Directions for Use

Prepare S-25-Supplemented M-25 Medium

To support the plating and proliferation of human large vessel endothelial cells, including HUVECs, supplement M-25 Medium with S-25 (included in the kit). S-25 is an endothelial supplement that has been optimized for angiogenesis applications, improved cell health, and increased growth rates.

1. Thaw the S-25 (50X) solution in a 37°C water bath or overnight at 4°C. If thawed in a water bath, do not leave the vial at 37°C after the solution has thawed; alternatively, aliquot the S-25 solution and refreeze only once.
2. Aseptically transfer the contents of the thawed S-25 solution to a bottle containing M-25 Medium at a ratio of 1:50 (20 µl S-25 supplement/1 ml M-25 Medium). Tightly cap the bottle and swirl to mix. Avoid causing medium to foam.
3. Thawed S-25 Supplement should be stored in the refrigerator, in the dark, at 4-7°C, and used within 3 weeks.
4. Prepare supplemented medium as needed and use immediately, and store unused S-25-supplemented medium in the dark at 4-7°C. Do not freeze supplemented medium. When stored properly, S-25-supplemented M-25 Medium is stable for up to 3 weeks.

Tube formation assay

The following procedure was designed for the seeding of cells onto a 24-well plate. Please note that the 4 wells in column 1 (A1 to D1) are not coated with SMS-ECM-1 and can serve as control wells if needed.

1. Remove the plate from the freezer and allow the plate to come to room temperature for 30-40 min.
2. Carefully remove the plastic cover inside of a class 2 biosafety hood.
3. Equilibrate a sufficient volume of M-25 medium without the S-25 supplement to wash the coated wells by incubating the unsupplemented M-25 medium at 37°C in humidified atmosphere containing 5% CO₂. Use approximately 35 ml M-25 medium for each 24-well plate, or 1.5 ml for each of the 20 coated wells.
4. Equilibrate a sufficient volume of M-25 medium with the S-25 supplement (+ antibiotics) by incubating the S-25-supplemented M-25 medium at 37 C° in the humidified atmosphere of 5% CO2. Use approximately 13 ml S-25-supplemented M-25 medium for each 24-well plate, or 0.5 ml for each of the 20 coated wells.
5. Wash the wells three times with 0.5 ml equilibrated unsupplemented M-25 medium. Do not touch the bottom of the plate with the vacuum tip. Instead, you may tilt the plate and remove excess fluid from the side.
6. Add 0.5 ml equilibrated S-25-supplemented M-25 medium (+ antibiotics) to each well.
7. Incubate at 24-well plats at 37°C in a humidified atmosphere containing 5% CO₂. The plate is now ready for the plating of HUVECs.

Plating HUVECs

1. Rapidly thaw cryopreserved HUVECs (0.5ml; >500k) in a 37°C water bath. Please ensure that the tube is incubated for less than 90 seconds and that the cell suspension is only marginally thawed. After 90 seconds in the water bath, mix the HUVECs and continue to thaw them gently at room temperature.
2. Use 20 µl of the suspension to determine the amount and viability of the cells.
3. Add 20-40 µl of the cell suspension to each well (>1000 k cells/ml; 5-10 k cells/cm² per well).
4. Cover the plate, and gently shake horizontally to disperse the cells within the well.

Incubating the 24-well plate

1. Incubate the 24-well plate at 37 °C in a humidified atmosphere containing 5% CO₂.
2. The following day, replace the medium in each well with 0.5 ml of fresh supplemented medium (+ antibiotics). Do not touch the bottom of the plate with the vacuum tip. Instead, you may tilt the plate and remove excess fluid from the side.
3. Remove 0.25 ml medium from each well, and add 0.5 ml fresh supplemented medium (+ antibiotics) to each well (final volume in each well should be 0.75 ml). Do not touch the bottom of the plate with the vacuum tip. Instead, you may tilt the plate and remove excess fluid from the side.
4. Remove 0.25 ml medium from each well, and add 0.5 ml fresh supplemented medium (+ antibiotics) to each well (final volume in each well should be 1 ml). Do not touch the bottom of the plate with the vacuum tip. Instead, you may tilt the plate and remove excess fluid from the side.
5. Remove 0.5 ml medium from each well, and add 0.5 ml fresh supplemented medium (+ antibiotics) to each well (final volume in each well should be 1 ml). Do not touch the bottom of the plate with the vacuum tip. Instead, you may tilt the plate and remove excess fluid from the side.
6. Repeat step 5 three times a week (every 2-3 days).

Summary Table

This table applies only to 24-well plates. *The intervals between steps 2 through 5 may be either 2 or 3 days.

Visualization of cells

Cells and tubule can be visualized using fluorescent or non-fluorescent dyes.

Expected results

These expected results apply to HUVECs that are seeded on a 24-well polystyrene plate precoated with SMS-ECM Matrix at 7,000 viable cells/cm², using S-25-supplemented Medium 200 and incubated at 37°C in a 5% CO₂ atmosphere.

• Day 0: Within 1 hour, most of the HUVECs should be embedded within the matrix and appear as rounded cells.
• Day 1: Cells begin to exhibit a spread shape; some will appear round and be dividing.
• Days 5-10: Cells begin to connect and form a stable microtubule.
• Day 10 (approximately): Cells begin to form a tissue.
• Days 14-21: The tissue remodels dynamically, forming suspended macrovessels; some will be visible to the naked eye. Macrovessels will continue to form, with some remaining stable for months.

Typically, several suspended macrovessels are formed in each well.

Product Cell Assay

Below are examples of embedded microvessels (left) and suspended macrovessels (right) formed after culturing HUVEC's on ANGIOstream™.

Safety and Documentation

Safety Data Sheet

Product Disclaimer

This product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.