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Updated: Nov 10, 2025
Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye
Published on: January 14, 2021
Separating mouse malignant cell line (EL4) from neonate spermatogonial stem cells utilizing microfluidic device in
Behnaz Ashtari1,2,3, Azar Shams4, Narges Esmaeilzadeh3
1Shahdad Ronak Commercialization Company, Pasdaran Street, Tehran, Iran.
Background:
Some children who have survived cancer will be azoospermic in the future. Performing isolation and purification procedures for spermatogonial stem cells (SSC) is very critical. In this regard, performing the process of decontamination of cancerous cells is the initial step. The major objective of the present study is to separate the malignant EL4 cell line in mice and spermatogonial stem cells in vitro.
Methods:
The spermatogonial stem cells of sixty neonatal mice were isolated, and the procedure of co-culturing was carried out by EL4 which were classified into 2 major groups: (1) the control group (co-culture in a growth medium) and (2) the group of co-cultured cells which were separated using the microfluidic device. The percentage of cells was assessed using flow cytometry technique and common laboratory technique of immunocytochemistry and finally was confirmed through the laboratory technique of reverse transcription-polymerase chain reaction (RT-PCR).
Results:
The actual percentage of EL4 and SSC after isolation was collected at two outlets: the outputs for the smaller outlet were 0.12% for SSC and 42.14% for EL4, while in the larger outlet, the outputs were 80.38% for SSC and 0.32% for EL4; in the control group, the percentages of cells were 21.44% for SSC and 23.28% for EL4 (based on t test (p ≤ 0.05)).
Conclusions:
The present study demonstrates that the use of the microfluidic device is effective in separating cancer cells from spermatogonial stem cells.
Insights
This study shows a microfluidic device effectively separates cancer cells from spermatogonial stem cells (SSC). This technique is crucial for preserving fertility in childhood cancer survivors by purifying SSC for future use.
Area of Science:
- Reproductive biology
- Cancer research
- Biotechnology
Background:
- Childhood cancer survivors often face future infertility due to azoospermia.
- Isolation and purification of spermatogonial stem cells (SSC) are critical for fertility preservation.
- Effective decontamination of cancerous cells is a necessary first step.
Purpose of the Study:
- To separate malignant EL4 cells from mouse spermatogonial stem cells (SSC) in vitro.
- To evaluate the efficacy of a microfluidic device for cell separation.
- To develop a method for preserving fertility in cancer survivors.
Main Methods:
- Sixty neonatal mouse SSC were isolated and co-cultured with EL4 cancer cells.
- Cells were divided into a control group and a microfluidic separation group.
- Cell percentages were analyzed using flow cytometry, immunocytochemistry, and RT-PCR.
Main Results:
- The microfluidic device achieved high purity separation: 80.38% SSC and 0.32% EL4 in the larger outlet.
- The smaller outlet yielded 0.12% SSC and 42.14% EL4.
- The control group showed lower SSC purity (21.44%) compared to the microfluidic method.
Conclusions:
- Microfluidic device technology is effective for separating cancer cells from SSC.
- This method offers a promising approach for fertility preservation in cancer survivors.
- The study highlights the potential of microfluidics in regenerative medicine and cancer aftercare.
