Fibroblast Cell Systems Instructions for Use
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Cryopreserved Cells (Single donor)
Proliferating Cells
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.
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.
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
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. ReagentPack TM (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.
Product Applications
Clonetics® Normal Human NHDF and NHLF are:
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 .
Cell Isolation
Fibroblast cultures are established at BioWhittaker's cell culture facility from normal human tissue .
Quality Control
NHDF and NHLF are cultured without antimicrobial agents and assayed to ensure the absence of microbial contamination after cryopreservation.
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:
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:
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:
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
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. 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:
Seeding
After cells are thawed:
NOTE: Do not dispense the entire contents of the cryovial into one T-25 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:
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.
Assessing Yield and Viability
Several factors contribute to low cell count and low cell viability. An example of yield viability assessment is provided in the chart below. To determine the reason for low yield/viability, 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 far, left side of the square. It indicates high yield, or a cell count of more than 250,000 for NHDF and more than 500,000 for NHLF.
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 optimal.
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.
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 13.
6. Passage again when the cells are 70-90% confluent. (If seeded at the recommended seeding density, this should take 5-9 days.)
Instructions for Proliferating Cells
Cell Preparation: Proliferating Cells
With the proliferating culture of NHDF or NHLF you received, do the following:
Subculturing
Examine your cultures microscopically every day.
1) 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.
2) Cytokeratin 18 & 19. Call your Clonetics® Technical Specialist for a reference on this procedure.
3) Wagner, D. D., Olmsted, J.B. and V.J. Marder. (1982) Immunolocalization of Von Willebrand Protein in Weibel-Palade Bodies of Human Endothelial Cells. Journal of Cell Biology ., 95:355-360.
4) 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.
BioWhittaker Inc.
Clonetics® Products
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San Diego, CA 92121
(800) 852-5663
INTERNATIONAL TECHNICAL SERVICE:
BioWhittaker Inc.
Clonetics Products
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301-898-7025
FAX: 301-845-2924
E-mail: techsup@biowhittaker.com
BioWhittaker, Inc.
Clonetics® Products
8830 Biggs Ford Road
Walkersville, MD 21793
(800) 344-6618
APPENDIX A
OVERVIEW OF FIBROBLAST MEDIA
500 ml Bottles (except where indicated)
NOTE: All Clonetics® Media can be custom formulated to meet your research needs. Contact your Technical Specialist for more information.
APPENDIX B
CELL COUNTING USING A HEMACYTOMETER
Proper use of a hemacytometer is critical for obtaining an accurate count of cells and is a procedure used by BioWhittaker to determine the suspension counts for Clonetics® 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.
1.Prepare a cell suspension as instructed in step 13 on page 16.
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.
b. Be sure that the suspension is thoroughly, but gently, mixed 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.
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 or right borders.
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 not achieved, contact your Clonetics® 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
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.
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 ml of 0.4% Trypan Blue into a clean tube.
3. Add 50 ml 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.
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.
a. Allow the suspension to settle in the chambers 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 / Total cells counted (stained + unstained) x 100%
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%
IMPROVING CELL YIELD AND VIABILITY
Several factors, or a combination of factors, contribute to low cell count and low cell viability. If cell yield or viability is unsatisfactory, use the following information to increase the success rate of future cultures.
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).
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 Multi-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 growth medium and counted. The desired number of cells is then added to wells of sterile Multi-well tissue culture plates. The plates are incubated in a 37oC, 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 Dermal Fibroblasts and Lung Fibroblasts of 10,000 cells/cm2 for all multiwell plates.
Required Materials:
Procedure
NOTE: Before using the Multi-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 assays.)
