Comparison of Analytical Methods for Quantitation of Isolated Porcine Hepatocyte Yields

Abstract

As cell-based therapies receive approval for clinical evaluation and use, the development of reliable methods to quantify cell number and control the dose of therapy delivered is becoming increasingly important. An example is the determination of the number and volume of primary porcine hepatocytes used in an extracorporeal treatment for patients with liver disease. Conventional cell counting using optical microscopy was compared against two alternate methods to quantify isolated porcine hepatocytes: (1) automated cell counting using a commercially available particle characterization instrument, and (2) quantitation by cell mass. Methods were compared based on accuracy, precision, specificity, linear range, and ruggedness. The automated method delivered substantially improved accuracy, precision, and ruggedness when compared to the conventional optical method. It also provided valuable information about the size distribution of cell preparations, which often contained clumps of cells, and showed that processing steps such as cryopreservation can alter the size characteristics of a cell population. The automated method was also faster, and was well suited to use in a commercial manufacturing process. The mass-based method was simple and inexpensive, but suffered from nonlinearity at low cell concentrations. Automated cell quantitation using a commercially available particle characterization instrument proved to be the preferred method for obtaining accurate and consistent porcine hepatocyte counts in a timely manner.

Transplantation of hepatocytes from genetically engineered pigs into baboons

“…As the outcome of Nagata's study was significantly better than ours, this suggests that rendering the recipient in hepatic failure may not be essential. Is compatibility between pig CD47 and primate SIRP‐α essential if phagocytosis of the hepatocytes is to be avoided? As porcine hepatocytes (20‐30 μm) and primate macrophages (20‐50 μm) are of similar size, we suggest that the hepatocytes are too large to be phagocytosed by the macrophages. However, fragile hepatocytes could fragment, possibly from direct activation of recipient macrophages resulting in injury to the hepatocytes, and then be phagocytosed.…”

The effects of aging on hepatic microsomal scaling factor and hepatocellularity number in the horse

“…The hepatocellularity number obtained for the horse is higher than other species. One possible explanation for this may be due to the diameter of horse hepatocytes (14.6±4.8 µm) being smaller than the diameter of hepatocytes from other species; 19.2±0.6 µm in rat, 18.4±0.8 µm in pigs, and between 20-30 µm in human (Engelmann et al 1981;Stegemann et al 2000;Boyer et al, 2017). On the other hand, the microsomal protein value obtained from the horse was similar to values obtained for dog and rat; however, it was higher than that obtained for human (Table 1).…”

Stereological analysis of size and density of hepatocytes in the porcine liver

“…However, Stegemann et al. () demonstrated with sufficient precision and accuracy that the average size of isolated porcine hepatocytes remained consistent among individuals. The number of animals in our study does not enable a reliable comparison between males and females; however, we speculate that sexual dimorphism may explain, at least in part, this variability.…”

“…5, healthy pigs of the same age and breed with a similar total liver volume of 813 AE 85 mL (mean AE SD, see Table S1), and identical diet and handling may have significant interindividual differences at the level of hepatocytes evaluated within their tissue context. However, Stegemann et al (2000) demonstrated with sufficient precision and accuracy that the average size of isolated porcine hepatocytes remained consistent among individuals. The number of animals in our study does not enable a reliable comparison between males and females; however, we speculate that sexual dimorphism may explain, at least in part, this variability.…”

“…Marcos et al (2016) quantified hepatocytes, Kupffer cells and hepatic stellate cells in the rat liver, thereby combining the advantages of stereology with cytometric analysis of cell ploidy. However, other methods and techniques have also been used for the quantification of hepatocytes, namely, automated image analysis, cell proliferation assays, protein concentration measurements, DNA content methods and cytochrome P450 content methods (Carlile et al 1997;Stegemann et al 2000;Haga et al 2005;Sohlenius-Sternbeck, 2006;Deng et al 2009;Miyaoka et al 2012;Garrido et al 2013;Best et al 2015;Asaoka et al 2016). Many of these techniques are based on the use of single cell suspensions or liver homogenates.…”