A three-dimensional in vitro liver model: Proteomics investigation of cellular adaptations in human hepatic spheroids

Joel Mario Vej-Nielsen

doi:10.21996/32a8eebd-750f-48e1-a9ae-03993f048c8f

A three-dimensional in vitro liver model: Proteomics investigation of cellular adaptations in human hepatic spheroids

Bidragets oversatte titel: En tredimensionel in vitro levermodel: Proteomics studie af cellulære adaptationer i humane leverspheroider

Joel Mario Vej-Nielsen

Publikation: Afhandling › Ph.d.-afhandling

Abstract

Medicin kurerer, letter symptomer og hæmmer udviklingen af sygdomme og i takt med at biomedicinskforskning udvikler sig, udvikles der også nye muligheder for at behandle sygdom. Udvikling af lægemidlerkræver hård kontrol for at hindre anvendelsen af lægemidler med seriøse bivirkninger. Denne kontrol sikresved hjælp af kliniske forsøg, der både er tidskrævende og dyre. Færre end 10% af påbegyndte kliniske forsøgender i godkendelse af lægemidlet og de resurser der investeres i afviste lægemidler, resulterer i øgede priserpå medicin of langsom udvikling af nye behandlinger. Dette problem kunne afhjælpes hvis præ-kliniskeundersøgelser af lægemidler, der på nuværende tidspunkt udføres i en kombination af cellekulturer ogforsøgsdyr. Resultater produceret i forsøgsdyr kan ikke altid overføres på mennesker og klassiskecellekulturer opfører sig markant anderledes end de samme celler i kroppen. Vores hypotese er atudviklingen af mere avancerede levermodeller kan forbedre effekten af præ-kliniske studier.

Leveren spiller en vigtig rolle i udviklingen af lægemidler da den både modificerer lægemidler og fjerner dem fra kroppen, hvilket påvirker effekten af medicin og gør leveren til et udsat organ for mange lægemidler. Hepatocytter er den mest hyppige celletype i leveren og de celler der producerer enzymerne der behandler
lægemidler. Hepatocytter til cellekulturer kan komme fra forskellige kilder, men udødelige cellelinier er reproducerbare, nemt tilgængelige og nemme at arbejde med, hvilket gør dem ideelle til farmakologiske undersøgelser. I tredimensionelle (3D) cellekulturer, bliver levercellelinier bedre til at opfører sig som de gør i leveren. Denne positive effect af 3D celleklutur antages at skyldes udvikling af et mikromiljø, der minder mere om det der findes i kroppen.

Formålet med dette studie var at undersøge de cellulære tilpasninger der sker i langvarige levercelle spheroidkulturer for at forstå hvordan 3D cellekultur forbedre cellernes adfærd. I denne tese undersøger jeg de cellulære tilpasninger der sker 3D cellekultur, af den humane hepatocytcellelinie HepG2/C3A, med
massespektometri-baseret proteomanalyse. For at påvirke udviklingen af extracellulær matrix, introducerer jeg endnu en levercellelinie, LX-2 celler, i systemet for at undersøge hvordan det påvirker mikromiljøet. Resultaterne viste at udviklingen af en nekrotisk kerne i spheroider følger en udviklingsfase efterfulgt af
dannelsen af en stabil kerne. Levende celler i spheroider med en stabil kerne havde højere udtryk af proteiner involveret i udviklingen af mikromiljøet. Endvidere viste resultaterne at kokultivering af flere hepatiske cellelinier resulterede i en øget mængde mikromiljøkomponenter og indikationer af mere interaktion med mikromiljøet, såvel som øget leverfunktion i langvarig cellekultur.

Bidragets oversatte titel	En tredimensionel in vitro levermodel: Proteomics studie af cellulære adaptationer i humane leverspheroider
Originalsprog	Engelsk
Bevilgende institution	Syddansk Universitet
Vejledere/rådgivere	Rogowska-Wrzesinska, Adelina, Hovedvejleder
Dato for forsvar	24. jan. 2023
Udgiver	Syddansk Universitet. Det Naturvidenskabelige Fakultet
DOI	https://doi.org/10.21996/32a8eebd-750f-48e1-a9ae-03993f048c8f
Status	Udgivet - 14. dec. 2022

Note vedr. afhandling

Afhandlingen kan læses på SDUs bibliotek. 

