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LATVIAN

BIOMEDICAL

RESEARCH AND STUDY CENTRE


RESEARCH AND EDUCATION IN BIOMEDICINE FROM GENES TO HUMAN

Project Title: „ Testing of patient-derived stem cell extracellular vesicles loaded with drugs in a personalized lung-cancer on-chip platform (LoC4Ev).”

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Support for applied research”

Project No.: 1.1.1.1/20/A/124

Period: 1 February 2021 – 30 November 2023

Project costs: 540 540.53 EUR

Principle Investigator: Dr. biol. Artūrs Ābols

Cooperation partner: SIA “Cellboxlab”

Project summary:

This project aims to develop a personalised NSCLC patient lung cancer on chip (LCoC) and lung on chip (LoC) as a model system where to test the efficacy of drug-loaded patient-derived MSC produced EVs compared to the drug by itself.

Over half of the people diagnosed with lung cancer die within a year of diagnosis and the 5-year survival rate is less than 18%. Non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases. Depending on the stage of lung cancer, patients are eligible for certain treatments ranging from surgery to radiation to chemotherapy as well as targeted therapy. Thanks to genetic screening, specific mutations have been identified as a better target treatment for individual patients in the last decade. Also, there are molecular targeted therapies, that are not yet tested in lung cancer but could be applied in NSCLC cases where tumours have certain genetic variation. While there are options for different treatment, there is still a room for improvements in drug delivery, that could increase specificity and therefore decrease necessary concentrations and subsequently side effects of drugs. Extracellular vesicles (EV) have a natural ability to carry functional biomolecules, such as RNA, DNA, proteins and different metabolites in their lumen. This property makes EVs attractive for use in drug delivery. Indeed, several studies are currently underway to develop methods of exploiting EV for use as efficient drug delivery vehicles especially those derived from mesenchymal stem cells (MSC) due to their natural tumour cell tropism. However, current approaches have been tested only in simple in-vitro models or animal models. Next step would be testing these approaches into more advanced – personalised in vitro systems such as organs on chip platform

Information published 01.02.2021.

Progress of the project:

1 February 2021 – 30 April 2021

This reporting period, we started to prepare CMEK permit application for patient sample collection. Agreed on protocols for patient sample collection with doctors. Planning of the protocols for the isolation of lung cancer cells and the formation of organoids from patient surgery samples and the isolation of normal fibroblasts and their reprogramming into induced pluripotent stem cells (iPSC). Protocols for iPSC differentiation into lung epithelium, endothelium, and induced mesenchymal stem cells are also being developed. Lists of reagents for the necessary experiments are compiled. In parallel, the introduction of stem cell-derived extracellular vesicles (ASC52telo) into the first lung cancer on chip (LCOC) prototypes developed using stable cell lines A549 and HUVEC are being tested.

Information published 30.04.2021.

Progress of the project:

1 May 2021 – 31 July 2021

This reporting period we have been working on the TEER electrode design in a computer-aided design suit. Furthermore, we have been evaluating literature on the most optimal thin film electrode thickness and subsequently have been done the initial tests of electrode deposition via thermal evaporation. Additionally, five LOC devices have been fabricated for ensuing biological testing. Next, developed devices were tested with extracellular matrix (ECM) treatment process, functionalisation with HUVEC (Human umbilical vein endothelial cells), HSAEC (Human small airway epithelial cells) and pre-ECM treatment, cell seeding protocol was optimised to get functional models. Next LOC models are cultivated for functional LOC model tests with biosensors and ALI (Air liquid interface) establishment.

Information published 30.07.2021.

Progress of the project:

1 August 2021 – 31 October 2021

This reporting period we have been working on the fabrication of the first version of lung on chip (LOC) devices made from Off-stoichiometry thiol-ene polymer (OSTE) and thermoplastics with thin-film TEER (trans epithelial electric resistance) electrodes. Optimisation of the interconnection between thin-film electrodes and spring-loaded connectors. We are currently testing interconnections made from silver-loaded epoxy that sets in low temperature (60C). Low-temperature reflow soldering paste (138C) is also being tested with the heat applied to the spring-loaded connectors, which are immersed into the paste. Next, we have started to test commercial hiPSC (human induced pluripotent stem cells) differentiation into endothelial cell protocols with STEMCELL Technologies reagents, that will be applied to personalise Loc4Ev devices. LOC devices with integrated TEER biosensor were functionalised with HUVEC (Human umbilical vein endothelial cells) and A549 cells to test TEER functionality. Lecture about organs on a chip including LOC was prepared and presented to the M.D. students.

Information published 30.10.2021.

Progress of the project:

1 November 2021 – 31 January 2022

During this reporting period, we worked on the integration of O2 biosensors into LOC developed from Off-stoichiometry thiol-ene polymer (OSTE) and thermoplastics. The sensors were tested in both fluids such as 1xPBS and cell culture medium and in gas mixtures in cell culture incubators under normoxia and hypoxia. The commercial hiPSC (human-induced pluripotent stem cell) differentiation protocol into endothelial cells with STEMCELL Technologies reagents was optimised, that will be used to personalize Loc4Ev devices. Currently we are working to optimize the cisplatin packaging into mesenchymal stem cell derived extracellular vesicles methodology.

Information published 31.01.2022.

Progress of the project

1 February 2022 – 30 April 2022

Throughout the reporting period we have optimised the design of the device to improve the OSTED filling in the sidewalls of the device, which has led to improved fabrication yield of TEER devices. An alternative material to the mold material has been selected, which has significantly reduced particle contamination onto the thin-film TEER electrodes, therefore the resistance variation from channel to channel is now <10% for the current protocol, tested across multiple devices. Furthermore, an improved device design has allowed incorporation of 3 oxygen measurement ports per chip as opposed to initial design of 2 ports per chip. Experiments with the oxygen sensing in the chips are now being biologically validated. Work has been completed on optimizing cisplatin packaging in mesenchymal stem cell secreted extracellular vesicles. In parallel, testing of cisplatin-filled extracellular vesicles in lung cancer on a chip and lung on chip made up of commercial primary cells and stable cell lines was initiated. Results are compared between EVs without cisplatin and cisplatin without EVs. In addition, during this period we participated in the publication of the project topic in an online interview organized by the Riga Technical University Student Council in the studio entitled “What if?”.

Information published 29.04.2022.

Progress of the project:

1 May 2021 – 31 July 2021

TEER containing devices were tested by growing commercial primary and stable cell lines during lung on chip and lung cancer on chip establishment and during establishing ALI. Oxygen sensor placement outside the device within custom jig has been designed. HPLC methodology has been optimised to measure cisplatin within EVs. Next, EV staining by SYTOX and uptake experiments have been also optimised. In addition, during this period we participated and presented research project topics within conversation festival LAMPA at Cēsis, Latvia to the general public.

Information published 29.07.2022.