A Novel Approach to Orthotopic Hepatocyte Transplantation Engineered With Liver Hydrogel for Fibrotic Livers, Enhancing Cell-Cell Interaction and Angiogenesis.

Hepatocyte transplantation (HCT) is a potential bridging therapy or an alternative to liver transplantation. Conventionally, single-cell hepatocytes are injected via the portal vein. This strategy, however, has yet to overcome poor cell engraftment and function. Therefore, we developed an orthotopic HCT method using a liver-derived extracellular matrix (L-ECM) gel. PXB cells (flesh mature human hepatocytes) were dispersed into the hydrogel solution in vitro, and the gel solution was immediately gelated in 37°C incubators to investigate the affinity between mature human hepatocyte and the L-ECM gel. During the 3-day cultivation in hepatocyte medium, PXB cells formed cell aggregates via cell-cell interactions. Quantitative analysis revealed human albumin production in culture supernatants. For the in vivo assay, PXB cells were encapsulated in the L-ECM gel and transplanted between the liver lobes of normal rats. Pathologically, the L-ECM gel was localized at the transplant site and retained PXB cells. Cell survival and hepatic function marker expression were verified in another rat model wherein thioacetamide was administered to induce liver fibrosis. Moreover, cell-cell interactions and angiogenesis were enhanced in the L-ECM gel compared with that in the collagen gel. Our results indicate that L-ECM gels can help engraft transplanted hepatocytes and express hepatic function as a scaffold for cell transplantation.

A thioacetamide-induced liver fibrosis model for pre-clinical studies in microminipig.

Drug-induced liver fibrosis models are used in normal and immunosuppressed small animals for transplantation and regenerative medicine to improve liver fibrosis. Although large animal models are needed for pre-clinical studies, they are yet to be established owing to drug sensitivity in animal species and difficulty in setting doses. In this study, we evaluated liver fibrosis by administering thioacetamide (TA) to normal microminipig and thymectomized microminipig; 3 times for 1 week (total duration: 8 weeks). The pigs treated with TA showed elevated blood cytokine levels and a continuous liver injury at 8 weeks. RNA-seq of the liver showed increased expression of fibrosis-related genes after TA treatment. Histopathological examination showed degenerative necrosis of hepatocytes around the central vein, and revealed fibrogenesis and hepatocyte proliferation. TA treatment caused CD3-positive T cells and macrophages scattered within the hepatic lobule to congregate near the center of the lobule and increased αSMA-positive cells. Thymectomized pigs showed liver fibrosis similar to that of normal pigs, although the clinical signs tended to be milder. This model is similar to pathogenesis of liver fibrosis reported in other animal models. Therefore, it is expected to contribute to research as a drug discovery and pre-clinical transplantation models.

Local elasticity evaluation of acid-denatured collagen by photoacoustic spectroscopy.

While there are various analytical methods for elasticity evaluation, those with micrometer-order spatial resolution are still under developing. As some of biological tissues such as capillary vessels and cochlea are very small and/or highly heterogeneous, development of analytical techniques with such high spatial resolution has been desired for biological and medical purposes. Especially, the elasticity of capillary vessels (several micrometer in diameter) would be an important indicator to find out early diseases. To measure the local elasticity for such small and/or heterogeneous samples, we have proposed an approach based on a temporal waveform of photoacoustic (PA) signal, i.e., time-domain PA. As the time-domain PA contains both the vibrating frequency and the sound propagation time after the excitation, it provides the information on the local elasticity (from the frequency) at a specific depth (from the propagation time) of samples. In the present study, the signal from collagen sheets were obtained and analyzed as models of blood vessel walls and scaffolds for regenerative medicine. In contrast to previous studies using the agarose gel which showed a single frequency peak, the signal from the collagen sheets was mainly composed of two frequency peaks, assignable to surface and bulk vibration. Further, the bulk vibration was found to sensitively reflect the elasticity of the samples. Since the PA effect can be induced only at the position where the light absorber exists, the analytical method proposed here would allow us to measure the local elasticity and its spatial distribution in blood vessels and other tissues.

