User Guide
Quick-Glia™ Astrocyte - Human iPSC-derived Astrocytes
Catalog Numbers:

AS-SeV-CW50065, AS-SeV-CW10149, AS-SeV-CW20300, AS-SeV-CW50023,
AS-SeV-CW70067, AS-SeV-CW50025, AS-SeV-CW50113, AS-SeV-CW50114,
AS-SeV-CW50115, AS-SeV-CW50137, AS-SeV-CW50147, AS-SeV-CW60130,
AS-SeV-CW60231, AS-SeV-CW60236, AS-SeV-CW20026, AS-SeV-CW20090, or
AS-SeV-CW10130

Introduction
Elixirgen Scientific’s proprietary transcription factor-based technology allows rapid and reproducible differentiation ofhuman iPSCs into astrocytes without sacrificing the purity of the cells. Our Quick-GliaTM Astrocyte - Human iPSC- derived
astrocytes display typical astrocyte morphology and express markers such as S100 Calcium Binding Protein β (S100β),
Chondroitin Sulfate Proteoglycan 8 (CD44), Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1), and matureastrocyte marker Glial Fibrillary Acidic Protein (GFAP). When handled and maintained according to the instructions in thisuser guide, the iPSC-derived astrocytes are viable long-term and are suitable for a variety of characterization and assays.
Scale:

Quick- Glia™ Astrocyte- Human iPSC-derived astrocytes are available in two sizes: (Small) 1
million viable cryopreserved cells and (Large) 5 x 1 million viable cryopreserved cells. The
instructions outlined in this user guide are for seeding 1 million viable cells at approximately 2.6 x
104 cells/cm2 into 2 wells of a 6-well plate (5 x 105 cells/well), 10 wells of a 24-well plate (1 x 105
cells/well), or 62 wells of a 96-well plate (1.6 x 104 cells/well).

Related Products:

Quick-Glia™ Astrocyte - SeV Kit, Catalog Number: AS-SeV

Contents
Upon receipt, immediately store the items at the indicated temperatures. Be especially careful to keep the frozen cells on
dry ice until placing them in liquid nitrogen and avoid any temperature fluctuation and slight thawing.
Contents

Amount (Small Size)

Amount (Large Size)

StorageThaw

Cryopreserved cells>1 million viable cells,(1 vial, 500 μl)

5 x >1 million viable cells,(5 vials, 5 x 500 μl)

Liquid nitrogen

37°C50 μl

3 x 50 μl-20°C or -80°C

Room temperature

Component P

Condition of Use
This product is for research use only. It is not approved for use in humans or for therapeutic or diagnostic use.

Technical Support
For technical support please refer to the FAQ on our website.
You may also contact us at cs@elixirgensci.com or call +1 (443) 869-5420 (M-F 9am-5pm EST).

Last revised: September 11, 2023

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1Required Consumables
ItemVendor

Catalog Number(Optional) 6-well tissue-culture-treated polystyrene plate(e.g., Corning Costar Flat Bottom Cell Culture Plates)

Fisher Scientific07-200-80(Optional) 24-well tissue-culture-treated polystyrene plate(e.g., Corning Costar Flat Bottom Cell Culture Plates)

Fisher Scientific07-200-740(Optional) 96-well tissue-culture-treated polystyrene plate(e.g., Thermo ScientificTM 96 Well Black/Clear Bottom Plate)Fisher Scientific12-566-70

ScienCell ResearchLaboratories

1801

Geltrex hESC-Qualified, Ready-To-Use,Reduced Growth Factor Basement Membrane Matrix

ThermoFisher

A1569601(Optional) Phosphate-buffered saline (without Ca++ Mg++)*

ThermoFisher

20012050(Optional) TrypLE Select Enzyme (1X)

ThermoFisher12563011(Optional) 0.02% EDTA in DPBS

Sigma-Aldrich

E8008-100ML(Optional) STEM-CELLBANKER**AMSBIO

11890

ScienCell Astrocyte Medium Kit:
• Basal Medium• Astrocyte Growth Supplement
• FBS
• P/S

*PBS should be used at room temperature unless otherwise specified.
**This is only required if you intend to cryopreserve the cells after differentiation.

