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 pipeting 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...

Instruction for Cryopreserved Cells

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 FGM? and FGM?-2 BulletKits?, do the following:

1. Decontaminate the external surfaces of the SingleQuot? cryovials and the basal medium bottle with ethanol or isopropanol.

2. Aseptically open each cryovial and add the entire amount to the basal medium 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 SingleQuot? 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 6). This supplemented medium will now be referred to as either FGM? or FGM?-2.

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 mm, low protein binding filter. Routine refiltration is not recommended.

Set Up 
To set up vessels for NHDF and NHLF 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 recommended seeding density of 3500 cells/cm2 for NHDF and 2500 cells/cm2 for NHLF.

No. of cells available / 3500 cells /cm2 = max. surface area that can be plated

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

Example: A cryovial with 520,000 cells

520,000 / 3500 = 148 cm2

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

148 cm2 / 25 cm2 = 5 flasks (rounded down to nearest whole number of flasks)

A typical cryovial can be plated into at least five T-25 flasks. The advantage of setting up five 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 more T-25 flasks to use.

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

Example: For first passage out of cryopreservation for lung fibroblasts with strain number 5099, the label might appear as follows:

3· NHLF 5099; 12/30/96

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.

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 the 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 cryovial 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.