- Email: [email protected]
Nucleus number in clusters of transplanted fetal liver cells increases by partial hepatectomy of recipient rats
Journal of Bioscience and Bioengineering VOL. 115 No. 5, 568e570, 2013 www.elsevier.com/locate/jbiosc
NOTE
Nucleus number in clusters of transplanted fetal liver cells increases by partial hepatectomy of recipient rats Nana Shirakigawa and Hiroyuki Ijima* Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan Received 31 August 2012; accepted 19 November 2012 Available online 21 December 2012
The growth of transplanted hepatocytes is required for the construction of tissue-engineered liver. In this study, cellembedded hydrogel-filled polyurethane foam plates were subcutaneously transplanted into rat. A liver tissue-like structure was formed by transplanted fetal liver cells in 70% partial hepatectomy treated rat. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Fetal liver cells; Partial hepatectomy; Liver tissue engineering; Hepatocytes; Cell transplantation; Proliferation; Cluster formation]
Twenty years has passed since tissue engineering was propounded by Langer and Vacanti (1). Various researchers have performed studies to reconstruct tissues in vivo (2). There are some reports that some tissues like skin (3) and cornea (4) were applied for patients. Particularly, the construction of a liver substitute by tissue engineering is desired because it is difficult to replace liver functions artificially (5). Ogawa et al. reported that the viability of transplanted hepatocytes is improved by transplantation into pre-vascularized sites (6). Ohashi et al. reported that a subcutaneously implanted sheet of hepatocytes exhibits liver functions in mice (7). Previously, we developed a hepatocyte-embedded hydrogel-filled macroporous scaffold culture system for the construction of large liver tissues in vivo (8,9). Furthermore, viability of transplanted hepatocytes was improved by 70% partial hepatectomy (PH) that induces growth factor secretion in recipient rats (10,11). Pre-vascularization of recipient rats (10) and transplantation of hepatocytes as spheroids (10) are effective for improving the viability of transplanted hepatocytes. However, these technologies are not sufficient to construct liver tissue with a clinically significant mass (12). Much more hepatocytes are required to construct liver tissue for medical treatment. However, the increase of inoculated hepatocyte density in the transplanted samples is impossible without the construction of a fine vascular network because the oxygen consumption rate of hepatocytes is high (13). The combination of the vascular network construction with gaps of less than 1 mm (12) and the liver tissue construction between the vascular networks will enable to produce the large liver tissue. The inoculated cells at high density as a native liver will die even there are vascular network with gaps of around 1 mm. Therefore, the formation of liver tissue containing capillary vessels by transplanted cells is very
* Corresponding author. Tel./fax: þ81 92 802 2758. E-mail addresses: [email protected] (N. Shirakigawa), [email protected] (H. Ijima).
important for construction of a liver tissue substitute. In this study, formation of a liver tissue-like structure by using fetal liver cells (FLCs) and PH treatment was evaluated. Inbred rats (F344) were used in this study. Hepatocytes were obtained from 7-week-old male rats. FLCs were obtained from fetal livers of embryonic day 14 rats (14,15). Dispersed cells were obtained by collagenase digestion and subsequent mesh filtration (40e45 mm pore size). Hydrophilic-treated polyurethane foam (PUF) was prepared by coating with F344 rat serum (10). The pore size of PUF was 500 mm. Hydrophilic-treated PUF was soaked in collagen sol containing cells by placing the cell suspensions onto the hydrophilic-treated PUF dropwise. The inoculated cell density was 1 106 cells/ml. For gelation of collagen sol, the samples were incubated at 37 C for more than 30 min. Then, cell-embedded hydrogel-filled PUF plates were obtained. Two samples were subcutaneously transplanted into the back of each rat. Concurrently, PH was performed to half of the rats. The transplanted samples were retrieved at 7 days after transplantation and evaluated by histology. The preparation protocol of hydrophilic-treated PUF and cells has been described in detail by Hou et al. (10). The animal experiments in this study were reviewed and approved by the Ethics committee on Animal Experiments of Kyushu University, Fukuoka, Japan. Fig. 1A shows the histological analysis of retrieved samples. The PUF was not seen uniformly because the thickness of transplanted samples was 1 mm and the pore size of PUF was 500 mm. The number of nuclei in an area (N/S) of sectioned samples was evaluated (Fig. 1B). The N/S of samples that were transplanted into PHtreated rats was higher than that in normal rats. This phenomenon was observed for transplanted hepatocytes and FLCs. These PH effects were similar to those in a previous study (10). Here, the FLCs are considered the mixture of various types of cells (10) because the FLCs were collected by collagenase digestion without any cell separation. In other words, the N/S of hepatocytes and FLCs could not be compared because the number of hepatocytes cluster derided by FLCs might be under estimated (Fig. 1C)
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.11.015
VOL. 115, 2013
NOTE
569
FIG. 1. Histological evaluation and quantitative analysis. (A) Hematoxylin and eosin staining of a retrieved sample at 7 days after transplantation. Hepatocytes in normal (a) and PHtreated (b) rats. FLCs in normal (c) and PH-treated (d) rats. Arrows indicate PUFs. Arrowheads indicate hepatocytes. Scale bars: 100 mm. (B) Average number of nuclei per area. (C) The number of clusters per area. *, **Statistically significant differences from hepatocytes in PH-treated rat using Dunnett’s test. *p < 0.05. ** p < 0.01. (D) Average number of liver cell nuclei in each cluster (N/C). *Statistical analysis was performed using Dunnett’s test. p < 0.01. The results are given as means SD.
