Yoshitaka Miyamoto

Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells.

Quantum dots (QDs) have been used to study the effects of fluorescent probes for biomolecules and cell imaging. Adipose tissue-derived stem cells, which carry a relatively lower donor site morbidity, while yielding a large number of stem cells at harvest, were transduced with QDs using the octa-arginine peptide (R8) cell-penetrating peptide (CPP). The concentration ratio of QDs:R8 of 1 x 10(4) was optimal for delivery into ASCs. No cytotoxicity was observed in ASCs transduced with less than 16 nM of QDs655. In addition, >80% of the cells could be labeled within 1 h and the fluorescent intensity was maintained at least for 2 weeks. The ASCs transduced with QDs using R8 could be differentiated into both adipogenic and osteogenic cells, thus suggesting that the cells maintained their stem cell potency. The ASCs labeled with QDs using R8 were further transplanted subcutaneously into the backs of mice or into mice through the tail vein. The labeled ASCs could be imaged with good contrast using the Maestro in vivo imaging system. These data suggested that QD labeling using R8 could be utilized for the imaging of ASCs.

Monitoring transplanted adipose tissue-derived stem cells combined with heparin in the liver by fluorescence imaging using quantum dots.

Adipose tissue-derived stem cell (ASC) transplantation, when used in combination with heparin, has proven to be an effective treatment for acute liver failure in mice. However, the behavior and organ-specific accumulation of transplanted ASCs alone or in combination with heparin is poorly understood. In this paper, we investigated whether quantum dots (QDs) labeling using octa-arginine peptide (R8) for ASCs could be applied for in vivo fluorescence imaging in mice with acute liver failure, and analyzed the behavior and organ-specific accumulation of ASCs that were transplanted alone or in combination with heparin using an IVIS(®) Spectrum analysis. Almost all of the transplanted ASCs were observed to accumulate in the lungs within 10 min without heparin. However, when heparin was used in combination with the ASCs, the accumulation of the transplanted ASCs was found not only in the lungs but also in the liver. The region of interest (ROI) analysis of ex vivo fluorescence imaging showed that the accumulation rate of transplanted ASCs in the liver increased to about 30%. In the time course analysis, the accumulation rate of ASCs in the liver was about 10% in 1 day and was maintained at that level for at least 2 day. We observed that heparin was effective for increasing the accumulation of transplanted ASCs in the liver using fluorescence imaging technology. We suggest that fluorescence imaging by means of QDs labeling using R8 can be useful for tracing the transplanted cells.

Improvement of hepatocyte viability after cryopreservation by supplementation of long-chain oligosaccharide in the freezing medium in rats and humans.

Factors affecting cell viability, plating efficiency, and survival of hepatocytes after cryopreservation have been investigated. We focused especially on the effect of including trehalose and related oligosaccharides in the cryopreservation fluid. This was supplemented with either glucose, trehalose, maltotriose, or other sugars, in addition to dimethyl sulfoxide (10%) and first tested with primary rat hepatocytes cooled in a controlled rate freezer. After thawing, viability by trypan blue exclusion of cells frozen in oligosaccharide-supplemented medium was significantly higher than for those cryopreserved without oligosaccharides. Use of oligosaccharides with higher molecular weights resulted in greatest improvement in viability. Moreover, attachment and survival rates in plastic dishes were approximately 1.2–1.8-fold greater after freezing in the presence of di-, tri-, and tetrasaccharides. Human hepatocytes isolated from untransplantable liver showed the same tendency regarding viability, but cell adherence was not similarly improved by the addition of oligosaccharides. Possible reasons for these differences may be prior cell damage during extended cold ischemia of the human liver, donor age, or cell degradation caused by progression of fatty liver in humans, and/or species differences.

Cryopreservation of human adipose tissue-derived stem/progenitor cells using the silk protein sericin.

