User Guide
Quick-Endothelium™ Vascular - Human iPSC-derived Cells
Catalog Numbers:

VE-mRNA-CW50065, VE-mRNA-CW10149, VE-mRNA-CW20300, VE-mRNA-CW50023,
VE-mRNA-CW70067, VE-mRNA-CW50025, VE-mRNA-CW50113, VE-mRNA-CW50114,
VE-mRNA-CW50115, VE-mRNA-CW50137, VE-mRNA-CW50147, VE-mRNA-CW60130,
VE-mRNA-CW60231, VE-mRNA-CW60236, VE-mRNA-CW20026, VE-mRNA-CW20090, orVE-mRNA-CW10130

Introduction
Elixirgen Scientific’s proprietary transcription factor-based stem cell differentiation method uses synthetic mRNAs toproduce highly pure populations of vascular endothelial cells (VECs) without a genetic footprint. Quick-Endothelium™
Vascular - Human iPSC-derived Cells display morphology similar to human umbilical vein endothelial cells (HUVECs) andexpress a variety of endothelial cell markers, such as platelet and endothelial cell adhesion molecule 1 (PECAM1, also
known as CD31), claudin 5 (CLDN5), and von willebrand factor (VWF). When handled and maintained according to theinstructions in this user guide, vascular endothelial cells are suitable for a variety of characterization and functional assays.
Scale:

Quick- Endothelium™ Vascular- Human iPSC-derived Cells are available in two sizes: (Small) 1
million viable cryopreserved cells and (Large) 5 x 1 million viable cryopreserved cells.

Related Products:

Quick-Endothelium™ Vascular - mRNA Kit, Catalog Number: VE-mRNA
Quick-Endothelium™ Vascular - Maintenance Medium, Catalog Number: VE-MMKit Contents
Upon receipt, immediately store the items at the indicated temperatures. Be especially careful to keep the frozen cells ondry ice until placing them in liquid nitrogen and avoid any temperature fluctuation and slight thawing.
Contents

Amount (Small Size)

Amount (Large Size)

Storage

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

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

Liquid nitrogen

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

Last revised: October 20, 2021

elixirgensci.com

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

iMatrix-511 silk

Elixirgen Scientific

NI511S

Minimum Essential Media (MEM) α, no nucleosides

ThermoFisher

12561056

KnockOut Serum Replacement (KSR)

ThermoFisher

10828010

Sodium Pyruvate (100 mM)

ThermoFisher

11360070

MEM Non-Essential Amino Acids Solution (100X)

ThermoFisher

11140050

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

D8418Nuclease-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 our Cell Dissociation Reagent (Solution D1), Catalog Number: CDR.

elixirgensci.com

2

Workflow* From Day 3, users may maintain differentiated cells in the maintenance medium best suited for their needs, though we recommend
Quick-Endothelium™ Vascular Maintenance Medium, Catalog Number: VE-MM.

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 10 mM 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. This mixture, hereafter referred to as 10 mM SB431542, can be stored 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 E
1. Prepare Medium E using the reagents listed in the table below.
○ All reagents should be warmed at room temperature for 20-30 minutes.
2. Store Medium E for up to 2 weeks at 4°C.

elixirgensci.com

3Medium E 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 μlMedium 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.
2. Store Medium VE for up to 2 weeks at 4°C.
Medium VE Reagents

VolumeMedium E25 ml

20 μg/ml VEGF-165

12.5 μl

10 mM SB431542 (1,000x)

25 μlExperiment 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 entire
plate. Please refer to the table below for plate formats and corresponding surface area of each well used for calculating
reagents in the following sections.
Plate format
Approximate cell growthsurface area per wellDay 0

6-well plate24-well plate

96-well plate

9.5 cm2

1.9 cm2

0.32 cm2

5-6 hours

Plate Preparation
IMPORTANT! Cells can be plated in 6-well or 24-well plates depending on the desired format. Refer to the table at thebottom of this page for the recommended volumes per well.
1. Thaw iMatrix-511 silk on ice for 20-30 minutes (or at 4°C overnight one day before Day 0).
2. Take 2 ml ice-cold PBS into a tube and add 6.6 µl iMatrix-511 silk to it. Mix them well. Store the rest of iMatrix-511
silk at 4°C for its use at Day 8.
3. Select a proper cell culture size suited for your experiment. Add diluted iMatrix-511 silk according to the volumes
listed in Table 1 or scale the volumes described in the protocol by the ratio between well sizes accordingly.

