Combining a microfluidic platform with LC-MS for the detection of induced hepatotoxicity using primary hepatic spheroids

Student thesis: Doctoral Thesis

Abstract

The success rate of bringing a new drug from conception to market is extremely low as well as time consuming and very costly. Simple sub-cellular models can provide high throughput data allowing early detection of many drugs doomed to fail, but do not encompass the complexity found in whole cell based models, which provide more in-depth data. Cell culture models have evolved from simple 2-dimensional cultures on hard substrates i.e. plastic dishes, to 3 dimensional cultures that promote and enhance in vivo-like function and architecture. The liver is the main organ of interest during the drug development stages, as it is the organ that metabolizes xenobiotics. Drug metabolism entails converting a compound into a more hydrophilic state that can be actively excreted.However, the process of metabolism does sometimes result in the creation of a toxic intermediate metabolite that can lead to drug-induced liver injury. Thus to increase the efficacy of drug development the models, which are often scare and / or costly, and screening methods employed would need to be optimized. Microfluidics enables miniaturization through the control and manipulation of fluids n the nano – to millilitre range. Combining microfluidics with appropriate 2 dimensional / 3 dimensional cell culture systems can lead to higher throughput data production using far less resources compared to conventional culturing methods; and when combined with a screening modality that allows collection of detailed information it could be used to improve the efficacy of the preclinical drug-development process. One such modality is liquid chromatography-mass spectrometry.The chromatography technology allows separation of compounds from a mixed sample based on the interactions between the compounds of interest and the mobile and stationary phase. This enables quantitative analysis based on the retention time and absorbance measured by the detector. Mass spectrometry allows identification of the chemical composition of a component based on the mass-to-charge ratio. Both technologies are highly-sensitive and specific, which is ideally suited for metabolic analysis. In this thesis the effects of a miniaturisation process on cell culture environment are investigated, specifically the effect of using far less cells to form functional, metabolically active 3-dimensional liver spheroids, compared with conventional methods. HepG2 and primary rat hepatocyte spheroids were cultured in a microfluidic platform and exposed to drugs known to induce toxicity.The cells were cultured for 4 days before being exposed to each drug for 24 hours. Half the cultures were analyzed the following day and the other half were allowed to recover for an additional 24 hours. The platforms were compared to a gold standard culture model, a collagen sandwich culture. Cell supernatant and lysate samples were analyzed using liquid chromatography-mass spectrometry. The results showed that similar data could be obtained from the microfluidic platforms compared to collagen sandwich configurations when screening phase I and phase II metabolites, using far less cells and culture media. In conclusion, metabolically active liver spheroids can be generated using far less cell than previously thought. Information about metabolism can be extracted when combining the microfluidic cell culture system with liquid chromatography-mass spectrometry.
Date of Award15 Sep 2020
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsEPSRC (Engineering and Physical Sciences Research Council)
SupervisorMichele Zagnoni (Supervisor) & Mary Grant (Supervisor)

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