Adipose tissue-derived stem/progenitor cells (ASCs) have attracted attention as a cell source that replaces marrow stromal cells (MSCs); ASCs may thus have applications in both regenerative medicine and cell transplantation. These medical treatments, however, require a high-quality supply of human ASCs. Therefore, the cryopreservation methods have been improved by changing a component of a cryopreservation medium. Sericin, a protein hydrolysate (with an average molecular weight of 30 kDa) is very rich in serine. The viability and the adipogenic/osteogenic potential of human ASCs were tested after freezing in a cryopreservation medium containing sericin. After thawing, the viability of the human ASCs frozen in the cryopreservation medium was found to be more than 95%. The proliferation rate of human ASCs frozen in CELLBANKER 2, and DMEM/Hams F-12 medium (serum free) + 10% DMSO, 0.1 mol/L maltose, and 1% sericin was higher than that of the cells frozen in the maintenance medium + 10% DMSO. The adipogenic/osteogenic differentiation capabilities of frozen human ASCs were examined by Oil Red O staining/Von Kossas method. The human ASCs were frozen using CELLBANKER 2, and DMEM/Hams F-12 medium (serum free) + 10% DMSO, 0.1 mol/L maltose, and 1% sericin were positive. In conclusion, the cryopreservation medium containing sericin is therefore considered to have a beneficial effect on freezing human ASCs. This serum-free cryopreservation medium should be widely used in regenerative medicine, cell transplantation, and biological research.

Cryopreservation in situ of cell monolayers on collagen vitrigel membrane culture substrata: ready-to-use preparation of primary hepatocytes and ES cells.

Cryopreservation is generally performed on cells in suspension. In the case of adherent cells such as hepatocytes, a loss of their ability to attach is a more serious problem than a decreased viability after cryopreservation. We herein report a novel technology of direct in situ cryopreservation of cells cultured on collagen vitrigel membranes, which have excellent mechanical strength and can be easily handled by tweezers even when coated with cultured cells. Rat primary hepatocytes, mitomycin C-treated mouse fibroblasts (feeder cells for ES cells), and mouse ES cells on the feeder cells were cultured on collagen vitrigel membranes for 1 day. The membranes with cells attached were then plucked up from the dish, soaked in cryopreservation medium containing 10% dimethyl sulfoxide, frozen using a controlled-rate freezer, and transferred to liquid nitrogen. The cells cultured on plastic cell culture dishes were also frozen as controls. After storage in liquid nitrogen for periods from 1 week to 3 months, the cryopreserved membranes with the cells still attached were thawed by adding warmed culture medium. Cell viability estimated by morphology and functional staining with calcein showed significant improvement in comparison to cells cryopreserved without the collagen vitrigel membrane. The recoveries of living cells after cryopreservation were 26.7%, 76.2%, and 58.6% for rat hepatocytes, mitomycin C-treated mouse fibroblasts, and mouse ES cells on collagen vitrigel membranes, respectively. In contrast, essentially no cells at all remained on the plastic cell culture dishes after thawing. Because adherent cell storage under these conditions is very convenient, the use of this technique employing collagen vitrigel membranes should be generally applicable to the cryopreservation of adherent cells that are otherwise problematic to store as frozen stocks.

Effect of Existence of Red Blood Cells in Trapping Performance of Microbubbles by Acoustic Radiation Force

We have proposed a method to control microbubbles by making use of acoustic radiation force, which is generated with acoustic propagation, to correspond to therapeutic applications of ultrasound. By preventing bubbles from passing through the desired target area, the local concentration of bubbles can be enhanced. However, we have never experimentally confirmed this phenomenon under in vivo conditions or close to those. Thus, we carried out an experiment to evaluate the trapping performance of bubbles using a suspension of red blood cells (RBCs) and an artificial blood vessel. By defining the trapping index to evaluate the amount of trapped microbubbles, we have confirmed that the trapping performance was enhanced according to the concentration of RBCs and the sound pressure, but not according to the central frequency of ultrasound. The results indicate that the existence of RBCs near microbubbles contributed to the increase in the size of aggregations propelled against the vessel wall.

Experimental Study to Produce Multiple Focal Points of Acoustic Field for Active Path Selection of Microbubbles through Multi-bifurcation

We previously reported our attempt to propel microbubbles in a flow by a primary Bjerknes force, which is a physical phenomenon where an acoustic wave pushes an obstacle along its direction of propagation. However, when ultrasound was emitted from the surface of the body, controlling bubbles in an against-flow was necessary. It is unpractical to use multiple transducers to produce the same number of focal points because single-element transducers cannot produce more than two focal points. In this study, we introduced a complex artificial blood vessel according to a capillary model and a two-dimensional (2D) array transducer to produce multiple focal points for the active control of microbubbles in an against-flow. From the results, about 15% more microbubbles were led to the desired path with multiple focal points of ultrasound relative to the no-emission case.

