Generating and Maintaining Slice Cultures from Human Glioblastoma Tissue

Abstract

Source: Parker, J. J., et al. A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs. J. Vis. Exp. (2017).

The video demonstrates the generation and maintenance of organotypic slice cultures from glioblastoma tissue. The isolated tissue is embedded in an agarose matrix, and thin sections are obtained using a vibratome. These sections are then placed on permeable membrane inserts within a microplate and cultured in a slice culture maintenance media for long-term maintenance.

Protocol

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

1. Pre-slicing Preparation

1. Prepare "tissue processing" media and "slice culture maintenance" media the day before planned tumor resection and tissue collection (or utilize previously generated media within 2 weeks). Add 5 mL of Penicillin-Streptomycin solution (10,000 U/mL) and 5 mL of 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) to 500 mL of a High-Glucose Dulbecco's modified Eagle medium (DMEM) to generate the tissue processing medium.
2. Prepare 250 mL of slice culture ma....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM High Glucose	Invitrogen (Gibco)	11960-044
Neurobasal-A Medium, minus phenol red	Invitrogen (Gibco)	12349-015
B-27 Supplement (50x), serum free	Invitrogen (Gibco)	17504-044
Penicillin-Streptomycin (10,000 U/ mL)	Invitrogen (Gibco)	15140-122
GlutaMAX Supplement	Invitrogen (Gibco)	35050-061
L-Glutamine (200 mM)	Invitrogen (Gibco)	25030-081
HEPES (1 M)	Invitrogen (Gibco)	15630-080
Nystatin Suspension	Sigma-Aldrich	N1638-20ML	10,000 unit/mL in DPBS, aseptically processed, BioReagent, suitable for cell culture
UltraPure Low Melting Point Agarose	Invitrogen (Gibco)	16520-050	Melts at 65.5 °C, Remains fluid at 37 °C, and sets rapidly below 25 °C.
Peel-A-Way Embedding Mold (Square - S22)	Polysciences, Inc.	18646A-1	Molds for tumor sample embedding
Stainless Steel Micro Spatulas	Fisher Scientific	S50823	Bend instrument 45 degrees at the neck of the spoon blade
Curved Fisherbrand Dissecting Fine-Pointed Forceps	Fisher Scientific	08-875
Single Edge Razor Blade (American Safety Razors)	Fisher Scientific	17-989-001	Blade edge is 0.009" thick. Crimped blunt-edge cover is removed before loading onto vibratome.
Leica VT1000 S Vibratome	Leica Biosystems	VT1000 S
Hydrophilic PTFE cell culture insert	EMD Millipore	PICM0RG50	30 mm, hydrophilic PTFE, 0.4 µm pore size
35 mm Glass Bottom Dishes	MatTek	P35G-1.5-20-C Sleeve	20 mm glass diameter. Coverslip glass thickness 1.5 mm
PECON Stagetop Incubator	PeCON German	(Discontinued)	Incubator PM 2000 RBT is a comprable product designed for use with Zeiss Microscopes.