Proliferation of rat small hepatocytes after long-term cryopreservation

Journal of Hepatology 37 (2002) 7–14 www.elsevier.com/locate/jhep

Proliferation of rat small hepatocytes after long-term cryopreservation Shinichiro Ikeda 1,2, Toshihiro Mitaka 1,*, Keisuke Harada 1,2, Shinichi Sugimoto 1,3, Kohichi Hirata 2, Yohichi Mochizuki 1 1

Department of Pathology, Cancer Research Institute, Sapporo Medical University School of Medicine, Chu-Ku, S-1, W-17, Sapporo, Japan 2 First Department of Surgery, Sapporo Medical University School of Medicine, Chu-Ku, S-1, W-17, Sapporo, Japan 3 Department of Gastroenterological Surgery, Kyoto University Medical School, Kyoto, Japan

See Editorial, pages 145–146

Background/Aims: The demand for clinical use of hepatocytes is escalating because cell transplantation will be an alternative to orthotopic liver transplantation and the shortage of liver donors is a serious problem throughout the world. However, the supply of fresh differentiated hepatocytes is limited and methods for cryopreservation of hepatocytes that can proliferate with hepatic functions are not satisfactorily established. Methods: Colonies of small hepatocytes were collected and then maintained at 2808C for more than 6 months. Albumin secretion and mRNA expression of thawed cells were measured by enzyme linked immunosorbent assay and Northern blotting, respectively, and the expression of hepatic functions was examined by immunoblotting. The ultrastructure of cryopreserved cells was also examined. Results: About 60% of the cryopreserved colonies attached on dishes and then proliferated. The average area of small hepatocyte colonies was about 7.5 times larger at day 15 than at day 1. Albumin production increased with time in culture. In addition, the cells produced other serum proteins such as transferrin and fibrinogen, and expressed carbamoyl phosphate synthetase I and tryptophan 2,3-dioxygenase. Conclusions: Small hepatocytes maintain growth ability and hepatic differentiated functions even after long-term cryopreservation. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Small hepatocytes; Cryopreservation; Proliferation; Differentiation; Extracellular matrix

1. Introduction The demand for clinical use of hepatocytes is escalating because cell transplantation will be an alternative to orthotopic liver transplantation for many patients suffering from liver cirrhosis, cancers, and genetic disorders [1–3]. The Received 18 July 2001; received in revised form 5 February 2002; accepted 1 March 2002 * Corresponding author. Tel.: 181-11-611-2111 ext. 2391; fax: 181-11615-3099. E-mail address: [email protected] (T. Mitaka). Abbreviations: CPS, carbamoylphosphate synthetase; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; FBS, fetal bovine serum; MH, mature hepatocyte; NPC, non-parenchymal cell; SH, small hepatocyte; TEM, transmission electron microscopy; TO, tryptophan-2,3dioxygenase.

shortage of liver donors is a serious problem throughout the world. Therefore, large quantities of healthy and differentiated hepatocytes are urgently required. In concept, a facility akin to a blood bank in which hepatocytes could be stored and utilized as needed would be ideal. In such a system large quantities of hepatocytes could be stored and utilized as required. However, the supply of fresh, differentiated hepatocytes is limited because, in addition to the shortage of liver donors, methods for the proliferation of primary human hepatocytes and the cryopreservation of hepatocytes have not been satisfactorily established [4]. Many researchers have tried to store intact hepatocytes with retention of metabolic activity by using various cryopreservation agents [5,6], encapsulation techniques [7–9], and regulating the freezing temperature [10,11]. Although

0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S 0168-827 8(02)00069-7

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the viability and hepatic functions of the cells following cryopreservation were similar to freshly prepared ones, the functions rapidly decreased with time in culture [12– 14]. Therefore, it has been thought that the cryopreservation of hepatocytes may be very difficult because liver cells are sensitive and are easily damaged by the freezing and thawing processes. Thus, the establishment of a method for cryopreservation is a long-pursued project in many laboratories. Recently, we showed that a single small hepatocyte (SH) could clonally proliferate and form a large colony [15,16]. The cells can grow and survive for more than 5 months while maintaining the secretion of albumin. In addition, SHs can differentiate into mature hepatocytes (MHs) that interact with hepatic non-parenchymal cells (NPCs) and extracellular matrix (ECM) [16]. The MHs can form three-dimensional structures and bile canalicular formation was observed in those cells. Thus, we think that SHs may be ‘committed progenitor cells’ that can further differentiate into MHs, and that hepatic NPCs and ECM may be largely involved in their maturation. In the present experiment, we show that SHs can survive at 2808C for more than 6 months and, after thawing, proliferate while maintaining hepatic differentiated functions. 2. Materials and methods 2.1. Isolation and culture of SHs Male Sprague–Dawley rats (Shizuoka Laboratory Animal Center, Hamamatsu, Japan), weighing 250–400 g were used. All animals received humane care and the experimental protocol was approved by the Committee of Laboratory Animals according to university guidelines. The methods used for the isolation of parenchymal cells and SH-rich fractions and for the culture of cells were previously described [16]. After the number of viable cells was counted, 6 £ 10 5 and 15 £ 10 5 cells were plated on 60- and 100mm dishes, respectively. Some of the isolated cells were used for cryopreservation as a control.