Injectable extracellular matrix hydrogels contribute to native cell infiltration in a rat partial nephrectomy model.

Decellularized extracellular matrix (dECM) hydrogels have cytocompatibility, and are currently being investigated for application in soft tissues as a material that promotes native cell infiltration and tissue reconstruction. A dECM hydrogel has broad potential for application in organs with complex structures or various tissue injury models. In this study, we investigated the practical application of a dECM hydrogel by injecting a kidney-derived dECM hydrogel into a rat partial nephrectomy model. The prepared dECM hydrogel was adjustable in viscosity to allow holding at the excision site, and after gelation, had an elastic modulus similar to that of kidney tissue. In addition, the migration of renal epithelial cells and vascular endothelial cells embedded in dECM hydrogels was observed in vitro. Four weeks after injection of the dECM hydrogel to the partial excision site of the kidneys, infiltration of renal tubular constituent cells and native cells with high proliferative activity, as well as angiogenesis, were observed inside the injected areas. This study is the first to show that dECM hydrogels can be applied to the kidney, one of the most complex structural organs and that they can function as a scaffold to induce angiogenesis and infiltration of organ-specific renal tubular constituent cells, providing fundamental insights for further application of dECM hydrogels.

Decellularized Organ-Derived Scaffold Is a Promising Carrier for Human Induced Pluripotent Stem Cells-Derived Hepatocytes.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for elucidating disease pathology and therapy. The mass supply of hiPSC-derived cells is technically feasible. Carriers that can contain a large number of hiPSC-derived cells and evaluate their functions in vivo-like environments will become increasingly important for understanding disease pathogenesis or treating end-stage organ failure. hiPSC-derived hepatocyte-like cells (hiPSC-HLCs; 5 × 108) were seeded into decellularized organ-derived scaffolds under circumfusion culture. The scaffolds were implanted into immunodeficient microminiature pigs to examine their applicability in vivo. The seeded hiPSC-HLCs demonstrated increased albumin secretion and up-regulated cytochrome P450 activities compared with those in standard two-dimensional culture conditions. Moreover, they showed long-term survival accompanied by neovascularization in vivo. The decellularized organ-derived scaffold is a promising carrier for hiPSC-derived cells for ex vivo and in vivo use and is an essential platform for regenerative medicine and research.

An organ-derived extracellular matrix triggers in situ kidney regeneration in a preclinical model.

It has not been considered that nephrons regenerate in adult mammals. We present that an organ-derived extracellular matrix in situ induces nephron regeneration in a preclinical model. A porcine kidney-derived extracellular matrix was sutured onto the surface of partial nephrectomy (PN)-treated kidney. Twenty-eight days after implantation, glomeruli, vessels, and renal tubules, characteristic of nephrons, were histologically observed within the matrix. No fibrillogenesis was observed in the matrix nor the matrix-sutured kidney, although this occurred in a PN kidney without the matrix, indicating the structures were newly induced by the matrix. The expression of renal progenitor markers, including Sall1, Six2, and WT-1, within the matrix supported the induction of nephron regeneration by the matrix. Furthermore, active blood flow was observed inside the matrix using computed tomography. The matrix provides structural and functional foundations for the development of cell-free scaffolds with a remarkably low risk of immune rejection and cancerization.

Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model.

Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.

Development of dark-field dynamic light scattering microscopy and its application: Tracking dynamics of particles in condensed slurries spreading on planar/nonplanar substrates.

