Endothelial Cell Systems - Instructions For Use

Table of Contents

• Endothelial Cell System Components
• General Information
• Product Applications
• Materials Not Provided
• Product Warranty
• Cell Isolation
• Medium Information
• Quality Control
• Subculture Reagent Storage
• Handling Precautions
• Safety Precautions
• Overview of Endothelial Cell Culture Process
• Instructions for Cryopreserved Cells
• Before you Begin
• Medium Preparation
• Set Up
• Thawing
• Seeding
• Maintenance After Seeding
• Overview of the Subculture Process
• Subculture Preparation
• Subculturing
• Assessing Cell Yield and Viability
• Maintenance After Subculturing
• Instructions for Proliferating Cells
• Cell Preparation, Proliferating Cells
• Subculturing
• Bibliography
• Appendix A Overview of Endothelial Cell Media
• Appendix B Cell Counting Using A Hemacytometer
• Appendix C Assessment of Cell Viability with Trypan Blue
• Appendix D Improving Cell Yield and Viability
• Appendix E Growth Area of Common Plasticware
• Appendix F Seeding into 96-Well Plates
• FAX Order Form

THESE INSTRUCTIONS APPLY TO ORDERS CONTAINING THE FOLLOWING CELL PRODUCTS

Cryopreserved Cells

Human Microvascular Endothelial Cells

Large Vessel

*AA-1001*
AA-1001 Rev. 04/98

Proliferating Cells

This instruction sheet contains culture information for all of the cell types listed previously in the following proliferating formats:

1. Check all containers for leakage or breakage.

2. For cryopreserved cells - If there is dry ice left in the package, place cryopreserved cell cryovials immediately into liquid nitrogen. If no dry ice is left in the package, thaw and use them immediately.

For proliferating cells - Swab down the flask of proliferating cells with 70% ethanol or isopropanol, then place the flask in 37·C, 5% CO2, humidified incubator and allow to equilibrate for three to four hours. After cells have equilibrated, remove shipping medium from the flask following instructions on page 18.

3. Store cell culture medium in a 4·C refrigerator.

4. If you plan to proceed within 3 days, store all growth supplements, HEPES Buffered Saline Solution (HEPES-BSS) and Trypsin Neutralizing Solution at 4·C. Trypsin/EDTA Solution has a limited shelf life or activation at 4·C. If, upon arrival, Trypsin/EDTA is thawed, immediately aliquot and refreeze at -20·C. If frozen, store at -20·C. If you do not plan to set up the cell culture within 3 days, store all growth supplements and subculture reagents in a -20·C freezer.

Please read and follow these instructions carefully and completely. BioWhittaker is not responsible for product loss due to improper receipt and handling of its products by customers. Replacement product will be sent at the customer's expense.

Endothelial Cell Systems

1. Normal Human Endothelial Cells

*Research results may vary depending upon medium selection. Contact your Technical Specialist for details.

The proliferating cultures (HUVEC secondary 2· - all others quarternary 4·) are shipped in a 25 cm2 flask, 75 cm2 flask or 96-well plate filled with medium. The cells should be between 30 and 80% confluent upon arrival. A Certificate of Analysis is provided with each cell strain and indicates QC performance results and donor information.

The cryopreserved cultures (HUVEC primary 1· - all others tertiary 3·) are shipped in a screw cap cryovial containing approximately 500,000 cells. A Certificate of Analysis is provided with each cell strain and indicates date of cryopreservation. QC performance results, donor information and the number of cells contained in the cryovial.

2. Endothelial Cell Growth Medium (EGM®), as either:

Endothelial Cell Growth Medium (EGM®), (CC-3024), a complete medium in a 500 ml bottle with attached Bovine Brain Extract (BBE) supplement. EGM® is a modified MCDB 131 formulation and is supplied fully supplemented with the following: (amounts indicate final concentration, except BBE)

10 ng/ml hEGF (human recombinant Epidermal Growth Factor)
1.0 µg/ml Hydrocortisone
50 µg/ml Gentamicin, 50 ng/ml Amphotericin B
3 mg/ml BBE (Bovine Brain Extract) (CC-4092) 2 ml
2% v/v FBS (Fetal Bovine Serum), producing EGM®

(or)

Endothelial Cell Growth Medium BulletKit® (EGM® BulletKit®) (CC-3124), or Microvascular Endothelial Cell Growth Medium (EGM®-MV BulletKit®) (CC-3125), which contains a 500 ml bottle of Endothelial Cell Basal Medium (EBM®), and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots®. (amounts indicate concentration of each SingleQuot®)

10 µg/ml hEGF (human recombinant Epidermal Growth Factor) (CC-4017), 0.5 ml
1.0 mg/ml Hydrocortisone (CC-4035), 0.5 ml
50 mg/ml Gentamicin, 50 µg/ml Amphotericin-B (CC-4081), 0.5 ml
3 mg/ml BBE (Bovine Brain Extract) (CC-4092), 2 ml
10 ml FBS (Fetal Bovine Serum) (CC-4101) EGM®, (or) 25 ml FBS (CC-4102) EGM®-MV

(or)

Endothelial Cell Growth Medium BulletKit®-2 (EGM-2® BulletKit®) (CC-3162), or Microvascular Endothelial Cell Growth Medium-2 (EGM2®-MV BulletKit®) (CC-3202), which contains a 500 ml bottle of Endothelial Cell Basal Medium-2 (EBM-2®), and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots®.

