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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 paranodal domain which is crucial for the insulating properties of myelin^1,3. In between the paranodes are small unmyelinated gaps called the nodes of Ranvier. These nodes are rich in voltage-gated sodium channels (Nav), allowing the regeneration and rapid propagation of action potentials through saltatory conduction⁴. This tight interaction also enables axonal energy support through neuronal uptake of lactate from OLs^5,6.

The maturation of oligodendroglial lineage cells and the myelination process are tightly regulated by their interactions with neurons⁷. Indeed, OLs and OPCs, also named NG2 cells, express an array of receptors for neurotransmitters, and can receive input from excitatory and inhibitory neurons, allowing them to sense neuronal activity that can trigger their proliferation and/or differentiation into myelinating cells². In turn, OPCs/OLs secrete microvesicles and proteins into the extracellular space which alone or synergistically mediate neuromodulative and neuroprotective functions^8,9,10,11,12. However, the molecular mechanisms controlling the multiple modes of interactions between oligodendroglial lineage cells and neurons are yet to be fully deciphered.

Moreover, in several CNS pathological conditions, OLs are primarily affected, thus disturbing their interaction with neurons. For instance, in Multiple Sclerosis (MS), neurological dysfunction is caused by focal demyelination in the CNS, secondary to OLs loss that can lead to axonal damage and related disability accumulation. Remyelination can take place, albeit insufficiently in most cases¹³. Progress in the last decade, due to the development of immunotherapies, have reduce the relapse rate but promoting remyelination remains to date an unmet need. As such, a better understanding of OLs role, functions and influences is of particular interest to the development of new therapies for a wide spectrum of CNS conditions.

Here, we describe the methods of OLs purification and culture. This enables precise examination of intrinsic mechanisms regulating their development and biology. In addition, such highly enriched OLs cultures allow the production of oligodendrocyte-conditioned medium (OCM), which can be added to purified neuron cultures to gain insight into the impact of OLs-secreted factors on neuronal physiology and connectivity. Furthermore, we describe how to implement an in vitro co-culture system where purified oligodendrocytes and neurons are combined together, allowing to address the mechanisms regulating (re)myelination.

Protokół

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 overnight.
3. Rinse coated flasks 3 times with sterile distilled water on the day of dissection.

2. Preparation of media (~10 min)

NOTE: Perform the steps in a laminar flow hood under sterile conditions.

1. Prepare 500 mL of culture medium, consisting of Dulbecco's modified Eagle medium (DMEM) supplemented with 10% of fetal calf serum (FCS) and penicillin-streptomycin (100 IU/mL).
2. Prepare 20.6 mL of the enzyme digestion medium in a 50 mL tube, consisting of 20 mL of DMEM, 200 µL of DNase (50 µg/mL), 200 µL of papain (30 U/mL) and 200 µL of L-cysteine (0.24 mg/mL).
3. Filter-sterilize the media using a 0.22 µm filter.
4. Keep the media in the laminar flow hood at room temperature (RT) until dissection.

3. Preparation for dissection (~10 min)

NOTE: Perform the steps in a laminar flow hood under sterile conditions.

1. Prepare 100 mL of phosphate-buffered saline without calcium and magnesium (PBS; 1x). Filter-sterilize using a 0.22 µm filter.
2. Prepare 50 mL of ice-cold 1x PBS solution supplemented with 750 µL of 45% glucose. Filter-sterilize using a 0.22 µm filter.
3. Fill a 100 mm Petri dish with 1x PBS for cleaning instruments and three 60 mm Petri dishes with ice-cold PBS-glucose for tissue harvesting. Put the petri dishes on ice until dissection.

4. Dissection

NOTE: Dissection is performed from male and female Wistar rat pups at postnatal day (P) 2.

