260/ 280 and 260/ 230 ratios after a time course of warm (A, C) or cold (B, D) ischaemia.

<p>Values represent means ± SEM. All values were <i>N</i> = 6 except for 3 values that were <i>N</i> = 5; 260/ 280 ratio for warm ischaemia at 30 min (snap frozen), 260/ 230 ratios for warm ischaemia at 0 min (RNAlater) and 30 min (snap frozen). ^aSignificantly different from corresponding 0 min condition, <i>p</i> < 0.05. ^bSignificantly different from corresponding 90 min condition, <i>p</i> < 0.05.</p

A representative electropherogram is shown for the highest (A) and lowest (B) average RNA Integrity Number (RIN) obtained in this study. RIN of ileum mucosa specimens collected by snap-freezing in liquid nitrogen or banked using RNA stabilization solution after a time course of warm (C) or cold ischaemia (D).

<p>Values represent means ± SEM with <i>N</i> = 6. *Significantly different from corresponding snap-frozen condition, <i>p</i> < 0.05.</p

Normalised gene expressions of <i>MYC</i> (A, B), <i>HIF1α</i> (C, D), <i>CDX2</i> (E, F), <i>HMOX1</i> (G, H) or <i>IL1β</i> (I, J) after a time course of warm or cold ischaemia.

<p>Values represent means ± SEM. All values were <i>N</i> = 6 unless otherwise stated. The following values were <i>N</i> = 5; <i>HMOX1</i> expression at 0 and 90 min warm ischaemia (RNAlater) and 0 min cold ischaemia (RNAlater), <i>IL1β</i> expression at 90 min warm ischaemia (RNAlater) and 30 min cold ischaemia (snap frozen). The following values were <i>N</i> = 4; <i>IL1β</i> expression at 0 min warm or cold ischaemia (RNAlater). Four technical replicates were done per donor for each data point. *Significantly different from corresponding snap-frozen condition, <i>p</i> < 0.05.</p

Experimental plan for the collection of human ileum mucosa specimens from hemicolectomy right surgeries to examine the effects of warm or cold ischaemia and different processing methods used before banking of the tissues at -80°C.

<p>Experimental plan for the collection of human ileum mucosa specimens from hemicolectomy right surgeries to examine the effects of warm or cold ischaemia and different processing methods used before banking of the tissues at -80°C.</p

Description of the TaqMan Gene Expression Assays used.

<p>Description of the TaqMan Gene Expression Assays used.</p

An Algorithm that Predicts the Viability and the Yield of Human Hepatocytes Isolated from Remnant Liver Pieces Obtained from Liver Resections

<div><p>Isolated human primary hepatocytes are an essential <i>in vitro</i> model for basic and clinical research. For successful application as a model, isolated hepatocytes need to have a good viability and be available in sufficient yield. Therefore, this study aims to identify donor characteristics, intra-operative factors, tissue processing and cell isolation parameters that affect the viability and yield of human hepatocytes. Remnant liver pieces from tissue designated as surgical waste were collected from 1034 donors with informed consent. Human hepatocytes were isolated by a two-step collagenase perfusion technique with modifications and hepatocyte yield and viability were subsequently determined. The accompanying patient data was collected and entered into a database. Univariate analyses found that the viability and the yield of hepatocytes were affected by many of the variables examined. Multivariate analyses were then carried out to confirm the factors that have a significant relationship with the viability and the yield. It was found that the viability of hepatocytes was significantly decreased by the presence of fibrosis, liver fat and with increasing gamma-glutamyltranspeptidase activity and bilirubin content. Yield was significantly decreased by the presence of liver fat, septal fibrosis, with increasing aspartate aminotransferase activity, cold ischemia times and weight of perfused liver. However, yield was significantly increased by chemotherapy treatment. In conclusion, this study determined the variables that have a significant effect on the viability and the yield of isolated human hepatocytes. These variables have been used to generate an algorithm that can calculate projected viability and yield of isolated human hepatocytes. In this way, projected viability can be determined even before isolation of hepatocytes, so that donors that result in high viability and yield can be identified. Further, if the viability and yield of the isolated hepatocytes is lower than expected, this will highlight a methodological problem that can be addressed.</p></div

Tissue processing and cell isolation variables that have significant relationships with the yield (million hepatocytes/gram liver) of hepatocytes after linear regression analyses.

<p>Figures show relationships between yield and (<b>A</b>) warm ischemia <i>ex vivo</i> (min) or (<b>B</b>) weight of perfused liver (g). Values were deemed significant when P<0.05.</p

Liver variables that have significant relationships with the viability (%) of hepatocytes after linear regression analyses.

<p>Figures show relationships between viability and (<b>A</b>) fibrosis, (<b>B</b>) liver fat or (<b>C</b>) Ludwig score. Values were deemed significant when P<0.05. For the variable of Ludwig score, variables not sharing the same alphabet are significantly different, <i>P</i><0.05.</p

Variables measured in the blood or serum that have significant relationships with the yield (million hepatocytes/gram liver) after linear regression analyses.

<p>Figures show relationships between yield and (<b>A</b>) alkaline phosphatase activity (AP; U/L), (<b>B</b>) aspartate aminotransferase activity (GOT; U/L), (<b>C</b>) gamma-glutamyltranspeptidase activity (GGT; U/L), (<b>D</b>) alanine aminotransferase activity (GPT; U/L), (<b>E</b>) bilirubin (mg/dL), (<b>F</b>) partial thromboplastin time (PTT; s) or (<b>G</b>) quick value (%). Values were deemed significant when P<0.05.</p

The number of replicates (<i>N</i>) and the <i>P</i> values obtained after linear regression of the individual variables listed below to viability (%) or yield (million hepatocytes/g liver) of isolated human hepatocytes.

<p>*Significant at <i>P</i><0.05. Data are transformed to follow a normal distribution by logit¹, fourth root² or natural logarithm³ transformation.</p><p>The number of replicates (<i>N</i>) and the <i>P</i> values obtained after linear regression of the individual variables listed below to viability (%) or yield (million hepatocytes/g liver) of isolated human hepatocytes.</p