0.5 ml hEGF (human recombinant Epidermal Growth Factor)
2.0 ml hFGF-B (human Fibroblast Growth Factor - Basic with heparin)
0.5 ml VEGF (Vascular Endothelial Growth Factor)
0.5 ml Ascrobic Acid (Vitamin C)
0.2 ml Hydrocortisone
0.5 ml Long R3-IGF-1 (Human Recombinant Insulin-like Growth Factor)
0.5 ml Heparin
10 ml FBS (Fetal Bovine Serum) 2% EGM-2, 25 mls in EGM-2-MV 5%
0.5 ml Gentamicin, Amphotercin

4. ReagentPack™ (CC-5034) contains one 100 ml bottle of each of the following subculture reagents:

HEPES Buffered Saline Solution (HEPES-BSS) (CC-5022)1 x 100 ml bottle
Trypsin/EDTA Solution (CC-5012) 1 x 100 ml bottle
Trypsin Neutralizing Solution (CC-5002) 1 x 100 ml bottle

NOTE: If you use a different Clonetics® medium, see Appendix A, Endothelial Cell Media

Endothelial Cell Systems Media Options

BioWhittaker strives to optimize it's Clonetics® media in order to supply it's customers with the best product available for the proliferation of Endothelial cells. Each component of the basal medium and each growth supplement is carefully titered for optimal growth. BioWhittaker currently offers four Clonetics® media choices for the growth of endothelial cells allowing for desired performance and flexibility. When selecting a medium to use refer to specific media recommendations or call your formulation Technical Specialist for assistance.

EGM® & EGM BulletKit® (Endothelial Growth Medium)

• Basal medium based on modified MCDB-131, developed for normal human endothelial cell in a low-serum environment.
• EGM® is supplemented growth medium and includes an attached aliquot of Bovine Brain Extract (BBE)
• EGM® BulletKit® includes basal medium with all supplements and growth factors in separate, frozen aliquots
• Final serum concentration 2%
• EGM® can be used to grow all of Clonetics® endothelial cells except microvascular and coronary artery.

EGM® -MV BulletKit®

• Developed for microvascular and coronary artery endothelial cells
• Same basal medium as in EGM®
• Final serum concentration 5%
• EGM®-MV can be used to grow all Clonetics® endothelial cells except HMVEC-L

EGM®-2 BulletKit®

• Refinements to basal medium (CCMD™130) and the growth factors
• Does not contain BBE
• Final serum concentration 2%
• Improved cell proliferation over EGM®
• EGM®-2 can be used to grow all of Clonetic® endothelial cells except microvascular and coronary artery

EGM® -2-MV BulletKit®

• Developed for the enhanced growth of lung microvascular endothelial cells
• Does not contain BBE
• Final serum concentration increased to 5%
• EGM®-2-MV can be used to grow all endothelial cells
• Same basal media as EGM®-2

EGM® (CC-3024) EGM® BulletKit® (CC-3124)
BBE (Bovine Brain Extract), w/ heparin
hEGF (Human Epidermal Growth Factor)
Hydrocortisone
GA-1000 (Gentamicin, Amphotericin B)
FBS (Fetal Bovine Serum) 10 ml

EGM®-2 BulletKit® (CC-3162)
No BBE (Bovine Brain Extract)
hEGF
Hydrocortisone
VEGF (Vascular Endothelial Growth Factor)
hFGF-B (w/heparin) (Human Fibroblast Growth Factor)
Long R3-IGF-1 (Human Recombinant Insulin-like Growth Factor)
Ascorbic Acid
Heparin
GA-1000 (Gentamicin, Amphotericin-B)
FBS (Fetal Bovine Serum) 10 ml
*CCMD stands for Clonetics Cell Media Development

EGM®-MV BulletKit® (CC-3125)
BBE (Bovine Brain Extract), w/heparin
hEGF
Hydrocortisone
GA-1000 (Gentamicin, Amphotericin B)
FBS (Fetal Bovine Serum) 25 ml

EGM®-2-MV BulletKit® (CC-3202)
No BBE (Bovine Brain Extract)
hEGF
Hydrocortisone
VEGF
hFGF-B (w/ heparin)
Long R3-IGF-1
Ascorbic Acid
GA-1000 (Gentamicin, Amphotericin-B)
FBS (Fetal Bovine Serum) 25 ml

General Information

Product Applications
Clonetics ® Normal Human Endothelial Cells are:

1. FOR RESEARCH ONLY
2. NOT approved for human or veterinary use, for application to humans or animals, or for in vitro diagnostic procedures.

Materials Not Provided
Clonetics ® Normal Human Cell Systems do not include plasticware, glassware or other laboratory equipment used in a cell culture laboratory. Individual components are available separately.

Product Warranty
CULTURES HAVE A FINITE LIFESPAN IN VITRO. BioWhittaker warrants Clonetics ® products only if Clonetics ® media and reagents are used.

1. Cryopreserved cultures are assured for experimental use for fifteen population doublings.
2. Proliferating cultures are assured for experimental use for ten population doublings.
3. Additional population doublings and subcultures are possible, but growth rate, biological responsiveness and function may deteriorate with subsequent passage.
4. Endothelial cells can become irreversibly contact-inhibited if maintained at confluence for more than two days. To avoid the loss of your cells and forfeiture of your warranty, we recommend that you subculture cells before they reach 90% confluence.

