Restoration of differentiated functions in multicellular aggregates of a human liver epithelial cell line

Materials Science and Engineering C 6 Ž1998. 249–252

Restoration of differentiated functions in multicellular aggregates of a human liver epithelial cell line T. Tokiwa

a,b,)

, J. Kano

a,c

, M. Noguchi c , M. Kodama a , T. Tateishi

a

a

Bionic Design Group, National Institute for AdÕanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan Department of Cell Physiology, Kohno Clinical Medicine Research Institute, 3-4-4 Kitashinagawa, Shinagawa, Tokyo 140-0001, Japan Department of Pathology, Institute of Basic Medical Sciences, UniÕersity of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8562, Japan

b c

Received 6 May 1998

Abstract Whether or not the differentiated functions of a SV40 T antigen-immortalized human liver epithelial cell line ŽTHLE-5b., most of which were lost after serial subcultivations, are restored was examined by culturing it in a three-dimensional system using a macroporous membrane material, expanded polytetrafluoroethylene ŽePTFE.. The morphology and differentiated functions of THLE-5b cells were influenced by the modification of the ePTFE surface. THLE-5b cells formed anchored multicellular aggregates on a PVA-coated ePTFE surface 3 to 4 days in culture, but formed cell monolayers on a PVA-uncoated ePTFE surface as did the cells on a type I collagen-coated polystyrene surface. Albumin and transferrin were expressed in multicellular aggregates, but was negative in the corresponding monolayer cultures. However, a1-antitrypsin was not expressed in multicellular aggregates as well as monolayer cultures. Although cytokeratin 18 was found both in monolayer cultures and in multicellular aggregates, cytokeratin 19 and g-glutamyltranspeptidase were negative in monolayers and in multicellular aggregates. The present data suggest that some of the differentiated functions of THLE-5b cells are restored by three-dimensional cell–cell interaction in multicellular aggregates and also suggest that the multicellular aggregates of THLE-5b, as immobilized on a PVA-coated ePTFE surface, will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Human liver; Epithelial cell line; Multicellular aggregate; Restoration of differentiated function; ePTFE surface

1. Introduction An expanded polytetrafluoroethylene ŽePTFE. membrane has a macroporous structure providing a high surface area to volume ratio and surface complexity, while it is chemically inert. It has been studied as a candidate material for vascular substitute w1x. Previously, we examined ePTFE as a culture substrate in terms of the morphology and albumin expression of rat hepatocytes cultured on its surface in vitro and arrived at the conclusion that functional hepatocyte cultures can be obtained in anchored multicellular aggregates immobilized on a PVA-coated ePTFE surface w2–8x. We have generated a SV40 T antigen-immortalized human liver epithelial cell line ŽTHLE-5b. w8,9x, which does not show tumorigenicity in nude mice. The cells retained such differentiated liver functions as albumin and )

Corresponding author. Tel.: q81-3-34741831 Ext. 354; Fax: q81298-546209. 0928-4931r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 9 8 . 0 0 0 5 8 - 7

transferrin in primary or secondary culture, but most of the functions were lost after serial subcultivations. In the present study, whether or not the differentiated functions of THLE-5b cells are restored when the cells are cultured on the PVA-coated ePTFE surface was examined. The results will provide an information on their applicability in tissue engineering such as for use in a hybrid-type artificial liver device.

2. Experimental 2.1. Materials The polyvinyl alcohol ŽPVA.-coated Žhydrophilic. and ŽPVA.-uncoated Žhydrophobic. ePTFE membranes were provided by Sumitomo Denko ŽOsaka, Japan.. The fibril length and thickness of ePTFE membranes used were approximately 30 mm and 100 mm, respectively. PVA in the membranes was cross-linked by electronic beam irradi-