Estimation of the distribution of intravenously injected adipose tissue-derived stem cells labeled with quantum dots in mice organs through the determination of their metallic components by ICPMS.

Adipose tissue-derived stem cells (ASCs) have shown promise in cell therapy because of their ability to self-renew damaged or diseased organs and easy harvest. To ensure the distribution and quantification of the ASCs injected from tail vein, several whole-body imaging techniques including fluorescence optical imaging with quantum dots (QDs) have been employed, but they may suffer from insufficient sensitivity and accuracy. Here, we report quantitative distribution of ASCs in various organs (heart, lung, liver, spleen, and kidney) of mice, which were intravenously injected with QDs-labeled ASCs (QDs-ASCs), through the detection of QDs-derived metallic components by inductively coupled plasma mass spectrometry (ICPMS). For accurate and precise determination, each organ was harvested and completely digested with a mixture of HNO(3) and H(2)O(2) in a microwave oven prior to ICPMS measurement, which was equipped with a microflow injection system and a laboratory-made capillary-attached micronebulizer. After optimization, 16 elements including major components (Cd, Se, and Te) of QDs and essential elements (Na, K, Mg, Ca, P, S, Mn, Fe, Co, Cu, Zn, Se, Sr, and Mo) were successfully determined in the organs. As compared to untreated mice, QDs-ASCs-treated mice showed significantly higher levels of Cd and Te in all organs, and as expected, the molar ratio of Cd to Te in each organ was in good agreement with the molar composition ratio in the QDs. This result indicates that the increment of Cd (or Te) can be used as a tracer for calculating the distribution of ASCs in mice organs. As a result of the calculation, 36.8%, 19.1%, 0.59%, 0.49%, and 0.25% of the total ASCs injected were estimated to be distributed in the liver, lung, heart, spleen, and kidney, respectively.

Spheroid Formation and Evaluation of Hepatic Cells in a Three-Dimensional Culture Device

In drug discovery, it is very important to evaluate liver cells within an organism. Compared to 2D culture methods, the development of 3D culture techniques for liver cells has been successful in maintaining long-term liver functionality with the formation of a hepatic-specific structure. The key to performing drug testing is the establishment of a stable in vitro evaluation system. In this article, we report a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be applied as a toxicity evaluation system for drug discovery. The TASCL device was created with an overall size of 10 mm × 10 mm, containing 400 microwells with a top aperture (500 µm × 500 µm) and a bottom aperture (300 µm diameter circular) per microwell. We evaluated the formation, recovery, and size of HepG2 spheroids in the TASCL device. The formation and recovery were both nearly 100%, and the size of the HepG2 spheroids increased with an increase in the initial cell seeding density. There were no significant differences in the sizes of the spheroids among the microwells. In addition, the HepG2 spheroids obtained using the TASCL device were alive and produced albumin. The morphology of the HepG2 spheroids was investigated using FE-SEM. The spheroids in the microwells exhibited perfectly spherical aggregation. In this report, by adjusting the size of the microwells of the TASCL device, uniform HepG2 spheroids were created, and the device facilitated more precise measurements of the liver function per HepG2 spheroid. Our TASCL device will be useful for application as a toxicity evaluation system for drug testing.

In Vivo Imaging of Transplanted Islets Labeled with a Novel Cationic Nanoparticle

To monitor pancreatic islet transplantation efficiency, reliable noninvasive imaging methods, such as magnetic resonance imaging (MRI) are needed. Although an efficient uptake of MRI contrast agent is required for islet cell labeling, commercially-available magnetic nanoparticles are not efficiently transduced into cells. We herein report the in vivo detection of transplanted islets labeled with a novel cationic nanoparticle that allowed for noninvasive monitoring of islet grafts in diabetic mice in real time. The positively-charged nanoparticles were transduced into a β-cell line, MIN6 cells, and into isolated islets for 1 hr. MRI showed a marked decrease in the signal intensity on T1- and T2-weighted images at the implantation site of the labeled MIN 6 cells or islets in the left kidneys of mice. These data suggest that the novel positively-charged nanoparticle could be useful to detect and monitor islet engraftment, which would greatly aid in the clinical management of islet transplant patients.