Differential attachment, chemicals and trypsinization to purify olfactory ensheathing cells: Comparison with differential attachment or chemicals alone

Boyu YANG; Feng WANG; Wei WANG

ABSTRACT

BACKGROUND:

Reports of culture condition of olfactory ensheathing cells (OECs) vary. And some methods have bad reproducibility, not appropriate for actual application.

OBJECTIVE:

To observe the effect of differential attachment, chemicals in combination with trypsin digestion to purify rat OECs in vitro and to compare the effect with differential attachment or chemicals alone.DESIGN, TIME AND

SETTING:

In vitro controlled observation of cytology was performed at the Laboratory of Department of Human Anatomy & Histology and Embryology, Fujian Medical University from June 2007 to June 2008.MATERIALS A total of 8 SD rats, 2 days old, were provided by the Laboratory Animal Center of Fujian Medical University.

METHODS:

The OECs were dissociated from the postnatal rat olfactory bulbs under sterile condition, and seeded in poly-L-lysine-coated culture flask at a density of 4.0×10~8 /L for primary culture. The cells were divided into 4 groups control group (not purified); chemicals group (5 μmol/L arabinose); differential attachement group (Nash differential attachement); combination group (Nash differential attachement to eliminate most of the fibroblasts, followed by arabinose; when the cells were confluent at 6 days, the cells were digested with 1.25 g/L trypsin for 1 minute until the processes were shrank, cells became round, with some cells floating).MAIN OUTCOME

MEASURES:

Morphological changes of the cultured OECs and NGFRp75 immunocytochemistry were observed.

RESULTS:

The OECs displayed a very characteristic morphological appearance. Most of OECs were bipolar or tripolar with long and slim processes. In the unpurified control group the rapidly proliferating fibroblasts were in the majority (60%) after 7 days in culture, and confluent at day 14. The OECs occupied the most in the other groups, and their appearance remained unchanged.The surviving bipolar or tripolar OECs were positive for NGFRp75. The purifity by chemicals and differential attacehment methods was low (75%), while the combination group was high (85%).

CONCLUSION:

The method of purifing OECs through a combination of differential attacehment, chemicals and trypsinization is effective.