Dokumenter og links

10.21996/32a8eebd-750f-48e1-a9ae-03993f048c8f

SDU link

Tjek adgangen til materialet i Syddansk Universitetsbiblioteks søgemaskine

2 Kapitel i bog

3D-ViaFlow: A Quantitative Viability Assay for Multicellular Spheroids
Vej-Nielsen, J. M. & Rogowska-Wrzesinska, A., 2021, Next Generation Culture Platforms for Reliable In Vitro Models: Methods and Protocols. Brevini, T. A. L., Fazeli, A. & Turksen, K. (red.). Humana Press, s. 159-171 (Methods in Molecular Biology, Bind 2273).
Publikation: Kapitel i bog/rapport/konference-proceeding › Kapitel i bog › Formidling
Method to Disassemble Spheroids into Core and Rim for Downstream Applications Such as Flow Cytometry, Comet Assay, Transcriptomics, Proteomics, and Lipidomics
Frandsen, H. S., Štampar, M., Vej-Nielsen, J. M., Žegura, B. & Rogowska-Wrzesinska, A., 2021, Next Generation Culture Platforms for Reliable In Vitro Models: Methods and Protocols. Brevini, T. A. L., Fazeli, A. & Turksen, K. (red.). Humana Press, s. 173-188 (Methods in Molecular Biology, Bind 2273).
Publikation: Kapitel i bog/rapport/konference-proceeding › Kapitel i bog › Formidling

Citationsformater

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title = "A three-dimensional in vitro liver model: Proteomics investigation of cellular adaptations in human hepatic spheroids",

abstract = "Medicine cures, alleviates, and prevents the progression of diseases and as medical science evolves so doesthe opportunity to treat diseases. Drug development, however, requires stringent control to prevent drugswith severe adverse effects from being utilized. This is ensured through clinical trials, which is both timeconsuming and costly. Less than 10\% of initiated clinical trials result in drug approval and resources investedin the large number of failed drugs ultimately result in increased medicine prices and slow development ofnew treatments. This could be alleviated by improved pre-clinical screening procedures, which is currentlycarried out in a combination of cell culture and animal models. Animal models, however, suffer from poorinterspecies translatability and conventional cell culture techniques poorly recapitulate in vivo functionalityof organs. We hypothesize that the development of an advanced liver model could improve the predictivepower of pre-clinical screenings. The liver plays a key role in drug development due to its modification and elimination of drugs, which directly impacts the efficacy of medicines and makes hepatotoxicity a common side effect of many drugs. Hepatocytes are the most abundant cell type in the liver and the primary producers of the enzymes involved in drug metabolism. There are a few different sources of hepatocytes used for in vitro liver models, but immortal cell lines are highly reproducible, easily available, and easy to work with, which makes them ideal for pharmacological applications. 3D cell culture techniques are capable of improving the ability of hepatic cell lines to recapitulate liver function. The observed benefits of 3D cell culture are believed to be caused by development of microenvironment that closely resembles in vivo conditions. The aim of this study was to investigate cellular adaptations in long-term cultures of hepatocyte-based spheroids to understand how 3D culture systems improve cellular behavior. In this thesis, I investigate the cellular adaptations that occur within a 3D cell culture model, made from the human hepatocyte cell line HepG2/C3A, with mass spectrometry-based proteomics. In order to influence the development of extracellular matrix, I introduced another liver cell line, LX-2 cells, to the model and investigated changes in the organization of the cellular microenvironment. The results showed that necrotic core development within spheroids is characterized by core-formation followed by establishment of a stable core. Live cells within stable core-containing spheroids showed an increase in proteins related to the formation of the cellular microenvironment. Further results indicated that 3D co-cultures, containing multiple hepatic cell lines, had increased production of- and interactions with the cellular microenvironment as well as improved liver function in long-term culture. ",

