User Guide
Quick-Glia™ Astrocyte - SeV Kit (Small)
Catalog Number: AS-SeV-S

Introduction
The Quick-Glia™ Astrocyte - SeV Kit (Small) facilitates rapid and efficient differentiation of human iPS or ES cells into
astrocyte cells in just 28 days. Our proprietary transcription factor-based stem cell differentiation method uses the Sendai
virus to produce astrocytes without a genetic footprint. Quick-Glia™ Astrocyte differentiated cell cultures display typicalastrocyte morphology and markers such as S100 Calcium Binding Protein β (S100β), Chondroitin Sulfate Proteoglycan 8(CD44), Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1), and mature astrocyte marker Glial Fibrillary Acidic
Protein (GFAP). When handled and maintained according to the instructions in this user guide, astrocytes are viablelong-term and are suitable for a variety of characterization and assays.
Scale:The Quick-Glia™ Astrocyte - SeV Kit (Small) contains a set of reagents for use with a total of 4
wells of a 24-well plate.

Related Products:

Quick-Glia™ Astrocyte - SeV Kit (Large), Catalog Number: AS-SeV-L
Quick-Glia™ Astrocyte - Human iPSC-derived Astrocytes, Catalog Number: AS-SeV-CW

Contents
Upon receipt, store the reagents at the temperatures indicated in the table below. All reagents are shipped on dry ice.
Contents

VolumeStorageThaw

QGA-SeV*120 μl-80°C

On iceComponent N1

830 μl

-20°C or -80°C

On ice or 4°C

Component GA1

65 μl-20°C or -80°C

On ice or 4°C

Component GA2

65 μl-20°C or -80°C

Room Temperature

Coating Material A

15.7 μl-20°C or -80°C

Room Temperature*IMPORTANT! This kit contains Sendai
virus (SeV) particles that are active at 33°C
and become inactive at 37°C. SeV is
non-pathogenic in humans, and humans are
not natural hosts of SeV; however, Biosafety
Level 2 (BSL-2) containment is required for
its use. Please use a biological safetycabinet, laminar flow hood, and proper
personal protective equipment in order toprevent
mucosalexposure.
Moreinformation on BSL-2 guidelines can befound at www.cdc.gov/labs/BMBL.html.

This kit contains iMatrix-511 silk (Nippi, Inc.)

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 19, 2023

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1Required Consumables
Item

VendorCatalog Number

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

Fisher Scientific07-200-740

DMEM/F12

ThermoFisher

21331020

Neurobasal Medium

ThermoFisher

21103049

GlutaMAX

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

ScienCell ResearchLaboratories

1801

TrypLE Select Enzyme (1X)

ThermoFisher

12563011

0.02% EDTA in DPBS

Sigma-Aldrich

E8008-100ML

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

ThermoFisher

20012050

ROCK inhibitor Y27632

Selleckchem

S1049

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D2650

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

ThermoFisher

A1569601AMSBIO

11890

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

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

Source hPSC Culture Conditions
The Quick-Glia™ Astrocyte - SeV Kit (Small) gives the best differentiation results when source human pluripotent stem cells(hPSCs) have been maintained in StemFit® Basic04, StemFit® AK02N, StemFlex™ Medium, or other similar culture
media which enable the maintenance of cultures by single-cell passaging. This protocol also assumes that the source
hPSCs are cultured in a 35-mm culture dish or one well of a 6-well plate. If iMatrix-511 silk is routinely used as a coating
substrate, prepare one culture dish or well precoated with 0.25 μg/cm2 iMatrix-511 silk diluted in 2 ml chilled PBS for this kit.
● The protocols and reagents for StemFit® Basic04 and iMatrix-511 silk culture conditions are available at Elixirgen
Scientific (Catalog Numbers: ASB04-C, NI511S).
● Differentiation should not be performed until the cells are at least 14 days post-thaw.
● We recommend preparing a minimum of 4.4 x 105 viable hPSC for use with this kit. This is usually obtained by using
1 well of a 6-well plate at 50-70% confluency.
● For optimal differentiation, hPSC confluency should be around 50% to 70%. Do not use wells more than 90%
confluent.

Workflow
IMPORTANT! This workflow requires a humidified 33°C, 5% CO2 incubator. Before starting this protocol, please makesure the temperature is stable at 33°C.

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Note: This protocol assumes that Day 0 is a Monday.

