[Experimental study of implantable engineered liver tissue using type I collagen gel as scaffold].
OBJECTIVE
To construct implantable engineered liver tissue (ELT) using type I collagen gel as scaffold.


METHODS
Type I collagen was obtained from the tail of a rat. Hepatocytes were collected from a Sprague-Dawley rat, mixed with liquid type I collagen and Dulbecco's modified Eagle's medium to create hepatocyte/collagen gel construct. The construct was inoculated in a 96-well plate. 0, 3, 5, 7, 9, 11, 13, and 15 days after the inoculation the viability of hepatocytes in vitro was measured by MTT assay. Phase contrast microscopy was used to observe the morphology of the hepatocyte/collagen gel construct. Three SD rats underwent injection of the hepatocyte/collagen gel construct into the subcutaneous space. One week later the implant was taken out. The morphology was conducted by routine H.E. staining and immunohistochemical staining. The morphology and function of hepatocytes was investigated by inverted microscopy, routine H.E. staining and immunohistochemical staining. The constructs were also implanted into subcutaneous space, and the differentiation of hepatocytes and the formation of engineered liver tissue were observed by routine H.E. staining and immunohistochemical staining.


RESULTS
Phase contrast microscopy showed that the hepatocytes were distributed evenly in the construct and remained round-shape throughout the in vitro culture. MTT assay demonstrated that the high viability of hepatocytes (87%) was maintained up to 7 days, and then decreased gradually. Albumin, the specific marker of hepatocytes remained positive by immunohistochemical staining after 15-day culture. One week after implantation into subcutaneous space, the implanted hepatocytes retained its hepatocyte-specific morphology, i.e. round shape, large nuclear/cytoplasm ratio as well as binuclear cells, and formed small engineered liver tissue containing blood vessels within and surrounding the tissue.


CONCLUSIONS
A novel approach to construct implantable engineered liver tissue using collagen gel as scaffold for growth and differentiation of hepatocytes has been dev eloped. This technique is an attractive tool for the development of liver tissue engineering.
Zhao
Yun-shan
YS
Department of Regenerative Medicine, Institute of Field Surgery, General Hospital of People's Liberation Army, Beijing 100853, China.
Xu
Ying-xin
YX
Zhang
Bo-feng
BF
Wu
Xin
X
Liu
Ju-chao
JC
Zhang
Lan
L
Tang
Li-an
LA
Hang
Zhi-qiang
ZQ
D000818:Animals; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D017690:Cell Transplantation / Q000379:methods; D024042:Collagen Type I / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005782:Gels; D022781:Hepatocytes / Q000166:cytology*; D019164:Liver, Artificial; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry
D024042:Collagen Type I; D005782:Gels