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N2 - Medicine cures, alleviates, and prevents the progression of diseases and as medical science evolves so doesthe opportunity to treat diseases. Drug development, however, requires stringent control to prevent drugswith severe adverse effects from being utilized. This is ensured through clinical trials, which is both timeconsuming and costly. Less than 10% of initiated clinical trials result in drug approval and resources investedin the large number of failed drugs ultimately result in increased medicine prices and slow development ofnew treatments. This could be alleviated by improved pre-clinical screening procedures, which is currentlycarried out in a combination of cell culture and animal models. Animal models, however, suffer from poorinterspecies translatability and conventional cell culture techniques poorly recapitulate in vivo functionalityof organs. We hypothesize that the development of an advanced liver model could improve the predictivepower of pre-clinical screenings. The liver plays a key role in drug development due to its modification and elimination of drugs, which directly impacts the efficacy of medicines and makes hepatotoxicity a common side effect of many drugs. Hepatocytes are the most abundant cell type in the liver and the primary producers of the enzymes involved in drug metabolism. There are a few different sources of hepatocytes used for in vitro liver models, but immortal cell lines are highly reproducible, easily available, and easy to work with, which makes them ideal for pharmacological applications. 3D cell culture techniques are capable of improving the ability of hepatic cell lines to recapitulate liver function. The observed benefits of 3D cell culture are believed to be caused by development of microenvironment that closely resembles in vivo conditions. The aim of this study was to investigate cellular adaptations in long-term cultures of hepatocyte-based spheroids to understand how 3D culture systems improve cellular behavior. In this thesis, I investigate the cellular adaptations that occur within a 3D cell culture model, made from the human hepatocyte cell line HepG2/C3A, with mass spectrometry-based proteomics. In order to influence the development of extracellular matrix, I introduced another liver cell line, LX-2 cells, to the model and investigated changes in the organization of the cellular microenvironment. The results showed that necrotic core development within spheroids is characterized by core-formation followed by establishment of a stable core. Live cells within stable core-containing spheroids showed an increase in proteins related to the formation of the cellular microenvironment. Further results indicated that 3D co-cultures, containing multiple hepatic cell lines, had increased production of- and interactions with the cellular microenvironment as well as improved liver function in long-term culture.

AB - Medicine cures, alleviates, and prevents the progression of diseases and as medical science evolves so doesthe opportunity to treat diseases. Drug development, however, requires stringent control to prevent drugswith severe adverse effects from being utilized. This is ensured through clinical trials, which is both timeconsuming and costly. Less than 10% of initiated clinical trials result in drug approval and resources investedin the large number of failed drugs ultimately result in increased medicine prices and slow development ofnew treatments. This could be alleviated by improved pre-clinical screening procedures, which is currentlycarried out in a combination of cell culture and animal models. Animal models, however, suffer from poorinterspecies translatability and conventional cell culture techniques poorly recapitulate in vivo functionalityof organs. We hypothesize that the development of an advanced liver model could improve the predictivepower of pre-clinical screenings. The liver plays a key role in drug development due to its modification and elimination of drugs, which directly impacts the efficacy of medicines and makes hepatotoxicity a common side effect of many drugs. Hepatocytes are the most abundant cell type in the liver and the primary producers of the enzymes involved in drug metabolism. There are a few different sources of hepatocytes used for in vitro liver models, but immortal cell lines are highly reproducible, easily available, and easy to work with, which makes them ideal for pharmacological applications. 3D cell culture techniques are capable of improving the ability of hepatic cell lines to recapitulate liver function. The observed benefits of 3D cell culture are believed to be caused by development of microenvironment that closely resembles in vivo conditions. The aim of this study was to investigate cellular adaptations in long-term cultures of hepatocyte-based spheroids to understand how 3D culture systems improve cellular behavior. In this thesis, I investigate the cellular adaptations that occur within a 3D cell culture model, made from the human hepatocyte cell line HepG2/C3A, with mass spectrometry-based proteomics. In order to influence the development of extracellular matrix, I introduced another liver cell line, LX-2 cells, to the model and investigated changes in the organization of the cellular microenvironment. The results showed that necrotic core development within spheroids is characterized by core-formation followed by establishment of a stable core. Live cells within stable core-containing spheroids showed an increase in proteins related to the formation of the cellular microenvironment. Further results indicated that 3D co-cultures, containing multiple hepatic cell lines, had increased production of- and interactions with the cellular microenvironment as well as improved liver function in long-term culture.

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DO - 10.21996/32a8eebd-750f-48e1-a9ae-03993f048c8f

M3 - Ph.D. thesis

PB - Syddansk Universitet. Det Naturvidenskabelige Fakultet

ER -

A three-dimensional in vitro liver model: Proteomics investigation of cellular adaptations in human hepatic spheroids

Abstract

Note vedr. afhandling

Dokumenter og links

SDU link

Fingeraftryk

Relaterede publikationer

3D-ViaFlow: A Quantitative Viability Assay for Multicellular Spheroids

Method to Disassemble Spheroids into Core and Rim for Downstream Applications Such as Flow Cytometry, Comet Assay, Transcriptomics, Proteomics, and Lipidomics

Citationsformater