* From Day 28, users may maintain differentiated cells in the maintenance medium best suited for their needs. We recommend
continuing with the ScienCell Medium without FBS for an additional 2 weeks for more mature astrocytes.

Preparation
10 mM ROCK inhibitor Y27632 (iROCK)
1. Dissolve 10 mg ROCK inhibitor Y27632 in 3.12 ml DMSO.
2. Make aliquots of a convenient volume (e.g., 100 μl).
3. This solution, hereafter referred to as iROCK, can be stored at -20°C.
0.5X TrypLE Select with EDTA (Solution D1)
1. Mix 1 ml TrypLE Select Enzyme (1X) with 1 ml 0.02% EDTA in DPBS.
2. This mixture, hereafter referred to as Solution D1, can be stored at 4°C for 2 weeks.
Medium N1
1.

Prepare Medium N1 using the reagents listed in the table below.
○ Thaw Component N1 for 20-30 minutes at the temperatures indicated in the “Contents” table on page 1.
○ Warm all other reagents at room temperature for 20-30 minutes.
○ Tap each Component tube 3 times and then briefly spin them down before use.
○ Keep Medium N1, and any subsequent media made with it, protected from light.
○ Store Medium N1 for up to 2 weeks at 4°C.
○ Leftover reagents can be discarded or saved at 4°C for up to 2 weeks.
Reagents

VolumeDMEM/F12

5.3 mlNeurobasal

5.3 mlGlutaMAX

55 μl

Penicillin-Streptomycin (10000 units/ml; 100x)

110 μlComponent N1

330 μlDay 0~4 hours

Note: This protocol assumes that Day 0 is a Monday.
Plate Preparation1.

Prepare diluted Coating Material A by mixing together the following components in a 15 ml conical tube.

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○
○

2.
3.
4.
5.

Thaw Coating Material A on ice for 20-30 minutes (or at 4°C overnight one day before Day 0).
Make sure chilled PBS is used for this mixture.
Reagents

VolumeCoating Material A

6.0 µlChilled PBS

1.8 ml

Add 400 µl diluted Coating Material A to each new well of 4 wells.
Incubate the plate at 37°C, 5% CO2 for 2 hours (or 4°C overnight one day before Day 0).
Aspirate the supernatant from each well and add 500 µl PBS.
Incubate the plate at 37°C, 5% CO2 until the hPSCs are ready for plating.

Treatment
1.

Prepare Medium iN1V by mixing together the following components in a 15 ml conical tube.
○ Warm Medium N1, iROCK, and Solution D1 at room temperature for at least 1 hour protected from light.
○ The rest of Medium N1 should be stored at 4°C for later use.
Reagents

VolumeMedium N1

3.5 mliROCK

3.5 µl

2.
3.
4.
5.

Thaw QGA-SeV on ice.
Aspirate old medium from hPSC culture and add 2 ml PBS.
Rock the plate 3 times, aspirate PBS from the culture, and add 300 μl of the cell dissociation reagent Solution D1.
Incubate the culture plate at 37°C, 5% CO2 for 5 minutes. If all the cells are not rounded under a microscope,
continue to incubate at 37°C, 5% CO2 in 1-2 minute increments (see images below).

6.
7.
8.
9.

Carefully pipet out Solution D1 from the culture and add 1 ml Medium iN1 to the well.
Disperse the medium over the bottom surface of the well by pipetting 8-15 times to detach cells.
Using the same pipet tip, collect the cell suspension in a 1.5 ml tube.
Count cells and determine viability.