Workflow
Note: This protocol assumes that Day 0 is a Tuesday.

*From Day 7, users may maintain differentiated astrocytes in the maintenance medium best suited for their needs, though werecommend ScienCell Medium (without FBS and with the addition of Component P).

Experiment Planning
Define the cell culture plate or dish format in advance and calculate the number of wells to be used for each format inadvance. For example, you may use only a certain number of wells of a 96-well plate. The following section describes
culture condition volumes per well as user needs may vary. When a 96-well plate is used, we recommend filling the edge
wells of the plate with an aqueous medium instead of cells and culture medium. This will maintain humidity on the entireplate. If performing an image-based analysis with a 96-well plate, we have found plating approximately 1-2 x 104 cells/well toyield the best results. Please refer to the table below for plate formats and corresponding surface area of each well used for
calculating reagents in the following sections.

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Plate format
Approximate cell growthsurface area per well
Recommended plating viable
cells per well

6-well plate24-well plate

96-well plate

9.5 cm2

1.9 cm2

0.32 cm2

5 x 105 cells

1 x 105 cells

1.6 x 104 cells

Preparation
ScienCell Medium
1.

Prepare ScienCell Medium using the reagents listed in the table below.
○ Warm Basal Medium, Astrocyte Growth Supplement (AGS), and Pen/Strep (P/S) from the ScienCell kit
according to manufacturer’s instructions.
○ Aliquot and store unused AGS at -20°C and the Basal Medium and P/S at 4°C.
Reagents

VolumeBasal Medium

29.5 ml

AGS

300 µlP/S300 µlNote: Although the ScienCell Astrocyte Medium Kit includes FBS, do not add it to the media and instead culture
Quick-GliaTM Astrocytes without FBS.

Day 05-6 hours

Plate Preparation
IMPORTANT! Cells can be plated in 6-well, 24-well, or 96-well plates depending on the desired format. Refer to the table atthe bottom of this page for the recommended volumes per well.
1.
2.
3.
4.
5.
6.
7.

Calculate the required volume of Geltrex by multiplying the number of wells by the required volume per well as
written in the table and add 10% extra. Aliquot the calculated volume of Geltrex into a new tube and keep on ice.
Add Geltrex to each well in the volume specified in the table below.
Incubate the plate at 37°C, 5% CO2 for at least 1 hour (or at 4°C overnight one day before plating).
While the plate is incubating, warm ScienCell Medium at room temperature for 20-30 minutes.
After the Geltrex incubation, aspirate most, but not all, of the supernatant.
Add ScienCell Medium to each well in the volume specified in the table.
Incubate the plate at 37°C, 5% CO2 until cells are ready for plating.
Recommended volume per well
Reagents

Corresponding
steps

6-well plate24-well plate

96-well plate

Geltrex1,21.5 ml

300 µl

50 µl

ScienCell Medium

61 ml

400 µl

35 µl

Thawing Cells
1.

Take out the vial of frozen cells from the liquid nitrogen storage tank.

