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Hepatocyte isolation from resected benign tissues: Results of a 5-year experience

View Article: PubMed Central - PubMed

ABSTRACT

Aim: To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease.

Methods: We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic duct calculi (n = 7) and liver hemangioma (n = 17). Only the samples weighing ≥ 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation.

Results: Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 106 hepatocytes/g liver, with 76.4% ± 10.7% viability. The 17 samples of liver hemangioma had significantly higher yield of cells (5.4 ± 1.71 × 106 cells/g vs 3.49 ± 2.31 × 106 cells/g, P < 0.05) than the samples of intrahepatic duct calculi. However, there seems to be no clear difference in cell viability (80.3% ± 9.67% vs 76.4% ± 10.7%, P > 0.05). We obtained a cell yield of 5.31 ± 1.87 × 106 hepatocytes/g liver when the samples weighed > 20 g. However, for the tissues weighing ≤ 20 g, a reduction in yield was found (3.08 ± 1.86 × 106 cells/g vs 5.31 ± 1.87 × 106 cells/g, P < 0.05).

Conclusion: Benign diseased livers are valuable sources for large-number hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermore, the size of the tissues can affect the outcome of hepatocyte isolation.

No MeSH data available.


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Samples of liver hemangioma can provide better results, in terms of cell yield, than intrahepatic duct calculi. In addition, the size of the tissues can affect the outcome of hepatocyte isolation.
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Figure 4: Samples of liver hemangioma can provide better results, in terms of cell yield, than intrahepatic duct calculi. In addition, the size of the tissues can affect the outcome of hepatocyte isolation.

Mentions: We processed and isolated human hepatocytes from 24 liver wedges. The WIT, i.e., the interval between clamping and bathing in ice-cold solution, averaged 17.5 ± 8.8 min (range: 5-35 min) (Table 1). The cold ischemia time, i.e., the interval between liver resection and perfusion, averaged 19.3 ± 3.3 min (range: 15-45 min). For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 106 hepatocytes/g liver, with 76.4% ± 10.7% viability. However, for the 17 samples of liver hemangioma, we got better results for the hepatocyte yield (5.4 ± 1.71 × 106 cells/g vs 3.49 ± 2.31 × 106 cells/g, P < 0.05) compared to the samples of intrahepatic duct calculi (Figure 4A). However, there seemed to be no clear difference in cell viability (80.3% ± 9.67% vs 76.4% ± 10.7%, P > 0.05) (Figure 4B). In our study, we obtained a cell yield of 5.31 ± 1.87 × 106 hepatocytes/g liver when the samples weighed > 20 g. However, for the tissues that weighed ≤ 20 g, a reduction in yields was found (3.08 ± 1.86 × 106 cells/g vs 5.31 ± 1.87 × 106 cells/g, P < 0.05) (Figure 4C). In addition, no difference in cell viability was observed (80.0% ± 9.85% vs 76.0% ± 10.5%, P > 0.05) (Figure 4D).


Hepatocyte isolation from resected benign tissues: Results of a 5-year experience
Samples of liver hemangioma can provide better results, in terms of cell yield, than intrahepatic duct calculi. In addition, the size of the tissues can affect the outcome of hepatocyte isolation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037086&req=5

Figure 4: Samples of liver hemangioma can provide better results, in terms of cell yield, than intrahepatic duct calculi. In addition, the size of the tissues can affect the outcome of hepatocyte isolation.
Mentions: We processed and isolated human hepatocytes from 24 liver wedges. The WIT, i.e., the interval between clamping and bathing in ice-cold solution, averaged 17.5 ± 8.8 min (range: 5-35 min) (Table 1). The cold ischemia time, i.e., the interval between liver resection and perfusion, averaged 19.3 ± 3.3 min (range: 15-45 min). For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 106 hepatocytes/g liver, with 76.4% ± 10.7% viability. However, for the 17 samples of liver hemangioma, we got better results for the hepatocyte yield (5.4 ± 1.71 × 106 cells/g vs 3.49 ± 2.31 × 106 cells/g, P < 0.05) compared to the samples of intrahepatic duct calculi (Figure 4A). However, there seemed to be no clear difference in cell viability (80.3% ± 9.67% vs 76.4% ± 10.7%, P > 0.05) (Figure 4B). In our study, we obtained a cell yield of 5.31 ± 1.87 × 106 hepatocytes/g liver when the samples weighed > 20 g. However, for the tissues that weighed ≤ 20 g, a reduction in yields was found (3.08 ± 1.86 × 106 cells/g vs 5.31 ± 1.87 × 106 cells/g, P < 0.05) (Figure 4C). In addition, no difference in cell viability was observed (80.0% ± 9.85% vs 76.0% ± 10.5%, P > 0.05) (Figure 4D).

View Article: PubMed Central - PubMed

ABSTRACT

Aim: To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease.

Methods: We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic duct calculi (n = 7) and liver hemangioma (n = 17). Only the samples weighing &ge; 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation.

Results: Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 &plusmn; 2.31 &times; 106 hepatocytes/g liver, with 76.4% &plusmn; 10.7% viability. The 17 samples of liver hemangioma had significantly higher yield of cells (5.4 &plusmn; 1.71 &times; 106 cells/g vs 3.49 &plusmn; 2.31 &times; 106 cells/g, P &lt; 0.05) than the samples of intrahepatic duct calculi. However, there seems to be no clear difference in cell viability (80.3% &plusmn; 9.67% vs 76.4% &plusmn; 10.7%, P &gt; 0.05). We obtained a cell yield of 5.31 &plusmn; 1.87 &times; 106 hepatocytes/g liver when the samples weighed &gt; 20 g. However, for the tissues weighing &le; 20 g, a reduction in yield was found (3.08 &plusmn; 1.86 &times; 106 cells/g vs 5.31 &plusmn; 1.87 &times; 106 cells/g, P &lt; 0.05).

Conclusion: Benign diseased livers are valuable sources for large-number hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermore, the size of the tissues can affect the outcome of hepatocyte isolation.

No MeSH data available.


Related in: MedlinePlus