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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.


Related in: MedlinePlus

Preparation of liver wedges. Only patients who had undergone left hemi-hepatectomies were deemed suitable for obtaining normal resected liver tissue from (A). Cut-off end of a suitable pipet tip to obtain an optimal size to match the vessel opening and cannulate the chosen vessel (B, C). Primary human hepatocytes must be isolated under stringent and rigorous sterile conditions (D).
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Figure 2: Preparation of liver wedges. Only patients who had undergone left hemi-hepatectomies were deemed suitable for obtaining normal resected liver tissue from (A). Cut-off end of a suitable pipet tip to obtain an optimal size to match the vessel opening and cannulate the chosen vessel (B, C). Primary human hepatocytes must be isolated under stringent and rigorous sterile conditions (D).

Mentions: Liver wedges were prepared as shown in Figure 2. Representative images of isolated primary human hepatocytes, in culture for the first week, are shown in Figure 3. Similar morphological changes in the cultured cells were observed during the first week. The cells maintained normal morphology for at least 1 wk during culture in William’s E Medium.


Hepatocyte isolation from resected benign tissues: Results of a 5-year experience
Preparation of liver wedges. Only patients who had undergone left hemi-hepatectomies were deemed suitable for obtaining normal resected liver tissue from (A). Cut-off end of a suitable pipet tip to obtain an optimal size to match the vessel opening and cannulate the chosen vessel (B, C). Primary human hepatocytes must be isolated under stringent and rigorous sterile conditions (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Preparation of liver wedges. Only patients who had undergone left hemi-hepatectomies were deemed suitable for obtaining normal resected liver tissue from (A). Cut-off end of a suitable pipet tip to obtain an optimal size to match the vessel opening and cannulate the chosen vessel (B, C). Primary human hepatocytes must be isolated under stringent and rigorous sterile conditions (D).
Mentions: Liver wedges were prepared as shown in Figure 2. Representative images of isolated primary human hepatocytes, in culture for the first week, are shown in Figure 3. Similar morphological changes in the cultured cells were observed during the first week. The cells maintained normal morphology for at least 1 wk during culture in William’s E Medium.

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.


Related in: MedlinePlus