Cell Isolation
Clonetics ® endothelial cell cultures are established at BioWhittaker's cell culture facility from normal human tissue. The cell type and the passage in which they are shipped are outlined below:

Endothelial cells are cryopreserved in EGM ® or EGM ®-2 supplemented with 10% v/v fetal bovine serum and 10% v/v dimethyl sulfoxide as a cryopreservation solution to improve cell viability and seeding efficiency upon thawing.

Medium Information
Preparation, storage, and shelf life differs for the following five products: 1) Fully Supplemented EGM ® (CC-3024), and 2) EGM ® BulletKit ® (CC-3124), and 3) EGM ®- MV BulletKit ® (CC-3125) and 4) EGM ®-2 BulletKit ® (CC-3162) and 5) EGM ®-2-MV BulletKit ® (CC-3202). See the table below.

Quality Control
Endothelial cells are cultured without antimicrobial agents and assayed to ensure the absence of microbial contamination after cryopreservation.

1.All cell strains test negative by PCR6 for HIV-1, hepatitis B and hepatitis C.

2.After recovery from liquid nitrogen, cells are tested for viability, growth rate, morphology, seeding efficiency, proliferative capacity, mycoplasma, yeast, fungus and bacteria. Each culture meets in-house specifications for proliferative capacity, (i.e., 15 cumulative population doublings after thaw).

3.HUVEC are characterized by morphological observation throughout serial passage. All other Clonetics® endothelial cells test Positive for von Willebrand Factor VIII and Acetylated LDL and test Negative for Alpha Smooth Muscle Actin.

4.Inaddition to the above staining, HMVEC-L also test positively for platelet endothelial cell adhesion molecule (PECAM).

5.Before shipping, all basal media and cell culture reagents are tested for proper pH, osmolality, sterility, and cell culture performance. Growth factors are tested for sterility and cell culture performance. EBM®, EBM ®-2, EGM ®, and BBE are also tested for endotoxin levels.

Subculture Reagent Storage
1.Subculture reagents are sterile-filtered and then stored at -20·C until shipped from BioWhittaker's Distribution Centers.

2.Subculture reagents may thaw during transport. They may be refrozen once.

3.Subculture reagents can be stored at -20·C for up to one year after thawing once and refreezing.

4.To keep Trypsin/EDTA fresh and active after thawing, you may aliquot it into five 20 ml sterile centrifuge tubes and refreeze at -20·C. Trypsin/EDTA may be stored frozen up to one year.

5.We recommend that HEPES-BSS and the Trypsin Neutralization Solution, once stored at 4·C, be used within one month.

Handling Precautions
Normal human cells are fragile, and require special handling:

1. Upon receipt, immediately store cryopreserved cells in liquid nitrogen. Properly stored cells remain viable indefinitely.
2. Upon receipt, immediately place proliferating cells in a 37·C, 5% CO2, humidified incubator.
3. Do not use the medium or reagents beyond the expiration date.
4. Normal human cells are very sensitive to impurities in commercially available Trypsin. Use only Clonetics® Trypsin; every lot of our Trypsin is tested on normal human cell cultures.
5. Use only Clonetics® media. Keep media refrigerated at 4·C. When using a medium, take just the amount you need and then return the bottle to the refrigerator.
6. Regularly wipe flasks, cryovials, bottles and gloves with 70% isopropyl alcohol or 70% ethyl alcohol.
7. Because cells are anchored to one side of a flask, always add all liquids by pipetting them down the opposite side from where the cells are attached.

Safety Precautions
BioWhittaker stresses the importance of the following precautions:

The flow chart on the following page illustrates the culture process. It is followed by the step-by-step instructions...

Instructions for Cryopreserved Cells Medium Preparation

Before You Begin
Perform the following steps before you begin medium or cell preparation:

*May not be necessary for all end-user assays.

Medium Preparation
Perform the steps below in a sterile field. "Sterile field" is defined above.

For the bottle of fully supplemented EGM®, do the following:

1.Add BBE to a 500 ml bottle of EGM®.

a.Detach the BBE supplement from the medium bottle.

b.Wipe the BBE cryovial and EGM® bottle with ethanol or isopropanol.

c.Add the entire contents of the BBE cryovial (approximately 2 ml) to the EGM® with a pipette. Rinse the BBE cryovial with EGM® and pipette the contents back into the 500 ml bottle.

d.Replace the cap and swirl the medium gently a few times to mix.

e.Record the date the BBE was added on the medium label.

Instructions for Cryopreserved Cells
Medium Preparation

For the EGM® BulletKit®, EGM®-MV BulletKit®, EGM®-2 BulleKit® or EGM®-2-MV BulletKit® do the following:

1. Decontaminate the external surfaces of the SingleQuot® cryovials and the EBM® bottle with ethanol or isopropanol.
2. Aseptically open each cryovial and add the entire amount to the EBM® with a pipette.
3. Rinse each cryovial with the medium. It may not be possible to recover the entire volume listed for each cryovial. Small losses, even up to 10%, should not affect the cell growth characteristics of the supplemented medium.
4. Transfer the label provided with each kit to the basal medium bottle being supplemented. Use it to record the date and amount of each supplement added. We recommend that you place the completed label over the basal medium label to avoid confusion or possible double supplementation.
5. Record the new expiration date on the label based on the shelf life (see table on page 8).
   
