Periodic Reporting for period 4 - ProLiCell (Engineered Protein Nanosheets at Liquid-Liquid Interfaces for Stem Cell Expansion, Sorting and Tissue Engineering)
Reporting period: 2023-03-01 to 2024-08-31
A long standing dogma in the field of cell-based technologies was that bulk mechanical properties of solid substrates were essential to enable cell spreading, proliferation and fate decision. The use of solid materials to culture adherent cells constituted an important hurdle for the scale up, automation and speed up of cell culture and recovery. Our recent results showed that bulk solid substrates are not necessary to promote cell adhesion, growth and fate regulation as adherent stem cells spread and proliferate readily at the surface of ultra-soft materials, even liquids. In such cases, cell adhesion is enabled by the formation of a mechanically strong layer (nanosheet) of proteins at the interface between the oil (liquid substrate) and aqueous medium. This key discovery opened the door to the engineering of protein nanosheets enabling the use of liquid, free-flowing substrates sustaining cell adhesion, expansion, isolation and recovery.
ProLiCell designed the biochemical and mechanical properties of extracellular matrix (ECM) protein nanosheets that can sustain the formation of adhesion protein complexes and support cell proliferation and culture on materials with very weak bulk mechanical properties (liquids). The engineered ECM nanosheets were applied to: 1. the design of 3D bioreactors based on emulsions, for the culture of stem cells; 2. the formation of stem cell sheets at oil-water interfaces for tissue engineering; 3. the isolation and purification of stem cells using emulsions presenting antibody-adsorbed interfaces. ProLiCell provided fundamental insights into ECM nanosheet design and advance our understanding of the mechanisms via which cells adhering to such interfaces sense and respond to nanoscale cues. Such fundamental understanding enabled liquid-liquid platforms to transform stem cell technologies by borrowing a wider range of processing and manufacturing concepts to the field of Chemical Engineering.
ProLiCell designed the biochemical and mechanical properties of extracellular matrix (ECM) protein nanosheets that can sustain the formation of adhesion protein complexes and support cell proliferation and culture on materials with very weak bulk mechanical properties (liquids). The engineered ECM nanosheets were applied to: 1. the design of 3D bioreactors based on emulsions, for the culture of stem cells; 2. the formation of stem cell sheets at oil-water interfaces for tissue engineering; 3. the isolation and purification of stem cells using emulsions presenting antibody-adsorbed interfaces. ProLiCell provided fundamental insights into ECM nanosheet design and advance our understanding of the mechanisms via which cells adhering to such interfaces sense and respond to nanoscale cues. Such fundamental understanding enabled liquid-liquid platforms to transform stem cell technologies by borrowing a wider range of processing and manufacturing concepts to the field of Chemical Engineering.
ProLiCell was proposing to investigate the design of self-assembled protein nanosheets at liquid-liquid interfaces, resulting in interfaces displaying strong mechanical properties able to resist cell-mediated contractile forces. Understanding how the molecular structure of constituents of nanosheets impact on the mechanics of the assemblies allowed us to refine the design of corresponding interfaces whilst conferring tailored bioactive properties (including cell adhesion and cell specific ligands).
In the first part of this project, we developed methodologies allowing us to examine how mechanical properties of nanosheets assembled at liquid-liquid interfaces are regulated by molecular composition. To this aim, we set up a very sensitive interfacial rheology rig allowing the exchange of mobile phases and temperature modulation. We developed an analytical model allowing to quantitatively extract interfacial moduli from force probe microscopy experiments. In addition, we have set up a Langmuir-Blodgett trough system for liquid-liquid interfaces and optimised deposition conditions allowing the transfer of nanosheet for higher resolution imaging. Finally, we developed cell-based assays to characterise the impact of nanosheet composition and interfacial mechanics on cell adhesion and stem cell phenotype.
Having established these methodologies, the ProLiCell team has investigated how the chemistry of proteins and co-surfactants used for the assembly of nanosheets impact on their interfacial rheological properties, and in turn regulates the proliferation of adherent cells, including primary keratinocytes, mesenchymal stem cells, the epidermal HaCaT cell line, dermal fibroblasts and an HEK293 cell line often used for the production of biotherapeutics. Specifically, we investigated how supercharging globular proteins such as albumin impacts on their interfacial viscoelasticity and how further assembly with co-surfactants modulate such behaviour. In addition, we established that covalent co-surfactants underpin physical crosslinking of nanosheets via Van der Waals interactions and pi-stacking, conferring sufficient elasticity to the resulting interfaces. In addition, we showed how modulating the molecular weight of the proteins assembled alters interfacial toughness, presumably by providing soft domains that are able to dissipate energy upon local fracture of hard sub-domains. This results in assemblies sufficiently strong to resist cell-mediated forces.
