Fibroblast Cell Systems
Instructions for Use

Table of Contents

• Fibroblast Cell System
• General Information
• Product Applications
• Materials Not Provided
• Product Warranty
• Cell Isolation
• Medium Information
• Subculture Reagents
• Quality Control
• Reagent Storage
• Handling Precautions
• Safety Precautions
• Overview of NHDF and NHLF 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 Fibroblast 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 (Single donor)

Proliferating Cells

Proliferating Cells in Preseeded® 96-well Plates

1. Check all containers for leakage or breakage. Fibroblasts are available in 6, 12, 24 and 48 well plates, T-150 and T-225 flasks. Please call your Technical Specialist for details and also prices.
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 20.
   
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.

*AA-1005*
AA-1005-1 Rev. 04/98

Fibroblast Cell System

1. Normal Human Dermal Fibroblasts and Normal Human Lung Fibroblasts from single donors, as either:

The proliferating cultures are shipped in flasks or plates filled with medium. The cells should be between 30 and 100% confluent upon arrival. A Certificate of Analysis is provided with each cell strain and indicates QC performance results and donor information.

The cryopreserved cultures 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. Fibroblast Growth Medium (FGM®), as either:

Fibroblast Growth Medium BulletKit® (FGM® BulletKit®) (CC-3130), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM®) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots® (amounts indicate concentration of each SingleQuot®)

1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
5 mg/ml Insulin (CC-4021) 0.5 ml
50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml

(or)

Fibroblast Growth Medium-2 BulletKit® (FGM®-2 BulletKit® ) (CC-3132), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM®) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots® (amounts indicate concentration of each SingleQuot®)

1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
5 mg/ml Insulin (CC-4021) 0.5 ml
50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml
10 ml FBS (Fetal Bovine Serum) (CC-4101)

3. ReagentPackTM (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 (T/E) (CC-5012) 1 x 100 ml bottle
Trypsin Neutralizing Solution (TNS) (CC-5002) 1 x 100 ml bottle

NOTE: If you use a different Clonetics® medium, see Appendix A.

General Information

Product Applications
Clonetics® Normal Human NHDF and NHLF are:

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

Materials Not Provided
Fibroblast Cell Systems do not contain 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 its Clonetics® cells in the following manner only if Clonetics® media and reagents are used.

1. NHDF and NHLF cryopreserved cultures are assured for experimental use for fifteen population doublings.
2. NHDF and NHLF 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 deteriorate with subsequent passage.
4. NHDF and NHLF 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
Fibroblast cultures are established at BioWhittaker's cell culture facility from normal human tissue.

1. NHDF are isolated according to proprietary procedures, incubated until they approach 90% confluence, harvested and cryopreserved as frozen primaries.
2. NHLF are isolated according to proprietary procedures, incubated until they reach 90% confluence, subcultured once and cryopreserved as frozen secondary cultures.
3. NHDF and NHLF are cryopreserved in FGM® (or) FGM®-2 supplemented with 10% v/v fetal bovine serum and 10% v/v dimethylsulfoxide as a cryopreservation solution to improve cell viability and seeding efficiency upon thawing.

Medium Information

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

1. All cell strains test negative by PCR(1) 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 Clonetics® product specifications for proliferative capacity, (i.e., 15 cumulative population doublings after thaw).
3. Additional Testing:
   

   Cell Type	Von Willebrand Factor	Smooth Muscle Alpha Actin	Cytokeratins 18 & 19
   NHLF	Negative	Negative	Negative

4. Before shipping, all basal media, growth factors and cell culture reagents are tested for sterility and cell culture performance.

Subculture Reagent Storage
1. Subculture reagents are 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 should 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 Neutralizing 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 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.

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 your fingers.
2. Using a micro-pipette with a 1000 ml tip set to 800 ml, 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 an equal amount of cells into the flasks. If five T-25 flasks were prepared, set micropipetter to 200 ml 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, pg. 11).
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 Fibroblasts are not tolerant of 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 that 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 they will suffer contact inhibition and will pop off the flask and/or be difficult to trypsinize.

Overview of Subculture Preparation

Subculture Preparation
NOTE: The following instructions are for a 25 cm2 flask. Adjust all volumes accordingly Preparation 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 throughout 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. For NHDF grown in FGM® use cold T/E (4·C) for subculturing.
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. Have flasks available for seeding cells.

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. For NHLF and NHDF grown in FGM®-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. For NHDF grown in FGM®, skip steps 2 and 3.
3. Aspirate the HEPES-BSS from the flask.
4. Cover the cells with 3 ml of Clonetics® T/E solution. Use cold (4·C) T/E solution for NHDF grown in FGM®.
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 1 to 2 minutes, under optimal conditions.
   
   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 four 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 growth 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 ml.
    b. Flick the cryovial with your finger to loosen the pellet.
    
13. Dilute the cells in 4-5 ml of growth medium and note the total volume of the diluted cell suspension.
14. During these procedures keep the cells in ice until they are plated.
    
    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. Evaluate on bright-field microscope. Follow these steps:
    
15. 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.
    
16. If necessary, dilute the suspension with HEPES Buffered Saline Solution (HEPES-BSS) to achieve the desired "cells/ml" and re-count the cells.
17. Assess cell viability using Trypan Blue (see Appendix C).
18. 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
    
19. 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 is 3500 cells/cm2 for NHDF and 2500 cells/cm2 for NHLF.
    
    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 3500 cells/cm2 = 2 T-75 flasks (rounded down to nearest whole number)
    
20. 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 / 2 T-75 flasks = 2.15 ml per T-75 flask
    
21. Prepare flasks by labeling each flask with the passage number, strain number, cell type, and date.
22. Carefully open the medium bottle and transfer growth medium to new culture vessels 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.
    
23. 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.
24. After dispensing the cells,