(10). Therefore, N/S values of hepatocytes and FLCs should be separately evaluated. Moreover, we evaluated the nuclear number of liver cells in each cluster (N/C) by histology (Fig. 1D). There were no differences among transplanted hepatocyte samples with or without PH. These results suggest that PH treatment improves the viability of transplanted hepatocytes, and has no effect on cell growth.
In contrast, the N/C of FLCs in normal rats was higher than that of hepatocytes. Furthermore, the N/C of transplanted FLCs in PH-treated rats was higher than that in normal rats. Because the cells were embedded in a gel under a dispersed condition, the migration of transplanted hepatocytes was inhibited. Therefore, we believe that PH treatment improves the survival and growth of FLCs in vivo.
FIG. 2. Distribution of N/C in samples. (A) Distribution of N/C of hepatocytes (circles) and FLCs (triangles). Samples were transplanted into normal rats (closed symbols) and PHtreated rats (open symbols). (B) Ratio of nuclei number in each cluster group divided at five N/C (R5 N/C). Closed bars indicate five N/C and open bars indicate >five N/C. *,**Statistically significant differences from five N/C using Student’s t-test. p < 0.01. a,b,c,dStatistically significant differences from closed bar of hepatocytes in normal rat (a), closed bar of hepatocytes in PH-treated rat (b), open bar of hepatocytes in normal rat (c), open bar of hepatocytes in PH-treated rat (d) using Dunnett’s test. p < 0.01. The results are given as means SD.
570
SHIRAKIGAWA AND IJIMA
J. BIOSCI. BIOENG.,
FIG. 3. Histological evaluation of native liver (A) and reconstructed liver tissue (B). Scale bars: 50 mm.
Liver cell clusters of various sizes were obtained, and the distribution of N/C was evaluated. The cluster size of hepatocytes was uniform, but the range of the cluster sizes of FLCs was broad (Fig. 2A). The clusters with several cells that mean from one to three cells would pass through a 45-mm filter. In addition, 45% hepatocytes have two nuclei (16). The nuclei number of filtrated cluster is possibly almost under five. Therefore, all clusters were divided into two groups at five N/C (Fig. 2B). About 90% of hepatocytes were located in small clusters (five N/C), indicating that almost all hepatocytes were alive under the initial condition. Conversely, more than half of the FLCs were located in large clusters (>five N/C). Therefore, FLCs were considered to proliferate in vivo (Fig. 2B). In a typical example of transplanted FLCs in PH-treated rats, hepatocytes were placed in a line, and a sinusoid-like structure including red blood cells was formed between lines (Fig. 3B). It resembled a primitive liver lobe structure in appearance (Fig. 3). Based on this study, transplantation of FLCs into PH-treated rats might be a promising way for reconstruction of large liver tissues with a sinusoid-like network. Therefore, the ensuring of cells with the ability to proliferate and differentiate/mature into functional hepatocytes like FLCs is important for reconstruction of functional liver tissue. Thus, it is desirable to establish the optimum condition for the differentiation of stem/progenitor cells, which has been actively studied in recent years (17). Additionally, it has been suggested that more transplanted liver cells will remain alive by performing PH of recipient rats because hepatocyte growth factor (HGF) is released into circulation after PH in rats (11). In the case of humans, some patients undergo PH because of liver cancer, while other patients cannot undergo PH treatment. Even in such cases, it has been reported that the serum concentration of HGF increases in patients with liver diseases (18). Therefore, the phenomenon that occurs in rats after PH is expected to be similar to that in patients with liver diseases. Thus, we believe that our study can be applied in practical use. This work was supported by a Grant-in-Aid for Scientific Research (B): 22360348 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References 1. Langer, R. and Vacanti, J. P.: Tissue engineering, Science, 260, 920e926 (1993).
