Astrocyte Cell Culture Protocol

Astrocyte cells can be removed from their original source organism (rat, mouse, etc), isolated, and cultured in vitro. Example protocols for this type of work can be found here: Jove, Nature.

Astrocyte cultures can be purchased from commercial sources: Neuromics.

Astrocyte transfection reagents, kits, and electroporation products are available from Altogen.

Astrocyte Cell Culture

  1. Coat a T75 flask with 1 mg/mL of PureCol and let sit overnight
  2. Aspirate PureCol and rinse flasks with sterile ddH20. Allow flasks to dry upright in a sterile cell culture hood for two hours.

Tissue Dissection:

  1. Remove brainstem, cerebellum and diencephalons. Immediately place in cold dissection buffer
  2. Peel off the meninges and transfer cortex to a tube containing cold dissection buffer; immediately place tube on ice
  3. Pour tissue into a dish and wash, using a syringe, with modified DMEM/F12 culture medium with 10% FBS, 1% glutamine, and gentamicin antibiotic
  4. Mechanically digest tissue by mincing with a sterile razor blade
  5. Transfer tissue to a tube containing 5 mL 1X trypsin and 50 uL DNase; incubate at 37ºC for 25 minutes and remove only to swirl tube every 5 minutes
  6. Wash tissue with Glial Medium twice
  7. Dissociate tissue into a cell suspension by gently titrating through a 5 mL pipette followed by a fire-polished Pasteur pipette. Transfer supernatant to a fresh tube during each titration. Do a total of three titrations using both pipettes
  8. Dilute suspended cells in 10 mL of Glial Medium, and pass the solution through a 40 uM strainer
  9. Centrifuge cells at 1700 rpm for 5 minutes
  10. Remove supernatant, and resuspend pellet with 10 mL Glial Medium
  11. Seed 2 x 106 cells/T75 flask in 15 mL Glial medium
  12. Incubate the flasks at 37ºC in 5% CO2 for 2-3 days without disruption. Change the medium in each flask every 2-3 days by aspirating and adding 15 mL fresh Glial Medium until confluency is achieved (after approximately 6-7 days)

Purify Culture:

  1. After 6-7 days, or once the primary cultures are confluent, change the medium and tighten flask caps. Wrap flasks in plastic and place on shaker platform horizontally with medium covering the cells
  2. Shake at 350 rpm for 6 hours at 37ºC to separate oligodendrocytes from astrocytes
  3. Change medium (10mL) and replace flasks on shaker for 18 more hours
  4. Remove flasks from shaker, and aseptically pour contents into a new T75; incubate flasks
  5. Change medium in flasks (10mL), tighten caps, cover in plastic, and shake, again, for 24 more hours (remember to change the medium at the 6 hour point)
  6. Aseptically pour contents into a new T75 and incubate until confluent
  7. Reseed at 3 x 105 cells in each T75 flask

Passage

  1. Sterilize petri dishes by coating with 1 mg/mL of PureCol Collagen, washing with sterile ddH20, and allowing to dry in culture hood for 30 minutes
  2. Wash glial cells with sterile 1x PBS
  3. Add 3-5 mL of trypsin to the culture flask; incubate at 37ºC for 5 minutes
  4. Add 5-7 mL of Glial Medium to the culture flask, and then transfer cells to a 50 mL tube
  5. Centrifuge cells at 1700 rpm for 5 minutes
  6. Remove the supernatant and resuspend the cells in 10 mL of Glial Medium
  7. Seed cells at 7.5 X 104 cells/6 cm dish in 6mL Glial Medium (using sterilized dishes from step 1)

Tips on Flasks and Petri Dishes

Flasks and dishes are both acceptable plastics to use for cell culture.  Using sterile techniques allows an experienced scientist to utilize either item.  Here are pros and cons here for each item:

  • Flasks
    • Narrow opening means there is a lesser chance of a contaminant entering the flask
    • Narrow opening is ideal for long term culture
    • Shape allows for ease in stacking into cramped incubator space
  • Dishes
    • Wide opening allows for easier manipulation of cells
    • Opening allows for complete removal of washes

Links

List of Companies Offering Astrocyte Laboratory Assays and Services

Hopkins Medicine Astrocyte Cell Culture

Astrocyte Research Articles and References

Measurement of nitric oxide production in astrocytes: Overproduction of nitric oxide in the brain has been correlated with neurotoxicity and some neurodegenerative diseases. It is theorized that suppressing excess nitric oxide production may be beneficial in treatment of these disorders, and so this study tested the capacity of dietary-derived compounds in suppressing release of nitric oxide, finding some compounds which may be valuable in inhibition of nitric oxide. LINK to article