CRYOBIOLOGY

27,576-W

(1990)

BRIEF COMMUNICATION A New Approach to the Cryopreservation of Hepatocytes in a Sandwich Culture Configuration HANS G. KOEBE,* JAMES C. Y. DUNN,* MEHMET TONER,+8 LAURA M. STERLING,* ALLISON HUBEL,$ ERNEST G. CRAVALHO,t$ MARTIN L. YARMUSH,*4 AND RONALD G. TOMPKINS* *Surgical Service, Massachuselts General Hospital, Department of Surgery. Harvard Medical School, Bostoq Massachusetts 02114; fDepartment of Biomedical Engineering, Massachusetts General Hospitnl, Boston, Mussachusetts 02114; SHarvard University-Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, Massachusetts 02139; and #Department of Chemical and Biochemical Engineering and the Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854 Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term preservation of hepatocellular functions. A novel culture method has recently been developed to culture liver cells in a sandwich configuration of collagen layers in order to stabilize the phenotypic expression of these cells in vitro (J. C. Y. Dunn, M. L. Yarmush, H. G. Koebe, and R. G. Tompkins, FASEB J. 3, 174, 1989). Using this culture system, rat hepatocytes were frozen with 15% (v/v) Me,SO to -7o”C, and stored at - - 100°C. Following rapid thawing, long-term function was assessed by measuring albumin secretion in culture for 7-14 days postfreezing. Comparison was made with cryopreservation of liver cells in single cell suspensions. Cryopreservation of liver cells in suspension resulted in only a 2% yieId of cells which could be successfully cultured; albumin secretion rates in these cultured cells over 48 hr were 26-30% of secretion rates for nonfrozen hepatocytes. Freezing cultured liver cells in the sandwich cotiguration after 3, 7, and 11 days in culture maintained 0, 26, and 19% of the secretion rates of nonfrozen hepatocytes, respectively. Morphology of the cryopreserved cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented -30% of the cells in culture. These results represent the first demonstration of any quantitative long-term preservation of hepatocellular function by cryopreservation, suggesting that cultured hepatocytes can survive freezing and maintain function. o 1990Academic PESS, hc.

Temporary liver support has been attempted using many approaches which combine hepatocytes or hepatic tissue with mechanical devices (10, 11, IS, 18, 20, 21). Although the use of hepatocytes represents a promising approach to temporary replacement of liver function, the ultimate success of these systems relies heavily on a readily available supply of large numbers of hepatocytes (-10” cells) when the necessity for liver support arises (20). Since hepatocytes do not generally undergo cell division in Received August 21, 1989; accepted November 29, 1989.

culture and, therefore, do not grow in tissue culture, maintaining a supply of hepatocytes by tissue culture techniques is not feasible. Also, it is not practical to obtain fresh liver cells in large numbers from animals and particularly from humans each time liver support is necessary, Therefore, cryopreservation is critical for providing a stable and available supply of these cells, and it allows pooling of isolates from multiple donors such that sufficient numbers of cells may accumulate over time. Controversy exists over the current state of the art for cryopreservation of hepatocytes. Novicki et al. (17) using standard

576 @Ill-2240/90 $3.00 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

BRIEF

577

COMMUNICATION

freezing methods, dye exclusion techniques, and quantitation of cytochromes found comparable viability and quantity of cytochromes for cryopreserved and nonfrozen cells. Some cryopreserved cells attached to dishes and survived at least 24 hr. Of the surviving cells, a relatively high quantity of cytochrome P450 per milligram of microsomal protein was observed. A similar experience in which no apparent ultrastructural damage was seen in hepatocytes frozen using a high freezing rate (39”Wmin) was reported by Gomez-Lechon et al. (6)+ Using vital dye staining, plating efficiency, and short-term rates of gluconeogenesis, ureogenesis, and protein synthesis, frozen cells were comparable to nonfrozen cells. In contrast, others have found significant alterations in the ultrastructure and functional activities of the hepatocytes after cryopreservation (3, 4, 8, 14). In particular, important synthetic processes such as gluconeogenesis (14) and protein synthesis (3) were diminished, and cellular attachment was dramatically impaired immediately following cryopreservation (8). Jackson et al. (9) observed that cytochrome P450 activity was maintained in hepatocytes surviving cryopreservation; however, only 40% of the cells were considered viable following freezing, Inabe et al. (7) showed that aminopyrine metabolism (cytochrome P450 activity) was reduced to 10% of the original activity after 7 days of freezing. Using another long-term measure of viability (successful transplantation of single cells into spleen), Fuller et al. (5) and Kusano et al. (13) demonstrated that only a sparsity of cells remained 1 month after transplantation and cryopreservation. The present report describes a cryopreservation method for hepatocytes in which the liver cells have been cultured in monolayers sandwiched between two layers of collagen gel. This technique has been demonstrated to preserve hepatocyte function and longevity in vitro (1). Using this culture system, cryopreservation techniques were

optimized with albumin secretion and morphology as viability markers for the cryopreserved cells. Hepatocyte morphology and 26% of albumin secretion were maintained after freezing following 7 days of prefreeze culture using this new approach. MATERIALS

