Melina Malinen

Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture

Over the recent years, various materials have been introduced as potential 3D cell culture scaffolds. These include protein extracts, peptide amphiphiles, and synthetic polymers. Hydrogel scaffolds without human or animal borne components or added bioactive components are preferred from the immunological point of view. Here we demonstrate that native nanofibrillar cellulose (NFC) hydrogels derived from the abundant plant sources provide the desired functionalities. We show 1) rheological properties that allow formation of a 3D scaffold in-situ after facile injection, 2) cellular biocompatibility without added growth factors, 3) cellular polarization, and 4) differentiation of human hepatic cell lines HepaRG and HepG2. At high shear stress, the aqueous NFC has small viscosity that supports injectability, whereas at low shear stress conditions the material is converted to an elastic gel. Due to the inherent biocompatibility without any additives, we conclude that NFC generates a feasible and sustained microenvironment for 3D cell culture for potential applications, such as drug and chemical testing, tissue engineering, and cell therapy.

Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels.

Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.

A critical assessment of in vitro tissue models for ADME and drug delivery.

Cultured cells are widely used in the evaluation of new drugs and drug delivery systems. Cells can be grown at different levels of complexity ranging from simple reductionist models to complex organotypic models. The models are based on primary, secondary or stem cell derived cell cultures. Generation of tissue mimics with cultured cells is a difficult task, because the tissues have well-defined morphology, complex protein expression patterns and multiple inter-linked functions. Development of organotypic cell culture models requires proper biomaterial matrix and cell culture protocols that are able to guide the cells to the correct phenotype. This review illustrates the critical features of the cell culture models and, then, selected models are discussed in more detail (epidermal, corneal epithelial, retinal pigment epithelium, and hepatocyte models). The cell models are critically evaluated paying attention to the level of characterization and reliability of in vivo translation. Properties of the cell models must be characterized in detail using multiple biological assays and broad sets of model drugs. Robust in vivo predictions can be achieved with well-characterized cell models that are used in combination with computational methods that will bridge the gap between in vitro cell experiments and physiological situation in vivo in the body.

Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix

Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs.

Regulation of Human Pluripotent Stem Cell-Derived Hepatic Cell Phenotype by Three-Dimensional Hydrogel Models.

Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes are anticipated as important surrogates for primary human hepatocytes in applications ranging from basic research to drug discovery and regenerative medicine. Although methods for differentiating hepatocyte-like cells (HLCs) from hiPSCs have developed remarkably, the limited yield of fully functional HLCs is still a major obstacle to their utility. A three-dimensional (3D) culture environment could improve the in vitro hepatic maturation of HLCs. Here we compare 3D hydrogel models of hiPSC-derived HLCs in agarose microwells (3D Petri Dish; 3DPD), nanofibrillar cellulose hydrogels (Growdex; 3DNFC), or animal extracellular matrix-based hydrogels (3D Matrigel; 3DMG). In all the tested 3D biomaterial systems, HLCs formed aggregates. In comparison with two-dimensional monolayer culture, 3DPD and 3DMG models showed both phenotypic and functional enhancement in HLCs over 2.5 weeks of 3D culture. Specifically, we found higher hepatocyte-specific gene expression levels and enhanced cytochrome P450 functions. Our work suggests that transferring HLCs into 3D hydrogel systems can expedite the hepatic maturation of HLCs irrespective of the biochemical nature of the 3D hydrogel. Both plant-based nonembedding and animal-based embedding 3D hydrogel models enhanced the maturation.

Multicellular dosimetric chain for molecular radiotherapy exemplified with dose simulations on 3D cell spheroids

PURPOSE Absorbed radiation dose-response relationships are not clear in molecular radiotherapy (MRT). Here, we propose a voxel-based dose calculation system for multicellular dosimetry in MRT. We applied confocal microscope images of a spherical cell aggregate i.e. a spheroid, to examine the computation of dose distribution within a tissue from the distribution of radiopharmaceuticals. METHODS A confocal microscope Z-stack of a human hepatocellular carcinoma HepG2 spheroid was segmented using a support-vector machine algorithm and a watershed function. Heterogeneity in activity uptake was simulated by selecting a varying amount of the cell nuclei to contain 111In, 125I, or 177Lu. Absorbed dose simulations were carried out using vxlPen, a software application based on the Monte Carlo code PENELOPE. RESULTS We developed a schema for radiopharmaceutical dosimetry. The schema utilizes a partially supervised segmentation method for cell-level image data together with a novel main program for voxel-based radiation dose simulations. We observed that for 177Lu, radiation cross-fire enabled full dose coverage even if the radiopharmaceutical had accumulated to only 60% of the spheroid cells. This effect was not found with 111In and 125I. Using these Auger/internal conversion electron emitters seemed to guarantee that only the cells with a high enough activity uptake will accumulate a lethal amount of dose, while neighboring cells are spared. CONCLUSIONS We computed absorbed radiation dose distributions in a 3D-cultured cell spheroid with a novel multicellular dosimetric chain. Combined with pharmacological studies in different tissue models, our cell-level dosimetric calculation method can clarify dose-response relationships for radiopharmaceuticals used in MRT.