IMPORTANT! In this protocol, users will treat hPSCs with QGA-SeV in a tube and then plate the cells onto 4 wells with
500 μl Medium iN1 (1.0 x 105 cells) per well. However, we recommend preparing a suspension of 2.2 ml to avoid
insufficiency. First, QGA-SeV should be mixed with 110 μl of a dense cell suspension to increase the chance that
QGA-SeV finds its host cells. After 10 minutes incubation at 33°C, the total volume will be brought up to 2.2 ml with
Medium iN1. Cell count may vary based on cell health, the method, and instrument used for cell counting.
10. Take out the volume of the cell suspension needed for 4 wells and include a 10% buffer (a total of 4.4 x 105 cells toplate 1.0 x 105 cells in each of the 4 wells). Transfer the determined volume of the cell suspension into a 15 ml
conical tube.
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411. Bring the volume of the cell suspension up to 110 μl with Medium iN1.
If the volume of the cell suspension needed to get 4.4 x 105 cells exceeds 110 μl, centrifuge the required○
volume of cell suspension at 200 x g for 4 minutes, remove the supernatant, and resuspend the pellet into
110 μl Medium iN1.
IMPORTANT! Before adding QGA-SeV, ensure that it is fully thawed. Do not centrifuge, vortex, or mix SeV with apipettor; SeV is highly sensitive to physical stress.
12. Add 110 μl QGA-SeV to the hPSCs and mix them by tapping with finger 2-3 times. Cap the tube loosely to allow gas
exchange.
13. Incubate the cell suspension at 33°C, 5% CO2 for 10 minutes with intermittent mixing, by finger tapping, every 2
minutes.
Plating
1. Bring up the volume of cell suspension to 2.2 ml with Medium iN1.
2. Aspirate PBS from only one coated well at a time and add 500 μl cell suspension to the well. Most of the PBS
should be aspirated but not completely to prevent the coated wells from drying before adding the cell suspension.
Likewise, the cell suspension should be added to the well immediately after PBS is removed. Handle one well after
another.
3. Move the plate in 5 cycles of quick back-and-forth and side-to-side motions to evenly distribute treated cells in thecultures.
4. Incubate the culture plate at 33°C, 5% CO2 overnight.

Day 1< 1 hourMedium Change
1.

Prepare Medium A1 by mixing together the following components in a 15 ml conical tube.
○ Warm Medium N1 at room temperature for 20-30 minutes.
○ Thaw Component GA1 on ice for 20 minutes.
○ Thaw Component GA2, protected from light, at room temperature for 20 minutes.
○ The rest of Medium A1 should be stored at 4°C for its use on Day 2.
Reagents

VolumeMedium N1

7 ml

Component GA1

14 µl

Component GA2

14 µl

2. Pipet out the medium from each well, leaving a small volume behind to avoid the cells drying out, and add 500 μl
Medium A1.
3. Incubate the culture plate at 33°C, 5% CO2 overnight.

Day 2< 1 hourMedium Change and Temperature Shift
Note: This should be performed in the late afternoon.
1. Warm Medium A1 at room temperature for 20-30 minutes.
2. Pipet out the medium from each well, leaving a small volume behind to avoid the cells drying out, and add 500 μl
Medium A1.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.
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5Day 3< 1 hour

IMPORTANT! Following this protocol, users will encounter cell death in the infected cultures. It will be most noticeable the
day after making the temperature shift. When monitoring the health of infected cultures, please refer to the images in the
appendix showing the recovery trajectory of a typical hPSC culture subjected to QGA-SeV-mediated differentiation. It is
optional, but recommended, to include the PBS wash if cell death/floating cells are observed.
Medium Change
1. Warm Medium A1 at room temperature for 20-30 minutes.
2. Pipet out the medium from each well, leaving a small volume behind to avoid the cells drying out, and* add 500 μl
Medium A1.
*(Optional) Slowly add 1 ml PBS alongside the wall of each well to avoid lifting attached cells. Gently pipet out PBS
before adding Medium A1.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 4< 2 hours

Medium Change
1.

Prepare ScienceCell Medium by mixing together the following components in a 50 ml conical tube.
○ Warm Basal Medium, Astrocyte Growth Supplement (AGS), and Pen/Strep (P/S) from the ScienCell kit atroom temperature for 1 hour away from light.
○ Aliquot the Basal Medium in 49 ml aliquots.
○ Aliquot the AGS in 0.5 ml aliquots.
○ Store unused AGS aliquots at -20°C, store the Basal Medium and P/S at 4°C.
Reagents

VolumeBasal Medium

49 ml

AGS

500 µlP/S500 µl2. Prepare ScienceCell Medium with 2% FBS by mixing together the following components in a 50 ml conical tube.
○ Thaw FBS from the ScienCell kit at room temperature for 1 hour.
○ Aliquot FBS in 0.55 ml aliquots and store at -20°C.
○ The rest of ScienCell Medium should be stored at 4°C for up to 1 month.
○ Store unused AGS aliquots at -20°C, store the Basal Medium and P/S at 4°C.
Reagents

VolumeScienCell Medium (Basal + AGS + P/S)

30 ml

FBS

600 µl3. Pipet out the old medium from each well , leaving a small volume behind to avoid the cells drying out.
4. Add 1 ml of ScienCell Medium with 2% FBS to each well, slowly along the wall of the well.
5. Incubate the cultures at 37°C, 5% CO2 for 3 days.

