原代神经细胞培养方法 Neuron Cell Culture
Our standard mass cultures are plated on astrocytes. Those, in turn, are plated on glass coverslips pre-coated with poly-D-lysine and laminin.
Materials:
. #1 coverslips
. coverslip racks in a water-tight container (we made ours)
. poly-D-lysine (PDL) stock solution (1mg/ml in dd water)
. laminin stock solution (20 mg/ml in Hank’s BSS)
. 35 mm plastic culture dishes
. culture hood equipped with UV lamp
. sterile dd water
Procedure:
. Place the coverslips in the racks and leave them in the culture hood under UV light for 2 hrs.
. Coat the coverslips with 12.5 mg/ml PDL (5ml PDL in 400ml sterile dd water) for 2hrs. in the culture hood.
. Wash the coverslips with sterile dd water five times, in the culture hood.
. Place the coverslips in the sterile 35mm dishes
. Add 0.4ml laminin on top of the coverslips. Wait for 45’, then aspirate the excess solution
1.2- Agarose-collagen microislands
This protocol is based on protocols by Segal and Furshpan. Although the following “macro-island” approach has allowed for greater neuronal survival, while still providing a high probability of connection between DRG and dorsal horn (DH) autapses or DH-DH connections can only be obtained with high probability in the conventional microislands.
Materials:
. #1 coverslips coated with PDL as above
. type II agarose
. Vitrogen 100 collagen, ~3 mg/ml
. 35 mm plastic culture dishes
. culture hood equipped with UV lamp
. sterile dd water
. atomizer
Procedure:
. Place coverslips in 35 mm dishes
. Melt agarose in dd water at 0.2%, and place a drop on the top surface of each coverslip. The height of each drop is diminished as much as possible by removing excess solution with a pipette before the agarose gels.
. Allow coated coverslips to air dry overnight in the culture hood at room temperature to form a thin film. For adequate drying, the dishes must be uncovered.
. Spray the collagen onto the coverslips with the atomizer. We use a glass perfume bottle which can be bought at Macy’s inNew York. In our experience that makes bigger droplets than the Fisher chromatography atomizer, and is much less expensive (and looks better too). The atomizer is held parallel to the bottom of the dishes, about 25 cm above and away from them. It is then pumped forcefully a few times.
. The collagen islands can be examined with an inverted microscope. Their size should be between 300 and 1000 mm in order to maximize the probability of connection between any two neurons.
. ADD A DROP OF COLLAGEN to the edge of the coverslip. That will serve as support for a “feeder culture” that helps the survival of the insular neurons
. The collagen is also allowed to dry overnight, under ultraviolet light.
We find that few dorsal horn neurons seem to survive on the “traditional” atomizer-generated microislands, apparently regardless of neuron density. Although we still do not know the reason for that, we developed a variation of the microisland method which generates bigger (est. 1-3 mm) islands. With this method, the probability of finding an usable island in a giver coverslip is very much increased. Although, with such big islands, interconnected DH neurons are about as easy to find as in a mass culture, DRG neurons, which seem to grow extremely long axons, are usually connected to most of the DH neurons present.
Materials:
same as in 2.2, except:
.instead of the atomizer, a fine painting brush (#3 or smaller)
Procedures:
. All identical to 2.2, except for the collagen spraying.
. Dip the paint brush in the collagen solution, then shake the excess solution off the brush.
. Gently tap the brush on the edge of each dish, rotating the dish a few degrees after each tap.
. Control the quality of the islands by looking at the dishes through an inverted microscope. Some practice is required to optimize the islands.
2. Dissection, plating, and maintenance of cells
In our system, Neurobasal + B-27 seems to improve the survival of the neurons, as well as increase neurite extension/branching. However, it does not favor astrocyte survival. Overall, we tend to use it for the co-cultures in “macro-islands” and for the mass cultures in general, but not for the microislands on astrocytes.
Prepare stocks:
. For Glucose/Glutamine/Pen-Strep solution, mix
60 g of glucose in 200 ml dd water
100 ml Glutamine 200 mM
100 ml Penniciline-Streptomycine
. Filter through a 0.22 mm filter, separate in 20 aliquots of 20 ml each and freeze
. For FVM, mix
15 mg 6,7-dimethyl-5,6,7,8-tetrahydropterine hydrochloride
75 mg glutathione
1.5 g ascorbic acid
into 300 ml dd water
. Adjust the pH to 5.5, filter through a 0.22 mm filter, divide in 60 aliquots of 5ml each and freeze
Final medium preparation
. Add
375 ml IMDM
100 ml Fetal Bovine Serum (20 %)
20 ml Glucose/Glutamine/Pen-Strep
5 ml FVM
. Filter through a 0.22 mm filter and store in fridge after use
Prepare stocks:
. Heat-inactivate Horse Serum at 60 C for 30 min; then, freeze in 10 ml aliquots
. Freeze 8ml aliquots of Glucose (200mg/ml dd water)
. Freeze 2 ml aliquots of Vitamins for MEM
. Freeze 1 ml aliquots of ……UFDU
. Freeze 500 ml aliquots of NGF (10 mg/ml Hank’s BSS)
Final medium preparation
. Add
180 ml MEM
10 ml Heat-inactivated Horse serum
8 ml glucose stock
2 ml Vitamins for MEM
. Add U/FDU and NGF as directed below, after plating the cells
. Filter through 0.22 mm filter
Prepare stocks
. Freeze B-27 supplement in 4 ml aliquots
. Freeze 0.5 ml Glutamine 200 mM aliquots
Final medium preparation
.Add
195.5 ml Neurobasal
4 ml B-27
0.5 ml Glutamine 200 mM
Materials:
. 70% ethanol
. Three 60 mm culture dishes with cold L-15 medium (keep on ice)
. One 35 mm culture dish with 2 ml S-MEM
. Large scissors
. Small iris scissors
. Coarse dented forceps
. Two fine forceps
. Small spatula
. 2.5% trypsin
. IMDM for astrocytes
. Sterile 15 ml centrifuge tube, with cap, and culture centrifuge