HYPOTHESIS: While dynamics of particles in slurries is usually evaluated by dynamic light scattering measurements, this technique had only been applicable to particles in the bulk slurries. Because this limitation is mainly owing to strong reflection of light, the dynamics of particles in slurries spreading/drying on solid substrates is to be obtained by spatially separating the reflection light from scattering (signal) light. This may allow us to track the particles in practical samples such as cosmetics or inks spreading on solid surfaces. EXPERIMENT: We developed novel "dark-field dynamic light scattering microscopy". The system was evaluated with test samples of polystyrene beads dispersed in several viscosities of bulk glycerol aqueous solutions. This setup was then applied to slurries spreading/drying on planar and nonplanar substrates. FINDINGS: The results for planar surface indicate that origin of coffee-ring are the particles flowing into the edge of the droplet just before complete drying. On a skin-modelled nonplanar substrate, the slurry on bumps was found to maintain semi-dry condition longer than that at dents. This suggests that the dispersive medium was supplied to bumps from dents. This unique flow was explained as effective drying from the bumps increased surface tension at the bumps to pull up the liquid around.

Characterization of the Elasticity of Small Objects Buried in Media Based on the Measurements of Photoacoustic Temporal Waveforms.

Since the elasticity of biological tissues is related to their pathological states, the development of new methods allowing for non-invasive measurements of the elasticity has been desired in the medical field. We present a characterization of the elasticity of objects buried in media from the temporal waveforms of photoacoustic signals. As the increment in Young's moduli of the objects, the frequency corresponding to the gravitational center of the power spectra obtained by the Fourier-transformation of the waveforms is increased. In our experiment configuration, the elasticity of buried objects is able to be identified up to about 1 MPa of Young's modulus from the frequency. These results suggest that measurements on the temporal waveforms of photoacoustic signals and the resultant power spectra would provide a useful method for evaluating the elasticity of deeply-situated microscopic pathological lesions, such as stage 0 or 1 mammary gland cancer, which is difficult by conventional ultrasound elastography.

Dynamic Light Scattering Measurements for Soft Materials on Solid Substrates: Employing Evanescent-wave Illumination and Dark-field Collection with a High Numerical Aperture Microscope Objective.

We developed an instrument that allows us to measure dynamic light scattering from soft materials on solid substrates by avoiding strong background due to the reflection light from substrates. In the instrument, samples on substrates are illuminated by evanescent-light field and the resultant scattered light from the samples is collected with a dark-field optical configuration by employing a high numerical aperture microscope objective. We applied the instrument to measure the dynamic properties of supported lipid bilayers (SLBs), which have been widely utilized in industries as functional materials such as biosensors. From the time course of the scattered light from the SLBs, the power spectrum with the broad peak ranging from 10 to 20 kHz is observed. The use of the microscope objectives enables us to apply the instrument to future light scattering imaging for dynamic properties of soft materials supported on various substrates by combining with conventional microscope systems.

Development of a Near-infrared Laser-induced Surface Deformation Microscope and Its Application to the Dynamic Viscoelastic Measurements of Single Living Plant Cell Surfaces.

A near-infrared laser-induced surface deformation (NIR-LISD) microscope is developed and is applied to the dynamic viscoelastic measurements of the surface of a living plant cell. In the microscope, the deformation of the surface is induced by an NIR laser beam, and then the change in intensity of the probe beam reflected from the surface reflects its viscoelasticity. The application of the NIR laser beam has a great advantage for the prevention of damage to the plant cell compared to the irradiation of a visible laser beam in LISD measurements. The NIR-LISD microscope allows for discriminating the differences in power spectra between the subapical and lateral regions of single rhizoids. It is a useful method for the dynamic viscoelastic measurements of cells, such as plant cells, that are damaged due to the strong absorption of ultraviolet or visible light.

Mechanical Properties of the Coat Protein Layer and Cortex in Single Bacillus subtilis Spores Studied with an Atomic Force Microscope and Laser-induced Surface Deformation Microscope.

The differences in the mechanical properties between the cortex and coat protein layer in a Bacillus subtilis spore were clarified using an atomic force microscope (AFM) and an originally developed laser-induced surface deformation (LISD) microscope. AFM force curve measurements show that the Young's modulus of the coat protein layer is ca. 66% lower compared with that of the cortex. It has been experimentally clarified that the cortex makes a greater contribution to the rigidity of spores than the coat protein layer. From comparisons of the LISD power spectra, it is revealed that the coat protein layer has two different viscoelastic regions, and that the cortex relatively has a higher viscous nature than the coat protein layer. Furthermore, the LISD power spectrum above 1 × 105 Hz in the coat protein layer suggests that the local region in the coat protein layer behaves as a more elastic body compared with the cortex.

