Preparing Free-Floating Slice Cultures from an Adult Human Brain

Abstract

Source: Fernandes, A., et al. Short-Term Free-Floating Slice Cultures from the Adult Human Brain. J. Vis. Exp. (2019).

This video demonstrates a technique for preparing free-floating slice cultures from the adult human brain. Thin slices are generated from a brain section using a vibratome. These slices are then suspended in a culture medium supplemented with growth factors to maintain tissue viability.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Sterilization of materials

NOTE: All material and solutions must be sterilized prior to use.

1. Sterilize all surgical tools and vibratome slicing material (knife holder, specimen disk, buffer tray) in a dry sterilizing oven for 4 h at 180 °C.
2. Sterilize temperature-sensitive material or equipment by UV or gamma irradiation.
3. Sterilize media and solutions by autoclavation or filtration through 0.22 µm pore membranes.

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Results

Figure 1: Sample collection, transport, slicing, and culturing of cortical tissue from adult humans. The procedure starts at the surgical room with collection of cortical tissue from temporal lobectomy for the treatment of pharmacoresistant epilepsy (A,B). (C).......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Sigma Aldrich	A9539
Ammonium persulfate	Sigma	A3678-25G
Amphotericin B	Gibco	15290-018
B27	Gibco	17504-044
BDNF	Sigma Aldrich	SRP3014
Bovine Serum Albumin	Sigma Aldrich	A7906
Glucose	Merck	108337
Glutamax	Gibco	35050-061
Hank's Balanced Salts	Sigma Aldrich	H1387-10X1L
Hepes	Sigma Aldrich	H4034
Neurobasal A	Gibco	10888-022
PBS Buffer pH 7.2	Laborclin	590338
Penicilin/Streptomicin	Sigma Aldrich	P4333
Equipment and Material
Carbogen Mixture	White Martins		95% O2, 5% CO2
CO2 incubator	New Brunswick Scientific	CO-24	Incubation of slices 5% CO2, 36ºC
Plastic spoon			Size of a dessert spoon
Razor Blade	Bic		Chrome Platinum, used in slicing with vibratome
Scalpel Blade	Becton Dickinson (BD)	Number 24	Used for slicing of tissue; recommended same size or smaller
Superglue (Loctite Super Bonder)	Henkel		Composition: Etilcianoacrilato; 2-Propenoic acid; 6,6'-di-terc-butil-2,2'-metilenodi-p-cresol; homopolymer
Vibratome	Leica	14047235612 - VT1000S