In the final part of the project, ProLiCell developed a novel concept for the engineering of bioactive protein nanosheets and associated bioemulsions, using recombinant proteins that display tensioactive properties and bioactivity. This not only enables the simplified formation and biofunctionalisation of microdroplets, but also enable to bring sensitive bioactive elements to the surface of microdroplets in a scalable format. Using this approach, we demonstrated that bioactive microdroplet technologies (or bioemulsions) can capture important ligands to stimulate stem cells or capture them for separation. We have demonstrated the application of this technology to integrate in iPSC-derived platforms, including cerebral organoids.
In the first part of this project, we developed methodologies allowing us to examine how mechanical properties of nanosheets assembled at liquid-liquid interfaces are regulated by molecular composition. To this aim, we set up a very sensitive interfacial rheology rig allowing the exchange of mobile phases and temperature modulation. We developed an analytical model allowing to quantitatively extract interfacial moduli from force probe microscopy experiments. In addition, we have set up a Langmuir-Blodgett trough system for liquid-liquid interfaces and optimised deposition conditions allowing the transfer of nanosheet for higher resolution imaging. Finally, we developed cell-based assays to characterise the impact of nanosheet composition and interfacial mechanics on cell adhesion and stem cell phenotype.
Having established these methodologies, the ProLiCell team has investigated how the chemistry of proteins and co-surfactants used for the assembly of nanosheets impact on their interfacial rheological properties, and in turn regulates the proliferation of adherent cells, including primary keratinocytes, mesenchymal stem cells, the epidermal HaCaT cell line, dermal fibroblasts and an HEK293 cell line often used for the production of biotherapeutics. Specifically, we investigated how supercharging globular proteins such as albumin impacts on their interfacial viscoelasticity and how further assembly with co-surfactants modulate such behaviour. In addition, we established that covalent co-surfactants underpin physical crosslinking of nanosheets via Van der Waals interactions and pi-stacking, conferring sufficient elasticity to the resulting interfaces. In addition, we showed how modulating the molecular weight of the proteins assembled alters interfacial toughness, presumably by providing soft domains that are able to dissipate energy upon local fracture of hard sub-domains. This results in assemblies sufficiently strong to resist cell-mediated forces.
In the final part of the project, ProLiCell developed a novel concept for the engineering of bioactive protein nanosheets and associated bioemulsions, using recombinant proteins that display tensioactive properties and bioactivity. This not only enables the simplified formation and biofunctionalisation of microdroplets, but also enable to bring sensitive bioactive elements to the surface of microdroplets in a scalable format. Using this approach, we demonstrated that bioactive microdroplet technologies (or bioemulsions) can capture important ligands to stimulate stem cells or capture them for separation. We have demonstrated the application of this technology to integrate in iPSC-derived platforms, including cerebral organoids.
We have identified important parameters regulating the proliferation of adherent stem cells at liquid interfaces. In particular the modulation of interfacial mechanics by the molecular composition of nanosheets during self-assembly is found to play an important role on the regulation of stem cell phenotype. This allows a significant step change in the design of biomaterials for tissue engineering and stem cell technologies as it implies that engineering the nanoscale of biomaterials rather than bulk mechanical properties is a key determinant controlling cell adhesion and stem cell phenotype. We also propose that liquid-based technologies will allow the simplification of stem cell manufacturing processes, the increase of their throughput and their parallelisation, together with a significant reduction in cost (oils used to generated droplets for cell culture are 100 to 10,000 times cheaper than current solid microcarriers and solid substrates used for adherent cell manufacturing. Liquid-liquid systems have been particularly successful in the field of chemical engineering, in which they have allowed the reduction of costs and increase in throughput for the manufacturing of fine chemicals, polymers and nanomaterials. ProLiCell proposes to enable a step change in the field of biotechnologies and stem cell technologies by allowing liquid-liquid systems to revolutionise associated manufacturing processes.