Discrimination between Normal and Cancerous Cells from Dynamic Viscoelastic Properties with a Laser-induced Surface Deformation Microscope.

We have clarified the differences in the power-law between normal and corresponding cancerous cells from dynamic viscoelastic measurements in a frequency range of 102 to 105 Hz with a laser-induced surface deformation (LISD) microscope. From the differences in the power spectra at higher frequencies, it has been clarified that a normal cell obeys the power-law with a single exponent, while a cancer cell with two exponents, indicating that the plasma membrane in the cancerous cell has at least two layers with different viscoelastic properties. In LISD measurements, the extension of the upper limit of the applied frequency up to 105 Hz allows us to clarify the existence of the two power-law exponents in the cancerous cell. Understanding the differences between normal and cancerous cells from the power-law in addition to conventional elasticity would be useful for the identification of cancerous cells and for the construction of a mechanical model for their invasion.

Laser-induced surface deformation microscope for the study of the dynamic viscoelasticity of plasma membrane in a living cell.

A laser-induced surface deformation (LISD) microscope is developed and applied to measurement of the dynamic relaxation responses of the plasma membrane in a living cell. A laser beam is tightly focused on an optional area of cell surface and the focused light induces microscopic deformation on the surface via radiation pressure. The LISD microscope not only allows non-contact and destruction-free measurement but provides power spectra of the surface responses depending on the frequency of the intensity of the laser beam. An optical system for the LISD is equipped via a microscope, allowing us to measure the relaxation responses in sub-cellular-sized regions of the plasma membrane. In addition, the forced oscillation caused by the radiation pressure for surface deformation extends the upper limit of the frequency range in the obtained power spectra to 106 Hz, which enables us to measure relaxation responses in local regions within the plasma membrane. From differences in power-law exponents at higher frequencies, it is realized that a cancerous cell obeys a weaker single power-law than a normal fibroblast cell. Furthermore, the power spectrum of a keratinocyte cell obeys a power-law with two exponents, indicating that alternative mechanical models to a conventional soft glassy rheology model (where single power-laws explain cells' responses below about 103 Hz) are needed for the understanding over a wider frequency range. The LISD microscope would contribute to investigation of microscopic cell rheology, which is important for clarifying the mechanisms of cell migration and tissue construction.

α-Melt Structure of 1,3-Dipalmitoyl-2-oleoyl-sn-glycerol (POP) under a Thermal Thawing Process Studied by Infrared Spectroscopy.

The temperature thawing, as called tempering, of triacylglycerols (TAGs) is an important processing method in food productions, such as chocolates, cream, confections, and spreads. Especially, melt-mediation by temperature thawing is famous in chocolate production for controlling the polymorphic crystalline forms and accelerating crystallization. In the present study, we investigated the α-melt structure of 1,3-dipalmitoyl-2-oleoyl-sn-glycerol (POP), one of the major continuants of cacao butter, under a phase transition from its melt to γ-crystal with in-situ attenuated total reflection-infrared (ATR-IR) spectroscopy. The differential IR spectrum between α-melt via temperature thawing (α-melt mediation) and melt via simple cooling revealed that crystal-like local ordered structures remained in part in the α-melt, and that they acted as nuclei for a rapid phase transition to the γ-crystal. The changes to the γ-crystal occur in the local ordered structures at first from the glycerol moiety to the acyl chains in the crystallization, providing an important suggestion concerning the mechanism for the acceleration of crystallization to the γ-form via α-melt mediation.

Structural Discrimination between Aß(1-40) and Aß(1-42) Peptides in Films with Vibrational Circular Dichroism Spectroscopy.