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2. Incubate the cryovial in a water bath set at 37°C (do not submerge the cap) until the most of the content is thawed
but a small ice crystal remains (~2 minutes).
3. Wipe the vial with a dry paper towel. Spray 70% ethanol to the vial and bring it inside a biosafety cabinet.
4. Transfer 4.5 ml room temperature ScienCell Medium to a new 15 ml conical tube.
5. Set a P1000 pipette to 1 ml but take approximately 500 μl ScienCell Medium from the 15 ml conical and add it to thecryo-vial dropwise at 1 drop per 1-2 seconds.
○ IMPORTANT! Use the same pipette tip for Steps 6-10.
6. Gently pipet the cell suspension up and down once.
7. Gently transfer all of the cell suspension to the 15 ml conical tube prepared in Step 5.
8. Take 1 ml of the cell suspension from the conical tube and add it to the original cryovial and pipet up and down 2-3
times and then transfer the whole contents back to the same 15 ml conical tube.
9. Mix the contents in the conical tube by gently pipetting cell suspension up and down 3 times.
10. Centrifuge the cell suspension at 200 x g for 4 minutes.
11. Use an aspirator to remove most of the supernatant from the conical tube, leaving a small volume of thesupernatant (<50 μl) to cover the pellet.
12. Tap the side of the conical tube up to 10 times to break up the cell pellet.
13. Add 1 ml room temperature ScienCell Medium to the conical tube using a P1000 pipettor and pipet up and down nomore than 2-3 times.
Plating Cells
1.

Count the cells to determine the volume of cell suspension needed for the chosen number of wells and include
10% extra for cell number and volume (e.g., for a 24-well plate scenario, a total of 1.1 x 106 cells to plate 1 x 105 cells
in each of the 10 wells). If the volume of the cell suspension needs to be adjusted, centrifuge the required volume ofcell suspension at 200 x g for 4 minutes, remove the supernatant, and resuspend the pellet with ScienCell Medium
to reach the multiplied volume of cell suspension with the number of wells.
2. Add cell suspension to the center of each well. Since each well already has ScienCell Medium, the total volume ofthe medium in each well is indicated in the table below.
Recommended amounts
6-well plate24-well plate

96-well plate5 x 105 cells

1 x 105 cells

1.6 x 104 cells

Required total volume of cell suspension/well● (Volume of cell suspension/well) + 10% extra

550 µl110 µl16.5 µl

Volume of cell suspension distributed/well

500 µl100 µl

15 µl

Total volume/well● ScienCell Medium + cell suspension

1 ml

300 µl

50 µlViable cells per well

3. Move the plate in 5 cycles of quick back-and-forth and side-to-side motions to evenly distribute cells in the cultures.
4. Incubate at 37°C, 5% CO2 overnight.

Day 1< 1 hour

Maintenance1.

Prepare ScienCell Medium (P) using the reagents listed in the table below.
○ Thaw Component P for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Warm ScienCell Medium at room temperature for 20-30 minutes.
○ Tap the tube of Component P 3 times and then briefly spin down before use.

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Reagents

VolumeScienCell Medium

40 ml

Component P20 µl

2. Pipet out the old medium from each well, leaving a small volume behind to avoid the cells drying out.
3. Add ScienCell Medium (P) to each well according to the table below.
Required volume per wellReagents
ScienCell Medium (P)6-well plate24-well plate

96-well plate

2.5 ml500 µl100 µl4. Incubate the culture at 37°C, 5% CO2 for 2 days.

Days 3-6< 1 hour

Maintenance
1. Warm ScienCell Medium (P) at room temperature for 20-30 minutes.
2. Pipet out the old medium from each well, leaving a small volume behind to avoid the cells drying out.
3. Add ScienCell Medium (P) to each well according to the table below.
Required volume per wellReagents
ScienCell Medium (P)6-well plate24-well plate

96-well plate

2.5 ml500 µl100 µl4. Incubate the plate at 37°C, 5% CO2.
5. Repeat steps 1-4 every 2-3 days.

Day 7< 1 hour

Assay or Continuous Maturation●●

CD44, S100β, GFAP, and ALDH1L1-positive cells can be detected on Day 7. For more mature astrocytes withincreased expression of GFAP and ALDH1L1, we recommend culturing cells until Day 14.
From Day 7, users may maintain differentiated astrocytes in the maintenance medium best suited for their needs,
though we recommend ScienCell Astrocyte Medium (without FBS and without the addition of Component P).

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