   This supplemented medium will now be referred to as EGM®, EGM®-MV, EGM ®-2 or EGM ®-2-MV.

NOTE: If there is concern that sterility was compromised during the supplementation process, the entire newly prepared growth medium may be refiltered to assure sterility. If you refilter, use a sterile 0.2 µm filter. Routine refiltration is not recommended.

Instructions for Cryopreserved Cells
Set Up

Set Up
To set up vessels for endothelial cells coming out of cryopreservation, do the following:

1.Calculate the number of vessels to be set up. Refer to your Certificate of Analysis for the exact number of cells in your cryovial. Refer to Appendix E, Growth Area of Common Plasticware, for help in adjusting this calculation.

NOTE: Flasks and multiwell plates are most effective to subculture these cells.

Use the following calculations to determine the number of vessels to be set up for the following recommended seeding density of 2500 cells/cm2 for HUVEC, HCAEC, HAEC, and HPAEC; 5000 cells/cm2 for HMVEC-d Neonatal, HMVEC-d Adult, and HMVEC-L.

No. of cells available / Recommended Seeding Density = max. no. of cm2 that can be plated

Max. no. of cm2 that can be plated / Effective growth area of flask = max. no. of flasks that can be set up

Example: A cryovial of HMVEC-L with 520,000 cells

520,000 / 5000 = 104 cm2 to be set up

If you use a T-25 with an effective growth area of 25 cm2

104 cm2 / 25 cm2 = 4 flasks (rounded down to nearest whole no. of flasks)

A typical cryovial can be plated into at least four T-25 flasks for HMVEC and eight T-25 flasks for all other endothelial cells. The advantage of setting up this number of T-25 flasks from the initial cryovial, as opposed to larger flasks, is that it reduces the risk of losing large numbers of cells. That is, if you experience difficulty trypsinizing the first T-25 flask, there are other remaining T-25 flasks to use.

2. Label each flask with the passage number, cell type, strain number, and date.

Example: For a primary cryovial of HUVEC with strain number 5658, the label might appear as follows:

1· HUVEC 5658; 12/12/97

3. In a sterile field, carefully open the supplemented bottle of growth medium, and aseptically transfer the medium to new culture vessels by adding 1 ml growth medium for every 5 cm2 surface area of the flask.

Example:5 ml growth medium for a 25 cm2 flask or 60 mm plate.

4. Place caps on vessels loosely if vented caps are not being used (i.e., twist caps until tight, then loosen about * turn). Allow the culture vessels to warm and equilibrate in a 37·C, 5% CO2, humidified incubator for at least 30 minutes.

Instructions for Cryopreserved Cells Thawing

Thawing
NOTE: If more than one cryovial is to be thawed, thaw one cryovial at a time and keep other cryovials in liquid nitrogen until ready for use.

After the flasks have equilibrated for 30 minutes:

1. Prior to thawing, locate a micropipetter.
2. Remove the cryovial of cells from storage. Wipe cryovial with ethanol or isopropanol before opening. In a sterile field, briefly twist the cap a quarter turn to relieve the internal pressure, then retighten. Do not open the cropvial completely.
3. Holding the cryovial, dip the bottom 3/4 of the cryovial in a 37·C water bath, and swirl gently for 1-2 minutes until contents are thawed. Watch your cryovial closely; when the last sliver of ice melts remove it. DON'T submerge it completely. Thawing the cells for longer than 3 minutes results in less than optimal results.
4. Remove the cryovial immediately, wipe it dry, and transfer to a sterile field where the equilibrated flasks should be waiting, ready to seed. Rinse the cryovial with 70% alcohol, then wipe to remove excess.
5. Note the color of the thawed cryovial. Ideally, the color of the thawed cryovial should be pink. If the color is not pink, still seed the cells, note the color and mention this fact to your Technical Specialist if seeding is not successful.

Seeding
After cells are thawed:

NOTE: Do not dispense the entire contents of the cryovial into one T-25 flask!!

1. Remove the cap, being careful not to touch the interior threads with you fingers.
2. Using a micro-pipette with a 1000 µl tip set to 800 µl, put the tip into the cryovial and resuspend the cells, with a gentle, slow and steady up and down pipetting motion no more than five times. DO NOT resuspend quickly, and keep the tip near the bottom to avoid making bubbles.
3. Dispense and equal amount of cells into the flasks set up earlier. If four T-25 flasks were prepared, set micropipetter to 250 µl and dispense. If eight T-25 flasks were prepared, set micropipetter to 125 µl and dispense.
4. Replace the cap or cover, and gently rock the vessels to evenly distribute the cells. Loosen caps if necessary to permit gas exchange (see "Set Up," step number 4).
5. Return the culture vessels to 37·C, 5% CO2 incubator. Lay them flat on the shelf, providing the largest surface for cells to attach. The cells will anchor to the bottom surface of the flask.

Maintenance After Seeding
Normal Human Endothelial Cells are not tolerant or rapid temperature fluctuations or nutrient-deficient medium. Feeding them with fresh growth medium that has been warmed will avert potential problems. (Remember to warm only the amount needed.) Check and feed the cells on the schedule below, even on weekends and holidays.

1.Change the growth medium the day after seeding (to remove residual DMSO and unattached cells), then every other day thereafter while examining them daily.

