A Novel Method of Cryopreservation of Rat and Human Hepatocytes by Using Encapsulation Technique and Possible Use for Cell Transplantation

Abstract

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.

Von der Gewebezüchtung zur individuellen Geweberegeneration – Der Mensch als Bioreaktor

“…Damit wird das Ziel erreicht, osteochondrale Strukturen herzustellen, die Knochen und Knorpel kombinieren. Dies Verfahren wurde bei einem Hund, der mit Knochenersatz behandelt wurden musste, erstmals angewandt ; ein entsprechendes Implantat wurde ihm in den Unterkieferknochen eingesetzt (Aoki et al , 2005). Bei diesem Patienten (Abb.…”

“…Diese können durch eine standardisierte Technik kryokonserviert und wieder aufgetaut werden (Abb. 9); sie sind also ohne große Probleme zum Einsatzort transportierbar und haben in ihrer Wirkung keinen gravierenden Funktionsverlust (Arikura et al, 2002;Aoki et al, 2005).…”

Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

“…Most of the previous studies of cryopreservation of microencapsulated hepatocytes used a cryopreservation medium consisting of culture medium, 10% fetal calf serum (FCS), and 10% dimethyl sulfoxide (DMSO) placed into cryovials and stored in liquid nitrogen. [8][9][10]13 Although some of these protocols provided relatively good viability and function after thawing, they are not acceptable for clinical transplantation.…”

“…[8][9][10][11] The challenges are the high water content (>90%) together with the relatively large size of the microbeads and the number of cells compared to single cells. This makes microbeads susceptible to cryodamage by icecrystal formation, leading to cell death.…”

Oxygenated Static Preservation of Donation after Cardiac Death Liver Grafts Improves Hepatocyte Viability and Function

“…However, cell therapy, such as hepatocyte transplantation (HTx), is promising for metabolic liver diseases or as a bridge to OLTx in patients with fulminant liver failure [5,6,7,8]. HTx is technically simple and minimally invasive, provides consistent results, and has been studied in laboratory settings for almost 3 decades [9,10,11,12,13]. Mito et al [14] attempted the first clinical HTx during the early 1990s.…”