User Guide

Quick-Endothelium™ Vascular - Maintenance Medium
Catalog Number: VE-MMIntroduction
Quick-Endothelium™ Vascular - Maintenance Medium may be used for the long-term maintenance of human pluripotent
stem cell-derived vascular endothelial cells following differentiation as outlined in the Quick-Endothelium™ Vascular mRNA Kit user guide. Quick-Endothelium™ Vascular differentiated cell cultures display typical endothelial morphology
and capillary tube formation, and express endothelial cell markers, such as cluster of differentiation 31 (CD31) and vonWillebrand factor (vWF) as well as the tight junction marker Claudin-5 (CLDN5). When handled and maintained 		
according to the instructions in this user guide, vascular endothelial cells are viable long-term and are suitable for avariety of characterization and toxicity assays.
Scale: 									The Quick-Endothelium™ Vascular - Maintenance Medium provides for the expansion andmaintenance of up to 4 wells of a 24-well plate to approximately 3 wells of a 6-well plate overthe course of 2 weeks.

Related Products:

Quick-Endothelium™ Vascular - mRNA Kit (Small), Catalog Number: VE-mRNA-S

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

VolumeStorage

Coating Material A

15.7 μl x 2-20°C or -80°C

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

Conditions 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 contact us at cs@elixirgensci.com or call +1 (443) 869-5420 (M-F 9am-5pm EST).

Last revised: April 22, 2021

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1Required Consumables
ItemVendor

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

Fisher Scientific07-200-740(Optional) 6-well tissue-culture-treated polystyrene plate(e.g., Corning Costar Flat Bottom Cell Culture Plates)*

Fisher Scientific07-200-80

Minimum Essential Media (MEM) α, no nucleosides

ThermoFisher

12561056

KnockOut Serum Replacement (KSR)

ThermoFisher

10828010

Sodium Pyruvate (100mM)

ThermoFisher

11360070

MEM Non-Essential Amino Acids Solution (100X)

ThermoFisher11140050

Glutamax (100x)

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

TrypLE Select Enzyme (1X)**

ThermoFisher

12563011

0.02% EDTA in DPBS

Sigma-Aldrich

E8008-100ML

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

ThermoFisher

20012050

β-mercaptoethanol (β-ME)

ThermoFisher

21985023

ROCK inhibitor Y27632

Selleckchem

S1049

SB431542

Selleckchem

S1067

VEGF-165

Peprotech

100-20Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D8418

Nuclease-free H20

Fisher ScientificAM9937

50% Glycerol solution, sterile

Fisher Scientific50-841-704

30% Bovine Serum Albumin (BSA) solution

Sigma-Aldrich

A9576*Can be substituted with 35-mm culture dishes.
**Can be substituted with our Cell Dissociation Reagent (Solution D1), Catalog Number: CDR.

Workflow

Note: Depending on the inital number of wells (1-4) from the 24-well plate and the confluence of the cells in those wells,
the day of passaging will vary and may be earlier or later than Day 8 indicated in the workflow above. We recommend a
1:3 split when passaging the cells. You may need to passage more than once during a 15 day period. Do not allow thecells to become more than 80-90% confluent.

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Media 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.
*Can be substituted with our Cell Dissociation Reagent (Solution D1), Catalog Number: CDR.
10 mM β-ME stock solution in PBS (10 mM β-ME)
1. Mix 80 µl 55 mM β-ME with 360 µl PBS.
2. Sterile filter the mixture, hereafter referred to as 10mM β-ME, and store at 4°C.
10 mM SB431542
1. Dissolve 10 mg SB431542 in 2.60 ml DMSO.
2. Make aliquots of a convenient volume (e.g., 100 μl).
3. Store at -80°C for up to two years.
20 μg/ml VEGF-165
1.
2.
3.
4.
5.

Dissolve 2 μg VEGF-165 in 20 µl of 0.1% BSA prepared with nuclease-free H2O.
Add 60 µl 0.1% BSA prepared with PBS.
Add 20 µl of 50% Glycerol solution.
Make aliquots of a convenient volume (e.g., 20 μl).
This mixture, hereafter referred to as 20 μg/ml VEGF-165, can be stored at -20°C.

Medium VE
1. Prepare Medium VE using the reagents listed in the table below.
• Thaw 20 μg/ml VEGF-165 on ice for 20-30 minutes.
• All other reagents should be warmed at room temperature for 20-30 minutes.
Medium VE Reagents

VolumeMinimum Essential Media (MEM) α, no nucleosides

28.8 ml

KnockOut Serum Replacement

1.6 mlSodium Pyruvate (100 mM)

320 μlMEM Non-Essential Amino Acids Solution (100X)

320 μl200 mM Glutamax (100x)

320 μlPenicillin-Streptomycin (10000 units/ml; 100x)

320 μl10 mM β-ME

320 μl20 μg/ml VEGF-165

16 μl

10 mM SB431542 (1,000x)

32 μl

2. Store Medium VE for up to 2 weeks at 4°C.

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~4 hr

Day 1

IMPORTANT! Cells should be passaged if cultures reach 80-90% confluency. After passaging, the new cultures may
take 7 days to reach confluency again.
Plate Preparation
1. Prepare diluted Coating Material A by mixing together the following components in a 15 ml conical tube.
• Thaw Coating Material A on ice for 20-30 minutes (or at 4°C overnight one day before Day 1).
• Make sure chilled PBS is used for this mixture.
• Keep the rest of Coating Material A at 4°C.

