The ultrastructural characteristics of porcine hepatocytes donated after cardiac death and preserved with warm machine perfusion preservation

Abstract

The effects of warm machine perfusion preservation of liver grafts donated after cardiac death on the intracellular three-dimensional ultrastructure of the organelles in hepatocytes remain unclear. Here we analyzed comparatively the ultrastructure of the endomembrane systems in porcine hepatocytes under warm ischemia and successive hypothermic and midthermic machine perfusion preservation, a type of the warm machine perfusion. Porcine liver grafts which had a warm ischemia time of 60 minutes were perfused for 4 hours with modified University of Wisconsin gluconate solution. Group A grafts were preserved with hypothermic machine perfusion preservation at 8°C constantly for 4 hours. Group B grafts were preserved with rewarming up to 22°C by warm machine perfusion preservation for 4 hours. An analysis of hepatocytes after 60 minutes of warm ischemia by scanning electron microscope revealed the appearance of abnormal vacuoles and invagination of mitochondria. In the hepatocytes preserved by subsequent hypothermic machine perfusion preservation, strongly swollen mitochondria were observed. In contrast, the warm machine perfusion preservation could preserve the functional appearance of mitochondria in hepatocytes. Furthermore, abundant vacuoles and membranous structures sequestrating cellular organelles like autophagic vacuoles were frequently observed in hepatocytes after warm machine perfusion preservation. In conclusion, the ultrastructure of the endomembrane systems in the hepatocytes of liver grafts changed in accordance with the temperature conditions of machine perfusion preservation. In addition, temperature condition of the machine perfusion preservation may also affect the condition of the hepatic graft attributed to autophagy systems, and consequently alleviate the damage of the hepatocytes.

Impact of human-derived hemoglobin based oxygen vesicles as a machine perfusion solution for liver donation after cardiac death in a pig model

“…The storage of energy in mitochondria during preservation is also important. Bochimoto et al [20] described the appearance of abnormal vacuoles and invagination of mitochondria in hepatocytes after 1 h warm ischemia by SEM. In the present study, the hepatocytes preserved by HMP showed extremely swollen mitochondria, while the SNMP+HbV groups had hepatocytes with healthy-looking mitochondria, abundant vacuoles, and membranous structures sequestrating cellular organelles.…”

“…Preparation of osmium macerated samples for observation by a scanning electron microscope was described previously [20]. In brief, a fixative mixture of 0.5% PFA and 0.5% glutaraldehyde in 0.1 MPB (pH 7.4) was macerated with liver samples for 30 min at 4°C.…”

Ultrastructural changes in colonic epithelial cells in a rat model of inflammatory bowel disease

“…Osmium maceration was applied as described (Bochimoto et al, ). Briefly, colon blocks were immersed in 1% OsO 4 for 6 hr followed by 25 and 50% dimethyl sulfoxide, frozen with a flat aluminum block precooled in liquid nitrogen, and broken into two pieces using a screwdriver and a hammer.…”

“…Osmium‐maceration scanning electron microscopy (SEM) allows visualization of three‐dimensional ultrastructural architectures after removing soluble protein (Bochimoto et al, ; Koga, Bochimoto, Kusumi, Ushiki, & Watanabe, ; Tanaka & Naguro, ). The present study compared colonic epithelial cells in rat models of IBD induced by TNBS and control rats using TEM and osmium‐maceration SEM to identified detailed ultrastructural changes.…”

Seasonal ultrastructural changes in apocrine gland cells in back skin of male brown bears (Ursus arctos)

“…The three‐dimensional (3D) ultrastructure of intracellular organelles has been investigated by scanning electron microscopy with osmium‐maceration (OM‐SEM) (Bochimoto et al, 2017; Koga, Bochimoto, Kusumi, Ushiki, & Watanabe, 2017). The 3D architecture of membranous organelles such as mitochondria, rough endoplasmic reticulum (rER), and Golgi apparatus can be assessed at high magnification after soluble protein is removed using a dilute solution of osmium.…”