2.2. The timing of the harvest To collect many SH colonies, we examined the timing for the harvest of the colonies. As previously described [16,17], in these culture conditions SHs rapidly proliferated and formed a colony. NPCs such as stellate cells and liver epithelial cells also proliferated and surrounded SH colonies. With the continuation of culture, SHs in the colony sometimes became large and piled-up. These morphological changes of SHs resulted in a decrease of growth ability and in the acceleration of maturation. In the present experiment 95.3 ^ 5.1% of colonies were monolayered and the average number of SHs in a colony was 33.2 ^ 3.7 at day 10. In addition, more than 90% of SH colonies were surrounded by NPCs at day 14 after plating. Preliminary experiments showed that many cells died during cryopreservation and few colonies grew after thawing when the colonies were harvested before 10 days of cultivation. On the other hand, as the period of the culture increased to more than 16 days, it became difficult to detach cells from dishes without damage. The trypan blue exclusion test showed that the number of dead cells increased when cells cultured for more than 16 days were harvested. Therefore, we decided to harvest cells at days 12–15.

2.3. Collection and cryopreservation of SH colonies Cells were washed with phosphate buffered saline (PBS) twice and then

treated with Hank’s balanced salt solution supplemented with 0.02% ethylene diamine tetraacetic acid (EDTA) for 1 min at room temperature (RT). Thereafter, the cells were incubated with cell dissociation solution (3 ml to 100-mm dish: Sigma Chem. Co., St. Louis, USA) in a 100% air incubator for 15 min at 378C. After the addition of 5 ml of Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, USA), SH colonies were physically detached with weak streams made of the solution several times and collected into 50 ml conical tubes. The suspension was then centrifuged at 50 £ g for 1 min. The pellet was suspended in the medium and recentrifuged. At this time, 99% of the cells were alive. The pellet was suspended in CellBanker (1.5 ml/100-mm dish; NZK Biochemicals, Koriyama, Japan) and 1 ml of the cell suspension was transferred to a cryotube. The tubes were first kept at 2308C in a freezer for 2 h and they were then stored at 2808C until use.

2.4. The efficiency of cell attachment and growth rate of SH colonies After cryopreservation, SH colonies were thawed by rapid immersion in a 378C water bath and then centrifuged at 50 £ g for 1 min. The pellet was suspended in the culture medium. The colonies looked like cell aggregates in the medium and the numbers of the aggregates were counted. Colonies (3 £ 10 3 aggregates/3 ml of medium) were plated on 60-mm dishes and cultured. One day after plating, the medium was changed to a fresh one supplemented with 1% dimethylsulfoxide (Aldrich Chemical Co. Inc., Milwaukee, USA) and then changed every other day. One hour after plating, the number of the colonies per viewing area was counted under a microscope (objective £ 10) and, 24 h after plating, the number of attached colonies using the same dish was counted again. The efficiency of cell attachment was expressed as the percentage of the number of attached colonies at day 1 divided by that of plated colonies at 1 h. The colonies were digitally recorded using a phase-contrast microscope (Olympus Optical Co., Tokyo, Japan) equipped with a CCD camera (Roper Scientific, Trenton, USA), and the area of each colony was measured (IP Lab Spectrume; Scanalytics, Billerca, USA). The growth rate of SH colonies was shown by the percentage of the average area of colonies to that of colonies at day 1. Three separate experiments were carried out and more than 30 colonies per experiment were monitored.

2.5. Enzyme-linked immunosorbent assay (ELISA) for rat albumin Every 48 h after replacement of the medium, the media of three dishes were separately collected into microcentrifuge tubes and they were centrifuged at 1 £ 10 4 rpm for 5 min. The supernatant was kept at 2358C until use. To quantify the secreted rat albumin, we used ELISA as previously described [18].

