In vitro purification of Schwann cells with double negative selection

Jinfeng SHI; Li LEI; Yin XIA

ABSTRACT

BACKGROUND:

Although cultured technique and purified method of Schwann cells (SCs) are changed successively,purity is still limited. Whether double negative selections can culture high-purified SCs still needs a further study.

OBJECTIVE:

To investigate the effects of double negative selections on growth and purity of SCs.

DESIGN:

Controlled animal study.

SETTING:

Department of Otolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University,and the Beijing Institution of Otolaryngology.MATERIALS The experiment was carried out in the Department of Otolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University and the Beijing Institution of Otolaryngology between May 2006 and July 2006. Twenty new-born Wistar rats of 3 days (without gender limitation) were provided by the Experimental Animal Center of the Chinese Medical Academy.

METHODS:

The sciatic nerves of rats were digested by 0.2% collagenase. ① Grouping All the rats were divided into 4 groups with 5 each. The experimental group was depurated by differential adhesion and Ara-c for 30 minutes. SCs in the control group 1 and 2 were managed by differential adhesion and Ara-c for 30 minutes, respectively. Cells in the control group 3 were not purified. ② Evaluation The cell growth was observed under invert microscope; growth curves were measured with MTT method in the experimental group and control group 3 to observe cell proliferation. In addition, the SCs were identified by immunocytochemistry; the culture purity was calculated (ratio of S100 positive cells in each sight).MAIN OUTCOME

MEASURES:

① Growth of SCs; ② immunocytochemistry stain of SCs; ③ growth curve of SCs; ④ purity of SCs.

RESULTS:

① Growth of SCs At 5 days after incubation, the SCs proliferated well. SCs were spindle, bipolar and sometimes tripolar, with obvious nuclei and a little cytoplasm. There was more fibroblast cell in the group 3 than that in experimental group. ② Growth curve The SCs in experimental group entered into the logarithm multiplication period at the third day. ③ Immunocytochemistry The positive-stained SCs were brown in its body and process, and arranged like swirl. The FB with spread and anomaly shape was slight blue. ④ The ratio of anti-S100 positive cells was higher in experimental group than that in group 3 (P ＜ 0.01).

CONCLUSION:

With the double negative selection, high-purified SCs are obtained effectively.