Isolation of rat Leydig cells with combined low-concentration type Ⅱ and Ⅳ collagenases

doi:10.3877/cma.j.issn.1674-0785.2017.14.005

Abstract

Abstract:

Objective

To establish a simple and effective method to isolate and purify rat Leydig cells.

Methods

Leydig cells were digested with a mixture of 0.1% types II and IV collagenases, purified by differential adherence, and identified by 3β-HSD immunofluorescence staining. The testosterone-secreting capability of Leydig cells was determined by ELISA.

Results

The optimal enzyme digestion time for Leydig cells was about 1 hour. The best adherence time for the purification of Leydig cells was 1-2 h. After 24 hours of culture, the purity of Leydig cells was (98.1 ± 1.5)% as revealed by 3β-HSD immunofluorescence staining, and the survival rate of Leydig cells was (97.3 ± 1.6)%. After incubation for 12 hours, the Leydig cells reached the peak of secretion.

Conclusion

In this study, a simple, stable, and reliable method for isolation and purification of primary Leydig cells was established. This method allows to obtain high-purity Leydig cells with high activity.

Key words: Leydig cell, Isolation, Purification, Identification, Rats

Yang Li, Yu Wang, Kang Cheng, Chun Liu. Isolation of rat Leydig cells with combined low-concentration type Ⅱ and Ⅳ collagenases[J]. Chinese Journal of Clinicians(Electronic Edition), 2017, 11(14): 2033-2037.

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酶解时间	每克睾丸获得睾丸间质细胞数目（×10⁶个）	睾丸间质细胞纯度（%）	培养24后睾丸间质细胞纯度（%）
0.5 h	0.72±0.31	83.5±1.9	98.2±1.8
1.0 h	2.27±0.34	76.1±2.4	98.1±2.1
2.0 h	2.61±0.28	70.2±3.1	97.6±1.6

酶解时间	每克睾丸获得睾丸间质细胞数目（×10⁶个）	睾丸间质细胞纯度（%）	培养24后睾丸间质细胞纯度（%）
0.5 h	0.72±0.31	83.5±1.9	98.2±1.8
1.0 h	2.27±0.34	76.1±2.4	98.1±2.1
2.0 h	2.61±0.28	70.2±3.1	97.6±1.6

贴壁时间	每克睾丸获得睾丸间质细胞数目（×10⁶个）	睾丸间质细胞纯度（%）
0.5 h	0.83±0.35	88.3±2.7
1.0 h	1.98±0.37	77.4±2.8
2.0 h	2.27±0.34	76.1±2.4
3.0 h	2.35±0.43	66.3±3.3

贴壁时间	每克睾丸获得睾丸间质细胞数目（×10⁶个）	睾丸间质细胞纯度（%）
0.5 h	0.83±0.35	88.3±2.7
1.0 h	1.98±0.37	77.4±2.8
2.0 h	2.27±0.34	76.1±2.4
3.0 h	2.35±0.43	66.3±3.3

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