Efficient human fetal liver cell isolation protocol based on vascular perfusion for liver cell-based therapy and case report on cell transplantation

Abstract

Although hepatic cell transplantation (CT) holds the promise of bridging patients with end-stage chronic liver failure to whole liver transplantation, suitable cell populations are under debate. In addition to hepatic cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being considered as alternative cell sources for initial clinical cell work. Fetal liver (FL) tissue contains potential progenitors for all these cell lineages. Based on the collagenase incubation of tissue fragments, traditional isolation techniques yield only a fraction of the number of available cells. We report a 5-step method in which a portal vein in situ perfusion technique is used for tissue from the late second trimester. This method results in the high viabilities known for adult liver vascular perfusion, addresses the low cell yields of conventional digestion methods, and reduces the exposure of the tissue to collagenase 4-fold. We used donated tissue from gestational weeks 18 to 22, Additional Supporting Information may be found in the online version of this article.

Clinical Hepatocyte Transplantation: Practical Limits and Possible Solutions

“…Hepatocytes have been transplanted into the liver [2,5,6,7,8,9,10,11,12,13,14], spleen [2,3,6,7,15,16,17,] and peritoneal cavity [6,18]. Intraportal infusion is the preferred and most common route for clinical HTx, unless the target organ's architecture is highly altered (e.g.…”

“…HTx has been proven as an effective therapy for a number of acute and chronic diseases, including acute liver failure, cirrhosis, and metabolic liver disease [2,3,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29], with documented improvements in the relevant clinical parameters after the procedure. HTx can be used not only to prolong and improve the quality of life in patients listed for OLT but also as a long-term stand-alone treatment, since it can provide support for metabolic and synthetic liver functions while the native liver regenerates and provide enzyme activity/functions in patients with metabolic liver disease.…”

Role of Chromatin Structural Changes in Regulating Human CYP3A Ontogeny

“…This problem was hard to overcome because of the difficulty in obtaining fetal primary hepatocytes combined with the number of cells needed to prepare sufficient chromatin for ChIP experiments. The fetal hepatic chromatin pool was prepared from second-trimester fetal livers that contained a heterogeneous mixture of hepatocytes, hepatocyte precursors, hematopoietic stem cells, and mesenchymal cells (Mahieu-Caputo et al, 2004;Nava et al, 2005;Gridelli et al, 2012), several of which exhibit low and undetectable CYP3A4 and 3A7 mRNA expression, respectively (Shao et al, 2007). This fetal hepatocyte dilution likely would increase the observed occupancy by modified histones associated with repressed transcription and result in an observed occupancy pattern inconsistent with known CYP3A7 ontogeny.…”

Cell Based Therapy for Chronic Liver Disease: Role of Fetal Liver Cells in Restoration of the Liver Cell Functions

“…Recently a new protocol for efficient and quick isolation of fetal liver cells from 18-24 weeks of gestation was developed. The protocol involved a 5 step portal vein in situ liver perfusion technique using both EGTA and limited exposure to collagenase that resulted in a greater and efficient cell yield [74].…”

“…Direct intrasplenic injection was suggested as a better method to transplant hepatocytes compared to the splenic artery infusion, since the latter led to vascular occlusion with hepatocytes, gastric erosion, and large areas of splenic necrosis [92]. In a recent study fetal cells were transplanted in a patient with end stage cirrhosis via the intrasplenic infusion through the splenic artery leading to improvement in the clinical condition [74]. Studies have demonstrated that after injection into the splenic pulp, most hepatocytes immediately translocate to splenic veins and then to hepatic sinusoids, although hepatocytes trapped in splenic sinusoids may engraft in the spleen itself [93].…”