AND

METHODS

Hepatocyte isolation, purification, culture, and viability assays. Hepatocytes were obtained from 2-month-old, female Lewis rats (200 g) by a modified procedure of Seglen (19). Using ether anesthesia, the liver, weighing -8 g, was perfused in situ with 300 ml of Ca’+-free Krebs Ringer bicarbonate buffer, containing 5.5 mM glucose and 20 mM Hepes + EDTA (1 m&f), pH 7.4, at 50 ml/min. The perfusate was maintained at 37°C and was equilibrated with 95% O2 and 5% CO*. The liver was subsequently perfused ex vivo with 0.05% collagenase (Type IV, Sigma, St. Louis, MO) solution containing 5 mM CaZ+ for 10 min in a recirculation system, The resulting cell suspension was filtered through two nylon meshes (Small Parts, Miami, FL), with grid sizes 210 and 62 km. The cell pellet was collected by centrifugation at 17g for 5 min. Hepatocytes were further purified by a modified procedure of Kreamer et al. (12). Briefly, the cell pellet was resuspended into 50 ml of Dulbecco’s Modified Eagle Medium (DMEM , Hazelton, Lenexa, KS) and 12.5 ml of cell suspension was added to 10.8 ml of Percoll (Pharmacia, Piscataway, NJ) and 1.2 ml of 10x DMEM (pH 7.4). The mixture was centrifuged at 50g for 5 min, and the cell pellet was washed twice with DMEM. Routinely, 200 to 300 million cells were isolated from an 8 g liver, with viability ranging from 90 to 98% as assessed by trypan blue exclusion. Hepatocytes were cultured on gelled rat tail tendon collagen which was prepared by a modified procedure of Eldsale et al. (2). Briefly, four tendons were dissected from each rat tail and were stirred in 200 ml of 3% acetic acid overnight at 4°C. The solu-

578

BRIEF

COMMUNICATION

tion was filtered through four layers of (I .4 ml) at 4°C. After 5 min and at 5-min cheese cloth and was centrifuged at 12,OOOg intervals thereafter, 0.15 ml of Me+0 at for 2 hr. The supernatant was precipitated 20” was added to the cultures until a total of with one-fifth volume of 30% NaCl, and the 0.6 ml of Me,SO had been added to the 4-ml pellet was collected by centrifugation at culture system (2 ml media and two I ml 4OOOg for 30 min. After two washes with 5% collagen gel layers). The final Me,SO conNaCl and 0.6% acetic acid, the pellet was centration was 15% (v/v). T-flasks were redissolved in 0.6% acetic acid. The solu- transferred to a programmable freezing unit tion was dialyzed against 1 mM HCl and (Cryomed, Model 1010) and held at 4°C for was sterilized with chloroform. A 5-ml ali- 10 min. The culture systems were cooled at quot was lyophilized to determine the con- S”C/min to -20°C which resulted in the centration. Generally, 150 mg was isolated spontaneous crystallization of the extracellular solution. After 30 min at - 20°C to alfrom one rat tail. Hepatocytes were cultured between two low for dissipation of latent heat of crystallayers of rat tail collagen in a sandwich con- lization, the unit temperature was defiguration as previously described in Ref. creased at 1”Clmin to -70°C. Cell cultures (1) and modified for T-flask cultures. were then stored in the vapor phase of liqBriefly, plates were prepared by distribut- uid nitrogen (7 - 1OO“C)for either 30 min ing 1 ml of collagen gel (1 mg/ml) evenly or 24 hr. T-flasks were removed from storover a 25 cm2 tissue culture T-flask at least age and DMEM containing 10% MezSO (4 one hour before use. Four million viable ml) at 37°C was added to the T-flask on a cells were seeded in 3 ml of complete me- hot plate at 37°C as rapidly as possible and dium, consisting of DMEM (4.5 giliter glu- thawing occurred in

A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration.

Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term pre...
2MB Sizes 0 Downloads 0 Views