The bioengineering of perfusable endocrine tissue with anastomosable blood vessels.

Organ transplantation is a definitive treatment for endocrine disorders, but donor shortages limit the use of this technique. The development of regenerative therapies would revolutionize the treatment of endocrine disorders. As is the case for harvested organs, the ideal bioengineered graft would comprise vascularized endocrine tissue, contain blood vessels that could be anastomosed to host vessels, have stable blood flow, and be suitable for transplantation into various sites. Here, we describe a transplantable endocrine tissue graft that was fabricated byex vivoperfusion of tricultured cell sheets (isletß-cells, vascular endothelial cells (vECs), and mesenchymal stem cells (MSCs)) on a vascularized tissue flap ofin vivoorigin. The present study has three key findings. First, mild hypothermic conditions enhanced the success ofex vivoperfusion culture. Specifically, graft construction failed at 37 °C but succeeded at 32 °C (mild hypothermia), and endocrine tissue fabricated under mild hypothermia contained aggregations of isletß-cells surrounded by dense vascular networks. Second, the construction of transplantable endocrine tissue byex vivoperfusion culture was better achieved using a vascular flap (VF) than a muscle flap. Third, the endocrine tissue construct generated using a VF could be transplanted into the rat by anastomosis of the graft artery and vein to host blood vessels, and the graft secreted insulin into the host's circulatory system for at least two weeks after transplantation. Endocrine tissues bioengineered using these techniques potentially could be used as novel endocrine therapies.

Networked lymphatic endothelial cells in a transplanted cell sheet contribute to form functional lymphatic vessels.

This study evaluated whether cell sheets containing a network of lymphatic endothelial cells (LECs) promoted lymphangiogenesis after transplantation in vivo. Cell sheets with a LEC network were constructed by co-culturing LECs and adipose-derived stem cells (ASCs) on temperature-responsive culture dishes. A cell ratio of 3:2 (vs. 1:4) generated networks with more branches and longer branch lengths. LEC-derived lymphatic vessels were observed 2 weeks after transplantation of a three-layered cell sheet construct onto rat gluteal muscle. Lymphatic vessel number, diameter and depth were greatest for a construct comprising two ASC sheets stacked on a LEC/ASC (3:2 ratio) sheet. Transplantation of this construct in a rat model of femoral lymphangiectomy led to the formation of functional lymphatic vessels containing both transplanted and host LECs. Further development of this technique may lead to a new method of promoting lymphangiogenesis.

External pressure dynamics promote kidney viability and perfusate filtration during ex vivo kidney perfusion.

Normothermic machine perfusion (NMP) has not yet been established as a technique for preserving organs for a day. A key contributing factor to the same is that the perfusing solutions cannot circulate continuously and evenly in the organs. Here, we conceived a method of applying intermittent air pressure from outside the organ to assist its circulatory distribution during perfusion. We used a perfusion culture system while applying external pressure to culture rat kidneys and compared the circulatory distribution in the kidneys, changes in tissue morphology due to injury, and perfusate filtration. The intermittent pressurization (IMP) (-) group showed markedly poorer circulation on the upper side compared with that in the lower side, alongside histological damage. On the other hand, the IMP (+) group showed improved circulation in the upper side and had lesser histological damage. Furthermore, the IMP (+) group maintained the ability to filter perfusate for 24 h. In transplantation medicine and regenerative medicine research, this method has the potential to contribute to more efficient organ preservation and more functional tissue regeneration in the future.

Organ Bioluminescence Imaging Under Machine Perfusion Setting for Assessing Quality of Harvested Organ Preservation.

Determination of organ viability over a period of time is a key technology in the process of organ preservation. However, a robust methodology to address this issue has not been established. Luciferase-expressing organs enable the assessment of the variances in organ viability over time as well as the visualization of a damaged tissue region. Herein, we introduce the assessment method for organ viability in detail using luciferase-expressing organs harvested from transgenic Lewis rats (Luc-LEW Tg rats). We exemplify the femoral muscle pedicle flap for the methods of tissue preparation, of setting up the machine perfusion system, and of measuring emitted light to assess organ viability. This evaluation method would be applicable to other organ-preservation studies as an innovative tool for developing a profound understanding of organ preservation.

Inhibitory effects of class I antiarrhythmic agents on Na+ and Ca2+ currents of human iPS cell-derived cardiomyocytes.

INTRODUCTION: Human induced pluripotent stem cells (hiPSCs) harboring cardiac myosin heavy chain 6 promoter can differentiate into functional cardiomyocytes called "iCell cardiomyocytes" under blasticidin treatment condition. While iCell cardiomyocytes are expected to be used for predicting cardiotoxicity of drugs, their responses to antiarrhythmic agents remain to be elucidated. We first examined electrophysiological properties of iCell cardiomyocytes and mRNA levels of ion channels and Ca handling proteins, and then evaluated effects of class I antiarrhythmic agents on their Na+ and Ca2+ currents. METHODS: iCell cardiomyocytes were cultured for 8-14 days (38-44 days after inducing their differentiation), according to the manufacturer's protocol. We determined their action potentials (APs) and sarcolemmal ionic currents using whole-cell patch clamp techniques, and also mRNA levels of ion channels and Ca handling proteins by RT-PCR. Effects of three class I antiarrhythmic agents, pirmenol, pilsicainide and mexiletine, on Na+ channel current (INa) and L-type Ca2+ channel current (ICaL) were evaluated by the whole-cell patch clamp. RESULTS: iCell cardiomyocytes revealed sinoatrial node-type (18%), atrial-type (18%) and ventricular-type (64%) spontaneous APs. The maximum peak amplitudes of INa, ICaL, and rapidly-activating delayed-rectifier K+ channel current were -62.7 ± 13.7, -8.1 ± 0.7, and 3.0 ± 1.0 pA/pF, respectively. The hyperpolarization-activated cation channel and inward-rectifier K+ channel currents were observed, whereas the T-type Ca2+ channel or slowly-activating delayed-rectifier K+ channel current was not detectable. mRNAs of Nav1.5, Cav1.2, Kir2.1, HCN4, KvLQT1, hERG and SERCA2 were detected, while that of HCN1, minK or MiRP was not. The class Ia antiarrhythmic agent pirmenol and class Ic agent pilsicainide blocked INa in a concentration-dependent manner with IC50 of 0.87 ± 0.37 and 0.88 ± 0.16 µM, respectively; the class Ib agent mexiletine revealed weak INa block with a higher IC50 of 30.0 ± 3.0 µM. Pirmenol, pilsicainide and mexiletine blocked ICaL with IC50 of 2.00 ± 0.39, 7.7 ± 2.5 and 5.0 ± 0.1 µM, respectively. CONCLUSIONS: In iCell cardiomyocytes, INa was blocked by the class Ia and Ic antiarrhythmic agents and ICaL was blocked by all the class I agents within the ranges of clinical concentrations, suggesting their cardiotoxicity.