Large-scale isolation of sinusoidal endothelial cells from pig and human liver.

OBJECTIVE: Hepatic in vitro studies, like those on hypoxia/reperfusion injury in liver transplants, demand large numbers of cultivated sinusoidal endothelial cells (SECs). In this article, we present and evaluate a new method for the isolation of SECs from porcine and human livers. METHODS: SECs were isolated employing a four-step collagenase perfusion. The sinusoidal character of the cells was validated by transmission and scanning electron microscopy, exclusion of Weibel-Palade bodies and factor VIII-related antigen, expression of scavenger receptor, and incorporation of latex beads. RESULTS: In 23 pigs, an average of 9 x 10(4) SECs were harvested from each liver. Cells were cultivated under standard conditions, as well as in multilayer cocultures of isolated SECs and hepatocytes in a "sandwich" configuration. Standard cultures showed an average of 90% SECs in primary cultures and 100% SECs after the first passage. The possibility of isolation of SECs from human livers was demonstrated in eight cases. CONCLUSION: With the four-step collagenase perfusion it is possible to easily isolate large numbers of viable and pure SECs from one organ. A further advantage is the possibility of isolating hepatocytes from the same organ.

Association of newly synthesized histones with replicating and nonreplicating regions of chromatin.

Histone deposition in HeLa cells has been studied by monitoring the fractionation and electrophoresis mobility of pulse-labeled histones under conditions that separate newly replicated from bulk chromatin DNA. The separation efficiency of these two methods is approximately 70%. Following micrococcal nuclease digestion, chromatin was fractionated by salt elution. 50-65% of the newly synthesized histones eluted with bulk chromatin at NaCl concentrations between 0.1 and 0.3 M and were further down to co-electrophorese with bulk chromatin DNA, not with the more extensively digested newly replicated chromatin DNA contained in those fractions. The remaining chromatin fractions, solubilized with 0.4-0.6 M NaCl, were several-fold enriched in nascent DNA (Annunziato, A. T., Schindler, R. K., Thomas, C. A., Jr., and Seale, R. L. (1981) J. Biol. Chem. 256, 11880-11886) and were correspondingly enriched for the balance (35-50%) of newly synthesized core histones. This fraction of newly synthesized core histone may be preferentially deposited onto newly replicated DNA. In contrast, histone H1 showed little tendency toward deposition onto new DNA. Within 15 min all new core histones attained the same solubility and electrophoretic mobility as bulk chromatin. We conclude that newly synthesized histones are deposited onto both replicating and nonreplicating regions of chromatin.

Dual nature of newly replicated chromatin. Evidence for nucleosomal and non-nucleosomal DNA at the site of native replication forks.

When chromatin is extracted from nuclease-digested nuclei by stepwise salt elution, two different classes of newly replicated chromatin can be distinguished. Nascent DNA eluted from nuclei under conditions of low to moderate ionic strength (0.1-0.3 M NaCl) exhibits nucleosomal periodicity and is found in particles which have the same electrophoretic mobility as bona fide H1- or high mobility group protein-containing mononucleosomes. Thus, factors believed to be involved with both the higher order coiling and transcriptionally active state of chromatin are rapidly complexed with newly synthesized DNA and may be retained on parental nucleosomes throughout replication. In contrast, approximately 40% of new DNA is resistant to extraction with solutions of moderate ionic strength. Most of this material is eluted from nuclei by 0.4-0.6 M NaCl. While bulk chromatin that is extracted by 0.4-0.6 M NaCl is organized into nucleosomes, most of the newly replicated "chromatin" from the same fractions lacks subunit structure, as determined by DNA size analyses in polyacrylamide gels, thereby distinguishing this nascent material from newly replicated chromatin eluted at lower ionic strength. Within 15 min all newly synthesized chromatin matures and exhibits the solubility and nucleosomal periodicity characteristics of bulk chromatin. The unusual properties of the "nonnucleosomal" fractions may reflect the structure of newly synthesized DNA prior to its assembly into nucleosomes.

Free-flow electrophoresis. III. An analytical version for a rapid, quantitative determination of electrophoretic parameters.

An analytical device based on the principle of free-flow electrophoresis is described. Electrophoretic mobility distributions of proteins or cells can be obtained quickly with field-strengths of about 140 V/cm. Up to 100 samples can be tested per hour. The accuracy and reproducibility of the device is illustrated by the example of the serum protein separation. Quantities in the picomole range can be quantitatively evaluated. In the course of mobility measurements of cells and cell mixtures, a reproducibility of +/- 1.4% was obtained. Therefore this device meets both the needs of electrokinetic research and the requirement of routine measurements in clinical diagnosis. Finally, the application of a pH gradient is described and the limitation due to methodical aspects is shown.