Cell shape regulation based on hepatocyte sheet engineering technologies.
The de novo engineering of a uniform hepatocyte sheet in vitro is considered as a novel approach for liver-directed therapeutics. Hepatocytes can be cultured on a temperature-responsive culture dishes coated with poly(N-isopropylacrylamide) (PIPAAm). Following multiple days of  culturing, the hepatocytes can be easily harvested as a uniform sheet by decreasing temperature from 37°C to 20°C. By modifying the sheet harvesting protocol, we have noticed that two different forms of the hepatocyte sheets, "extended" and "shrinking," were  obtained. This study describes the methods for harvesting the two different forms of sheets, and their cellular structure and hepatocyte-specific functions. To obtain an "extended sheet" form, a cluster of hepatocytes covered with a support membrane was harvested by the temperature  reduction. For the "shrinking sheet" form, the hepatocyte sheet was floated after reducing the culture temperature, and the floating process allowed the sheet to shrink spontaneously. Histological analysis revealed that the hepatocytes in the extended sheet form were predominantly  flat, whereas the shrinking sheet contained cuboidal shaped hepatocytes. The preservation of hepatocyte-specific ultrastructures was confirmed in both types of sheets. To investigate hepatocyte-specific functionality, the harvested hepatocyte sheets were recultured on Matrigel-coated dishes.  Assessment of protein production levels and chemical metabolizing activities showed the similar functionalities for each form. In contrast, the recalculation of these values per sheet versus per square centimeter of sheet surface demonstrated that the function of the shrinking sheet was significantly  higher than that of the extended sheets. This study demonstrated that the hepatocyte sheets created on the PIPAAm dish could spontaneously shrink in size, but retain their hepatocyte functionality. This type of hepatocyte sheet could be utilized for the engineering of liver tissue in limited  areas that are unable to give adequate transplant space.
Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Shinjuku-ku, Tokyo, Japan.
Soichi
S
Takagi
Maki
M
Ohno
Kazuo
K
Ohashi
Rie
R
Utoh
Kohei
K
Tatsumi
Teruo
T
Okano
D000180:Acrylic Resins / Q000737:chemistry; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D048430:Cell Shape / Q000187:drug effects*; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000295:instrumentation / Q000379:methods*; D003094:Collagen / Q000737:chemistry / Q000494:pharmacology; D004338:Drug Combinations; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007797:Laminin / Q000737:chemistry / Q000494:pharmacology; D008012:Lidocaine / Q000494:pharmacology; D008297:Male; D008567:Membranes, Artificial; D051379:Mice; D008822:Mice, Transgenic; D011509:Proteoglycans / Q000737:chemistry / Q000494:pharmacology; D013696:Temperature; D023822:Tissue Engineering; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D000180:Acrylic Resins; D004338:Drug Combinations; D007797:Laminin; D008567:Membranes, Artificial; D011509:Proteoglycans; D000515:alpha 1-Antitrypsin; D000643:Ammonium Chloride; C059485:matrigel; C052970:poly-N-isopropylacrylamide; D003094:Collagen; D008012:Lidocaine