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T. Tokiwa et al.r Materials Science and Engineering C 6 (1998) 249–252

ation by the manufacturer. The presence of PVA in the PVA-coated ePTFE surface was confirmed by FTIR-ATR as described earlier w7,10x. Multiwell culture plate inserts were purchased from Millipore ŽBedford, MA.. Type I collagen-coated polystyrene 6-well plates were purchased from Iwaki ŽTokyo, Japan.. Rabbit anti-rat albumin, mouse monoclonal anti-human a1-antitrypsin, anti-human transferrin, mouse monoclonal anti-human CK18, mouse monoclonal anti-human CK19 antibodies, and LsAB kit were purchased from Cappel ŽDurham, NC., Zymed ŽSouth San Francisco, CA., Dako-Japan ŽKyoto, Japan., Progen ŽHeidelberg, Germany., Progen, and Dako-Japan, respectively. HuH-7 cells and Ac2F cells were provided by Japanese Cancer Research Resources Bank, JCRRB. CHOK1 cells were purchased from ATCC ŽRockville, MD.. 2.2. Culture of THLE-5b cells Normal human liver tissues were subjected to primary culture and then transfected with SV40 large T antigen gene. The transfected cells were cytokeratin 18-positive and exhibited colony-like growth. The cells could be passaged and were established as a cell line, THLE-5b. The detailed procedure of an establishment of THLE-5b cells is described elsewhere w8,9x. The cells grew with about 0.62

population doublingrday. No tumorigenicity is noticed by injecting 5 = 10 6 cells into nude mice and observing the mice for 6 months. THLE-5b cells retained such differentiated liver functions as albumin and transferrin in primary or secondary culture, but most of the functions were lost after serial subcultivations. Cell monolayers or multicellular aggregates were prepared by plating cells onto 30-mm multiwell culture plate inserts attached with ePTFE membranes. The inserts with these membranes were placed in six well plates. Culture medium Ž1 and 1.5 ml., RPMI 1640 supplemented with 5% fetal bovine serum ŽFBS., were added to the upper and the lower chamber of the plates, respectively. The cells were cultured in a humidified atmosphere of 5% CO 2 and 95% air at 378C. The culture medium was changed routinely every 2 days. Usually, cell monolayers or multicellular aggregates were formed 3 to 4 days after plating. 2.3. Morphology Cell morphology in the culture was observed by phase contrast microscope and by ordinary microscope after sectioning and staining. The anchored multicellular aggregates were removed with a scraper from the ePTFE membranes and spun down by centrifugation at 800 rpm for 3 min,

Fig. 1. Morphology of THLE-5b cells cultured on a PVA-coated and -uncoated ePTFE surface. Cells were cultured on a PVA-coated Ža,c. and -uncoated Žb,d. ePTFE surface. Ža,b. Phase contrast. =200 Ž7 days after seeding.; Žc,d. H.E. =400 Ž28 days after seeding.. Scale bar: 10 mm.

T. Tokiwa et al.r Materials Science and Engineering C 6 (1998) 249–252 Table 1 Phenotypic characteristics of THLE-5b cells and other cells cultured on a PVA-coated and -uncoated ePTFE surface THLE-5b a

Albumin Alfa 1-anti trypsin Transferrin CK18 CK19 GGT

PVA

Uncoated

q y q q y y

y y y q " y

HuH-7 b

Ac2F ne c ne ne ne y q

cells, the method of Rutenberg et al. w14x was employed. Ac2F cells were used as a control.

CHO-K1

Uncoated Uncoated Uncoated q " q q ne ne

251

y y y y y y

a

PVA-coated ePTFE; b PVA-uncoated ePTFE; c not examined. q: Positive staining; ": slightly positive; y: negative staining.

fixed with buffered 10% formalin, embedded in paraffin and sectioned. 2.4. Immuno- and non-immunocytochemistry The cell monolayers formed on a type I collagen-coated polystyrene or a PVA-uncoated ePTFE surface were fixed in a buffered 10% formalin solution at appropriate times during culture for albumin, a1-antitrypsin, and transferrin and in an absolute ethanol for cytokeratin 18 ŽCK18. and cytokeratin 19 ŽCK19. at y308C. Sections were prepared from multicellular aggregates formed on a PVA-coated ePTFE surface as mentioned in ‘Morphology’. For immunocytochemistry, rabbit anti-rat albumin, mouse monoclonal anti-human a1-antitrypsin, anti-human transferrin, mouse monoclonal anti-human CK18, and mouse monoclonal anti-human CK19 antibodies were used as primary antibodies. We used LsAB method with 3-amino-9-ethylcarbazole as the substrate of horseradish peroxidase ŽLsAB kit.. Immunocytochemistry was carried out as described before w11,12x. A human hepatoma cell line, HuH-7 cells, a normal rat liver epithelial cell line, Ac2F cells that possess a biliary epithelial cell nature w13x, and CHO-K1 cells were used as controls. For the enzyme-cytochemical demonstration of g-glutamyltranspeptidase ŽGGT. in the cultured

