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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.


Flow diagram of the preparation of isolated human hepatocytes with a modified four-step retrograde perfusion technique. Buffers: PBE: Perfusion buffer with EDTA; PB: Perfusion buffer; PBD: Perfusion buffer with dispase; PBC: Perfusion buffer with collagenase; WB: Washing buffer; WBD: Washing buffer with DNase.
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Figure 1: Flow diagram of the preparation of isolated human hepatocytes with a modified four-step retrograde perfusion technique. Buffers: PBE: Perfusion buffer with EDTA; PB: Perfusion buffer; PBD: Perfusion buffer with dispase; PBC: Perfusion buffer with collagenase; WB: Washing buffer; WBD: Washing buffer with DNase.

Mentions: We adopted a rigorous and stringent isolation protocol for all liver tissues. Precise time between tissue resection and isolation commencement was recorded. Liver wedges were usually obtained from segments II/III and were carefully cut and weighed. Primary human hepatocytes were isolated under strict sterile conditions using a modified four-step retrograde perfusion technique (Figure 1). To eliminate interpersonal variability, all of the subsequent isolation procedures were carried out by single individual (Meng FY).


Hepatocyte isolation from resected benign tissues: Results of a 5-year experience
Flow diagram of the preparation of isolated human hepatocytes with a modified four-step retrograde perfusion technique. Buffers: PBE: Perfusion buffer with EDTA; PB: Perfusion buffer; PBD: Perfusion buffer with dispase; PBC: Perfusion buffer with collagenase; WB: Washing buffer; WBD: Washing buffer with DNase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Flow diagram of the preparation of isolated human hepatocytes with a modified four-step retrograde perfusion technique. Buffers: PBE: Perfusion buffer with EDTA; PB: Perfusion buffer; PBD: Perfusion buffer with dispase; PBC: Perfusion buffer with collagenase; WB: Washing buffer; WBD: Washing buffer with DNase.
Mentions: We adopted a rigorous and stringent isolation protocol for all liver tissues. Precise time between tissue resection and isolation commencement was recorded. Liver wedges were usually obtained from segments II/III and were carefully cut and weighed. Primary human hepatocytes were isolated under strict sterile conditions using a modified four-step retrograde perfusion technique (Figure 1). To eliminate interpersonal variability, all of the subsequent isolation procedures were carried out by single individual (Meng FY).

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.