1. In order to provide a sterile environment, make sure to clean the bench with 100% ethanol. Sterilize all surgical tools with 100% ethanol.
2. Gently spray the neck of the pup with 70% ethanol.
3. Use large surgical scissors to decapitate the animal and place the head in a 100 mm Petri dish containing ice-cold PBS-glucose.
4. Use curved forceps to maintain the head of the animal at eye level. Use small surgical scissors to make a small incision at the base of the skull and cut the skull following the brain midline.
5. Use forceps to gently peel off the two parts of the skull from the midline.
6. Use a small surgical spoon to remove the brain from the head cavity. Put the brain in a 60 mm Petri dish containing ice-cold PBS-glucose on ice.
7. Viewing under a steromicroscope, use fine forceps to remove the cerebellum, the brainstem and olfactory bulbs from cerebral hemispheres.
8. Use fine forceps to separate the two cerebral hemispheres. Use fine forceps to peel off the meninges. Put the cerebral cortices in a 60 mm-petri dish on ice.
   NOTE: Ice-cold PBS is critical for correct meninges removal.

5. Tissue dissociation

NOTE: Perform the steps in a laminar flow hood under sterile conditions.

1. Use a sharp scalpel to finely chop the cerebral cortices. Transfer the minced tissue into a 50 mL tube containing enzyme digestion medium.
2. Incubate for 30 min in a humidified incubator at 37 °C under 5% CO₂.
3. Use a p1000 micropipette to gently remove the enzyme digestion medium while making sure that the cortical tissue remains at the bottom of the 50 mL tube.
4. Use a p1000 micropipette to add 1 mL of DMEM-10% FCS and gently triturate the tissue.
5. Use a 70 µm filter and a piston of a 1 mL syringe to filter the cortical tissue into a 50 mL tube.
   NOTE: One can rinse residual tissue on the inner tube wall several times with DMEM-10% FCS.
6. Fill the 50 mL tube with DMEM-10%FCS. Centrifuge at 423 x g for 5 min at RT. Carefully remove supernatant and resuspend cell pellet with 2 mL of DMEM-10%FCS.
7. Gently triturate cell pellet with a p1000 micropipette and then with a p200 micropipette. Dilute the cell suspension with the appropriate volume of DMEM-10%FCS.
   NOTE: Two brains = one T150 = 5 mL of DMEM-10%FCS.
8. Plate 5 mL of the cell suspension on a T150 at a density of 1 x 10⁵ cells/cm². Add 20 mL of warm DMEM-10%FCS to each T150. Incubate in a humidified incubator at 37 °C under 5% CO₂.
9. Renew half of the culture medium after 6 days in vitro (DIV) with warm DMEM-10%FCS.

6. Shaking preparation

1. Perform shaking preparation on the day before shaking in a laminar flow hood under sterile conditions.
2. Renew half of culture medium by adding fresh warm culture medium into the flask and incubate at 37 °C under 5% CO₂.

7. Shaking

1. Coat three 100 mm Petri dishes with PEI. Store them at 4 °C overnight.
2. Cover the flasks cap with paraffin film and put the flasks into a plastic bag. Shake flasks containing glial cells overnight at 250 rpm at 37 °C.
   NOTE: First shaking is performed at 8 DIV and one can perform up to three different shakings (see Figure 1 for timing).

8. OL lineage cells harvesting and culture

NOTE: These steps should be performed in a laminar flow hood under sterile conditions.