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6Days 7-16

< 1 hour

Maintenance
1.
2.
3.
4.
5.

Warm 2.2 ml of ScienCell Medium with 2% FBS at room temperature for 30 minutes.
Pipet out 500 µl old medium from each well.
Add 500 µl of ScienCell Medium with 2% FBS to each well, slowly along the wall of the well.
Incubate the cultures at 37°C, 5% CO2.
Repeat steps 1-4 every 2-3 days.

Day 17~ 2 hours

New Plate Preparation
1. Aliquot 6.5 ml of Geltrex into a prechilled 15 ml conical tube and keep on ice.
2. Add 300 µl Geltrex to 20 wells of a new 24-well plate.
3. Incubate the plate at 37°C, 5% CO2 for at least 1 hour or until cells are ready for plating. Alternatively, coating can be
performed by incubating the plate at 4°C overnight.
Passaging Cells
IMPORTANT! For the following steps, gently pipet and add solutions. Differentiating cells are delicate and should be
handled with great care. Steps 2-10 below are critical. Perform these steps one well at a time. Refer to the images below
to successfully manage cell treatment and dissociation.

1.
2.
3.
4.
5.
6.

Warm Solution D1 at room temperature for at least 1 hour before use.
Pipet out the old medium from one well and add 1 ml PBS and gently rock the plate.
Pipet out the PBS from the well and add 100 μl Solution D1.
Rock the plate 3 times to spread the Solution D1 evenly.
Incubate the culture plate at 37°C, 5% CO2 for 5 - 10 minutes.
Carefully pipet out Solution D1 from the well and add 500 µl ScienCell Medium (without FBS) to the well along thewall of the well.
7. Disperse the medium quickly over the bottom surface of the well by pipetting 6-8 times to detach cells.
8. Observe cells and/or cell aggregates floating in the well under a microscope. It is normal that 10-20% of cells
remain attached to the well bottom after pipetting. These clusters of cells are not supposed to be lifted. Do not
attempt to detach all of the cells remaining on the well bottom.
9. Gently pipet the cell suspension up and down in the well up to 5 times to break the cell aggregates. Excessive
pipetting can damage the already-suspended neuronal cells.
10. Collect 500 μl cell suspension from the well in a tube using the same pipette tip.
11. Repeat steps 2-10 for the rest of the wells.
12. Count cells and determine viability.
13. Prepare 1 x 105 viable cells/ml cell suspension using ScienCell Medium.

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714. Working two wells at a time, aspirate most, but not all, Geltrex from the plate and directly add 500 µl cell suspension(5 x 104) to each well.
15. Repeat step 2 until the cell suspension has been used up.
Note: The density recommended may not be optimal for all hPSC cell lines as growth rates can vary depending on thehPSC cell line.
16. Move the plate in 5 cycles of quick back-and-forth and side-to-side motions to evenly distribute treated cells in thecultures.
17. Allow cells to attach by incubating the plate at room temperature for 10 minutes.
18. Incubate the cultures at 37°C, 5% CO2 overnight.

Day 18< 1 hourMedium Change
1.
2.
3.
4.

Warm ScienCell Medium (without FBS) at room temperature for 30-40 minutes.
Pipet out the old medium from each well, leaving a small volume behind to avoid the cells drying out.
Add 1 ml of ScienCell Medium (without FBS) to each well.
Incubate the cultures at 37°C, 5% CO2 for 2-3 days.

Days 20-27< 1 hourMedium Change
1.
2.
3.
4.
5.

Warm ScienCell Medium (without FBS) at room temperature for 30-40 minutes.
Pipet out 500 µl of old medium from each well, leaving a small volume behind to avoid the cells drying out.
Add 500 µl of ScienCell Medium (without FBS) to each well.
Incubate the cultures at 37°C, 5% CO2.
Repeat steps 3-5 every 2-3 days, making more ScienCell Medium as needed following the instructions above.