3. Results and discussion Morphological observation shows that THLE-5b cells spread poorly, retaining their cuboidal shape and 3 to 4 days later reorganized themselves into anchored multicellular aggregates when cultured on a PVA-coated ePTFE surface as shown in Fig. 1. However, no such multicellular aggregates were noted when the cells were cultivated on a PVA-uncoated ePTFE surface and formed monolayers ŽFig. 1. as did the cells cultured on a type I collagen-coated polystyrene surface Ždata not shown.. Similar results were obtained in the culture of rat hepatocytes w2–7x. After 3 to 4 weeks in culture, the cells cultured on a PVA-uncoated ePTFE surface were observed both outside Žmembrane surfaces. and inside the membranes. The mechanism of anchored multicellular aggregates formation on a PVA-coated ePTFE surface remains to be studied. It is well known that texture, porosity and other physical properties and surface charge, hydrophobicity and other chemical properties of the polymer substrates can influence cell behaviours such as cell adhesion and growth w15x. It is also noted that limiting cell spreading andror growth on substrates may be important to maintain or express liver functions w16,17x. It is likely that hydrophilic nature of a PVA-coated ePTFE surface, probably its porous structure, and such serum components in the medium as proteoglycan limit cell spreading andror growth and affect subsequent cell differentiation or formation of anchored multicellular aggregates. Hepatocytes-related markers were examined immuno and non-immunocytochemically and the results were summarized in Table 1. Strong positive staining for albumin was seen in nearly all cells cultured on a PVA-coated ePTFE surface, though most of the cells cultured on a PVA-uncoated ePTFE

Fig. 2. Immunocytochemistry of albumin of THLE-5b cells cultured on a PVA-coated and -uncoated ePTFE surface. Cells were cultured on a PVA-coated Ža. and -uncoated Žb. ePTFE surface and stained immunocytochemically. ŽInsert. HuH-7. Scale bar: 10 mm.

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surface were negative, while HuH-7 was clearly positive ŽFig. 2.. Similarly, strong positive staining for transferrin was observed in nearly all cells cultured on a PVA-coated ePTFE surface. However, few cells cultured on a PVA-uncoated ePTFE surface were positive for transferrin. Staining intensity for transferrin was more heavier than that for albumin. THLE-5b cells cultured on neither a PVA-coated nor -uncoated ePTFE surface were negative for a1-antitrypsin, though staining was positively seen in HuH-7 cells. Positive staining for CK18 was seen when THLE-5b cells were cultured on both a PVA-coated and -uncoated ePTFE surface. The staining intensity, however, was much lighter when cultured on the former than when cultured on the latter. These data suggest that some of the different functions of THLE-5b were restored by three-dimensional cell–cell interaction in multicellular aggregates. CK19 and GGT were examined as biliary cell markers. THLE-5b cells cultured on a PVA-coated ePTFE surface were negative for CK19 staining, but some cells cultured on a PVA-uncoated ePTFE surface were positive. Ac2F cells were negative for CK19. THLE-5b cells cultured on not only a PVA-coated, but also-uncoated ePTFE surface were negative for GGT stain. Ac2F cells were positive for GGT as expected. The present results in regard to biliary cell markers suggest that three-dimensional cell–cell interaction by the formation of spheroids or multicellular aggregates may force cells along a pathway toward only hepatocyte differentiation.

4. Conclusion Some of the differentiated functions of a SV40 T antigen-immortalized human liver epithelial cell line ŽTHLE-5b. were restored by culturing them in a three-dimensional system using a macroporous membrane material, ePTFE. Since ePTFE has such advantageous characteristics as it is chemically and immunologically inert,

highly insoluble and physically strong, the present results suggest that ePTFE is useful as a substrate for applications in tissue engineering such as for use in a hybrid-type artificial liver device.

Acknowledgements THLE-5b was kindly provided by Dr. C.C. Harris, National Cancer Institute, NIH ŽBethesda, MD, USA.. A part of this work was supported by the New Energy Industrial Technology Development Organization ŽNEDO..

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