline here a simple method for combining peptide self-assembly with emulsion processing to produce spherical microgels composed of laterally associated -sheet fibrils. The extreme salt-sensitivity of -sheet fibrillization was a useful means for triggering the gelation of initially soluble short peptides into micron-scale particles within the aqueous phase of water-in-oil emulsions. Size control over the microgels was achieved by specification of blade type, speed, and additional shear steps. Microgels constructed in this way could then be embedded within other self-assembled matrices by mixing pre-formed microgels with un-assembled peptides and inducing gelation of the entire composite. In this way, spatially heterogeneous synthetic ECMs could be produced. The gels themselves were cytocompatible, as was the microgel fabrication procedure, enabling the embedding of cells within the microgels with excellent viability. Functional readout.

, providing a novel route for constructing encapsulated cell systems or co-cultures with multiple spatially resolved regions of ECM formulations.


Micron-scale gels, particles, and beads have been employed for some time in a wide rage of biomedical and biotechnological applications, including therapeutic release, solid-phase synthetic or separation processes, bead-based assays, and ___. Recently, microgels have received particular attention as means for encapsulating cells into ___ that can be more easily manipulated than individual cells, allowing their implantation, organization into cocultures, _____. (ref sefton, etc). Microgels such as these can be produced using a variety of approaches, including polymerization within one phase of an oil-in-water or water-in-oil emulsion, or by triggering the gelation of responsive biopolymers such as agarose or collagen within emulsions (ref).
As a biocompatible, chemically defined way of producing hydrogels, peptide self-assembly has received increasing attention, particularly within biomedical applications such as regenerative medicine [2-4]. In comparison with covalent polymerization or materials based on whole proteins, self-assembling systems offer routes for precisely integrating and manipulating several different molecular features necessary for controlling complex biological behaviors and interactions [3, 6]. Previously, we have investigated scaffolds based on short -sheet fibrillizing peptides for constructing multi-peptide, chemically defined synthetic extracellular matrices (ECMs) [1, 5, 7]. More on this.
Self-assembly processes for constructing gels tend to have favorable biocompatibility, as they do not depend on solvents, temperature changes, or covalent chemistry. In addition, many self-assembly processes, including peptide fibrillization, show extreme sensitivity to the presence of salts. In previous work, solutions of -sheet fibrillizing peptides were gelled by releasing salts from light- or temperature-sensitive liposomes. In the work reported here, the salt-sensitivity of fibrillizing peptides was exploited in order to gel the peptides within the aqueous phase of water-in-oil emulsions simply by controlling the addition of physiological buffers during the emulsification process. In this way, the matrices and ultimately encapsulated cells were not exposed to any harsh processing conditions such as temperature changes, covalent chemistries, or potentially denaturing solvents. This is in contrast to other previously reported methods for producing microgels, which depend on temperature changes for biopolymers such as agarose or ___ (ref), covalent polymerization processes for in-situ polymerizing synthetic polymers (ref), or organic solvents in the case of _____ (ref).
The system we utilized is based on the short fibrillizing sequence (Ac-QQKFQFQFEQQ-Am), previously named Q11 [1, 5, 7].

Peptide synthesis. Peptides Q11 (QQKFQFQFEQQ) and NBD-RGD-Q11 were synthesized in-house on a CS Bio 136 peptide synthesizer as previously reported using standard Fmoc protocols [5, 7].
Microgel synthesis. Peptides were mixed in deionized water to make a 30mM solution, which was then mixed with USP mineral oil using either a stator-blade homogenizer or a paddle-type mixer for 1 minute at variable shear rate. To induce self-assembly of the peptides in the aqueous phase, PBS was added into the homogenate. To reduce the size of the microgels, some groups were subjected to further shearing. Microgels were extracted in PBS.
Microgel Characterization. Gels were either stained by Congo red or by incorporating an NBD-labeled Q11 derivative during synthesis. Image J software was employed to measure the number and diameters of the microgels.
Synthesis and characterization of spatially heterogeneous synthetic ECMs. Microgels were stained with Congo red, and unbound CR and buffer salts were removed by dialysis. Stained microgels were then suspended in a solution of 30mM Q11 containing 50μM NBD-RGD-Q11, and the mixture was gelled by overlaying with PBS. Macroscale gels with embedded microgels were observed using a Zeiss LSM 510 confocal microscope.

Cell encapsulation. NIH/3T3 cells were cultured in DMEM with 10% FBS and 5% glutamine. Cells were passaged by trypsinization, suspended in 10% sucrose solution to avoid
premature gelation of the peptide, and mixed with the Q11 solution. The mixture was then emulsified with a paddle-type stirrer at 500 RPM as described above. Microgels were collected in DMEM and cultured in 96-well plates. To assess viability, cells were stained with calcein-AM, a live-cell marker.


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