Day 28Assay or Continuous Maturation
●
●

●
●

CD44, S100β, GFAP, and ALDH1L1-positive cells can be detected on Day 28.
For more mature astrocytes with increased expression of GFAP and ALDH1L1, we recommend culturing cells until
Day 42. When cells approach confluence they can be passaged following the instructions on Day 17, though
maintaining them at higher confluency results in higher GFAP expression.
From Day 28, users may maintain differentiated astrocytes in the maintenance medium best suited for their needs,
though we recommend ScienCell Astrocyte Medium without FBS.
Cells can be frozen following the instructions in Appendix B.

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Appendix A

Figure 1. Representative images of Quick-Glia™ Astrocyte - SeV Kit (Small) cell cultures on days 1, 4, 7, 14, 21, 28, 35 and42 post-differentiation (scale bar = 100 μm).

Figure 2. (A) Gene expression profiles of iPSCs and Quick-Glia™ - Astrocyte SeV Culture on day 28 were compared withthe profile of human primary astrocytes and the results are shown as scatter plots. The horizontal axis indicates theexpression levels of genes in human primary astrocytes purchased from ScienCell (Catalog Number: 1800-5), whereas thevertical axis indicates the expression levels of genes in iPSCs (left) and in Quick-Glia™ - Astrocyte SeV Culture on day 28(right). The levels of gene expression are shown based on transcripts per million (TPM) in the log10 scale. Blue and green
dots represent upregulated and downregulated genes (FDR<0.05), respectively, relative to their levels in human primary
astrocytes. (B) Similarities of gene expression profiles of human iPSCs and Quick-Glia™ - Astrocyte SeV Culture on days
14, 28 and 42 to the profile of human primary astrocytes are shown as a bar chart. The vertical axis indicates Pearson
correlation (r) based on median-subtracted logTPM.

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9Figure 3. Real-time quantitative PCR analysis of expression levels of astrocyte-associated genes CD44, GFAP, S100β
and ALDH1L1 were examined. Graphs show comparison of gene expression in Quick-Glia™ - Astrocyte SeV Culture
on day 28 (A) and day 42 (B) with gene expression in human brain total RNA (TaKaRa, Catalog Number: 636530). The
relative gene expression is normalized to phosphoglycerate kinase 1 (PGK1), and then calculated as a fold induction
relative to undifferentiated hPSCs as a control. Error bars show standard deviation. Primers used are listed in Table 1.
Table 1. List of PCR primers used in Figure 4
Gene

Forward primer

Reverse Primer

Primer Concentration

CD44

CTGCCGCTTTGCAGGTGTA

CATTGTGGGCAAGGTGCTATT

250 nM

GFAP

ATCGAGAAGGTTCGCTTCCTG

TGTTGGCGGTGAGTTGATCG

250 nM

S100β

GGCTGGTCTCAAACTTCCTG

TCCACAACCTCCTGCTCTTT

250 nM

ALDH1L1TCACAGAAGTCTAACCTGCC

AGTGACGGGTGATAGATGAT

250 nM

PGK1

GTATGCTGAGGCTGTCACTCG

CCTTCCAGGAGCTCCAAACTGG

250 nMelixirgensci.com

10Appendix B
Freezing Cells Down
Note: After thawing frozen cells, approximately 80% of cells will be viable.
1.
2.
3.
4.

Follow the instructions in steps 1-12 of the “Passaging Cells” section of Day 17.
Determine the volume of the cell suspension and number of cryovials needed to freeze 1 ~ 4 x 106 cells per cryovial.
Centrifuge at 200 x g for 4 minutes.
While waiting for the centrifugation, label each cryovial. We recommend writing the name of the iPSC line used, thetype of neurons, harvesting day and date, and the number of cells in the vial.
5. Aspirate the supernatant and resuspend the pellet with 0.5 ml/vial STEM-CELLBANKER.
6. Distribute 0.5 ml of the suspension to each cryovial.
7. Make sure that the caps are closed tightly and transfer the cryovials into a Mr. Frosty Freezing Container. Make surethat Mr. Frosty contains 250 ml isopropanol.
8. Loosely close the lid of Mr. Frosty with cryovials, put it into a -80°C freezer and leave it overnight or up to a few days.
9. Transfer the cryovials into a liquid nitrogen storage tank.
10. Follow the thawing instructions in the user guide for Quick-Glia™ Astrocyte - Human iPSC-derived Astrocytes,
Catalog Number: AS-SeV-CW.

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