2. Baddour, J. A., Sousounis, K., and Tsonis, P. A.: Organ repair and regeneration: an overview, Birth Defects Res. C Embryo Today, 96, 1e29 (2012). 3. Green, H.: The birth of therapy with cultured cell, Bioessays., 30, 897e903 (2008). 4. Pellegrini, G., Traverso, C. E., Franzi, A. T., Zingrian, M., Cancedda, R., and De Luca, M.: Long-term restration of damaged corneal surfaces with autologous cultivated corneal epithelium, Lancet, 349, 990e993 (1997). 5. Funatsu, K., Ijima, H., Nakazawa, K., Yamashita, Y., Shimada, M., and Sugimachi, K.: Hybrid artificial liver using hepatocyte organoid culture, Artif. Organs, 25, 194e200 (2001). 6. Ogawa, K., Asounuma, K., Inomata, Y., Kim, I., Tabata, Y., and Tanaka, K.: The efficacy of prevascularization by basic FGF for hepatocyte transplantation using polymer devices in rats, Cell Transplant., 10, 723e729 (2001). 7. Ohashi, K., Yokoyama, T., Yamato, M., Kuge, H., Kanehiro, H., Tsutsumi, M., Amanuma, T., Iwata, H., Yang, J., Okano, T., and Nakajima, Y.: Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets, Nat. Med., 13, 880e885 (2007). 8. Ijima, H.: Practical and functional culture technologies for primary hepatocytes, Biochem. Eng. J., 48, 332e336 (2010). 9. Ijima, H., Hou, Y. T., and Takei, T.: Development of hepatocyte-embedded hydrogel-filled macroporous scaffold cultures using transblutaminase, Biochem. Eng. J., 52, 276e281 (2010). 10. Hou, Y. T., Ijima, H., Takei, T., and Kawakami, K.: Growth factor/heparinimmobilized collagen gel system enhances viability of transplanted hepatocytes and induces angiogenesis, J. Biosci. Bioeng., 112, 265e272 (2011). 11. Kinoshita, T., Hirao, S., Matsumoto, K., and Nakamura, T.: Possible endocrine control by hepatocyte growth factor of liver regeneration after partial hepatectomy, Biochem. Biophys. Res. Commun., 177, 330e335 (1991). 12. Shirakigawa, N., Ijima, H., and Takei, T.: Decellularized liver as a practical scaffold with a vascular network template for liver tissue engineering, J. Biosci. Bioeng., 114, 546e551 (2012). 13. Rotem, A., Toner, M., Tompkins, R. G., and Yarmush, M. L.: Oxygen uptake rates in cultured rat hepatocytes, Biotechnol. Bioeng., 40, 1286e1291 (1992). 14. Oertel, M., Menthena, A., Chen, Y. Q., and Shafritz, D. A.: Comparison of hepatic properties and transplantation of thyþ and thy cells isolated from embryonic day 14 rat fetal liver, Hepatology, 46, 1236e1245 (2007). 15. Oertel, M.: Fetal liver cell transplantation as a potential alternative to whole liver transplantation? J. Gastroenterol., 46, 953e965 (2011). 16. Ijima, H., Matsushita, T., and Funatsu, K.: Development of a hybrid type artificial liver using PUF/spheroid culture system of adult hepatocytes, Jpn. J. Artif. Organs, 22, 171e176 (1993). 17. Christ, B. and Stock, P.: Mesenchymal stem cell-derived hepatocytes for functional liver replacement, Front. Immunol., 3, 1e10 (2012). 18. Tsubouchi, H., Niitani, Y., Hirono, S., Nakayama, H., Gohda, E., Araaki, N., Sakiyama, O., Takahashi, K., Kimoto, M., Kawakami, S., and other 5 authors: Levels of human hepatocyte growth factor in serum of patients with various liver diseases determined by an enzyme-linked immunosorbent assay, Hepatology, 13, 1e5 (1991).
