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Endothelial Cell Systems Instructions
 

Endothelial Cell Systems - Instructions For Use

Table of Contents

THESE INSTRUCTIONS APPLY TO ORDERS CONTAINING THE FOLLOWING CELL PRODUCTS

Cryopreserved Cells

Human Microvascular Endothelial Cells

CC-2505 HMVEC-d Neo Dermal - Neonatal   500,000 cells/amp
CC-2516 HMVEC-d Neo Dermal - Neonatal (Pooled from 500,000 cells/amp
      several donors)  
CC-2543 HMVEC-d Ad Dermal - Adult   500,000 cells/amp
CC-2527 HMVEC-L Lung   500,000 cells/amp
CC-2564 UtMVEC-Myo Uterine-Myometrial   500,000 cells/amp

Large Vessel

CC-2517 HUVEC Human Umbilical Vein   500,000 cells/amp
CC-2519 HUVEC Human Umbilical Vein (Pooled from 500,000 cells/amp
      several donors)  
CC-2585 HCAEC Human Coronary Artery   500,000 cells/amp
CC-2535 HAEC Human Aortic Artery   500,000 cells/amp
CC-2530 HPAEC Human Pulmonary Artery   500,000 cells/amp
CC-2545 HIAEC Human Illac Artery   500,000 cells/amp
CC-2520 HUAEC Human Umbilical Artery   500,000 cells/amp

*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:

T - 25 FLASK 6 - Well Plate 96-Well Plate
T - 75 FLASK 12 - Well Plate  
T - 150 FLASK 24 - Well Plate  
T - 225 FLASK 48 - Well Plate  

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
ACRONYM
DESCRIPTION
MEDIUM*
HUVEC

HPAEC

HAEC

HUAEC

Umbilical Vein

Pulmonary Artery

Aortic Artery

Umbilical Artery

EGM ®-2 BulletKit® CC-3162

(or)

EGM® BulletKit® CC-3124

HCAEC

HIAEC

HMVEC-d Neo

HMVEC-d Ad

HMVEC-L

UtMVEC-Myo

Coronary Artery

Iliac Artery

Dermal - Neonatal

Dermal - Adult

Lung

Uterine-Myometrial

EGM ®-2 MV BulletKit® CC-3202

(or)

EGM® BulletKit® CC-3125

*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)

EGM® -MV BulletKit®

EGM®-2 BulletKit®

EGM® -2-MV BulletKit®

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:

Product Name
Passage For Shipment
 
 
Cryopreserved
Proliferating
All Microvascular Endothelial Cells 3rd or 4th 4th or 5th passage
HUVEC primary 2nd passage
All Large Vessel Endothelial Cells 3rd 4th passage

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.

FULLY SUPPLEMENTED EGM® (CC-3024)    
How Prepared
Storage Requirements
Shelf Life
The pH is approximately 7.8 and osmolality approximately 294 mOsm/kg.

Prior to shipping, basal medium is supplemented with epidermal growth factor, hydrocortisone, insulin, gentamicin, amphotericin-B and FBS.

After receiving, you will add BBE immediately before use to complete the EGM® formulation.

EGM® is stored at 4·C until shipped. The attached BBE will thaw during shipment.

Fully supplemented EGM® should be stored at 4·C.

Avoid repeated warming and cooling. If the entire contents are not needed for a single procedure, transfer only the required volume to a sterile secondary container. Do not freeze.

EGM® has an optimum shelf life of 5 months from date of manufacture.
  EGM® BulletKit® (CC-3124) and EGM®-MV BulletKit® (CC-3125) and

EGM®-2 BulletKit® (CC-3162) and EGM®-2- MV BulletKit® (CC-3202)

 
How Prepared
Storage Requirements
Shelf Life
All EGM ® , EGM ®-MV BulletKit ® , EGM®-2 and EGM®-2-MV BulletKit® components have been tested against Clonetics ® Normal Human Cells. All solutions are sterile-filtered by passage through a 0.2 µm filter. Basal medium is stored at 4-8·C, and growth factors are stored at -20·C until shipment. If thawed upon arrival, growth factors can be stored at 4·C and added to EBM® or EBM®-2 within 72 hours of receipt.

If thawed and will NOT be used within 72 hours, growth factors must be refrozen. They may be refrozen only once and then stored at -20·C for up to one year.

Store EBM® or EBM ®-2 at 4·C. Store fully supplemented EGM® and EGM®-MV and EGM®-2 and EGM®- 2-MV at 4·C.

Avoid repeated warming and cooling. If the entire contents are not needed for a single procedure, transfer only the required volume to a sterile secondary container. Do not freeze.

EGM ® and EGM ®-MV and EGM ®-2 and EGM®-2-MV BulletKit® shelf life is limited by the shelf life of the EBM® and EBM ®-2, respectively, which is 1 year from the date of manufacture. When growth factors are added at any time within this time period we recommend use within 1 month.

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:

Safety Precautions
As a precaution against contamination, follow all procedures for handling products of human origin outlined in "Guidelines to Avoid Personnel Contamination By Infective Agents in Research Laboratories That Use Human Tissues," from the J. Of Tissue Culture Methods.2 (See Bibliography. Page 25)
Always wear gloves and safety glasses when working with all materials. Exercise caution when working with cryopreserved cells; rapid temperature changes may cause splattering of liquid nitrogen.
Wash hands thoroughly after performing all procedures.
Never mouth pipet.
Do not smoke, eat or drink in areas where reagents or cells are handled.
Products of human origin are potentially biohazardous. Although each cell strain tests negative by PCR for HIV-1, hepatitis B and hepatitis C, proper precautions must be taken to avoid inadvertent exposure.

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:

Step
Explanation
Prepare a sterile field. A sterile field consists of a Class II biological safety cabinet with a front access opening and filtered laminar airflow, or other such equivalent device.
Determine the amount of medium required. Review the Growth Area of Common Plasticware Chart (Appendix E) to determine the amount of medium to be used.
Collect sterile instruments and vessels.
  • Sterile disposable serological pipettes
  • Micropipetters and sterile pipette tips
  • Adjustable multichannel pipetter or repeating pipetter*
  • Sterile reservoirs for use with multichannel pipetter*
  • Sterile 15 ml centrifuge tubes
  • Cell culture flasks
  • Multi-well, flat-bottom tissue culture plates*
  • Hemacytometer or cell counter
Collect other supplies.
  • 70% alcohol (ethanol or isopropanol)
  • Growth medium (cell-type specific)
  • Protective gloves and garments
  • Trypan Blue
Plan and prepare for initial set up. Base your set up on the number of cells indicated on the accompanying Certificate of Analysis. (See Appendices B and C).
Check the calibration on humidified incubator. Incubator should be a 5% CO2/95% air, humidified incubator, set to 37·C.

*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.

Cryopreserved cells are very delicate. Thaw and return them to culture as quickly as possible with minimal handling!

Wear eye protection when handling frozen cells. Rapid temperature changes may cause splattering of liquid nitrogen.

Centrifugation should not be performed to remove cells from the cryoprotectant cocktail. This action is more damaging than the effects of DMSO residue in the culture.

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!!