NOTE: A change of medium requires removal of the medium by aspirating with a sterile pipette on the opposite side of the flask from where the cells are attached. Then warm, fresh medium is added down the same side.

2.Successfully recovered cultures will exhibit the following:

a. Cells with clear non-granular cytoplasm.

b. Numerous mitotic figures after day 2.

3.Feed the cells a larger volume of medium as they become more confluent. Use this table as a guideline:

4.Continue feeding the cells until 70 - 90% confluence. If the cells are allowed to become over-confluent and stay at confluence for more than 2 days, they can suffer irreversible contact inhibition and may pop off the flask and/or be difficult to trypsinize.

Instructions for Cryopreserved Cells Subculturing

Subculture Preparation
NOTE: The following instructions are for a 25 cm2 flask. Adjust all volumes accordingly for other size flasks.

Preparation for subculturing the first flask:

1. Subculture the cells when they are 70-90% confluent and contain many mitotic figures throughtout the flask.
2. For each 25 cm2 of cells to be subcultured, allow 3 ml of Clonetics® Trypsin/EDTA (T/E) to thaw and come to room temperature.
3. For each 25 cm2 of cells to be subcultured, allow 5 ml of Clonetics® HEPES Buffered Saline Solution (HEPES-BSS) to come to room temperature.
4. For each 25 cm2 of cells to be subcultured, allow 3 ml of Trypsin Neutralizing Solution (TNS) to come to room temperature.
5. Remove growth medium from 4·C storage and allow to start warming to room temperature.
6. Prepare new culture vessels:
   a. Prepare five to ten T-75 flasks. The number of flasks needed depends upon confluence and total yield. Larger flasks may be used to save plasticware and time spent on subsequent subcultures. Smaller flasks reduce the risk of losing a substantial part of your culture.
   b. As before, label each flask with the passage number, strain number, cell type, and date.
   c. In a sterile field, carefully open the bottle and transfer growth medium to new culture vessels by adding 1ml growth medium for every 5 cm2 surface area of the flask.
   
   Example: 15 ml growth medium for a 75 cm2 flask.
   
   d. If not using vented caps, loosen caps of flasks. Place the new culture vessels into a 37· C humidified incubator with 5% CO2 and equilibrate the flask for at least 30 minutes.

Subculturing
Subculture one flask at a time. All flasks following the first flask will be subcultured following an optimization of this protocol (explained later in this procedure), based on calculated cell count, cell viability, and seeding density.

In a sterile field:

1. Aspirate the medium from one culture vessel.
2. Rinse the cells with 2 - 3 ml of room temperature HEPES-BSS. DON'T forget this step. The medium contains complex proteins that neutralize the trypsin, making it ineffective.
3. Aspirate the HEPES-BSS from the flask.
4. Cover the cells with 3 ml of Clonetics® T/E solution.
5. Rock the flask to make sure all cells come into contact with the trypsin.
6. Tighten the cap and begin monitoring the flask under the microscope.
7. Continue to examine the cell layer microscopically.
   a.Allow the trypsinization to continue until 3 90% of the cells are rounded up.
   
   NOTE: Rounded up cells are spherical, have smooth edges and are refractile or shiny. If the cells still have protruding nubs which are still attached to the flask, they need more time to trypsinize. This entire process takes about 5-6 minutes.
   
   b.At this point, rap the flask against the palm of your hand to release the majority of cells from the culture surface. If only a few cells detach, you may not have let them trypsinize long enough. Wait 30 seconds and rap again. If cells still do not detach, wait and rap every 30 seconds thereafter.
   
   NOTE: Don't try to get all cells to detach by rapping them severely. This action may damage the cells.
   
   
8. After cells are released, neutralize the trypsin in the flask with 3 ml of room temperature TNS.
   
   If the majority of cells do not detach within seven minutes, the trypsin is either not warm enough or not active enough to release the cells. Harvest the culture vessel as described above, and either re-trypsinize with fresh, warm Clonetics® Trypsin/EDTA Solution (or) rinse with Clonetics® Trypsin Neutralizing Solution and then add fresh, warm medium to the culture vessel and return to an incubator until fresh trypsinization reagents are available.
   
   
9. Quickly transfer the detached cells to a sterile 15 ml centrifuge tube.
10. Rinse the flask with a final 2 ml of HEPES-BSS to collect residual cells, and add this rinse to the centrifuge tube.
11. Examine the harvested flask under the microscope to make sure the harvest was successful by looking at the number of cells left behind. This should be less than 5%.
12. Centrifuge the harvested cells at 220 x g for 5 minutes to pellet the cells.
    a. Aspirate most of the supernatant, except for 100-200 µl.
    b.Flick the cryovial with your finger to loosen the pellet.
13. Dilute the cell pellet in 4-5 ml of growth medium and note the total volume of the diluted cell suspension.
    
    To obtain the best results from your cells, you will assess cell yield and viability with Trypan Blue. Trypan Blue is a dye used to highlight dead cells. Dead cells take up the dye and appear blue, instead of refractile and colorless. Follow these steps:
14. Count the cells with a hemacytometer or cell counter and calculate the total number of cells. (see Appendix B). Make a note of your cell yield for later use.
    
    The cell suspension should contain between 250,000 to 1,000,000 cells/ml for greatest accuracy.
    