NOTE
Nucleus number in clusters of transplanted fetal liver cells increases by partial hepatectomy of recipient rats Nana Shirakigawa and Hiroyuki Ijima* Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan Received 31 August 2012; accepted 19 November 2012 Available online 21 December 2012
The growth of transplanted hepatocytes is required for the construction of tissue-engineered liver. In this study, cellembedded hydrogel-filled polyurethane foam plates were subcutaneously transplanted into rat. A liver tissue-like structure was formed by transplanted fetal liver cells in 70% partial hepatectomy treated rat. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Fetal liver cells; Partial hepatectomy; Liver tissue engineering; Hepatocytes; Cell transplantation; Proliferation; Cluster formation]
Twenty years has passed since tissue engineering was propounded by Langer and Vacanti (1). Various researchers have performed studies to reconstruct tissues in vivo (2). There are some reports that some tissues like skin (3) and cornea (4) were applied for patients. Particularly, the construction of a liver substitute by tissue engineering is desired because it is difficult to replace liver functions artificially (5). Ogawa et al. reported that the viability of transplanted hepatocytes is improved by transplantation into pre-vascularized sites (6). Ohashi et al. reported that a subcutaneously implanted sheet of hepatocytes exhibits liver functions in mice (7). Previously, we developed a hepatocyte-embedded hydrogel-filled macroporous scaffold culture system for the construction of large liver tissues in vivo (8,9). Furthermore, viability of transplanted hepatocytes was improved by 70% partial hepatectomy (PH) that induces growth factor secretion in recipient rats (10,11). Pre-vascularization of recipient rats (10) and transplantation of hepatocytes as spheroids (10) are effective for improving the viability of transplanted hepatocytes. However, these technologies are not sufficient to construct liver tissue with a clinically significant mass (12). Much more hepatocytes are required to construct liver tissue for medical treatment. However, the increase of inoculated hepatocyte density in the transplanted samples is impossible without the construction of a fine vascular network because the oxygen consumption rate of hepatocytes is high (13). The combination of the vascular network construction with gaps of less than 1 mm (12) and the liver tissue construction between the vascular networks will enable to produce the large liver tissue. The inoculated cells at high density as a native liver will die even there are vascular network with gaps of around 1 mm. Therefore, the formation of liver tissue containing capillary vessels by transplanted cells is very
* Corresponding author. Tel./fax: þ81 92 802 2758. E-mail addresses: [email protected] (N. Shirakigawa), [email protected] (H. Ijima).
important for construction of a liver tissue substitute. In this study, formation of a liver tissue-like structure by using fetal liver cells (FLCs) and PH treatment was evaluated. Inbred rats (F344) were used in this study. Hepatocytes were obtained from 7-week-old male rats. FLCs were obtained from fetal livers of embryonic day 14 rats (14,15). Dispersed cells were obtained by collagenase digestion and subsequent mesh filtration (40e45 mm pore size). Hydrophilic-treated polyurethane foam (PUF) was prepared by coating with F344 rat serum (10). The pore size of PUF was 500 mm. Hydrophilic-treated PUF was soaked in collagen sol containing cells by placing the cell suspensions onto the hydrophilic-treated PUF dropwise. The inoculated cell density was 1 106 cells/ml. For gelation of collagen sol, the samples were incubated at 37 C for more than 30 min. Then, cell-embedded hydrogel-filled PUF plates were obtained. Two samples were subcutaneously transplanted into the back of each rat. Concurrently, PH was performed to half of the rats. The transplanted samples were retrieved at 7 days after transplantation and evaluated by histology. The preparation protocol of hydrophilic-treated PUF and cells has been described in detail by Hou et al. (10). The animal experiments in this study were reviewed and approved by the Ethics committee on Animal Experiments of Kyushu University, Fukuoka, Japan. Fig. 1A shows the histological analysis of retrieved samples. The PUF was not seen uniformly because the thickness of transplanted samples was 1 mm and the pore size of PUF was 500 mm. The number of nuclei in an area (N/S) of sectioned samples was evaluated (Fig. 1B). The N/S of samples that were transplanted into PHtreated rats was higher than that in normal rats. This phenomenon was observed for transplanted hepatocytes and FLCs. These PH effects were similar to those in a previous study (10). Here, the FLCs are considered the mixture of various types of cells (10) because the FLCs were collected by collagenase digestion without any cell separation. In other words, the N/S of hepatocytes and FLCs could not be compared because the number of hepatocytes cluster derided by FLCs might be under estimated (Fig. 1C)
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.11.015
VOL. 115, 2013
NOTE
569
FIG. 1. Histological evaluation and quantitative analysis. (A) Hematoxylin and eosin staining of a retrieved sample at 7 days after transplantation. Hepatocytes in normal (a) and PHtreated (b) rats. FLCs in normal (c) and PH-treated (d) rats. Arrows indicate PUFs. Arrowheads indicate hepatocytes. Scale bars: 100 mm. (B) Average number of nuclei per area. (C) The number of clusters per area. *, **Statistically significant differences from hepatocytes in PH-treated rat using Dunnett’s test. *p < 0.05. ** p < 0.01. (D) Average number of liver cell nuclei in each cluster (N/C). *Statistical analysis was performed using Dunnett’s test. p < 0.01. The results are given as means SD.
