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Layer-by-Layer cellulose nanofibril coating for spheroid formation combined with decellularized extracellular matrix for 3D tumor modeling
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.ORCID iD: 0000-0002-6544-9955
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.ORCID iD: 0000-0002-5444-7276
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.ORCID iD: 0000-0002-0242-358X
(English)Manuscript (preprint) (Other academic)
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:kth:diva-321149OAI: oai:DiVA.org:kth-321149DiVA, id: diva2:1708997
Note

QCR 20221108

Available from: 2022-11-07 Created: 2022-11-07 Last updated: 2022-11-08Bibliographically approved
In thesis
1. Decellularized liver extracellular matrix as a 3D scaffold for bioengineering applications
Open this publication in new window or tab >>Decellularized liver extracellular matrix as a 3D scaffold for bioengineering applications
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The increasing global burden of end-stage liver disease has increased the need for liver transplantation, the definitive cure. However, there is a huge discrepancy between the number of available organ donors and the number of patients waiting for transplantation, resulting in the deaths of a significant number of patients on the waiting list as only 10% of the global need for transplantation is met. Liver tissue engineering is a promising alternative solution to this problem, which utilizes bioengineering techniques to create an ex vivo microenvironment niche for liver cells embedded in a liver-specific extracellular matrix (ECM) for cell growth and function. Despite many advances in this field, the scarcity of appropriate ECM-mimicking biomaterials with good mechanical properties for biofabrication technique remains limited. To address this, different biofabrication techniques, such as bioprinting and biomaterial scaffolds, are studied to simulate liver microarchitecture for different applications. This thesis presents the development and application of a decellularized liver extracellular matrix hydrogel combined with the liver cell line HepG2 (papers 1-3). It also focuses on the decellularized whole liver scaffold to differentiate amniotic epithelial cells (paper 4). The decellularized liver extracellular matrix (dLM) is a cell-free scaffold that retains liver-specific components to direct cell growth and functions. The dLM can be digested to form hydrogel for 3D bioprinting applications, or it can be used as a biomaterial scaffold to seed the cells directly. In paper I, porcine dLM hydrogel was modified with gelatin and a PEG-based crosslinker to induce a cytocompatible gelation mechanism to generate a robust bioink with a 16-fold increment in viscosity and a 32-fold increment in storage modulus as compared to unmodified dLM hydrogel. This work established the application of dLM with other biofabrication methods, such as Indirect bioprinting, where a sacrificial biopolymer is 3D printed, and the scaffold material is subsequently added. In paper II, a 3D-printed polyvinyl alcohol framework resembling the liver lobules was used as a sacrificial scaffold to impart its structure to the dLM hydrogel modified with PEG-based crosslinker and mushroom tyrosinase. The crosslinked dLM hydrogel with co-culture of HepG2 and NIH 3T3 fibroblasts cell line retained the structure of PVA to create a scaled-up liver-like microarchitecture with lobules. The PVA dissolved with cell culture media leaving behind a robust 3D construct of dLM hydrogel. In paper III, cellulose nanofibril-coated HepG2 spheroids incorporating dLM hydrogel were studied for tumor modeling. The dLM incorporation affected the spheroid formation and growth depending on the time of addition. In paper IV, the functional differentiation of amniotic epithelial cells into hepatocyte-like cells was performed in a decellularized rat liver scaffold in a perfusion bioreactor with dynamic oxygenation and media exchange. This dLM perfusion technology supported the maturation and proliferation of amniotic epithelial cells into hepatocyte-like cells. This is a preliminary step into developing a liver-like organ model in a laboratory setting. 

To conclude, this thesis presents different bioengineering approaches, such as 3D bioprinting and perfusion decellularization, to study the 3D dLM scaffolds for HepG2 and amniotic epithelial cell culture. 3D bioprinting technique utilized a robust dLM hydrogel to create a scaled-up microarchitecture, whereas perfusion decellularization retained the natural 3D architecture of the whole liver ECM and the native vascular system for recellularizing the scaffold with stem cells. We successfully modified and characterized the dLM hydrogel to enhance its printability to develop complex structures such as liver lobules and microchannels. We utilized different cell systems, including monoculture, co-culture, and spheroids, to analyze the biocompatibility, cell proliferation, and liver-specific functions of the dLM scaffold. Ultimately, the advancement of dLM as a biomaterial presented in this thesis could improve the application and modification of various decellularized tissues to generate larger-scale models for in vitro testing and organ transplantation.

