Endothelial Cell Systems - Instructions For Use

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 Hydrocortisone50 µg/ml Gentamicin, 50 ng/ml Amphotericin B3 mg/ml BBE (Bovine Brain Extract) (CC-4092) 2 ml2% 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 ml1.0 mg/ml Hydrocortisone (CC-4035), 0.5 ml50 mg/ml Gentamicin, 50 µg/ml Amphotericin-B (CC-4081), 0.5 ml3 mg/ml BBE (Bovine Brain Extract) (CC-4092), 2 ml10 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 Hydrocortisone0.5 ml Long R3-IGF-1 (Human Recombinant Insulin-like Growth Factor)0.5 ml Heparin10 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 bottleTrypsin/EDTA Solution (CC-5012) 1 x 100 ml bottleTrypsin 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/ heparinhEGF (Human Epidermal Growth Factor)HydrocortisoneGA-1000 (Gentamicin, Amphotericin B)FBS (Fetal Bovine Serum) 10 ml

EGM®-2 BulletKit® (CC-3162)No BBE (Bovine Brain Extract)hEGFHydrocortisoneVEGF (Vascular Endothelial Growth Factor)hFGF-B (w/heparin) (Human Fibroblast Growth Factor)Long R3-IGF-1 (Human Recombinant Insulin-like Growth Factor)Ascorbic AcidHeparinGA-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/heparinhEGFHydrocortisoneGA-1000 (Gentamicin, Amphotericin B)FBS (Fetal Bovine Serum) 25 ml

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

General Information

Product Applications Clonetics ® Normal Human Endothelial Cells are:

FOR RESEARCH ONLY 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 laboratoryequipment used in a cell culture laboratory. Individual components are available separately.

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

Cryopreserved cultures are assured for experimental use for fifteen population doublings. Proliferating cultures are assured for experimental use for ten population doublings. Additional population doublings and subcultures are possible, but growth rate, biological responsiveness and function may deteriorate with subsequent passage. 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 InformationPreparation, 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 ControlEndothelial cells are cultured without antimicrobial agents and assayed to ensure the absence of microbial contamination after cryopreservation.

1.All cell strains test negative by PCR6for 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 Storage1.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 PrecautionsNormal human cells are fragile, and require special handling:

Upon receipt, immediately store cryopreserved cells in liquid nitrogen. Properly stored cells remain viable indefinitely. Upon receipt, immediately place proliferatingcells in a 37・C, 5% CO2, humidifiedincubator. Do not use the medium or reagents beyond the expiration date. 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. 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. Regularly wipe flasks, cryovials, bottles and gloves with 70% isopropyl alcohol or 70% ethyl alcohol. 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 PrecautionsBioWhittaker 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 BeginPerform the following steps before you begin medium or cell preparation: