F344 njcl rnu rnu

Extracellular field potential (EFP) of cell sheet constructs before and after TX was measured by the difference between electric potentials of two sensor electrodes with 1 mm distance (Research electrode SCR-2; unique medical co). The electric potential was amplified (bio-signal amplifier unit; unique medical co) and recorded (UAS-3088; unique medical co). Just before TX of 5-GHM constructs, EFP was measured by direct attachment of the sensor electrodes to the cell sheet constructs in a medium (sheet EFP (in vitro)). Then, the constructs were transplanted to the normal hearts of male athymic nude rats (F344/N Jcl-rnu/rnu, CLEA Japan, Inc., Tokyo, Japan) aged 12 weeks. EFP of the transplanted cell constructs was measured by direct attachment of the electrodes on the surface of the cell constructs (sheet EFP (heart)) with simultaneous recording of the rat electrocardiogram (ECG).

For the human fecal sample, we used a 15% (wt/vol) suspension of feces, obtained at the acute phase, from a Japanese patient who contracted a domestic infection of genotype 3b HEV (JE03-1760F), which was successfully used for the development of a cell culture system for HEV [41 (link)]. For the rat fecal sample, we used a 15% suspension of feces obtained from a nude rat (F344/NJcl-rnu/rnu (CLEA Japan, Inc., Tokyo, Japan)) experimentally infected with a rat HEV strain (ratELOMB-131), which was successfully used to develop a cell culture system for rat HEV [42 (link)]. A 15% fecal suspension in 10 mM phosphate-buffered saline, with a pH of 7.5 (PBS), was centrifuged at 6200× g for 5 min at 4 °C, and the resulting clear supernatants were aliquoted and stored at −80 °C until use. They were then thawed on ice, filtrated through microfilters with a pore size of 0.2 mm (Millex-GV; Merck Millipore, Billerica, MA, USA), and subjected to assays.

The plate-shaped tissue was fabricated on one layer of needles on a large size pinholder with a central lumen. After 5 days of cultivation (2 days of spheroid formation and 3 days of cultivation on needles), the liver-like tissue was transplanted onto the transection plane of the liver in nude rats (F344/NJcl-rnu/rnu, 200–250 g, male, CLEA Japan, Tokyo, Japan). For the functional analysis of the transplanted liver tissue, blood samples were collected to measure the amount of human-specific albumin that was produced in the recipient rats. For the histological analysis, the livers of the recipient rats were removed, and the animals were euthanized.

All animal experiments and care were conducted humanely in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The left anterior descending artery of athymic nude rats (F344/NJcl-rnu/rnu; CLEA Japan, Tokyo, Japan) was permanently ligated through left thoracotomy under general anesthesia administered via endotracheal intubation. After 2 weeks, rats underwent re-thoracotomy for the exposed pericardial space. Rats were randomly divided into a sham group or two cell-sheet-transplanted groups divided as per myoblasts or NMCs (n = 11 each). Myoblasts or NMCs were cultured on temperature-responsive culture dishes (CellSeed, Tokyo, Japan) for 1 day at 37 °C in the growth medium, and cell sheets were harvested by reducing the temperature to room temperature. Cell sheets were transplanted over the epicardium of the anterior and lateral left ventricular walls.

Male nude rats (F344/NJcl-rnu/rnu (CLEA Japan, Inc.); 8–10 weeks of age) underwent left coronary artery ligation as previously described [37 (link), 40 (link)]. The rats were anesthetized by inhalation of isoflurane and placed on a respirator during surgery to maintain ventilation. After 2 weeks, echocardiography was performed to evaluate the extent of MI, and rats with an EF greater than 30% and less than 50% were used as MI model rats. The MI model rats were randomly divided into three treatment groups: a fresh group (cell sheets used without further treatment, n = 11), vitrification group (n = 11), and sham-operation group (n = 10). The cell sheets were transplanted onto the infarct area in the fresh and vitrification groups. In the sham-operation group, rats were subjected to thoracotomy, and the chest was closed without transplantation. The effect of the transplantation of various sheets on cardiac function was evaluated by echocardiography, including estimates of the EF, FS, ΔLVDd, ΔLVDs, and ΔIVSd at 2 and 4 weeks after transplantation. The rats were anesthetized by inhalation of isoflurane to alleviate suffering and the hearts were quickly procured to determine the extent of fibrosis of the left ventricle (%), the length of the minor axis of the cells (μm), and the number of microvessels (per mm²).