Primary cell culture recipes

Note: This page describes the procedures used by Jung Choi on 2/12/04. Most of these have been arrived at after considerable testing and trial and error. YMMV.

Materials:

1. Glass Coverslips: German 12mm (Electron microscopy sciences, Cat. # 72196-12)
   1. Wash - use sonicator
      1. In ethanol (95% +) for 1 hr; rinse with dd-H₂O. Repeat.
      2. In dd-H₂O - 30min (3x)
   2. Separate and dry. (Separating: use tweezers to separate coverslips in water; take out one by one.) (Drying - a rack of for coverslips is ideal; if not available, may do it on wrinkled foil)

2. Silicon dish:
   1. Drill a hole in the bottom (3/8 - 1/2" diameter) of 35mm culture dish.
   2. Cut a piece of silicon and carefully glue (silicon rubber) onto the bottom of the dish so that the shiny part is shown through the opening. Let it dry overnight
   3. Wash the silicon surface with soap and water.
   4. Fill up the dish with ethanol and leave it in for 15min.
   5. Rinse with dd-H₂O. Dry in the hood before applying substrate.

3. Enzyme solution
   1. 50µl of cysteine stock (40mg/ml)
   2. 100µl EDTA stock (50mM)
   3. 150µl of 100mM CaCl₂ (there is 1M CaCl₂ stock too)
   4. 200 units of Papain
   5. 10ml HBSS
   6. 50µl of Dnase stock (0.2mg/ml) - add just before digestion then filter.

4. Modified HBSS Soln ("HBSS")
   1. 0.35g NaHCO₃
   2. 1.3g HEPES
   3. HBSS (Sigma H2387) in 1L H₂O.

5. Culture Medium

   	a.	Basal Medium Eagle (Invitrogen)	430ml
   	b.	Glucose	1.8g
   	c.	Na-Pyruvate (100mM, Invitrogen)	5ml
   	d.	FBS (Hyclone)	50ml
   	e.	Mito serum extender (Invitrogen)	500µl
   	f.	B27 (Invitrogen)	10ml
   	g.	1 M HEPES	5ml

6. Substrate (a or b)
   1. Collagen/PDL mix - for Post-natal island cultures
      1. 4ml DD-H2O
      2. 2ml 17mM Acetic Acid
      3. Rat tail collagen (BD Scientific) - 3.6mg
      4. 40µl of PDL stock soln (1mg/ml)
   2. Matrigel (BD biociences, 356234) - micro dots - for embryonic
      1. Make 1:100 dilution - keep it in ice!!!
      2. Spray using atomizer

7. 24 well plates - use inner 8 wells only and fill up the outer ones with water

General Procedure

1. If needed, apply 100µl of collagen/PDL mix onto coverslip or silicon. Incubate during the dissection. For embryonic, use matrigel (see materials section)
2. Prepare the enzyme solution.
3. Perform dissection (E18 or P1-3); keep tissues in HBSS
4. Collect Hippocampus, transfer to 15ml conical tube
5. Wash 3x with HBSS. Add Dnase to enzyme solution.
6. Filter and add enzyme solution to the tube, then incubate 45 - 50 min.
7. Wash 3x with Culture medium
8. Add 1-2ml of Conditioned culture medium and mechanically dissociate using 1ml pipette. Be gentle.
9. Count the cells. (# x 10K / ml)
10. Make appropriate dilutions for desired density with the conditioned culture medium
    • Embryonic - 15k/well
    • Postnatal neurons on glia - 10k/well
    • Postnatal neurons on silicon dish - 50~75k/ml x 2ml
    • Glial islands preparation - 5k/well
11. Add 500µl to each inner well. Fill up the outer wells with water to minimize evaporation

Feeding

1. Prepare 24µM Ara-C soln (Sigma C6645) in conditioned culture medium. Leave the medium in the incubator for at least half an hour with the cap loose.
2. Feeding Schedule:

   Glial islands

   One day after the culture, add 100µl of feeding medium to each well. Monitor the size of the islands throughout the week and add Ara-C as needed. If the neurons remain in the culture after the 2nd week, put the plate in the refrigerator overnight. Islands will be used on 3rd or 4th week.

   Postnatal neurons on glial islands

   Regardless of the growth, add feeding medium the day after. No feeding needed until the recording. Postnatal Neurons on silicon: after 2nd day of culture, add 400µl of feeding medium. Replace ½ medium with 4µM Ara-C feeding medium weekly.

   Embryonic neurons

   Wait until the neurons and glia cells have grown to form islands (usually 3-4 days, on matrigel is quicker). Then add 100µl. No more feeding needed.

3. Alternate method: if the culture has too much debris, prepare 4µM-Ara-C solution in conditioned culture medium and replace the entire (~90%) medium with it. Works just as good.

Conditioned Medium

1. Prepare cells at 10k/ml in culture medium and place 20ml in a culture flask (BD353136)
2. Replace the medium with 4µM Ara-C containing medium when glias formed smooth islands that cover roughly ½ of area.
3. In 2-3 weeks most neurons should die. If not, incubate in the refrigerator overnight.
4. When the glias are neuron-free, they are ready to be used to condition the medium
5. Simply incubate fresh medium overnight and use it the next day. When not in use, incubate them in 4µM Ara-C culture medium. Glias are good up to 4 months, but are generally replaced every 1-2 months.