Protocol Guide: Astrocytes Monoculture

What are Astrocytes?

Specialized glia that are the majority of the central nervous system (CNS), astrocytes do not conduct electrical signals but promote the health of the CNS through regulating the blood-brain barrier (BBB), excess neurotransmitter clearance, and maintaining ion balance, and more. Astrocytes are important targets for drug discovery and development for disorders such as epilepsy, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal cord injury.

In partnership with BrainXell, we provide both cortical and spinal astrocytes. Our cortical astrocytes are energy and structural support glial cells from a forebrain lineage that when co-cultured with neurons, promote and improve neurite outgrowth and synaptic firing. They are fully differentiated and express GFAP at DIV 7. Our spinal astrocytes are energy and structural support glial cell from a spinal cord lineage that when co-cultured with spinal motor neurons, exhibit earlier and higher levels of synchronous firing. They are fully differentiated and express GFAP at DIV 7.

Here we describe a protocol for seeding and culturing astrocytes in monoculture.

Section Overview

• Astrocyte Culture Materials
• Astrocyte Culture Protocol
• Related Products

Astrocyte Culture Materials

Astrocyte cryovials must be stored immediately in a liquid or vape nitrogen storage system. Store the BrainFast supplements at -20°C for up to 6 months or -80°C for up to 18 months. Return astrocyte cryovial to -20°C between each use to maintain astrocyte stability.

• BrainXell Human Astrocytes (BX-0600-30-1M or BX-0650-30-1M)
• BrainFast Astrocyte Supplement (BX-2600-100uL)
• BrainFast SK/Supplement K (BX-2020-100uL)
• DMEM/F12 Medium, +L-glutamine, +HEPES (D0697)
• Neurobasal Medium (Thermo Fisher Scientific #21103049)
• N-2 Supplement (Thermo Fisher Scientific #17502048)
• GlutaMAX Supplement (Thermo Fisher Scientific #35050061)
• Fetal Bovine Serum (Corning #35016CV)
• PDL-coated 96-Well Plate (M0812, LPDL001)
  

Astrocyte Culture Protocol

Day 0: Seeding Preparation

Seed 2.2-2.8 million live cells for a 96-well plate; additional cells can be used depending on experimental needs. For further viability and seeding information, see the Certificate of Analysis (CoA).

1. Using the components listed below, make the Basal Medium in the cell culture hood or biological safety cabinet. Allow medium to come to room temperature for 15 minutes - do not place the medium in a 37°C water bath. Store at 4°C for up to 3 weeks. Culture plates should be equilibrated at room temperature.

2. Prepare cell seeding solutions - add 3ml of Basal Medium to a 50ml conical tube and 25µl of Trypan Blue solution to a separate microcentrifuge tube for cell counting.

Day 0: Seeding Astrocytes

1. Remove a cryovial of astrocytes from the liquid nitrogen storage and place in a 37°C water bath to thaw. Avoid submerging the cap to minimize contamination.
2. Remove the vial from the water bath immediately as the last of the ice melts and disinfect with 70% ethanol. Transfer the vial to the cell culture hood.
3. Transfer 500µl of the Basal Medium from the 50ml conical tube using a P1000 pipette at a rate of 2-3 drops/sec to the astrocyte vial (the transfer process should take about 10 seconds).
4. Transfer the 1ml astrocyte cryovial contents to the 50ml conical tube.
5. Centrifuge the cells at 465xg for 5 minutes.
6. Aspirate the supernatant and resuspend the cell pellet by gently pipette up and down in 950µl fresh Basal Medium with a P1000 pipette.
7. Based on the CoA value, resuspend the cells to 1.0 x 10⁶ live cells/ml final concentration. Slowly add Basal Medium to the existing 1ml in the conical tube to get to the desired concentration.
   a. For example, 2.3 million viable cells/vial is diluted to 2.3 final volume.
8. Count the cells using the Trypan Blue solution from Step 2. Pipette up and down 3-5 times to evenly suspend astrocytes in Basal Medium. Transfer 25µl of the suspension to the 25µl Trypan Blue solution microcentrifuge tube and mix by pipetting. Count the number of viable and dead cells using a hemocytometer. Determine the viability and live cell concentration.
9. Calculate the volume needed to create an astrocyte seeding suspension. The typical seeding density is 20,000 - 25,000 viable cells/100µl/well for a 96-well plate (~62,500 - 78,000 viable cells/cm²), but the recommended seeding density may vary (refer to the CoA for lot-specific information). Dilute the astrocytes to the desired seeding concentration based on Trypan Blue cell count determined above.

Example dilution calculation:

10. In a new 50ml conical tube, mix the previously calculated volumes of the astrocytes and the Basal Medium to obtain 11ml of the astrocyte seeding suspension.
11. Make the Seeding Medium for the astrocytes:

12. Transfer the final, fully mixed Seeding Medium to a PDL-coated 96-well plate at 100µl/well. Do NOT move the plate during the seeding process, as movement can lead to uneven attachment.
13. Allow cells to settle for 10 minutes and transfer plate to the humidified incubator at 37°C with 5% CO₂.

This protocol should not exceed 1 hour. The post-thaw viability of the astrocytes can be impacted if the process takes too long.

Day 1: Astrocyte Medium Replacement

1. Prepare fresh Day 1 Medium:

2. Remove 100µl medium/well and replace with 100µl medium/well of the Day 1 Medium – complete one column or row at a time so the wells do not dry out.

Day 4: Astrocyte Medium Addition

1. Prepare fresh Day 4 Medium:

2. Add 100µl medium/well of the Day 4 Medium to each well for a final volume of 200µl medium/well.

Day 7 and Onward: Astrocyte Medium Changes

1. Change half of the media twice weekly using the Basal Medium that was made in Step 1. Gently remove 100µl/well and add 100µl/well of Basal Medium slowly to the entire plate.

NOTE: Adding BrainFast SK at low concentrations (0.1-0.5X) in the medium may be helpful for long-duration cultures.

Astrocytes can be maintained in culture under the above conditions for at least 3 weeks post-seeding.

These products are available in only the US and UK.