2.6. Northern blot analysis For electrophoresis, total RNA was extracted from the cells and 20 mg of it was loaded on 1% agarose gel containing 0.5 mg/L of ethidium bromide. For the detection of albumin mRNAs, 32P-labeled RNA probes were prepared from rat albumin cDNA (partial 1.0 kb Pst-I fragment; a gift from M. Sakai) using an RNA labeling kit (Takara, Tokyo, Japan). The details were previously described [16].

2.7. Western blot analysis After washing with PBS twice, the cells were scraped in lysis solution (20 mM Tris–HCl [pH 7.4], 150 mM NaCl, 2.5 mM ethylene diammine tetraacetic acid (EDTA), 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10% glycerol, 1% aprotinin, 20 mg/ ml leupeptin, 1 mM phenylmethylsulfonylfluoride (PMSF), 50 mM NaF, 1 mM Na3VO4, 20 mM Na4P2O4). The cells were kept on ice for 30 min and

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Fig. 1. Thawed SH colonies after 7-month cryopreservation. (A) Colonies after thawing were plated on a culture dish and immediately photographed. Bar shows 100 mm. (B, C) Toluidin blue staining of 7-month cyropreserved cells mounted in agar. Arrows show ECM and arrowheads show NPCs. Stars show dead cells. The nuclei and cytoplasm of dead cells are faintly stained and nuclei are irregularly shaped. The single cell indicated by a red arrow is dead. Bar shows 20 mm.

centrifuged at 15 £ 10 3 rpm for 20 min. The supernatant was kept at 2808C until use and the protein content was measured using a BCA assay kit (Pierce, Rockford, USA). Western blot analysis was carried out as previously described [16]. Samples (20 mg/lane) separated by SDS-PAGE were transferred electrophoretically to a polyvinylidine difluoride membrane (Immobilon-P; Millipore Corp., Bedford, USA) with a Semi-dry Transfer Cell (BioRad Lab.). Rabbit anti-rat transferrin, fibrinogen (Cappel, West Chester, USA), carbamoylphosphate synthetase I (CPS; a gift from Dr W.H. Lamers), and tryptophan 2,3-dioxygenase (TO; a gift from Dr T. Nakamura) antibodies were used. Supersignale West Dura Extended Duration substrate (Pierce) was used as the substrate for the chemiluminescence.

2.8. Ultrastructure of cultured cells The frozen cells (7 months) were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at RT for 30 min, and mounted in warm 2% agar (DIFCO, Detroit, USA). The gels were cut into pieces and then postfixed in 2% osmium tetroxide in the buffer. The details of the method for transmission electron microscopy (TEM) were previously described [16]. Semithin sections were stained with 1% toluidin blue and examined with a light microscope.

3. Results The condition of SH colonies during cryopreservation was examined by phase-contrast microscopy and TEM. As shown in Fig. 1A, the cells soaked in the cryopreservant looked like aggregates and had various ball-shaped forms. The cutsurface of the cell aggregates showed that the colonies curled into in a ball-shaped or a jar-shaped form (Fig. 1B,C). SHs existed inside the structure and linear materials covered the cells. Some NPCs and MHs usually attached to the edge of the curled cells. Many cells inside were alive, whereas the cells on the edges were faintly stained with toluidin blue and had cell membrane and nuclei damage, which showed that the cells were dead. As shown in Fig. 2, SHs had a high nuclei/cytoplasm ratio but organelles such as mitochondria and rough endoplasmic reticulum were abundant in their cytoplasm. They also possessed peroxisomes and maintained

cell-to-cell junctions such as tight junctions and desmosomes even after 7-month cryopreservation. When the frozen cells were thawed and plated on dishes, the centers of SH colonies first attached on the dish surface forming spherical structures. As shown in Fig. 3, after live cells attached, the cells at the edge of the colony attached within 1 day and started to proliferate. Most expanding cells were small mononucleate cells and most colonies were maintained in a monolayer. Some NPCs, which attached to SHs and survived during cryopreservation, also proliferated from the edges of SH colonies. Many solitary cells and aggregates consisting of small numbers of cells died during cryopreservation, or even if they survived, they could not proliferate after plating. We examined the efficiency of the attachment of the plated colonies. At day 1, about 60% of colonies tightly attached on dishes and the cells could grow (Table 1). The length of the period of cryopreservation did not influence the attachment rate of the colonies. However, when isolated cells from a liver were cryopreserved without culture, no growth of SHs was observed. Once SH colonies could attach on the dish, SHs could continue proliferating. When SHs actively proliferated, the growth of NPCs had a tendency to be suppressed (Fig. 3C,D). In addition, most colonies maintained a monolayer. Few colonies with piled-up cells were observed during the culture of thawed cells although an aggregated lesion often remained in the center. To examine the growth rate of 6-month cryopreserved cells, we measured the areas of colonies (Fig. 4). The average area of SH colonies remarkably increased and was about 7.5 times larger at day 15 than that at day 1. SH colonies continued proliferating and reached near confluence at around 30 days after plating, though this was dependent on the number of attached SH colonies. To examine whether the proliferating cells were differentiated hepatocytes, the ability of albumin secretion and the expression of albumin mRNA were investigated in the cells cultured after more than 6-month cryopreservation (Fig. 5).