elixirgensci.com

44. Incubate the plate at 37°C, 5% CO2 for 2 hours (or 4°C overnight one day before Day 0).
5. Aspirate the supernatant from each well and add a volume of PBS to it according to Table 1.
6. Incubate the plate at 37°C, 5% CO2 until the cell suspension is ready for plating.
Table 1. Recommended volumes per well for different plate formats.
Recommended volume per well
Reagents

Correspondingsteps

6-well plate24-well plate3

2.0 ml400 µlDiluted iMatrix-511 silk

Thawing Cells
1. Warm Medium E and Medium VE to room temperature for 20-30 minutes.
2. Take 4.5 ml Medium E into a 15 ml conical tube. Take out the vial of Quick-Endothelium™ Vascular (Frozen) fromthe liquid nitrogen storage tank.
3. 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 (approximately 1-2 minutes).
4. Wipe the vial with a dry paper towel. Spray 70% ethanol to the vial and bring it inside a biosafety cabinet.
5. Take 0.5 ml Medium E from the conical tube using a P1000 pipettor and add into the cryovial dropwise at 1 drop /1-2 seconds.
6. Using the same P1000 tip, gently pipet up and down the cell suspension once.
7. Transfer all cell suspension using the same P1000 tip to the 15 ml conical tube containing 4 ml Medium E. Mix themwell by pipetting 3 times.
8. Centrifuge the cell suspension at 200 x g for 4 minutes.
9. While centrifugation is running, take 2 ml Medium VE into a tube and add 2 μl 10 mM ROCK inhibitor to it. Mix themwell. This medium is referred to as Medium iVE.
10. Aspirate most of the supernatant from the centrifuged conical tube but leave a small volume of the supernatant (<50 μl) to cover the pellet.
11. Simply tap the side of the conical tube 4-5 times to break the cell pellet.
12. Add 1 ml fresh Medium iVE to the conical tube with the cell pellet using a P1000 pipettor and pipet it up and down
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 a
10% buffer for cell number and volume. If the volume of the cell suspension needs to be adjusted, centrifuge therequired volume of cell suspension at 200 x g for 4 minutes, remove the supernatant, and resuspend the pellet withMedium iVE 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 Medium iVE, the total volume of themedium in each well is indicated in the table below.
3. Incubate at 37°C, 5% CO2 overnight.
Table 2. Recommended cell plating density
Recommended amounts
6-well plate24-well plate

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

550 µl110 µlVolume of cell suspension distributed/well

500 µl100 µlTotal volume/well● Medium iVE + cell suspension

1 ml

300 µlelixirgensci.com

5Day 1+ (or until the culture reaches nearly 80-90% confluency)< 1 hour

Maintenance
1. Warm Medium VE at room temperature for 20-30 minutes.
2. Pipet out the old medium from each well and add Medium VE to each well along its wall according to the tablebelow.
Required volume per well
Medium VE

6-well plate24-well plate

4 ml

800 µl3. Incubate the culture at 37°C, 5% CO2 for 2 days.
4. Repeat steps 1-3 every 2 days until the culture reaches close to 90% confluency. Do not let the culture overgrow.
Cells can be passaged 2 more times.

Day 3+< 1 hour

Assay or Continuous Maturation●●●

Differentiated vascular endothelial cells can be observed on Day 1. For more mature cells, we recommend culturing
cells until Day 3. From Day 3, users may maintain differentiated cells in the medium best suited for their needs,
though we recommend Quick-Endothelium™ Vascular Maintenance Medium, Catalog Number: VE-MM.
Differentiation into vascular endothelial cells after using Quick-Endothelium™ Vascular - Human iPSC-derived
cells can be confirmed with the endothelial cell markers CD31, CLDN5, and VWF.
Cells can be cultured for up to 3 passages post-thaw.

elixirgensci.com

6

Appendix

Figure 1. Representative phase contrast images of Quick-Endothelium™ Vascular - Human iPSC-derived vascular endothelial cell
cultures on post-thaw days 1, 2, and 3 post-thaw (scale bar = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Endothelium™ Vascular - Human iPSC-derived vascular endothelial- mRNA- CW Kit cell
cultures shows typical vascular endothelial cell morphology and expression of CD31 on day 3 post-thaw (scale bar = 100 μm). Staining
conditions:Purified anti-human CD31 antibody (Biolegend, Catalog number: 303102, 1:50 dilution) was used in combination with asecondary antibody (Invitrogen, Catalog number: A11032 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody,
Alexa Fluor 594, 1:500 dilution). Nuclei were counterstained with Hoechst 33342.

elixirgensci.com7