Comparative cryopreservation of bovine and porcine primary hepatocytes

Abstract

The isolation of primary hepatocytes from liver tissue of farm animals yields a very high number of cells, and a part of them can be stored by cryopreservation for future experiments. As no experience exists with the cryopreservation of hepatocytes from cattle, our study aimed at the cryopreservation of bovine hepatocytes by use of different protocols compared with the cryopreservation of hepatocytes from pig. We tested different freezing media (William's Medium E vs. University of Wisconsin solution), cryoprotectants (dimethyl sulfoxide with vs. without trehalose as additional additive), freezing systems (standard freezing container vs. controlled-rate freezer) and freezing times (4 vs. 28 d). These tests identified a general influence of species and freezing systems, whereas the influence of freezing media, trehalose additive and freezing time was less or not obvious. In this regard, we determined a mean recovery of 30% of bovine hepatocytes and 55% of porcine hepatocytes cryopreserved in a controlled-rate freezer, whereas the rates were about 10% less when hepatocytes were frozen in a standard freezing container. In accordance with this observation, the cultivation of cryopreserved hepatocytes from cattle was less effective than that of porcine hepatocytes. Hepatocytes from cattle can be successfully cryopreserved and partially cultured after cryopreservation but with lower percentage than porcine hepatocytes.

Keywords: cattle; controlled-rate freezer; freezing medium; liver; pig; primary cells; trehalose.

Figure 1

In suspension hepatocytes immediately after enzymatic isolation from the liver tissue of cattle or pig. Arrows indicate selected spheroids formed by the primary hepatocytes.

Figure 2

Comparative influence of cryopreservation by uncontrolled (propan-2-ol-filled freezing container) or controlled (controlled-rate freezer) techniques on the recovery rate of primary hepatocytes from cattle or pig, which have been frozen with (+) or without (−) addition of trehalose (T) in DMSO/FCS-containing William’s medium E (WE) or University of Wisconsin solution (UW) and stored below −135°C for 4 or 28 days. This evaluation only included hepatocyte with intact cell membrane after staining with trypan blue solution. Data are means ± SD (n = 6) with p < 0.001 for species and freezing process (multiple three-way ANOVA tests) and p < 0.05 vs. ^*uncontrolled freezing process, ^#cattle, and ⁺without trehalose of the individual test group (Bonferroni post-hoc test).

Figure 3

Mean influences of freezing process and species on the recovery rate of primary hepatocytes after cryopreservation in different solutions for 4 and 28 days. We only counted hepatocytes with intact cell membrane after cell staining with trypan blue solution. Data are means ± SD (n = 48 using 6 animals each species) with p < 0.001 vs. ^*uncontrolled and ^#cattle (two-way ANOVA with Bonferroni post-hoc test).

Figure 4

Comparative images showing primary hepatocytes from cattle or pig seeded on a collagen layer directly after cell isolation (without cryopreservation) or after controlled cryopreservation in DMSO/FCS-containing University of Wisconsin solution with trehalose. Cryopreservation was performed for 4 or 28 d. All cells were imaged 2 days after seeding.