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Fig. 2. Electron micrographs of cells thawed after 7-month cryopreservation. (A) Live SHs are shown in photographs. Arrows show NPCs and arrowheads show ECM. Scale bar, 2 mm. (B) The enlargement of the square area shown in (A). The arrow shows a peroxisome with a crystalline nucleoid. Scale bar, 0.5 mm. (C) Gap junctional structure between SHs is shown by the arrow. Scale bar, 0.5 mm.

The albumin secretion into culture medium increased as SH colonies grew. The amount of secretion was about five times larger at day 13 than at day 3. In addition, the expression of albumin mRNA also increased with time in culture. The expression of other proteins related to hepatic differentiated functions was also examined by Western blot analysis (Fig. 6). Cells after more than 1 year of cryopreservation could express transferrin, fibrinogen and CPS, and the expression increased with time in culture. Although the expression of TO, which is an enzyme expressed only in MHs, was initially low in the cells, its protein was detected in the cells at days 14 and 35 after thawing.

4. Discussion Until now, no hepatocytes, even fetal ones, have been reported to be able to proliferate following replating after long-term cryopreservation. Considering why many SHs could survive and proliferate, we hypothesized the following reasons, as shown in the illustration of a frozen SH colony (Fig. 7): (1) the colony curls into in a ball-shaped or a jar-shaped form and SHs are located inside. Furthermore, the entrance is thin or nearly closed. In addition, NPCs and MHs are located in the periphery of the colony and their location creates the entrance of the jar-shaped

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Fig. 3. Phase-contrast photographs of proliferating SHs after 6-month cryopreservation. After 6-month cryopreservation, a SH colony was plated on a culture dish and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 10 mM nicotinamide, 10 ng/ml epidermal growth factor, 1 mM ascorbic acid 2-phosphate, 10 27 M dexamethasone, 0.5 mg/ml insulin and antibiotics. A colony in the area marked by the tip of a needle was photographed at day 3 (A), day 7 (B), day 14 (C), day 19 (D), day 25 (E), and day 35 (F) after thawing. Arrows show hepatic NPCs. The bar shows 100 mm.

structure. Therefore, many NPCs die and SHs inside the jarshaped structure may be protected from the physical and/or physiological influence of the cryopreservation solution and change of temperature. (2) Hepatocytes are epithelial cells. Cell–cell contact may be very important for the cells to maintain a stable condition. Therefore, although most previous experiments have been performed to preserve separately isolated hepatocytes, many thawed cells died or, even after the cells survived, they rapidly lost their functions with time in culture [4]. On the other hand, in this study, more than 30 SHs gathered and formed a colony. When SHs tightly attach to each other, it may result in a decrease of the surface area exposed to the cryopreservant

and in the strength of cell membranes. (3) A thin layer of ECM surrounded the colony. As previously reported [16], ECM such as type I and type IV collagens and laminin were produced and secreted by the cells during cultivation. Although we did not examine which ECM covered the colony, photos of the cutsurface (Fig. 1B,C) and TEM (Fig. 2) revealed that some ECM attached to the outside of the cell aggregates. Thus, SHs might have been protected from physical and physiological damage due to being covered by ECM. For these three major reasons, SHs could maintain the capacity for growth and differentiation into MHs after long-term cryopreservation. Recently, it has been reported that the transplantation of

Table 1 The rates of the colony attachment on the dishes after long-term cryopreservation Experiment no.

At the day of harvest (days after plating)

Periods of freezing (weeks)

Rate of attachment (%)

1 2 3 4 5 6 7 8 9 10 11

12 13 13 14 14 14 14 15 15 16 16

70 35 35 95 88 63 45 72 52 40 52

33.0 54.9 62.8 68.3 44.4 59.2 75.0 83.6 65.3 53.7 47.5

Average

14.2

58.8

58.9 ^ 14.4

0

1

Primary cells a

0a

Three independent experiments were carried out. Although the attached cells were cultured for 1 week, no colony formation was observed.