The secondary structure of full-length Aß(1-40) and Aß(1-42) peptides in films has been investigated with IR and vibrational circular dichroism (VCD) spectroscopy. From IR spectra, it is shown that the prepared films of Aß(1-40) and Aß(1-42) mainly comprise the ß-sheet conformation that is characteristic of aggregated and fibrous Aß. In the VCD spectra, the Aß(1-42) film shows an intense and sharp band with left-handed optical activity at around 1625 cm-1, while the Aß(1-40) film shows a weak and broad band with right-handed activity at around 1630 cm-1. The wavenumbers are characteristic of the ß-sheet conformation. It has been clarified that the aggregated Aß(1-42) adopts the ß-sheet conformation with reverse optical activity compared with the aggregated Aß(1-40). The left-handed optically active ß-sheet of the aggregated Aß(1-42) may contribute to the formation of protofibrils, which are a cause for the higher neurotoxicity of Aß(1-42) fibrils than Aß(1-40) fibrils.

Structural Transition of Bombyx mori Liquid Silk Studied with Vibrational Circular Dichroism Spectroscopy.

We investigated the structural transition from liquid silk to silk fibers with vibrational circular dichroism spectroscopy. Liquid silk showed a major right-handed optically active band at around 1650 cm(-1) and a minor one at around 1680 cm(-1). The former disappeared over time, while the intensity in the latter increased. With the former wavenumber, liquid silk mainly adopted a random-coil structure. In contrast, the latter may reflect an intermediate structure in the transition. Furthermore, two right-handed bands at around 1630 and 1660 cm(-1) appeared with the disappearance of the major band, and then the wavenumber of the former shifted to around 1620 cm(-1). The shift results from the decrease in the frequency of the CO stretching mode due to the stacking of the ß-sheet that comprises fibers. The band at 1660 cm(-1) may reflect another intermediate structure due to its strong correlation with that at 1620 cm(-1) in terms of their temporal change in intensity.

Dehydration-induced initial conformational change of hydrated proteins detected by the changes in vibrational circular dichroism activity.

Conformational changes of hydrated proteins induced by gradual dehydration were monitored by vibrational circular dichroism (VCD) spectroscopy. In myoglobin and casein, representative α-helix-rich and random-coil proteins, respectively, an increase in left-handed optical activity in the amide I band was detected at the initial stage of dehydration, followed by an increase in opposite right-handed activity in both the amide I and II bands with further dehydration. Because the second step was observed with an increase in the turbidity of the proteins, it can be attributed to their aggregation. In contrast, because the increase in left-handed optical activity is induced by the conformational change of the proteins and is followed by the aggregation, it may derive from the increase in the regularity of the local structure in individual myoglobin or casein that triggers the aggregation.

Analysis of aquaporin-mediated diffusional water permeability by coherent anti-stokes Raman scattering microscopy.

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P(f)), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P(d)). Here, we report an accurate and instantaneous method for measuring the P(d) of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H(2)O/D(2)O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τ(CARS)) for the external solution H(2)O/D(2)O exchange was 16.1 ms, whereas the intracellular H(2)O/D(2)O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τ(CARS) for the intracellular H(2)O/D(2)O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P(d). The average P(d) values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10(-3) and 8.3 ± 2.6 × 10(-4) cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs.

Drosophila big brain does not act as a water channel, but mediates cell adhesion.

The neurogenic gene Drosophila big brain (bib) has a high sequence homology to aquaporin-4. However, its cellular functions in Drosophila neurogenesis have remained elusive. Here we investigated cell adhesion, and the ion and water permeability of Bib. The adhesive function was examined by a cell aggregation assay using L cells. Bib-transfected L cells formed aggregated clusters, while control-L cells remained as a single cell suspension. Ion permeation was not confirmed in L cells stably expressing Bib. When expressed in COS7 cells, Bib exhibited limited water permeability. This newly found cell adhesive function of Bib may be important for Drosophila neurogenesis.