    
15. If necessary, dilute the suspension with HEPES Buffered Saline Solution (HEPES-BSS) to achieve the desired "cells/ml" and re-count the cells.
16. Assess cell viability using Trypan Blue (see Appendix C).
17. Use the following equation to determine the total number of viable cells:
    
    Total # of Viable Cells = Total cell count x percent viability / 100
    
    Example:1,000,000 cells x 60 / 100 = 600,000 viable cells
    
18. Determine the total number of flasks to inoculate by using the following equation: The number of flasks needed depends upon cell yield and seeding density. Larger flasks may be used to save plasticware and time spent on subsequent subcultures. Smaller flasks reduce the risk of losing a substantial part of your culture if contamination occurs.
    
    NOTE: recommended seeding density of 2500 cells/cm2 for HUVEC, HCAEC, HAEC, and HPAEC; 5000 cells/cm2 for HMVEC-d Neonatal, HMVEC-d Adult, and HMVEC-L.
    
    Total # of flasks to inoculate = Total # of viable cells / Growth area of flask x Recommended Seeding Density
    
    Example: 600,000 viable cells / 75 cm2 x 2500 cells/cm2 = 3 T-75 flasks (rounded down to nearest whole number)
    
19. Use the following equation to calculate the volume of cell suspension to seed into your flasks.
    
    Seeding volume = Total volume of diluted cell suspension / # of flasks as determined in step 18
    
    Example: 4.3 ml of diluted cell suspension / 3 T-75 flasks = 1.43 ml per T-75 flask
    
20. Prepare flasks by labeling each flask with the passage number, strain number, cell type and date.
21. Carefully open the medium bottle and transfer growth medium to new culture vessels to by adding 1 ml growth medium for every 5 cm2 surface area of the flask (1ml/5cm2).
    
    Example:15 ml growth medium for a 75 cm2 flask.
    
22. After mixing the diluted cells with a 5 ml pipet to ensure a uniform suspension, dispense the volume of suspension calculated above into the prepared subculture flasks.
23. After dispensing the cells, gently rock flask to promote even distribution.
24. If not using vented caps, loosen caps of flasks. Place the new culture vessels into a 37·C humidified incubator with 5% CO2.

Assessing Cell Yield and Viability
Several factors contribute to low cell count and low cell viability. An example of yield and viability assessment is provided in the chart below. To determine the reason for low yield/visibility, follow these steps:

1.Study the sample chart below. It is a sample of high yield, high viability.

a.Note the "solid dot" on the Y axis or far, left side of the square. It indicates high yield, or more than 500,000 cell count.

b.Note the "solid dot" on the X axis or bottom line of the square. It indicates high viability, or more than 50% viability.

c.Extend a line from each dot as shown in the chart. The point where the lines intersect (the bold "X") is located in the High Yield/High Viability quadrant. Thus, the sample is optional.

2.Now, using the blank diagram below, plot your cell yield and cell viability. Follow these steps:

a.Mark a (*) on the Y axis to indicate the total cell count of your culture.

b.Mark a (*) on the X axis to indicate the calculated percent viability of your culture.

3.If your result falls into any quadrant other than the "High Yield/High Viability" quadrant, refer to Appendix D, Improving Cell Yield and Viability, before proceeding to your next trypsinization.

Maintenance After Subculturing
After 24 hours:

1. Examine the cells microscopically. At least 60% of the cells should have attached to the culture flask.
   
   Some cells will be loosely adherent, but most will have spread out on the culture flask surface. At this stage, most cells will be single or in small colonies.
   
2. Change the culture medium to remove residual trypsin and non-attached cells.
3. Incubate for an additional 24 hours, and re-examine the culture.
   a.At this stage, the vessel should have several mitotic figures indicating that the cells have resumed active growth.
   b.If few mitotic figures are observed, contact your Clonetics® Technical Specialist for assistance.
   
4. Change the medium again 48 hours after the day 1 feeding, and every 48 hours thereafter while examining the culture daily.
5. Feed with volumes as outlined in the table on page 16.
6. Passage again when the cells reach 70-90% confluence. (If seeded at recommended seeding density, this should take 5-9 days).

Instructions for Proliferating Cells

Cell Preparation: Proliferating Cells
With the proliferating culture of epidermal cells you received, do the following:

1. Examine the culture microscopically for any signs of distress during shipment (i.e., detachment, rounding-up or atypical morphology). Check the relative cell density and estimate "% confluency." The culture should be 30-100% confluent upon receipt. Some cellular detachment is normal. Please call your Technical Specialist immediately if cells look severely distressed.
2. Decontaminate the external surface of the cell culture flask by wiping with 70% ethanol or isopropanol.
3. Incubate the sealed flask at 37·C, 5% CO2 for three to four hours to equilibrate temperature.
4. Warm an appropriate amount of growth medium (see table on page 16) to 37·C in a sterile container. Warming the entire bottle can shorten the life of the medium. Never warm medium under hot running water or any other uncontrolled temperature source. NEVER MICROWAVE!
5. In a sterile field, carefully open the endothelial cell culture flask, remove the medium and replace it with the warmed, fresh medium. Aseptically remove any medium inside the neck or cap area because it can facilitate microbial contamination.
6. If you are using a nonvented cap, loosen the cap, and return the flask to the 37·C humidified incubator with 5% CO2 for at least 24 hours.

