M.Sc. Krupansh Desai

In ophthalmology, living biomaterials have appeared as promising drug delivery and biosensor devices to tackle dynamic sensing and delivery of compounds. Their living character complicates their assessment with the also dynamic ocular tissues. The use of animal experiments increases complexity, and most animal ocular models are anatomically different from humans. Thus, in vitro ocular systems applied specifically to living biomaterials are required to assess their safety, compatibility and efficacy. Here, we report on an in vitro cornea model for co-cultures with Corynebacterium glutamicum-polyvinyl alcohol living biomaterials, which are reported as suitable living contact lenses, to study their cytocompatibility to the eye. We co-cultured this living biomaterial with human primary corneal cells (epithelial and fibroblasts) for 7 days, mimicking contact lens extended wear. We studied bacterial proliferation, biocontainment and biosafety. We investigated potential cytotoxicity and pro-inflammatory responses of living biomaterials to corneal cells. Our results revealed that the living biomaterial does not trigger cytotoxicity or pro-inflammatory phenotypes on corneal cells during the 7-day co-culture. We placed the living biomaterial on top of the corneal epithelium, observing no cytotoxic effects. Overall, these findings highlight the potential of in vitro investigations for living biomaterials and the applicability of these devices for ophthalmology purposes.

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.

Advances in the past decades in materials science, biofabrication methods, and synthetic biology have given rise to new fields like living materials. A living material is a class of biohybrid composite with living elements, including bacteria, yeasts, fungi, and mammalian cells, integrated with nonliving components. (1−6) These materials combine the advantages of both living and nonliving components to generate novel functions such as responses to environmental parameters and syntheses of complex biomolecules. (7) The nonliving aspect combines diverse chemistries and manufacturing techniques to support or enhance the functions of the living part. (6) Living materials as therapeutics (Living Therapeutic Materials, LTMs) bring revolutionary options to diagnostic and therapeutic practice, offering a solution to life-concerning issues by life itself (Figure 1). Living Therapeutic Materials are revolutionizing classical drug delivery devices, as they can produce therapeutics long-term, in situ, and on demand. This represents a more sustainable way for treatment. To realize Living Therapeutic Materials in the clinic, more preclinical studies need to be carried out so the concerns in terms of safety are well understood and their capacity as a more efficient delivery system is assessed. In the past decade, there has been a rise in the number of proof-of-concept LTMs and yet, the preclinical investigation of these materials is just starting.

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.