Organotypic Culture

Monday, May 03, 2004

Description
Organotypic Culture

Procedure
Organotypic Hippocampal Slice Cultures

1. MED probe coating: Polyethylenimine (PEI) method

a. Sterilize the MED probe with 70% ethanol (DO NOT autoclave).

b. Dry up the MED probe in a clean bench and continue to sterilize it with UV radiation for 15 min.

c. Store it in the 100 mm sterile Petri dish for easy handling.

d. Treat the surface of the MED probe with a sterile 0.1% polyethylenimine in 25 mM borate buffer, pH 8.4, for 8 hours at room temperature.

e. Remove the PEI solution. Dry and rinse 3 times with sterile distilled water.

f. Pour culture medium into the probe and keep it in a CO2 incubator until use.

2. How to make slice cultures

g. Prepare slices using standard methods. Thin slice (~200 mm) is better for long culture.

h. Place the slice to cover the 8x8 array. Reposition if necessary.

i. Remove all solution and gently add the culture medium up to the slice interface level (approximately 250 ml).

j. Add sterile distilled water (~5 ml) directly to the 100 mm Petri dish (not to the probe) to enrich humidity and prevent over drying of the culture medium in the MED probe.

k. Store MED probe in the covered Petri dish in the CO2 incubator. Exchange the medium with half volume every day.

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Organotypic Culture