Culture the spinal cord diffuse astrocytoma cells and establish nude mice model

Jianjun Sun¹, Zhenyu Wang¹, Lingsong Li², Haiyan Yu³, Yongsheng Xu³, Yi Luo¹, Bin Liu¹, Jinlong Mao⁴, Li Shen², Yingjie Lin²

1 Neurosurgical Department, Peking University Third Hospital, Neural Malignant Tumor Stem Cell Research Center, Peking University, Beijing 100191, China; 2 China Stem Cell Research Center; Peking University Health Science Center; 3 Clinical Stem Cell Center, Peking University Third Hospital; 4 Neurosurgical Department, Peking Union Medical College Hospital

Objective This basic study was to isolate and culture original spinal cord diffuse astrocytoma cells, and to extract CD133+ glioma stem cells, and to initiate glioma in the brain of nude mice model.

Methods The original astrocytoma cells were isolated from a surgical specimen of a patient with recurrent intramedullary spinal cord diffuse astrocytoma. The CD133- negative and CD133+ positive cells were separated by magnetic sorting using the CD133 Microbead kit. The nude mice model with brain glioma was established with different kind glioma cells. 10000 cells were implanted in brain for every mouse. Five mice implanted with CD133+ glioma cells were grouped as Tumor, and another five mice implanted with CD133- glioma cells as Control. In the six weeks, weigh and head circumference of mice were measured for two times every week. Between seven and eleven weeks, a couple of mice, including one Tumor mouse and one Control mouse, were selected to accept MRI scan according to its obviously enlarged head circumference and fluctuated weigh every week. Whole skull and brain of mice was checked under anatomy microscope. The slice of mice brain was stained with HE and immunohistochemistry.

Results Glioma stem cells cultured in special culture media and aggregated in suspension cells ball. Up to second generation, the rate of CD133 positive cells in whole glioma cells was evaluated in 49.44 percent. Up to third generation, the number of cells was enough to be implanted for nude mice model. In Tumor group with CD133+ cells, the xenograft formed surely. Under anatomy microscope, some show red xenograft nodule with abundance blood supply, and some show implicitly with red-brown lesion around the insert spot. On the histological slice, some glioma cells grow infiltrative into Alba. On the MRI scan, the xenograft was shown in contrast. In Control group with CD133- cells, no xenograft was observed in most mice brain, only inserted spot kept there. However, small xenograft was found into deep Alba in No. A329C3 mouse.

Conclusion CD133 antibody is not the unique spinal cord astrocytomas stem cells marker, CD 133 negative tumor cells can also be proliferation into the xenograft.

Key Words: Glioma; Astrocytoma; Cancer stem cell; CD133 antibody; Nude mice model