A new method for culturing highly enriched microglia in vitro

Guirong WEI; Min ZHANG; Jihua DONG; Yuanwu MEI; Rengang LIU

ABSTRACT

BACKGROUND:

Microglial cells are prominently involved in certain neurologic diseases such as Parkinson disease and Alzeheimer disease. In vitro primary culture is commonly used in studies on the functions of microglia.However, these classical culture methods have some defects including complex procedures and low out-put.

OBJECTIVE:

To establish a simplified high-output primary culture of microglia.

DESIGN:

An explorative experiment with microglial cells as the single sample.

SETTING:

Department of Neurology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS The study was finished at the Central Laboratory of Union Hospital from April to October 2004. Microglial cells were obtained from 10 newborn(one day) male Kunming mice that were selected.

METHODS:

The author' s culture method was based on McCarthy method, we developed a new culture method and made some improvements,including the increased cell density for primary culture and nutritional deprivation. The microlglial cells were isolated with low-concentration trypsin-EDTA(ethylene diamine tetraacetic acid) digestion and immunochemically labeled with MAC-1 antibody, so as to measure the output and purity of microglia.MAIN OUTCOME

MEASURES:

① Morphologic features of microglial cells, observed with inverted microscope; ② Purity and activity of microglia cultured with these two methods, were measured immunohistochemically.

RESULTS:

For microglia cultured with McCarthy method, the culture cycle was 20 days and the output was 2 × l05 cells per flask with a purity of 95% -97%. The new method shortened the culture cycle to 15 days and the output reached 1 × 106 cells per flask with a purity of 96-98%. Cell purity and activity had no significant difference between these two culture methods.

CONCLUSION:

The new method has a similar purity and activity with classical method; however, it may simplify procedures, shorten cycle, and increase output, and therefore can be a useful method for studies on microglia function and for nerve repair.