2.
3.
4.
5.

Diluted Coating Material A Reagents

VolumeCoating Material A

6.6 µlChilled PBS

2 ml

Add 2 ml diluted Coating Material A to 1 well of a 6-well plate.
Incubate the plate at 37°C, 5% CO2 for 2 hours (or 4°C overnight one day before Day 1).
Aspirate the supernatant from the well and add 500 µl PBS.
Incubate the plate at 37°C, 5% CO2 until the cells are ready for plating.

Plating
1. Prepare Medium iVE by mixing together the following components in a 15 ml conical tube.
• Warm Medium VE, iROCK, and Solution D1 at room temperature for 1 hour.
Medium iVE Reagents

VolumeMedium VE

2 mliROCK

2 µl

2. Tap the Solution D1 tube 5 times with a finger and centrifuge at maximum speed for 1 minute.
3. Pipet the old medium out from each of up to 4 wells of vascular endothelial cell culture (on a 24-well plate) using
P1000 pipettor and add 500 μl PBS.
4. Rock the plate 3 times, pipet out PBS from each well using a P1000 pipettor, and add 75 μl of the cell dissociation
reagent Solution D1. Keep the rest of Solution D1 at 4°C for its use on subsequent passages.
5. Incubate the cultures at 37°C, 5% CO2 for 5 minutes. If all the cells are not rounded under a microscope, incubate at37°C, 5% CO2 for up to 5 more minutes in 1-2 minute increments.
6. Carefully pipet out Solution D1 from each well using a P1000 pipettor and add 250 μl Medium iVE.
7. Disperse the medium over the well bottom surface by pipetting 8-15 times to detach cells.
8. Collect the cell suspension from each well into a tube.
9. Add Medium iVE to bring up the volume to 1.5 ml.
10. Aspirate PBS from the coated well and add 1.5 ml cell suspension.
11. Incubate the culture at 37°C, 5% CO2 overnight.

Days 2-8~1 hr

Maintenance
1.
2.
3.
4.

Warm Medium VE at room temperature for 20-30 minutes.
Pipet out the old medium from the well using a P1000 pipettor and add 2 ml Medium VE.
Incubate the culture at 37°C, 5% CO2 for 2-3 days.
Repeat Steps 1-3 every 2-3 days until cells are ready for passaging.

Note: If the cell confluency is more than 60% use 3 ml medium per well.

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Day 8 (Or When Cells Reach 80-90% Confluency)~4 hr

Plate Preparation
1. Prepare diluted Coating Material A by mixing together the following components in a 15 ml conical tube.
• Thaw Coating Material A on ice for 20-30 minutes (or at 4°C overnight one day before Day 8).
• Make sure chilled PBS is used for this mixture.

2.
3.
4.
5.

Diluted Coating Material A Reagents

VolumeCoating Material A

21.5 µl

Chilled PBS

6.5 ml

Add 2 ml diluted Coating Material A to 3 wells of a 6-well plate.
Incubate the plate at 37°C, 5% CO2 for 2 hours (or 4°C overnight one day before Day 8).
Aspirate the supernatant from the well and add 500 µl PBS.
Incubate the plate at 37°C, 5% CO2 until the cells are ready for plating.

Plating
1. Prepare Medium iVE by mixing together the following components in a 15 ml conical tube.
• Warm Medium VE, iROCK, and Solution D1 at room temperature for 1 hour.
Medium iVE Reagents

VolumeMedium VE

5 mliROCK5 µl

2. Tap the Solution D1 tube 5 times with a finger and centrifuge at maximum speed for 1 minute.
3. Pipet out old medium from the well using a P1000 pipettor and add 500 μl PBS.
4. Rock the plate 3 times, pipet out PBS from each well using a P1000 pipettor, and add 300 μl of the cell dissociation
reagent Solution D1.
5. Incubate the cultures at 37°C, 5% CO2 for 5 minutes. If all the cells are not rounded under a microscope, incubate
at 37°C, 5% CO2 for up to 5 more minutes in 1-2 minute increments.
6. Carefully pipet out Solution D1 from the well using a P1000 pipettor and add 1 ml Medium iVE to it.
7. Disperse the medium over the well bottom surface by pipetting 8-15 times to detach cells.
8. Collect the cell suspension from the well into a tube.
9. Add Medium iVE to bring up the volume to 4.5 ml.
10. Aspirate PBS from each coated well and add 1.5 ml cell suspension to each well.
11. Incubate the culture at 37°C, 5% CO2 overnight.
Continuing Culture
1. Continue Maintenance as in the “Maintenance” section on the previous page.
2. Passage as needed when the cells reach 80-90% confluency following the instructions above. We recommend a 1:3
split as above.
3. Make more Medium VE following the instructions above as needed for expansion and maintenance of the cells.

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