(10). Therefore, N/S values of hepatocytes and FLCs should be separately evaluated. Moreover, we evaluated the nuclear number of liver cells in each cluster (N/C) by histology (Fig. 1D). There were no differences among transplanted hepatocyte samples with or without PH. These results suggest that PH treatment improves the viability of transplanted hepatocytes, and has no effect on cell growth.
In contrast, the N/C of FLCs in normal rats was higher than that of hepatocytes. Furthermore, the N/C of transplanted FLCs in PH-treated rats was higher than that in normal rats. Because the cells were embedded in a gel under a dispersed condition, the migration of transplanted hepatocytes was inhibited. Therefore, we believe that PH treatment improves the survival and growth of FLCs in vivo.
FIG. 2. Distribution of N/C in samples. (A) Distribution of N/C of hepatocytes (circles) and FLCs (triangles). Samples were transplanted into normal rats (closed symbols) and PHtreated rats (open symbols). (B) Ratio of nuclei number in each cluster group divided at five N/C (R5 N/C). Closed bars indicate five N/C and open bars indicate >five N/C. *,**Statistically significant differences from five N/C using Student’s t-test. p < 0.01. a,b,c,dStatistically significant differences from closed bar of hepatocytes in normal rat (a), closed bar of hepatocytes in PH-treated rat (b), open bar of hepatocytes in normal rat (c), open bar of hepatocytes in PH-treated rat (d) using Dunnett’s test. p < 0.01. The results are given as means SD.
570
SHIRAKIGAWA AND IJIMA
J. BIOSCI. BIOENG.,
FIG. 3. Histological evaluation of native liver (A) and reconstructed liver tissue (B). Scale bars: 50 mm.
Liver cell clusters of various sizes were obtained, and the distribution of N/C was evaluated. The cluster size of hepatocytes was uniform, but the range of the cluster sizes of FLCs was broad (Fig. 2A). The clusters with several cells that mean from one to three cells would pass through a 45-mm filter. In addition, 45% hepatocytes have two nuclei (16). The nuclei number of filtrated cluster is possibly almost under five. Therefore, all clusters were divided into two groups at five N/C (Fig. 2B). About 90% of hepatocytes were located in small clusters (five N/C), indicating that almost all hepatocytes were alive under the initial condition. Conversely, more than half of the FLCs were located in large clusters (>five N/C). Therefore, FLCs were considered to proliferate in vivo (Fig. 2B). In a typical example of transplanted FLCs in PH-treated rats, hepatocytes were placed in a line, and a sinusoid-like structure including red blood cells was formed between lines (Fig. 3B). It resembled a primitive liver lobe structure in appearance (Fig. 3). Based on this study, transplantation of FLCs into PH-treated rats might be a promising way for reconstruction of large liver tissues with a sinusoid-like network. Therefore, the ensuring of cells with the ability to proliferate and differentiate/mature into functional hepatocytes like FLCs is important for reconstruction of functional liver tissue. Thus, it is desirable to establish the optimum condition for the differentiation of stem/progenitor cells, which has been actively studied in recent years (17). Additionally, it has been suggested that more transplanted liver cells will remain alive by performing PH of recipient rats because hepatocyte growth factor (HGF) is released into circulation after PH in rats (11). In the case of humans, some patients undergo PH because of liver cancer, while other patients cannot undergo PH treatment. Even in such cases, it has been reported that the serum concentration of HGF increases in patients with liver diseases (18). Therefore, the phenomenon that occurs in rats after PH is expected to be similar to that in patients with liver diseases. Thus, we believe that our study can be applied in practical use. This work was supported by a Grant-in-Aid for Scientific Research (B): 22360348 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References 1. Langer, R. and Vacanti, J. P.: Tissue engineering, Science, 260, 920e926 (1993).
