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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Refeed (Customised lipid supplementation of culture media for efficient in vitro cell culture applications)

Teaser

Summary

The system of cellular membranes is a cornerstone in cell physiology, representing more than 50% of the entire cell by weight and volume and with more than 90% of events happening in or nearby it. Thanks to their selective permeability to ions and organic molecules, they control the movement of substances in and out of cells. The membrane itself is composed by a lipid bilayer : lipids constitute around 50% of the total membrane weight, with proteins (45% in weight) embedded in the bilayerMembrane lipids influence most aspects of cell physiology: membrane fluidity and curvature; protein functionality, diffusion and clustering; lipid rafts and signalling; peroxidation and radical stress; trafficking processes; micro-vesicle number and properties, cell motility; stemness and differentiation, etc. Notwithstanding, currently the lipid composition and, consequently, the biophysical properties of cell membranes of in-vitro cultures are highly neglected and extremely altered . Media manufacturing companies do not have the possibility to put high quality/quantity lipid formulations inside their products for two main reasons: 1) lipids are not soluble in a aqueous environment and 2) lipids generate oxidative stress by reacting with water oxygen, thus heavily lowering product shelf-life. As a result, media currently available on the market are very limited in lipid quality and quantity, and cultured cells always display different membrane lipid profiles when compared to their respective in-vivo counterparts. Differences up to 250% exist between in-vivo and in-vitro membranes in terms of individual lipids, lipid sums and lipid indexes in any cell type (primary cells, stem cells and immortalized cell lines) used worldwide in bio-laboratories

Refeed is a customizable ethanol-based lipid supplement for in-vitro cultures, capable of addressing major culturing issues and/or boost specific bioprocesses. Tailored Refeed formulations can be adapted to any cell culture conditions and can be animal-free, fully synthetic and, upon completing the Phase 2 project, GMP-grade.

Remembrane reviewed the overall objectives outlined to scale-up its business, and adjusted them on the basis of the interaction with prospect stakeholders. The goal of the product development plan is to generalise and validate the process for the deployment of customized Refeed supplements in order to serve small, medium and large players on a global dimension in extremely reduced time (3 working days for production and worldwide shipment).

Work performed

Remembraneâ€™s founders have been working for many years on membrane lipid profiles of in-vitro culture, and they have discovered that current cultured cells do not represent an optimal situation for mimicking in-vivo membrane environment. With his PhD thesis in Cell Biology, â€˜Membrane lipidomics: the reorganization of fatty acids as a biomarker of cell conditionâ€™ (2006-08), Dr. Alexandros Chatgilialoglu, CEO and co-founder of Remembrane, condensed a decade of research in the realisation of a disruptive concept and foundation of the company: Remembrane has started to design and commercialise tailored lipid supplements, called Refeed, for in-vitro applications: such supplements were found to be successful in preserving a lipid profile almost identical to that of their in-vivo counterparts. Refeed use is quite simple: supplements are added to the growth medium as cell feeders, able to develop membranes that mimic realistic membrane environments or create personalised membrane phenotypes. Being Refeed ethanol-based lipid solutions, solubility and oxidative stress generation issues disappear, giving to the Refeed technology a great advantage over competition.

Between 1st January 2017 and 30th April 2017, Remembrane undertook a full feasibility study of its Refeed business project, planned the strategy for its scale up and commercialization, and contacted many stakeholders in the biotechnology industry to assess the technical and economic viability of its product.
The first result was the identification of the Cell Therapy industry as the most appealing market sector for Refeed, given the market condition and the possibility of fine-tuning the Refeed supplement for each therapy under development: in the Cell Therapy industry Refeed can be highly competitive, the market itself is boosting, and the players in the field are easier to be reached, since they are often medium-size enterprises, instead of big pharma companies as it is in the vaccine market, for instance. Another important result was the recognition that, as a supplier of cell therapy companies, each with their own specific needs, Remembrane will need flexible scale-up capabilities for the Refeed production, and a GMP (Good Manufacturing Practice) compliant production process. This led to the redefinition of a few objectives of the feasibility assessment. During Phase 1 project a great effort was made by Remembrane to identify preliminary features and potential stakeholders to fully engineer the automated production line for Refeed, and the steps required for the GMP compliance were mapped.

Final results

During Phase 1, Remembrane conducted a thorough competition analysis of companies producing reagents and supplements for cell culture media. The most important finding is the complete absence of attention to membrane lipidomics in the existing in-vitro protocols: as a result, lipid supplementation is always ignored during in-vitro cultivation. Remembrane is the only company offering a solution of this type on the market. The use of polyunsaturated and trans fatty acids (solubilised with ethanol under inert gas to prevent oxidative stress) allows targeting of cell membrane lipid composition through customised lipid supplementation. Other lipid supplements available on the market are low quality and not functional in modulating membrane lipid composition and related properties. The main companies operating in the cell culture media market are: BD Biosciences, Lonza Group, Sigma-Aldrich Corporation, GE healthcare and Thermo Fisher Scientific Inc. All these companies are huge players in the market; however, no one of them sell cell-specific and/or lipid-specific tailored supplements; their standard off-the-shelf products lack in customisation and flexibility. Moreover, media manufacturing companies are not able to use high quality/quantity lipid formulations inside their products for two main reasons: 1) lipids are not soluble in a aqueous environment and 2) lipids generate oxidative stress by reacting with water oxygen, thus heavily lowering product shelf-life. As a result, media currently available on the market are very limited in lipid quality and quantity, and cultured cells always display different membrane lipid profiles when compared to their respective in-vivo counterparts. Differences up to 250% exist between in-vivo and in-vitro membranes in terms of individual lipids, lipid sums and lipid indexes in any cell type (primary cells, stem cells and immortalized cell lines) used worldwide in bio-laboratories.