A method for isolation of mouse liver cells by a two-step perfusion with calcium and magnesium-free Hanks' salt solution followed by a medium containing collagenase is described. Several variations of the commonly used procedure for rat liver cell isolation were quantitatively compared with respect to cell yield and viability. The optimal isolation technique involved perfusion through the hepatic portal vein and routinely produced an average of 2.3 x 10(6) viable liver cells/g body weight. Optimal perfusate collagenase concentration was found to be 100 U of enzyme activity per milliliter of perfusate. Light and electron microscopic evaluation of liver morphology after several steps of the isolation showed distinct morphologic changes in hepatocytes and other liver cells during perfusion. After perfusion with Hanks' calcium- and magnesium-free solution, many hepatocytes exhibited early reversible cell injury. These changes included vesiculation and slight swelling of the endoplasmic reticulum as well as mitochondrial matrix condensation. Subsequent to perfusion with collagenase, the majority of hepatocytes appeared connected to one another only by tight junctional complexes at the bile canaliculi. Multiple evaginations were seen on the outer membrane resembling microville and probably represented the remains of cell-to-cell interdigitations between hepatocytes and sinusoidal lining cells from the space of Disse. The cytoplasmic injury seen after Hanks' perfusion was reversed after collagenase perfusion. After mechanical dispersion, isolated mouse hepatocytes were spherical in shape and existed as individual cells; many (80 to 85%) were binucleated under hase contrast light microscopy. By electron microscopy, cells appeared morphologically similar in cytoplasmic constitution to that seen in intact nonaltered liver cells.

http://mouselivercells.com/Images/1981_Mouse%20Liver%20Cell%20Culture%20I_Hepatocyte%20Isolation_In%20Vitro.pdf

Mouse liver cell culture. I. Hepatocyte isolation.

https://www.cell.com/cell/pdf/0092-8674(79)90364-7.pdf

Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation

The culture conditions found to result in stable proliferation of normal rat hepatocytes are: (i) subconfluent cell densities; (ii) serum-free medium; (iii) hormonally defined medium containing epidermal growth factor, insulin, glucagon, prolactin, and other growth factors; and (iv) substrata of liver extracellular matrix depleted of growth inhibitors. Serum was found deleterious to parenchymal cells: it was inhibitory to the expression of liver-specific functions, cytostatic to parenchymal cells at all seeding densities, and cytotoxic to them at low seeding densities. These studies emphasize the relevance of synergies in the influences of hormones and extracellular matrix in regulating hepatocellular physiology.

https://www.pnas.org/content/pnas/81/5/1411.full.pdf

Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix.

A major advance in the study of mature hepatocytes ex vivo was the development of methods for dispersing the liver with proteolytic enzymes and harvesting intact viable cells. Howard and coworkers introduced bacterial collagenase for this purpose,' an approach that was superceded in the intact organ by collagenase perfusion' because of substantially increased yields of viable cells by this method. This was followed by the demonstration that collagenase-isolated he atoc tes, in the appropriate nutrient medium, could be established in c~l ture .~ ' Because the perfusion technique made available large numbers of cells, preparation of mass primary cultures was possible, an approach that has attracted attention because it involves a homogeneous preparation of hepatic parenchymal cells and also sustains cell viability well beyond that obtained with other methods for mature differentiated hepatocytes.' Primary hepatocyte cultures can be prepared with nonproliferating cells (from normal liver) and are established within 4 h of cell plating (see below). This proximity, both physiologically and temporally, to the intact liver has encouraged numerous studies of the retention of liver-specific function in culture-a problem raised frequently in the past with respect to proliferating cell lines from liver.5. ' The first studies of adult rat hepatocytes in primary monolayer culture were concerned with quantitation of specific function, comparing cultured hepatocytes to the intact parent liver, and demonstrated diminished specific function in some instance^.^' Subsequent studies in several laboratories have established that phenotypic change occurs in primary hepatocyte culture, not only in mammalian but also in amphibian hepatocytes7; it is distinct from a process of senescence or impaired viability; and it may be quantitatively impressive, involving key hepatic functions. The discussion that follows will be concerned with the documentation of phenotypic change in various hepatocyte culture systems, with the available information concerning its prevention or reversal and with its implications for studies using hepatocyte culture. The scope of this review will be limited largely to primary culture of mature hepatocytes, to the exclusion of fetal hepatocyte culture and propagated P Y

Phenotypic stability of adult rat hepatocytes in primary monolayer culture.

Epidermal growth factor (EGF) especially in combination with insulin and glucagon, has been shown to stimulate DNA synthesis in liver cells, both in the whole animal and in cell cultures. As a further development we have found that in primary monolayer cultures of freshly isolated adult rat liver parenchymal cells, in which contamination with nonparenchymal cells was negligible, DNA synthesis was substantially stimulated by these substances. In control cultures, incorporation of [3H]thymidine into DNA and labeling of nuclei in autoradiographs was low. The stimulation by EGF was enhanced by insulin and glucagon, whereas these hormones by themselves exhibited only limited activity. These observations were made in cultures of hepatocytes that were never exposed to serum, even during cell isolation and plating. Hence for stimulation of DNA synthesis under these conditions neither serum factors nor interactions with other types of cells or their products were required. The effects of glucagon were reproduced by substances that elevate intracellular concentration of cyclic-AMP, including cholera toxin, isoproterenol, and methylisobutylxanthine. These various substances, especially EGF, glucagon, or cyclic-AMP, altered the morphological characteristics of the cultures during early stages, promoting cellular spreading and aggregation.

DNA synthesis in primary cultures of adult rat hepatocytes in a defined medium: effects of epidermal growth factor, insulin, glucagon, and cyclic-AMP.

A proliferation-competent adult rat liver cell monolayer system has been analyzed for tissue-specific functions during its growth cycle. High levels of the adult (L type) form of pyruvate kinase (EC 2.7.1.40) and glutathione S-transferase B ("ligand," EC 2.5.1.18) are observed during the early lag phase; they decline markedly during the logarithmic phase and reappear during the stationary phase. By contrast, elevated levels of the fetal (K type) form of pyruvate kinase and alpha1-fetoprotein production appear only after proliferation begins; this pattern diminishes slightly during stationary phase as the adult phenotype is restored. Albumin production continues throughout the entire growth cycle. These in vitro findings simulate those observed during hepatoproliferative transitions in the intact animal and, as such, constitute a developmental program for normal epithelial cells in primary culture.

K Ibsen

Papers

Growth state-dependent phenotypes of adult hepatocytes in primary monolayer culture.