Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilization of Bioemulsions Enabling Adherent Cell Expansion

Abstract

Bioemulsions are attractive platforms for the scalable expansion of adherent cells and stem cells. In these systems, cell adhesion is enabled by the assembly of protein nanosheets that display high interfacial shear moduli and elasticity. However, to date, most successful systems reported to support cell adhesion at liquid substrates have been based on coassemblies of protein and reactive cosurfactants, which limit the translation of bioemulsions. In this report, we describe the design of protein nanosheets based on two globular proteins, bovine serum albumin (BSA) and β-lactoglobulin (BLG), biofunctionalized with RGDSP peptides to enable cell adhesion. The interfacial mechanics of BSA and BLG assemblies at fluorinated liquid-water interfaces is studied by interfacial shear rheology, with and without cosurfactant acyl chloride. Conformational changes associated with globular protein assembly are studied by circular dichroism and protein densities at fluorinated interfaces are evaluated via surface plasmon resonance. Biofunctionalization mediated by sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) is studied by fluorescence microscopy. On the basis of the relatively high elasticities observed in the case of BLG nanosheets, even in the absence of cosurfactant, the adhesion and proliferation of mesenchymal stem cells and human embryonic kidney (HEK) cells on bioemulsions stabilized by RGD-functionalized protein nanosheets is studied. To account for the high cell spreading and proliferation observed at these interfaces, despite initial moderate interfacial elasticities, the deposition of fibronectin fibers at the surface of corresponding microdroplets is characterized by immunostaining and confocal microscopy. These results demonstrate the feasibility of achieving high cell proliferation on bioemulsions with protein nanosheets assembled without cosurfactants and establish strategies for rational design of scaffolding proteins enabling the stabilization of interfaces with strong shear mechanics and elasticity, as well as bioactive and cell adhesive properties. Such protein nanosheets and bioemulsions are proposed to enable the development of new generations of bioreactors for the scale up of cell manufacturing.

Matrix Nanoscale Mechanics Regulates Exosome Production by Mesenchymal Stem Cells

“…To support the growth of MSCs on scalable bioemulsions, microdroplets stabilised by β-lactoglobulin (βLG) were investigated (Figure 1A) [49]. As this protein is not bioactive, at least from the point of view of integrin ligation and the promotion of cell adhesion, the resulting droplets were further functionalised with RGD peptides.…”

“…The upper liquid phase was aspirated and replaced with PBS six times. The droplet surface was functionalised as described before [49]. Briefly, sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC, Sigma, at a concentration of 2 mg/mL in PBS) were added to the emulsion and kept to react for 1 h at room temperature.…”

Engineered Protein Nanosheets for the Scale up of Mesenchymal Stem Cell Culture on Bioemulsions

“…This retention in overall globular conformation is an excellent indicator that functionalisation levels should not interfere with self-assembly properties and the ability of corresponding albumins to stabilise emulsions. A globular architecture and striking changes in secondary structure were indeed associated with assembly at liquid-liquid interfaces, including hydrophobic and fluorophilic oil interfaces, and the stabilisation of associated emulsions [25,46,47]. The assembly of functionalised proteins at the surface of the fluorinated oil Novec 7500 was next examined, using interfacial rheology.…”

“…Recently, liquid substrates, in the form of oil microdroplets, were proposed to replace solid microcarriers. This has enabled the culture and retention of phenotype of a broad range of cell types, from fibroblasts, keratinocytes and HEK293 cells, to mesenchymal stem cells (MSCs), induced pluripotent stem cells and neural cells [23][24][25][26][27][28]. Underpinning these technologies, it was demonstrated that protein nanosheets self-assembling at corresponding liquid-liquid interfaces and presenting strong interfacial mechanical properties enabled the adhesion, spreading and proliferation of cells [28][29][30][31][32].…”

Enhancement of Immune Response of Bioconjugate Nanovaccine by Loading of CpG through Click Chemistry

“…Considering that CpG needs to enter antigen-presenting cells to perform its biological function, researchers also tried to improve the utilization of CpG by coupling it with antigens [ 30 , 31 ]. At present, many CpG and antigen connectors, such as NHS-PEG n -maleimide (SMCC) and the His-hydrophobic-His (HUH) superfamily of endonucleases, have been explored [ 32 , 33 , 34 ]. Among them, SMCC, which contains an N-hydroxysuccinimide-active ester and maleimide, is used most frequently.…”