2. Baddour, J. A., Sousounis, K., and Tsonis, P. A.: Organ repair and regeneration: an overview, Birth Defects Res. C Embryo Today, 96, 1e29 (2012). 3. Green, H.: The birth of therapy with cultured cell, Bioessays., 30, 897e903 (2008). 4. Pellegrini, G., Traverso, C. E., Franzi, A. T., Zingrian, M., Cancedda, R., and De Luca, M.: Long-term restration of damaged corneal surfaces with autologous cultivated corneal epithelium, Lancet, 349, 990e993 (1997). 5. Funatsu, K., Ijima, H., Nakazawa, K., Yamashita, Y., Shimada, M., and Sugimachi, K.: Hybrid artificial liver using hepatocyte organoid culture, Artif. Organs, 25, 194e200 (2001). 6. Ogawa, K., Asounuma, K., Inomata, Y., Kim, I., Tabata, Y., and Tanaka, K.: The efficacy of prevascularization by basic FGF for hepatocyte transplantation using polymer devices in rats, Cell Transplant., 10, 723e729 (2001). 7. Ohashi, K., Yokoyama, T., Yamato, M., Kuge, H., Kanehiro, H., Tsutsumi, M., Amanuma, T., Iwata, H., Yang, J., Okano, T., and Nakajima, Y.: Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets, Nat. Med., 13, 880e885 (2007). 8. Ijima, H.: Practical and functional culture technologies for primary hepatocytes, Biochem. Eng. J., 48, 332e336 (2010). 9. Ijima, H., Hou, Y. T., and Takei, T.: Development of hepatocyte-embedded hydrogel-filled macroporous scaffold cultures using transblutaminase, Biochem. Eng. J., 52, 276e281 (2010). 10. Hou, Y. T., Ijima, H., Takei, T., and Kawakami, K.: Growth factor/heparinimmobilized collagen gel system enhances viability of transplanted hepatocytes and induces angiogenesis, J. Biosci. Bioeng., 112, 265e272 (2011). 11. Kinoshita, T., Hirao, S., Matsumoto, K., and Nakamura, T.: Possible endocrine control by hepatocyte growth factor of liver regeneration after partial hepatectomy, Biochem. Biophys. Res. Commun., 177, 330e335 (1991). 12. Shirakigawa, N., Ijima, H., and Takei, T.: Decellularized liver as a practical scaffold with a vascular network template for liver tissue engineering, J. Biosci. Bioeng., 114, 546e551 (2012). 13. Rotem, A., Toner, M., Tompkins, R. G., and Yarmush, M. L.: Oxygen uptake rates in cultured rat hepatocytes, Biotechnol. Bioeng., 40, 1286e1291 (1992). 14. Oertel, M., Menthena, A., Chen, Y. Q., and Shafritz, D. A.: Comparison of hepatic properties and transplantation of thyþ and thy cells isolated from embryonic day 14 rat fetal liver, Hepatology, 46, 1236e1245 (2007). 15. Oertel, M.: Fetal liver cell transplantation as a potential alternative to whole liver transplantation? J. Gastroenterol., 46, 953e965 (2011). 16. Ijima, H., Matsushita, T., and Funatsu, K.: Development of a hybrid type artificial liver using PUF/spheroid culture system of adult hepatocytes, Jpn. J. Artif. Organs, 22, 171e176 (1993). 17. Christ, B. and Stock, P.: Mesenchymal stem cell-derived hepatocytes for functional liver replacement, Front. Immunol., 3, 1e10 (2012). 18. Tsubouchi, H., Niitani, Y., Hirono, S., Nakayama, H., Gohda, E., Araaki, N., Sakiyama, O., Takahashi, K., Kimoto, M., Kawakami, S., and other 5 authors: Levels of human hepatocyte growth factor in serum of patients with various liver diseases determined by an enzyme-linked immunosorbent assay, Hepatology, 13, 1e5 (1991).