Abstract [sv]

Den global ökningen av skrumplever, eller levercirros, har ökat behovet av levertransplantationer, det definitiva botemedlet. Det finns dock en stor skillnad mellan antalet organdonatorer och antalet patienter som väntar på transplantation. Detta leder till att många patienter på väntelistan dör i väntan på transplantation, eftersom endast 10 % behovet tillgodoses. Vävnadsrekonstruktion av levervävnad erbjuder en alternativ lösning på detta problem, som använder bioteknik för att skapa en rätt mikromiljö för levercellergenom att bädda in dem i en leverspecifik s.k extracellulär matris (ECM). Trots många framsteg är tillgången på lämpliga ECM-liknande biomaterial med goda mekaniska egenskaper för vävnadsrekonstruktion fortfarande begränsad. Därför har vi studerat olika biotekniska tillverkningsmetoder, t ex bioprinting och ramverk av biomaterial för att simulera leverns mikroarkitektur. 

Denna avhandling presenterar utvecklingen och tillämpningen av ett gelmaterial tillverkat av extracellulär matrix från lever (artikel 1-3) kombinerat med levercellinjen HepG2 (artikel 4) eller leverliknande celler, för vävnadsrekonstruktion. Efter att levercellerna avlägsnats behåller leverns extracellulära matrix (dLM) leverspecifika komponenter som kan styra celltillväxt och funktioner. I 3D bioprinting kan en robust dLM-baserad hydrogel användas för att skapa en leverliknande mikroarkitektur. Dessutom bibehålls den naturliga 3D-arkitekturen och kärlstrukturen hos leverns ECM och kan inympas med önskvärda celler. I artikel I modifierades dLM-hydrogel från gris med gelatin och en PEG-baserad tvärbindare för att resultera i ett robust biobläck för 3D utskrifter med en goda mekaniska egenskaper, jämfört med omodifierad dLM hydrogel. Detta arbete etablerade användningen av dLM för nya tillverkningsmetoder för utskriven vävnad t ex Indirekt bioprinting, där en offer-biopolymer 3D-printas och vävnads-ramverket därefter läggs till. I artikel II användes ett 3D-utskrivet ramverk av polyvinylalkohol som tillfällig gjutform för en modifierad dLM-hydrogel. dLM-hydrogelen stelnade på PVA-strukturen, där lever- och fibroblastceller kunde odlas. PVAt löstes upp när cellodlingsmedia tillsattes, och lämnade efter sig en rekonstruerad vävnadsstruktur. I artikel III studerades en nanocellulosabelagd sfäroidmodell av lever- och tjocktarms celler innehållande dLM för läkemedelsscreening. I artikel IV utfördes den funktionella differentiering av stamceller från fostervatten till lever-liknande celler i ett vävnadsramverk från råtta i en perfusionsbioreaktor med dynamisk syresättning och mediautbyte.

Sammanfattningsvis presenterar denna avhandling olika biotekniska metoder såsom 3D bioprinting och perfusionsdecellularisering, för att studera 3D ramverk för vävnadsrekonstruktion av dLM, och odling av celler i dessa. Vi har framgångsrikt modifierat och karakteriserat dLM-hydrogelen för att förbättra dess 3D utskriftsegenskaper i komplexa strukturer som leverlobuli och mikrokanaler. Vi använde olika cellsystem inklusive monokultur, samodling och sfäroider för att karakterisera celltillväxt och leverspecifika funktioner i dLM-ramverket. Utvecklingen av dLM som biomaterial att kommer att öka förutsättningarna för vävnadsrekonstuktion för att skapa uppskalade organmodeller för läkemedelstestning och organtransplantation.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2022. p. 91
Series
TRITA-CBH-FOU ; 2022:59
Keywords
liver decellularization, decellularized liver matrix bioink, bioprinting, sacrificial scaffold, viscoelasticity, bioengineering, tumor modeling, stem cell differentiation, bioreactor
National Category
Biomaterials Science Engineering and Technology Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-321180 (URN)978-91-8040-421-1 (ISBN)
Public defence
2022-12-02, Karolina, Widerströmska huset, Tomtebodavägen 18a, via Zoom: https://kth-se.zoom.us/j/69659607478, Solna, 10:00 (English)
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Note

QC 2022-11-08

Available from: 2022-11-08 Created: 2022-11-08 Last updated: 2022-11-29Bibliographically approved

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Abbasi Aval, NegarKhati, VamakshiPettersson, TorbjörnRussom, Aman

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