Optimized conditions for growth andin vitropurification of neonatal rat Schwann cells*,*,⋆

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Abstract

BACKGROUND:

Schwann cells can promote the growth, development and repair of peripheral nerve. The reports on the method of Schwann cells cultured in vitro are various presently.

OBJECTIVE:

To study the optimized conditions of Schwann cells cultured in vitro.

DESIGN, TIME AND SETTING:

The cytology in vitro study was performed at the Central Laboratory of Renmin Hospital of Wuhan University from June to October in 2008.

MATERIALS:

Twenty-four 3-4-day-old Sprague-Dawley rats were provided by Experimental Animal Center, Medical College, Wuhan University.

METHODS:

Bilateral sciatic nerve was sterilely dissociated from rats. Schwann cells were prepared and purified by low-concentration enzyme fast digestion and differential adherence. At exponential growth phase, cells at 1×107/L were incubated in 6-well plate. There were 4 conditions: medium pH (6.8-8.8), fetal bovine serum (2%-20%), incubator temperature (34-39 °C), and incubator CO2 concentration (3%-8%). Firstly, volume fraction of fetal bovine serum, incubator temperature and incubator CO2 concentration were fixed. Medium pH value was changed for cell culture to determine an optimal pH value. Subsequently, volume fraction of fetal bovine serum, incubator temperature and incubator CO2 concentration were changed in order to determine optimal values. Clone formation was observed under a microscope. One clone contained at least five cells. Cell clone was counted under an inverted microscope.

MAIN OUTCOME MEASURES:

The following parameters were measured: Schwann cell growth and clone formation, optimal pH value, volume fraction of fetal bovine serum, incubator temperature and incubator CO2 concentration.

RESULTS:

The cell-state of purified Schwann cells cultured in DMEM/F12 medium (1:1) was good, at pH value of 7.0-7.2. At ph value of <7.0 or >7.2, clone formation significantly decreased. Schwann cells with 10% fetal bovine serum had good growth, and clone number obviously increased. When serum concentration was higher or lower, clone formation would be poor. Cell growth was markedly inhibited between 34-36 °C, with a few clones. At 37 °C, clones were the most. At > 37 °C, cell growth became poor, and clone formation decreased. At 39 °C, cells could not adhere. Under incubator CO2 concentration between 3% and 8%, there was no significant difference in cell clone formation.

CONCLUSION:

The optimized culture condition of Schwann cells were 7.2 pH, 10% fetal bovine serum, 37 °C of temperature and 5% of CO2(v/v) of incubator.

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