Subculturing
Examine your cultures microscopically every day.

1. Subculture the cells when they reach 70-90% confluence. Endothelial cultures should have numerous mitotic figures throughout the flask. Cells should be ready to subculture within 24 to 48 hours, however, shipping conditions such as temperature fluctuations may affect the actual time at which the cells are ready for subculture.
2. See pages 18-21 for detailed subculturing instructions.

Further Information on Culture of Endothelia Cells

BIBLIOGRAPHY

1) Gimbrone, M.A. (1976) Culture of vascular endothelium. Prog. Hemost. Thromb., 3:1-29.

2) Grizzle, W.E., and S.S. Polt. (1988) Guidelines to Avoid Personnel Contamination By Infective Agents in Research Laboratories That Use Human Tissues, J. of Tissue Culture Methods, Vol. 11, No. 4.

3) Hoshi, H. and W.L. McKeehan. (1986) Isolation, growth requirements, cloning, prostacyclin production and life span of human adult endothelial cells in low serum culture medium. In Vitro Cellular and Developmental Biology, 22 (1), 51-56.

4) Voyta, J.C., Netland, P.A., Via, D.P. and B.R. Zetter. (1984) Specific labeling of endothelial cells using fluorescent acetylated-low density lipoprotein. J. Cell Biology, 81(A), 99.

5) Maciag, T., Cerundolo, J., Ilsley, S., Kelley, P.R. and R. Forand. (1979) An Endothelial Cell Growth Factor from Bovine Hypothalamus: Identification and Partial Characterization. Proc. Natl. Acad. Sci., U.S.A., 79:5674-5678.

6) Polymerase Chain Reaction (PCR) technology is covered by U.S. Patents 4,683,195, 4,683,202, and 4,965,188 owned by Hoffman La-Roche, Inc.

TECHNICAL SERVICE:

BioWhittaker Inc.
Clonetics® Products
9245 Brown Deer Road
San Diego, CA 92121
(800) 852-5663

INTERNATIONAL TECHINICAL SERVICES

BioWhittaker, Inc.
Clonetics® Products
8830 Biggs Ford Road
Walkersville, MD 21793
(800) 898-7025
FAX: 301-845-2924
E-mail: techsup@biowhittaker.com

ORDERS:

BioWhittaker, Inc.
Clonetics® Products
8830 Biggs Ford Road
Walkersville, MD 21793
(800) 344-6618

APPENDIX A
Keratinocyte Media

OVERVIEW OF ENDOTHELIAL CELL MEDIA

500 ml Bottles (except where indicated)

Our media formulation laboratory can provide custom formulations of any Clonetics® medium. Minimum order of ten liters (20 bottles). Call your Technical Specialist for more information.

APPENDIX B
Cell Counting Using a Hemacytometer

CELL COUNTING USING A HEMACYTOMETER

Background

Proper use of a hemacytometer is critical for obtaining an accurate count of cells and is a procedure used by Clonetics to determine the suspension counts for all cell strains. A hemacytometer consists of a thickened glass slide into which a small chamber has been cut to allow for the introduction of cells to be counted. The floor of the chamber is divided (etched) into nine sections; usually only the four corner sections are used in cell counting (See Figure 1 below). With a coverslip in place, each square of the hemacytometer represents a total volume of 0.1 mm3 or 10-4 cm3. Since 1 cm3 is approximately equivalent to 1 ml, the cell concentration per ml (and the total number of cells) can be determined.

Procedure

1. Prepare a cell suspension as instructed in step 13 on page 20.
2. Prepare a hemacytometer for use.
   a.Carefully clean all surfaces of the hemacytometer and coverslip.
   b.Take care to ensure that all surfaces are completely dry using non-linting tissue.
   c.Center the coverslip on the hemacytometer.
3. Pipet approximately 9 microliters (this volume will vary slighting with the brand of hemacytometer) of the cell suspension into one of the two counting chambers.
   a.Use a clean pipet tip.
   b.Be sure that the suspension is thoroughly, but gently, mixed before drawing the samples.
   c.Fill the chamber slowly and steadily.
   d.Avoid injecting bubbles into the chambers.
   e.Do not overfill or underfill the chambers.
4. Count the Cells.
   a.Allow the cell suspension to settle for at least 10 seconds.
   b.Count all of the cells in each of the four 1 mm3 corner squares labeled A thru D in Figure 1 on the next page.
   1) DO count the cells touching the top or left borders
   2) DO NOT count the cells touching the bottom and right borders.

Hemacytometer Reference Figure 1

5. Determine the Cell Count.
   a.Calculate the total cells counted in the four corner squares.
   
   1)If the total cell count is less than 100, or if more than 10% of the cells counted appear to be clustered, carefully re-mix the original cell suspension and repeat steps 2 through 4 (above).
   2)If the total cell count is greater than 400, dilute the suspension so the count will be 100-400 cells. Then repeat Steps 2-4 (above).
   
   NOTE: If satisfactory results are note achieved, contact your Technical Specialist by telephoning 800-852-5663
   
   b.Calculate the cell count using the equation: cells/ml = (n) x 104, where: n = the average cell count per square of the four corner squares counted.
   
   Example:If the calculated average (n) of cells in the four 1 mm corner squares of the hemacytometer is 30:
   
   cells/ml = (n) x 104 (or) cells/ml = 30 x 10,000 = 300,000 cells/ml.
   
   c.Determine the total number of cells in the total suspension volume.
   
