JD900 cerebral cortical neuron primary culture

Neuron primary culture is part of the neuronic system of the embryonic mammal, such as brain, hippocampus, spinal cord The method of tissue is directly taken out and inoculates the cultivation. At present, there are many ways to cultivate neuron culture in China, but there are still some urgent problems in the purity and yield of neurons in primary culture. Since neuron is a highly differentiated cell, neurons are more difficult to survive and grow in vitro relative to other cells. Therefore, the culture methods and nutrient conditions for culturing neuron requirements in vitro are particularly special.

In vitro culture technology of neuronal cells is an important tool for studying the pathogenesis and process of neurogenic diseases. Very good, intuitive reflecting the development of the varying gathline and physiological activity, how to establish a stable mature neural cell culture system is the basis for the smooth progress of the experimental study of neurological diseases. With the gradual depth of basic medicine and clinical medicine, in vitro culture technology of neural cells has extensively valued and popularized in brain injury, neurological disorder disease, aging.

Second, the experimental step

(1) 75% (volume) Score) Alcohol-toxic pregnant, take the tires, separated and cut into the spinal cord in sterile conditions, and placed in a plate of cold calcium-free, magnesium HBSS (next ice bag).
(2) Anatomy microscope is carefully peeled off the ridge film and blood vessels.

(3) Scissilizing the spinal cord into 1 cm3 size, digestion 15-20 min at 37 ° C, oscillating once every five minutes;

[ 123] (4) Remove the supernatant, add the termination fluid to terminate digestion, blow 10-15 times, can not have bubbles, collect the superficial, and the remaining organization is digested once.

(5) 4 ° C 1000 rpm centrifuge for 10 min, discarded, adding planting 1 resuspend, incubator within 30 min;

(6) Collecting the supernatant, Tanpan blue staining count, press 6 * 105Cells / hole into the incubator of hexagonal plate (planting 1), 37 ° C, 5% CO2, 95% saturated humidity;

(8) Cultivate the fourth day, half amount Exchangement is added to 5 uM of cytidine, 24h full amount;

(9) After each week is changed twice.