Cryopreservation of primary human hepatocytes: The benefit of trehalose as an additional cryoprotective agent

Abstract

Problems with the limited availability of human hepatocytes for cell transplantation may be overcome by efficient cryopreservation techniques and formation of appropriate cell banking. In this study we investigated the effect of the disaccharide trehalose on the cryopreservation of human hepatocytes. For analysis, liver cells were frozen in culture medium containing 10% dimethyl sulfoxide (DMSO) that was supplemented with varying concentrations of trehalose. During the postthawing culture period, viability, plating efficiency, total protein, cell proliferation, enzyme leakage, albumin and urea formation, as well as phase I and II metabolism were analyzed. In the pilot study, among the concentrations investigated, 0.2 M trehalose showed the best overall outcome. Compared to the use of DMSO alone, we found significant improvement in postthaw cell viability (62.9 Ϯ 13 vs. 46.9 Ϯ 11%, P Ͻ 0.01) and plating efficiency (41.5 Ϯ 18 vs. 17.6 Ϯ 13%, P Ͻ 0.01) in the trehalose group. The use of trehalose as an additive for cryopreserving human hepatocytes resulted in a significantly increased total protein level in the attached cells, higher secretion of albumin and a lower aspartate aminotransferase (AST) level after thawing. In conclusion, the use of trehalose as cryoprotective agent significantly improves the outcome of human hepatocyte cryopreservation. Liver Transpl 13:38-45, 2007.

Sensing and Sensibility: Single‐Islet‐based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules

“…[ 54 ] Due to these unique properties, trehalose has been applied in cryopreservation and freeze-drying of biospecimens in the last few decades. [22][23][24][55][56][57][58][59] In this work, we further investigated the infl uence of trehalose for cryopreservation of islet microcapsules. When trehalose was absent in the cryopreservation medium (culture medium with 10% (v/v) DMSO), freezing and thawing process decreased the ATP values of the islet signifi cantly ( P < 0.05), for both bare islets and encapsulated islets being cryostored for 1 or 7 d (see red and green bars in Figure 3 ).…”

Quality and Availability Evaluation of Human Hepatocytes Isolated from Resected Partial Livers for Toxicology and Drug Metabolism Studies in Korea

“…하지만, 초대배양 1 인체 간세포는 정상 간 조직에서 분리해야 하기 때문에 얻기 가 매우 어렵고, 냉동 보존 효율이 낮아 [2] 현재 국내에서의 활용성이 매우 낮은 상태이다. 대체 세포원으로써 동물 간세 포나 HepG2 세포와 같은 간암 세포주가 사용되고 있지만, 인체 특이적인 약리 특성을 반영하지 못하는 한계를 가지고 있다 [3].…”

Hepatocyte Transplantation

“…21 The addition of trehalose or other related oligosaccharides to the cryopreservation medium improves the post-thaw cell viability and plating efficiency of both rat and human hepatocytes. 23 Trehalose is a naturally occurring (though not found in the human body) disaccharide containing two glucose molecules. This saccharide was tested because lower organisms such as fungi, yeast, bacteria and insects, which have the ability to survive complete freezing and/or drying, accumulate a large amount of trehalose.…”