   1)Determine the total volume of the cell suspension.
   2)Multiply the volume of the cell suspension by the "cells/ml" value calculated above.
   
   Example:If the initial suspension volume is 2 ml:
   
   cells/ml x total volume = 300,000 cells/ml x 2 ml = 600,000 cells.

APPENDIX C
Assessment of Cell Viability with Trypan Blue

Background

Trypan blue is a dye that enables easy identification of dead cells. Dead cells take up the dye and appear blue with uneven cell membranes. By contrast, living cells repel the dye and appear refractile and colorless.

Using Trypan Blue

1.Prepare the hemacytometer for use.

a.Carefully clean all surfaces of the hemacytometer and cover slip.

b.Take care to ensure that all surfaces are completely dry using non-linting tissue.

c.Center the cover slip on the hemacytometer.

2.Transfer 50 µl of 0.4 % Trypan Blue into a clean tube.

3.Add 50 µl of the prepared cell suspension into the tube containing the stain.

4.Mix the solution thoroughly, but gently. Take care to avoid making excessive bubbles.

5.Allow the mixture to sit for 2-3 minutes after mixing. (Do not let the cells sit in the dye for more than five minutes because both the living and dead cells will begin to take-up the dye after five minutes).

6.Pipet approximately 9 microliters of the Trypan Blue/cell suspension mixture (this volume will vary with brand of hemacytometer) into one of the two counting chambers.

a.Use a clean pipet tip.

b.Be sure that the suspension is mixed thoroughly but gently before drawing the samples.

c.Fill the chambers slowly and steadily.

d.Avoid injecting bubbles into the chambers.

e.Do not overfill or underfill the chambers.

7.Determine Cell Viability.

a. Allow the suspension to settle in the chamber for at least 10 seconds.

b.Count all of the stained cells in each of the four corner squares of the hemacytometer.

c.Separately count all of the unstained cells in the same squares.

d.Calculate the cell viability using the equation:
% Cell Viability = number of unstained (living) cells x 100 / Total cells counted (stained + unstained)

Example: If a total of 300 cells (stained + unstained) are counted and 200 are identified as living cells (unstained), then the viability is calculated as:

% Cell viability = 200 / 300 x 100% = 67%

APPENDIX D
Improving Cell Yield and Viability

Background

Several factors, or a combination of factors, contribute to low cell count and low cell viability. If cell yield or viability if unsatisfactory, use the following information to increase the success rate of future cultures.

Improving Cell Yield

If your cell yield is low (less than 50%), determine the cause(s) and possible solution(s) using the table below. Then subculture one more flask applying the appropriate solution(s).

Improving Cell Viability

If your cell viability is low (less than 50%), determine the possible cause(s) and solution(s) using the table below. Then subculture one more flask applying the appropriate solution(s).

Once you have determined how to achieve high yield and high viability, subculture the remaining flasks.

APPENDIX E
Growth Area of Common Plasticware

APPENDIX F
Seeding Into 96-Well Plates

Overview

A culture flask of normal human cells is harvested by trypsinization and subsequent trypsin inhibitor treatment. The cells are centrifuged, resuspended in qrowth medium and counted. The desired number of cells is then added to wells of sterile 96-well tissue culture plates. The plates are incubated in a 37·C, 5% CO2 humidified incubator for one to three days to allow for cell adherence and growth. Seeding densities will vary somewhat with your experimental requirements. We recommend a density for Endothelial Cells of 10,000 cells/cm2 for all multiwell plates.

Required Materials:

1. T-25 flask of proliferating normal human cells between 70% and 90% confluence.
2. 96-well, flat-bottom, tissue culture plates
3. 37·C humidified incubator with 5% CO2/95% air
4. Laminar flow hood or other sterile environment
5. Adjustable multichannel pipetter (8- or 12-channel) or repeating pipetter
6. Sterile reservoir(s) for use with multichannel pipetter

Procedure

1. Follow the steps on pages 18-21 for subculture preparation and subculturing. Then follow steps 2-4 below.
2. Since the cells/ml calculation computed on p. 20 is "per ml", one must increase the cell concentration by 4 times before seeding 96 well plates (to accommodate the 1:4 dilution when adding 250 ul of suspended cells per well). When making the cell suspension, adjust the cell concentration with growth medium.
3. Transfer the diluted cell suspension to a sterile reservoir. Using a multichannel (8- or 12-channel) pipetter equipped with sterile pipette tips, add 250 µl of the diluted cell suspension to each well of the labeled 96-well, flat bottom, tissue culture plates(s). RESUSPEND THE CELL SUSPENSION OFTEN DURING THE SEEDING PROCEDURE TO ENSURE A UNIFORM NUMBER AND DISTRIBUTION OF CELLS INTO EACH WELL BY PIPETTING UP AND DOWN A FEW TIMES BETWEEN EVERY OTHER DISPENSING.
4. Cover and incubate the plates for 1 to 3 days at 37·C/5% CO2. (Incubation periods exceeding 3 days are generally not recommended because of evaporation of medium from the edge wells of the plate).
   
   NOTE: Before using the 96-well plate culture in a bioassay, examine them microscopically for the presence of mitotic figures as a confirmation that the cells have resumed active growth. (Does not apply for all end-user plate.)

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