Primary Cultures Of Adult Hepatocytes Research Articles

Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation*1

Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation*1

Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation

Cryopreserved hepatocytes from various animal species and human beings were tested for their ability to survive and function in primary culture. The freeze/thaw protocol primarily designed for rat hepatocytes was used with slight modifications for the cells of all other species; it consisted of suspending parenchymal cells in the Leibovitz L15 medium containing 10% fetal calf serum and 10% to 16% dimethyl sulfoxide. After transient storage at 4 degrees C cell suspensions were transferred to -20 degrees C and then to -70 degrees C before being plunged in liquid nitrogen. Hepatocytes were stored for a few weeks to 4 yr. Prolonged storage did not augment loss of cell viability and function. Cell viability after thawing was estimated by the trypan blue exclusion test, and attachment efficiency to plastic was estimated by measuring intracellular lactate dehydrogenase content. Similar values were obtained for most species tested; after cryopreservation cell viability and attachment were decreased by 10% to 25% and by 40% to 50%, respectively. A lower attachment rate was found with dog hepatocytes. Total cytochrome P-450 and protein synthesis were compared in fresh and cryopreserved cells from four species after 4, 24, 48 or 72 hr of culture. Similar values were found in both cells after 24 or 48 hr of culture. In addition, drug-metabolizing activities were measured in human hepatocytes from five donors. In most cases phenacetin deethylation activity was decreased whereas procainamide N-acetylation and paracetamol sulfoconjugation and glucuronidation were increased in cryopreserved cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of rat P-450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion.

The onset of the sexually dimorphic pattern of GH secretion and increased hepatic GH-binding capacity in rats at puberty is temporally correlated with the developmental induction of three hepatic cytochrome P-450s with steroid hydroxylase activity, P-450 IIC11, P-450 IIC12, and P-450 IIC13, and one cytochrome P-450 with vitamin A hydroxylase activity, P-450 IIC7. In this study we demonstrate that expression of the 2C11, 2C12, and 2C13 genes is modulated by GH at the level of transcriptional initiation both in vivo and in primary cultures of adult hepatocytes. In an effort to define the minimum sequence responsible for the inductive effects of GH, we have analyzed the ability of a 0.7-kilobase fragment isolated from the 5'-flank of the 2C12 gene, including the natural promoter, to drive transcription of a 320-basepair G-less cassette in vitro. We were unable to detect any substantial difference in RNA polymerase-II-dependent transcriptional efficiency toward the 2C12 promoter between liver nuclear extracts from normal and hypophysectomized rats of both sexes. This observation supports the assumption that the sequence information contained between bases -700 and 1 is sufficient to support basal transcription of the 2C12 gene. Sequence information residing 5' or 3' of the 0.7-kilobase 5'-flank or a higher ordered chromatin structure may be necessary for the sex-specific transcriptional activation of the 2C12 gene.

Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments

Adult rat hepatocytes formed floating multicellular spheroids in primary culture in an uncoated plastic dish with a positively charged surface. Cells in the spheroids formed in such a simple way were similar to those formed in dishes coated with proteoglycan fraction isolated from rat liver reticulin fibers; in both cases, cells maintained high ability to produce albumin and poor ability to proliferate in response to epidermal growth factor. Coating dishes with albumin was also helpful in spheroid formation; coating with 2-hydroxymethyl methacrylate resulted in formation of incomplete spheroids. Elimination of serum factors was essential for the formation of spheroids; when cells were washed with serum-containing medium before seeding or if the medium was replaced with a serum-containing medium, spheroid formation was completely inhibited. Collagens, fibronectin, and laminin, all of which promote the adhesion and spreading of hepatocytes on substrates, inhibited spheroid formation. Furthermore, collagens disintegrated spheroids, and cells in the monolayer initiated proliferation. Thus, two distinct, mutually exclusive features of primary culture of adult hepatocytes apparently exist; monolayer culture with proliferative activity in an adherent environment and spheroid culture with poor proliferative activity and high albumin-producing ability in a nonadherent environment.

Alpha-fetoprotein synthesis in primary cultures of adult mouse hepatocytes

Synthesis of the embryo-specific serum protein alpha-fetoprotein (AFP) is renewed in the regenerating mouse liver [i]. Studies of the cellular basis for AFP synthesis in the regenerating mouse liver have shown that it is renewed in mature, differentiated, pre-existing hepatocytes, quite unconnected with and, indeed, in the total absence of proliferation [4, 5, 8]. The main sign which distinguishes AFP-containing cells from the remaining hepatocytes in th~ mouse liver after exposure to hepatotoxins or after local mechanical injury is the localization of these cells in the perinecrotic regions [5, 8]. On these grounds the writer suggested that renewal of AFP synthesis is directly linked with local disturbances of interaction between hepatocytes and that the basic condition for repression of AFP synthesis at the tissue level is integrity of the trabecular structure of the liver [3]. It was accordingly decided to determine whether AFP is synthesized in primary cultures of adult hepatocytes in which the normal trabecular structure is completely disturbed.

Alpha 2 micro-Globulin in the rat. The regulation of the appearance of multiple forms in vivo and in primary cultures of adult hepatocytes.

alpha 2 micro-Globulin in the rat. The regulation of the appearance of multiple forms in vivo and in primary cultures of adult hepatocytes.