1. On the day after shaking, prepare Bottenstein-Sato (BS) medium according to Table 1.
2. Rinse coated Petri dishes 3 times with sterile distilled water.
3. Harvest flasks' supernatant containing mainly OL lineage cells but also some microglial cells and plate it on non-coated 100 mm Petri dishes.
   NOTE: This step allows removal of microglial cells through differential fast adhesion on the dish surface.
4. Incubate the Petri dishes for 15 min in a humidified incubator at 37 °C under 5% CO₂.
5. Fill each T150 flask with 25 mL of warm freshly prepared culture medium and incubate in a humidified incubator at 37 °C under 5% CO₂ until the second shaking.
6. Transfer the supernatant from the Petri dishes into new non-coated 100 mm Petri dishes to allow adhesion of residual microglial cells.
7. Incubate the Petri dishes for 15 min in a humidified incubator at 37 °C under 5% CO₂.
8. Remove the supernatant, which contains non-adherent OL lineage cells, and transfer it into 50 mL tubes (supernatant from 2 Petri dishes for a 50 mL tube). Discard Petri dishes plated with microglia.
9. Centrifuge the supernatant for 5 min at 423 x g. Carefully remove supernatant and resuspend cell pellet with 1 mL of BS medium. Pool all pellets in a common 50 mL tube and adjust volume to 10 mL with BS medium.
10. Determine cell density counting cells under a microscope.
    NOTE: A cell density between 3 x 10⁵/mL and 5 x 10⁵/mL should be obtained.
11. Add 20 mL of BS if cell density is higher than or equal to 4 x 10⁵/mL to obtain a final volume of 30 mL, or add only 10 mL of BS if cell density is less than 4 x 10⁵/mL to obtain a final volume of 20 mL.
12. Plate two or three pre-coated 100 mm Petri dishes with 10 mL of cell suspension. Incubate in a humidified incubator at 37 °C under 5% CO₂.
13. Clear the debris from the Petri dishes by refreshing all of the BS medium 2 h later.
    NOTE: Examine the culture under the microscope before and after clearing to verify cell density and efficiency of debris removal.
14. Incubate for 2 days in BS medium in a humidified incubator at 37 °C under 5% CO₂.
    NOTE: Examine the culture under the microscope. The confluence should be 70% to 80%.

9. OCM production

NOTE: Perform these steps in a laminar flow hood under sterile conditions.

1. Prepare NB-B27low medium according to Table 2.
2. Renew culture medium with 10 mL of warm NB-B27low medium. Incubate for 2 days in a humidified incubator at 37 °C under 5% CO₂.
3. Harvest the OCM, i.e., supernatant containing OL secreted factors. Filter-sterilize OCM using a 0.22 µm filter.
   NOTE: Store OCM at 4 °C for a maximum of 2 months.

10. OCM addition

NOTE: Steps should be performed in a laminar flow hood under sterile conditions. OCM can be added to purified hippocampal neuron cultures prepared according to the following protocol¹⁴, and obtained by adding, 24 h after isolation, the anti-mitotic agents uridine and 5- fluorodeoxyuridine (5 µM) for 36 h.

1. At 3 DIV, remove all neuron culture medium containing anti-mitotic agents and add 500 µL of fresh warm OCM.
2. Renew half of medium every 3 days with freshly made warm NB-B27.
   NOTE: Such cultures can be maintained up to 21 DIV.

11. Addition of OL to purified hippocampal neuron culture

NOTE: Perform the following steps in a laminar flow hood under sterile conditions. OLs can be added to purified hippocampal cultures obtained the same way as described above.

1. Prepare co-culture medium according to Table 3.
2. Retrieve the OL culture at 70% to 80% confluence. Rinse with 2 mL of warm 1x PBS.
3. To detach the cells, add 2 mL of 0.25% trypsin to a 100 mm Petri dish.
4. Incubate for 5 min in a humidified incubator at 37 °C under 5% CO₂.
5. Add 2 mL of DMEM-10% FCS to block the enzymatic reaction. Harvest the supernatant containing OL lineage cells.
6. Centrifuge at 423 x g for 5 min at RT. Carefully remove supernatant and resuspend the cell pellet in warm co-culture medium to obtain a concentration of 1.25 x 10⁵ cells/mL.
7. Add OL to purified hippocampal neurons culture by removing 200 µL of neuron culture medium and adding 200 µL of cell suspension per well (2.5 x 10⁴ cells/well) of a 24-well plate.
8. Refresh half of co-culture medium every 2-3 days.
   NOTE: Co-cultures can be maintained up to 24 DIV.

Wyniki

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 ...

Dyskusje

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...

Materiały

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