Congenital and chronic liver diseases have a substantial health burden worldwide. The most effective treatment available for these patients is whole organ transplantation; however, due to the severely limited supply of donor livers and the side effects associated with the immunosuppressive regimen required to accept allograft, the mortality rate in patients with end-stage liver disease is annually rising. Stem cell-based therapy aims to provide alternative treatments by either cell transplantation or bioengineered construct transplantation. Human amnion epithelial cells (AEC) are a widely available, ethically neutral source of cells with the plasticity and potential of multipotent stem cells and immunomodulatory properties of perinatal cells. AEC have been proven to be able to achieve functional improvement towards hepatocyte-like cells, capable of rescuing animals with metabolic disorders; however, they showed limited metabolic activities in vitro. Decellularised extracellular matrix (ECM) scaffolds have gained recognition as adjunct biological support. Decellularised scaffolds maintain native ECM components and the 3D architecture instrumental of the organ, necessary to support cells’ maturation and function. We combined ECM-scaffold technology with primary human AEC, which we demonstrated being equipped with essential ECM-adhesion proteins, and evaluated the effects on AEC differentiation into functional hepatocyte-like cells (HLC). This novel approach included the use of a custom 4D bioreactor to provide constant oxygenation and media perfusion to cells in 3D cultures over time. We successfully generated HLC positive for hepatic markers such as ALB, CYP3A4 and CK18. AEC-derived HLC displayed early signs of hepatocyte phenotype, secreted albumin and urea, and expressed Phase-1 and -2 enzymes. The combination of liver-specific ECM and bioreactor provides a system able to aid differentiation into HLC, indicating that the innovative perfusion ECM-scaffold technology may support the functional improvement of multipotent and pluripotent stem cells, with important repercussions in the bioengineering of constructs for transplantation.

Bioprinting is an acclaimed technique that allows the scaling of 3D architectures in an organized pattern but suffers from a scarcity of appropriate bioinks. Decellularized extracellular matrix (dECM) from xenogeneic species has garnered support as a biomaterial to promote tissue-specific regeneration and repair. The prospect of developing dECM-based 3D artificial tissue is impeded by its inherent low mechanical properties. In recent years, 3D bioprinting of dECM-based bioinks modified with additional scaffolds has advanced the development of load-bearing constructs. However, previous attempts using dECM were limited to low-temperature bioprinting, which is not favorable for a longer print duration with cells. Here, we report the development of a multi-material decellularized liver matrix (dLM) bioink reinforced with gelatin and polyethylene glycol to improve rheology, extrudability, and mechanical stability. This shear-thinning bioink facilitated extrusion-based bioprinting at 37 degrees C with HepG2 cells into a 3D grid structure with a further enhancement for long-term applications by enzymatic crosslinking with mushroom tyrosinase. The heavily crosslinked structure showed a 16-fold increase in viscosity (2.73 Pa s(-1)) and a 32-fold increase in storage modulus from the non-crosslinked dLM while retaining high cell viability (85-93%) and liver-specific functions. Our results show that the cytocompatible crosslinking of dLM bioink at physiological temperatures has promising applications for extended 3D-printing procedures.

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.

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.

3D bioprinting is an acclaimed technology to develop architecturally significant tissue models, however most bioinks require a secondary support structure to create a clinically relevant sized model. In this work, we develop a 3D sacrificial support structure of polyvinyl alcohol (PVA) with decellularized liver extracellular matrix (dECM) bioink with HepG2 cells. The PVA backbone imparts its 3D structure to dECM and dissolves in cell culture media. We evaluated the PVA dissolution using refractometry and microscopic observation. We further tested the crosslinking of dECM with a PEG-based crosslinker using scanning electron microscopy (SEM). Alamar blue assay and gene expression analysis results demonstrated an increase in cell proliferation within the 3D structure.

The liver exhibits complex geometrical morphologies of hepatic cells arranged in a hexagonal lobule with an extracellular matrix (ECM) organized in a specific pattern on a multi-scale level. Previous studies have utilized 3D bioprinting and microfluidic perfusion systems with various biomaterials to develop lobule-like constructs. However, they all lack anatomical relevance with weak control over the size and shape of the fabricated structures. Moreover, most biomaterials lack liver-specific ECM components partially or entirely, which might limit their biomimetic mechanical properties and biological functions. Here, we report 3D bioprinting of a sacrificial PVA framework to impart its trilobular hepatic structure to the decellularized liver extracellular matrix (dLM) hydrogel with polyethylene glycol-based crosslinker and tyrosinase to fabricate a robust multi-scale 3D liver construct. The 3D trilobular construct exhibits higher crosslinking, viscosity (182.7 ± 1.6 Pa·s), and storage modulus (2554 ± 82.1 Pa) than non-crosslinked dLM. The co-culture of HepG2 liver cells and NIH 3T3 fibroblast cells exhibited the influence of fibroblasts on liver-specific activity over time (7 days) to show higher viability (90–91.5%), albumin secretion, and increasing activity of four liver-specific genes as compared to the HepG2 monoculture. This technique offers high lumen patency for the perfusion of media to fabricate a densely populated scaled-up liver model, which can also be extended to other tissue types with different biomaterials and multiple cells to support the creation of a large functional complex tissue.

Decellularized extracellular matrix is a tissue-specific biomaterial that recapitulates the complexity of the inherent tissue environment to elicit cellular response. Here, a multi-material decellularized liver (dLM)-based bioink with gelatin is developed and polyethylene glycol crosslinking is used to enhance the viscoelasticity of the dLM. We evaluated the necessity of a post-printing secondary cross-linker mushroom tyrosinase to improve robustness and long term stability. We further demonstrate it's biocompatibility using liver specific gene analysis of HepG2 cells.