Materials Host Interactions
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Scientific Unit
Living Therapeutic Materials (LTMs) are a promising alternative to polymeric drug carriers for long term release of biotherapeutics. LTMs contain living drug biofactories that produce the drug using energy sources from the body fluids. To clarify their application potential, it is fundamental to adapt biocompatibility and cytotoxicity assays applied from non-living biomaterials and therapeutics to evaluate how LTMs interact with host cells. Here, we have established a first step in this direction, by developing a practical workflow to parallelize in vitro assessment of minimal safety and cytocompatibility properties of bacterial LTMs. It allows systematic monitoring and quantification of the dynamic evolution of the bacterial population (growth, metabolic activity) in parallel to quantify the response of different mammalian cells to LTM supernatants with regards to cytotoxicity and release of pro-inflammatory cytokines over a period of 7 days using a maximum of 10 samples. The protocol was tested with a Pluronic-based thin film containing ClearColi. The results show no cytotoxic effects of ClearColi containing hydrogels in three mammalian cell lines, and no induction of pro-inflammatory cytokines under the tested conditions. This workflow represents a first step in establishing a roadmap for the safety assessment of LTMs, and investigation of biocompatibility potential of future living therapeutic devices.
Engineered living materials (ELMs), which usually comprise bacteria, fungi, or animal cells entrapped in polymeric matrices, offer limitless possibilities in fields like drug delivery or biosensing. Determining the conditions that sustain ELM performance while ensuring compatibility with ELM hosts is essential before testing them in vivo. This is critical to reduce animal experimentation and can be achieved through in vitro investigations. Currently, there are no standards that ensure ELM compatibility with host tissues. Towards this goal, we designed a 96-well plate-based screening method to streamline ELM growth across culture conditions and determine their compatibility potential in vitro. We showed proliferation of three bacterial species encapsulated in hydrogels over time and screened six different cell culture media. We fabricated ELMs in bilayer and monolayer formats and tracked bacterial leakage as a measure of ELM biocontainment. After screening, an appropriate medium was selected that sustained growth of an ELM, and it was used to study cytocompatibility in vitro. ELM cytotoxicity on murine fibroblasts and human monocytes was studied by adding ELM supernatants and measuring cell membrane integrity and live/dead staining, respectively, proving ELM cytocompatibility. Our work illustrates a simple setup to streamline the screening of compatible environmental conditions of ELMs with the host.
Engineered living materials (ELMs), which usually comprise bacteria, fungi, or animal cells entrapped in polymeric matrices, offer limitless possibilities in fields like drug delivery or biosensing. To determine the conditions that sustain ELM performance while ensuring ELM-host compatibility is essential before testing them in vivo. This is critical to reduce animal experimentation and can be achieved through in vitro investigations. Towards this goal, we designed a 96-well plate-based screening method to streamline ELM growth across culture conditions and determine their compatibility potential in vitro. We showed proliferation of three bacterial species encapsulated in hydrogels over time and screened six different cell culture media. We fabricated ELMs in bilayer and monolayer formats and tracked bacterial leakage. After screening, an appropriate medium was selected that sustained growth of an ELM, and it was used to study cytocompatibility in vitro. ELM cytotoxicity on murine fibroblasts and human monocytes was studied by adding ELM supernatants and measuring cell membrane integrity and live/dead staining, respectively, proving ELM cytocompatibility. Our work illustrates a simple setup to streamline the screening of compatible environmental conditions of ELMs with the host.
The increasing prevalence of dry eye syndrome in aging and digital societies compromises long-term contact lens (CL) wear and forces users to regular eye drop instillation to alleviate discomfort. Here a novel approach with the potential to improve and extend the lubrication properties of CLs is presented. This is achieved by embedding lubricant-secreting biofactories within the CL material. The self-replenishable reservoirs autonomously produce and release hyaluronic acid (HA), a natural lubrication and wetting agent, long term. The hydrogel matrix regulates the growth of the biofactories and the HA production, and allows the diffusion of nutrients and HA for at least 3 weeks. The continuous release of HA sustainably reduces the friction coefficient of the CL surface. A self-lubricating CL prototype is presented, where the functional biofactories are contained in a functional ring at the lens periphery, outside of the vision area. The device is cytocompatible and fulfils physicochemical requirements of commercial CLs. The fabrication process is compatible with current manufacturing processes of CLs for vision correction. It is envisioned that the durable-by-design approach in living CL could enable long-term wear comfort for CL users and minimize the need for lubricating eye drops.
Methylsulfone derivatized poly(ethylene) glycol (PEG) macromers can be biofunctionalized with thiolated ligands and cross-linked with thiol-based cross-linkers to obtain bioactive PEG hydrogels for in situ cell encapsulation. Methylsulfonyl-thiol (MS-SH) reactions present several advantages for this purpose when compared to other thiol-based cross-linking systems. They proceed with adequate and tunable kinetics for encapsulation, they reach a high conversion degree with good selectivity, and they generate stable reaction products. Our previous work demonstrated the cytocompatibility of cross-linked PEG-MS/thiol hydrogels in contact with fibroblasts. However, the cytocompatibility of the in situ MS-SH cross-linking reaction itself, which generates methylsulfinic acid as byproduct at the cross-linked site, remains to be evaluated. These studies are necessary to evaluate the potential of these systems for in vivo applications. Here we perform an extensive cytocompatibility study of PEG hydrogels during in situ cross-linking by the methylsulfonyl-thiol reaction. We compare these results with maleimide–thiol cross-linked PEGs which are well established for cell culture and in vivo experiments and do not involve the release of a byproduct. We show that fibroblasts and endothelial cells remain viable after in situ polymerization of methylsulfonyl-thiol gels on the top of the cell layers. Cell viability seems better than after in situ cross-linking hydrogels with maleimide–thiol chemistry. The endothelial cell proinflammatory phenotype is low and similar to the one obtained by the maleimide–thiol reaction. Finally, no activation of monocytes is observed. All in all, these results demonstrate that the methylsulfonyl-thiol chemistry is cytocompatible and does not trigger high pro-inflammatory responses in endothelial cells and monocytes. These results make methylsulfonyl-thiol chemistries eligible for in vivo testing and eventually clinical application in the future.
The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels.
