Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

Summary

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

Abstract

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 °C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Introduction

Oligodendrocytes (OLs) are glial cells of the central nervous system (CNS) that generate myelin wrapping around axons. OLs originate from oligodendrocyte precursor cells (OPCs) which proliferate within the ventricular zones of the embryonic CNS and then migrate and differentiate into fully mature OLs (i.e., myelin-forming cells)¹. OPCs are abundant during early development, but also persist in the adult brain where they represent the major proliferative cell population². A single OL ensheathes multiple axons in non-excitable sections (i.e., internodes), and the edge of each myelin loop attaches to the axon forming the pa....

Protocol

The care and use of rats in this experiment conforms to institutional policies and guidelines (UPMC, INSERM, and European Community Council Directive 86/609/EEC). The following protocol is established for a standard litter of 12 pups.

1. Preparation of the flasks (~5 min)

NOTE: Perform the following steps the day before dissection in a laminar flow hood under sterile conditions.

1. Coat the 150 cm² flasks (T150) with filter cap (1 flask for 2 pups) using 5 mL of polyethylenimine (PEI, 100 mg/L, see protocol in Supplementary File 1).
2. Store the flasks at 4 °C over....

Results

In this protocol, OL lineage cells are purified from glial cultures by shaking off astrocytes and microglia. Purity and phenotypic examination of OL cultures can be assessed by immunostaining with glial markers¹⁵. Analysis of the expression of different markers indicated that OL cultures were mostly pre-OLs with 90% ± 4% of O4⁺ cells, 85% ± 7% NG2⁺ cells, and 4.7% ± 2.1% of PLP⁺ cells, while 7.2% ± 2.5% of cells were GFAP⁺

Discussion

Here, we provide a detailed protocol to obtain highly enriched oligodendroglial lineage cell cultures from mixed glial cultures, adapted from a previously published method¹⁶, and the subsequent production of OL-conditioned medium. This shaking technique is not expensive, can be repeated three times and is optimal to obtain high quantity of purified OLs, as cells cultured in Bottenstein-Sato (BS) medium containing PDGFα proliferate. Glial cells are prepared using cerebral cortices of Wistar ra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-fluorodeoxyuridine	Sigma	F0503
B27 supplement	ThermoFisher	17504044
D-(+)-Glucose solution	Sigma	G8769
DNase (Deoxyribonuclease I)	Worthington	LS002139
Dulbecco's Modified Eagle Medium	ThermoFisher	31966021
Ethanol 100%	Sigma	32221-M
Ethanol 70%	VWR Chemicals	83801.360
Fetal Calf Serum	ThermoFisher	10082147
L-cysteine	Sigma	C7352
Neurobasal	ThermoFisher	21103049
Papain	Worthington	LS003126
Penicillin-Streptomycin	ThermoFisher	15140122
Phosphate Buffered Saline without calcium and magnesium	ThermoFisher	A1285601
Polyethylenimine(PEI)	Sigma	P3143
Tetraborate decahydrate	Sigma	B9876
Trypsin	Sigma	Sigma
Uridine	Sigma	U3750
Bottenstein-Sato (BS) media
apo-Transferrin human	Sigma	T1147
BSA (Bovine Serum Albumin)	Sigma	A4161
Dulbecco's Modified Eagle Medium	ThermoFisher	31966021
Insulin	Sigma	I5500
PDGF	Peprotech	AF-100-13A
Penicillin-Streptomycin	ThermoFisher	15140122
Progesterone	Sigma	P8783
Putrescine dihydrochloride	Sigma	P5780
Sodium selenite	Sigma	S5261
T3 (3,3',5-Triiodo-L-thyronine sodium salt)	Sigma	T6397
T4 (L-Thyroxine)	Sigma	T1775
Co-culture media
apo-Transferrin human	Sigma	T1147
B27 supplement	ThermoFisher	17504044
Biotin	Sigma	B4639
BSA (Bovine Serum Albumin)	Sigma	A4161
Ceruloplasmin	Sigma	239799
Dulbecco's Modified Eagle Medium	ThermoFisher	31966021
Hydrocortisone	Sigma	H4001
Insulin	Sigma	I5500
N-Acetyl-L-cysteine	Sigma	A8199
Neurobasal	ThermoFisher	21103049
Penicillin-Streptomycin	ThermoFisher	15140122
Progesterone	Sigma	P8783
Putrescin	Sigma	P5780
Recombinant Human CNTF	Sigma	450-13
Sodium selenite	Sigma	S5261
T3 (3,3',5-Triiodo-L-thyronine sodium salt)	Sigma	T6397
Vitamin B12	Sigma	V6629
Tools
0.22 µm filter	Sartorius	514-7010
1 mL syringe	Terumo	1611127
100 mm Petri dish	Dutscher	193100
15 mL tube	Corning Life Science	734-1867
50 mL tube	Corning Life Science	734-1869
60 mm Petri dish	Dutscher	067003
70 µm filter	Miltenyi Biotec	130-095-823
Binocular microscope	Olympus	SZX7
Curved forceps	Fine Science Tools	11152-10
Fine forceps	Fine Science Tools	91150-20
Large surgical scissors	Fine Science Tools	14008-14
Scalpel	Swann-morton	233-5528
Shaker	Infors HT
Small surgical scissors	Fine Science Tools	91460-11
Small surgical spoon	Bar Naor Ltd	BN2706
T150 cm2 flask with filter cap	Dutscher	190151
Animal
P2 Wistar rat	Janvier	RjHAn:WI