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Fig. 4. Growth of SH colonies thawed after 6-month cryopreservation. After 6-month cryopreservation, the cells were thawed and cultured. Photos of colonies were digitally recorded every 3 days and the area of the colonies was calculated as described in Section 2. The average area of the colonies at day 1 after thawing was 0.123 ^ 0.02 mm 2 and is shown as 100%. Bars show the average ^ standard deviation of more than 30 colonies per experiment and three independent experiments were performed. After day 15, some colonies became too large to observe under the same magnification. Therefore, we stopped measuring the area of the colonies though the cells continued to grow.

Fig. 6. Western blotting of proteins expressed in cultured cells thawed after 67-week cryopreservation. Samples (20 mg/lane) separated by SDS-polyacrylamide gel electrophoresis in 10% (transferrin, CPS, and TO) and 7.5% (fibrinogen) gels were transferred electrophoretically to a PVDF membrane. After transfer, the blots were stained with antibodies against transferrin, fibrinogen, CPS and TO. P shows 3-h cultured primary hepatocytes isolated from an adult rat liver.

system to provide cells rapidly and in sufficient numbers must be available. In this respect, cryopreservation of hepa-

primary hepatocytes can improve the impaired liver functions in rodent models [19–22] and that the transplantation of human hepatocytes was performed for patients as a bridge to orthotopic liver transplantation [3] or for improvement of metabolic defects [23,24]. Given the importance that this form of surgery may have in the clinic, a reliable

Fig. 5. Albumin secretion of SH colonies thawed after 6-month cryopreservation. The secretion of albumin into culture medium was measured by ELISA and the expression of albumin mRNA is shown inside the graph (0 shows the cells just after thawing). Bars show the average ^ standard deviation of three independent experiments.

Fig. 7. The typical condition of a SH colony in cryopreservation is illustrated. The colonies in the solution usually form ball-shaped or jar-shaped structures and each colony is surrounded by a thin layer of ECM. Dead cells are usually located in the neck of the structure.

S. Ikeda et al. / Journal of Hepatology 37 (2002) 7–14

tocytes is particularly attractive. Thus, cell tranplantation using cryopreserved hepatocytes has been performed [8,25–27]. Considering these reports, for mature hepatocytes treated with or without cryopreservation to proliferate and repopulate the liver, a substantial selection advantage such as extensive ongoing liver damage or the inability of endogenous hepatocytes to proliferate is necessary for host hepatocytes [28]. However, such strong selection may not be applicable for patients suffering from severe liver failure. If stem or progenitor cells continue to proliferate long after their transplantation, these cells may not need great selection pressure to ultimately repopulate the liver. Therefore, many studies of transplantation using hepatic stem/progenitor cells have been performed [29–33]. SHs may be hepatic progenitor cells and possess not only growth potential but also hepatic functions [15,16]. In addition, the growth and maturation of SHs may be regulated by ECM produced by NPCs. We reported that the addition of Matrigele to SHs can suppress the growth of the cells and rapidly induce the maturation [17]. On the other hand, as shown in the present experiment, when SHs were cocultured with a small number of NPCs, they rapidly proliferated without maturation. Recently, Katayama et al. [34] reported that transplanted SHs could settle into hepatic lobules in rats treated with retrorsine and 2/3 partial hepatectomy and that the growth potential was much higher than that of MHs. Therefore, cryopreserved SHs may have the potential to grow in the liver when they are transplanted. We need further experiments to prove the possibility of cryopreserved SH transplantation. In addition, the establishment of an isolation method for human SHs will be important, and the application of the method shown in this article may be useful for a hepatocyte bank and development of an artificial liver.

Acknowledgements The authors wish to thank Dr M. Sakai (Hokkaido University, Sapporo, Japan) for rat albumin cDNA, Dr T. Nakamura (Osaka University, Osaka, Japan) for anti-rat TO antibody, and Dr W.H. Lamers (University of Amsterdam, Amsterdam, The Netherlands) for anti-rat CPS antibody. We thank Ms M. Kuwano, Ms Y. Tanaka and Mr H. Itoh for technical assistance. We also thank Mr K. Barrymore for help with the manuscript. This study was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan (10670213, 12670211 for T.M., 1247243 for Y.M.).

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