Supercharged Protein Nanosheets for Cell Expansion on Bioemulsions

Chrysanthou, Alexandra; Kanso, Hassan; Zhong, Wencheng; Li, Shang; Gautrot, Julien E.

doi:10.1101/2022.06.21.497058

Cited by 2 publications

(3 citation statements)

References 59 publications

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Section: Introductionmentioning

confidence: 99%

“…Recently, the modification of globulins such as albumins with charged residues was found to impact their assembly at liquid–liquid interfaces and resulting nanosheet mechanics. 11 These supercharged protein nanosheets enabled the simple adsorption of cell-adhesive proteins such as fibronectin and collagen, but the strengthening of their mechanical properties still required introduction of PFBC in order to sustain stem cell proliferation. This study demonstrated that simple protein modification may allow to modulate both nanosheet mechanics and bioactivity, however the identification of proteins and oils that enable the achievement of high interfacial elasticity in the absence of cosurfactant was not possible.…”

Section: Introductionmentioning

confidence: 99%

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

2023

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

2023

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recently, the modification of globulins such as albumins with charged residues was found to impact their assembly at liquid-liquid interfaces and resulting nanosheet mechanics 12 . These supercharged protein nanosheets enabled the simple adsorption of cell-adhesive proteins such as fibronectin and collagen, but the strengthening of their mechanical properties still required introduction of PFBC in order to sustain stem cell proliferation.…”

Section: Introductionmentioning

confidence: 99%

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

Chrysanthou

Bosch‐Fortea

Gautrot

2022

Preprint

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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 co-assemblies of protein and reactive co-surfactants, 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), biofunctionalised 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 co-surfactant 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. Biofunctionalisation 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 co-surfactant, 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 co-surfactants 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.

show abstract

Supercharged Protein Nanosheets for Cell Expansion on Bioemulsions

Cited by 2 publications

References 59 publications

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

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

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

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