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.

Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells.

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.

The use of nanofibrillar cellulose hydrogel as a flexible three-dimensional model to culture human pluripotent stem cells.

Human embryonic stem cells and induced pluripotent stem cells have great potential in research and therapies. The current in vitro culture systems for human pluripotent stem cells (hPSCs) do not mimic the three-dimensional (3D) in vivo stem cell niche that transiently supports stem cell proliferation and is subject to changes which facilitate subsequent differentiation during development. Here, we demonstrate, for the first time, that a novel plant-derived nanofibrillar cellulose (NFC) hydrogel creates a flexible 3D environment for hPSC culture. The pluripotency of hPSCs cultured in the NFC hydrogel was maintained for 26 days as evidenced by the expression of OCT4, NANOG, and SSEA-4, in vitro embryoid body formation and in vivo teratoma formation. The use of a cellulose enzyme, cellulase, enables easy cell propagation in 3D culture as well as a shift between 3D and two-dimensional cultures. More importantly, the removal of the NFC hydrogel facilitates differentiation while retaining 3D cell organization. Thus, the NFC hydrogel represents a flexible, xeno-free 3D culture system that supports pluripotency and will be useful in hPSC-based drug research and regenerative medicine.

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.