II. Preparation for Culturing
- Ensure the Class II Biological Safety Cabinet, with HEPA filtered laminar airflow, is in proper working condition.
- Sterilize the Biological Safety Cabinet with 70% alcohol.
- Turn the Biological Safety Cabinet blower on for 10 minutes before beginning cell culture work.
- Make sure all serological pipettes, pipette tips and reagent solutions are sterile.
- Follow the standard sterilization technique and safety rules:
a. Do not pipette by mouth.
b. Always wear gloves and safety glasses when working with human cells even though all the strains have been
tested negative for HIV, Hepatitis B and Hepatitis C.
c. Handle all cell culture work in a sterile hood.
III. Culturing HLMVEC
A. Preparing Cell Culture Flasks for Culturing HLMVEC
- Take the Extracellular Matrix Protein Coated Flasks (121-25-02) and Microvascular Endothelium Cell Growth Medium (111-500) from the refrigerator. Decontaminate the bottle with 70% alcohol in a sterile hood.
- Prepare two Extracellular Matrix Protein Coated Flasks (121-25-02) for culturing HLMVEC by pipetting 7.5 mL of Microvascular Endothelium Cell Growth Medium (111-500)* to each flask.
*Keep the medium to surface area ratio at 1-1.5mL per 5 cm2.
- 5 mL for a T-25 flask (SIAL0639) or a 60 mm tissue culture dish (SIAL0166).
- 15 mL for a T-75 flask (SIAL0641) or a 100 mm tissue culture dish (SIAL0167).
B. Thawing and Plating HLMVEC
- Remove the cryopreserved vial of HLMVEC from the liquid nitrogen storage tank using proper protection for your eyes and hands.
- Turn the vial cap a quarter turn to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
- Thaw the cells quickly by placing the lower half of the vial in a 37 °C water bath and watch the vial closely during the thawing process.
- Remove the vial from the water bath when only a small amount of ice is left in the vial. Do not let cells thaw completely.
- Decontaminate the vial exterior with 70% alcohol in a sterile Biological Safety Cabinet.
- Remove the vial cap carefully. Do not touch the rim of the cap or the vial with your hands to avoid contamination.
- Resuspend the cells in the vial by gently pipetting the cells 5 times with a 2 mL pipette. Be careful not to pipette too vigorously as to cause foaming.
- Pipette 0.5 mL of the cell suspension from the vial into each Extracellular Matrix Protein Coated Flask (121-25-02) containing 7.5 mL of Microvascular Endothelium Cell Growth Medium (111-500).
- Cap the flask and rock gently to evenly distribute the cells.
- Place the T-25 flask in a 37 °C, 5% CO2 humidified incubator. Loosen the cap to allow gas exchange. For best results, do not disturb the culture for 24 hours after inoculation.
- Change to fresh Microvascular Endothelium Cell Growth Medium (111-500) after 24 hours or overnight to remove all traces of DMSO.
- Change Microvascular Endothelium Cell Growth Medium (111-500) every other day until the cells reach 60% confluency.
- Double the Microvascular Endothelium Cell Growth Medium (111-500) volume when the culture is >60% confluent or for weekend feedings.
- Subculture the cells when the HLMVEC culture reaches 80% confluency.
IV. Subculturing HLMVEC
A. Preparing Subculture Reagents
- Remove the Trypsin-EDTA solution (T3924) and Trypsin Inhibitor (T6414) from the -20 °C freezer and thaw overnight in a refrigerator.
- Make sure all the subculture reagents are thawed. Swirl each bottle gently several times to form homogeneous solutions.
- Store all the subculture reagents at 4 °C for future use.
- Aliquot Trypsin/EDTA solution (T3924) and store the unused portion at -20 °C if only a portion of the Trypsin/EDTA (T3924) is needed.
B. Preparing Culture Flask
- Swirl the Attachment Factor Solution (123-100) bottle a few times to form a homogenous solution.
- Decontaminate the bottle with 70% alcohol in a sterile hood.
- Coat T-75 flasks (SIAL0641) by adding 7.5 mL of Attachment Factor Solution (123-100)* and rock the flasks gently to distribute the solution evenly to cover the whole culture surface.
- Coat the culture ware for 30 minutes at 37 °C or for 2 hours to overnight at room temperature.
- Remove the Attachment Factor Solution (123-100) by aspiration in a sterile hood.
- The coated flask can be used immediately or stored at 4 °C for up to 2 weeks.
- Prepare the coated flask for culturing HLMVEC by pipetting 15 mL of Microvascular Endothelium Cell Growth Medium (111-500)* into this coated T-75 flask and wait for seeding.
*The coating concentration is 1 mL per 10 cm2 surface area of culture ware.
- 2.5 mL for coating a T-25 flask (SIAL0639) or a 60 mm tissue culture dish (SIAL0166).
- 7.5 mL for coating a T-75 flask (SIAL0641) or a 100 mm tissue culture dish (SIAL0167).
C. Subculturing HLMVEC
Trypsinize Cells at Room Temperature. Do Not Warm Any Reagents to 37 °C.
- Remove the medium from culture flasks by aspiration.
- Wash the monolayer of cells with HBSS (H6648) and remove the solution by aspiration.
- Pipette 2.5 mL of Trypsin/EDTA Solution (T3924) into the T-25 flask. Rock the flask gently to ensure the solution covers all the cells.
- Remove 1.5 mL of the solution immediately.
- Re-cap the flask tightly and monitor the trypsinization progress at room temperature under an inverted microscope. It usually takes about 45 seconds for the cells to become rounded.
- Release the rounded cells from the culture surface by hitting the side of the flask against your palm until most of the cells are detached.
- Pipette 5 mL of Trypsin Inhibitor Solution (T6414) to the flask to inhibit further tryptic activity.
- Transfer the cell suspension from the flask to a 50 mL sterile conical tube.
- Rinse the flask with an additional 5 mL of Trypsin Inhibitor Solution (T6414) and transfer the solution into the same conical tube.
- Examine the T-25 flask under a microscope. If there are >20% cells left in the flask, repeat Steps 2-9.
- Centrifuge the conical tube at 220 x g for 5 minutes to pellet the cells.
- Aspirate the supernatant from the tube without disturbing the cell pellet.
- Flick the tip of the conical tube with your finger to loosen the cell pellet.
- Resuspend the cells in 5 mL of Microvascular Endothelium Cell Growth Medium (111-500) by gently pipetting the cells to break up the clumps.
- Count the cells with a hemocytometer or cell counter. Inoculate at 5,000-6,000 cells per cm2 for regular subculturing.