Growth, Maintenance and Transfection of Suspension Adapted 293-EBNA cells

Procedure
I. INTRODUCTION

The 293 EBNA cell line is established from primary embryonal human kidney cells transformed with sheared human adenovirus type 5 DNA and Epstein-Barr virus nuclear antigen 1, allowing episomal amplification of plasmids containing the viral EBV origin of replication. This protocol describes the instructions for the growth, maintenance and transfection of suspension adapted 293-EBNA cell line using the PEI expression system. Cells can be purchased from ATCC


II. REFERENCES

•Transfectol-LS Mammalian Transient transfection Kit. 
GeneChoice; TR10v2 KT-011106

III. EQUIPMENT, MATERIALS, AND REAGENTS 
• CO2 incubator, Forma Scientific incubator
• BioSafety Cabinet, 
• Orbital Shaker, Forma Orbital Shaker 
• Water Bath, VWR Scientific
• 500 ml Vented Cap Erlenmymer Flask, 
• 125 ml Vented Cap Erlenmymer Flask, Corning product
• Pro293S-CDM, 1 L (Cambrex Biosciences, Cat 12-765Q) 
• Penicillin-Streptomycin, Invitrogen 
• Transfection Reagent, TR-10 10-Liter Transient transfection 
• BrightLine Hemacytometer, Sigma, 
• Trypan Blue Solution (0.4%), Sigma, 
• 0.22 um GP Express Membrane Filter, Millipore
IV. GENERAL CONSIDERATIONS

A. Prior to Startup
Since 293 cells tend to aggregate in suspension culture, 293-E cells are cultured in Cambrex Pro293 suspension chemically defined media system (Pro293s-CDM), which helps support single cell suspensions. Media does not contain L-Glutamine and should be added prior to use. While the use of antibiotics in antibody production is not recommended, their use should be considered dependent on downstream application. Antibiotics should be used in culture maintenance (5 ml/L Penicillin-Streptomycin). For general cell maintenance split cells when they have reached a density of 1-3 x 106 cells/ml (every 3 to 4 days). Trypan Blue exclusion is used to determine viability and density. 
. 

V. PROCEDURES

A. Thawing and Establishing Cells

1. Prepare 1L Pro293S media (5 mls Penicillin-Streptomycin and 10 ml/L L-Glutamine) 
2. Remove cryovial from Liquid Nitrogen and quickly thaw in 37oC water bath.
3. Add 19 mls pre-warmed complete media to 125 ml polycarbonate shaker flask.
4. When cryovial has thawed, decontaminate outside of vial with 70% ethanol. 
5. Transfer cells to shaker flask containing completed media under biosafety cabinet.
6. Incubate cells in incubator (37oC, 8% CO2) on orbital shaker rotating at 125 RPM. 

B. Passaging Cells

Subculture cells when density is approximately 1-3X106 viable cells/ml, which typically occurs every 3 to 4 days. In order for cells to properly recover after thawing cells should be subcultured for two passages prior to use in transfection. 

1. Determine cell density and viability using Trypan Blue exclusion Method (see Method below)
2. Using cell density determined in step 1, calculate the split ratio needed to seed the new shaker flask at 3x105 cells/ml according to the desired final volume of shaker flask
(3x105 cells/ml)(desired final volume)/cell density = cell volume needed
3. Transfer appropriate volume of cells to correct final volume fresh media
(desired final volume) – (cell volume needed) = fresh media volume
4. Gently pipette cells to break up cell clusters
5. Return cells to incubator (37oC, humidified 8% CO2, orbital shaker 125 RPM). 
6. Repeat steps as necessary to maintain or expand cells

C. Freezing Cells
Cells may be frozen directly in Pro293S media + 10% DMSO. Fresh media should be used when freezing cells and cell density should be 5-8 x 106 cells/ml. 

1. Continue to grow 293E cells until their density reaches 1X106 cells/ml. 
2. Determine cell density and viable cell count (page 4) and calculate the volume of cells needed to yield a final density of 5X106 cells/ml. 
3. Transfer cells to conical tubes and centrifuge 100xg for 5 minutes 
4. Prepare enough freezing media (90% Pro293S media + 10% DMSO) to resuspend cells at a final density of 5X106 cells/ml.
5. Aliquot 1ml of cell suspension to cryovials on ice. 
6. Transfer cells to -70oC overnight
7. Transfer cells to liquid nitrogen freezer for long term storage.

D. Transfecting Cells 

293E cells can be transfected with a variety of methods. Described below is transfection using Transfectol LS (GeneChoice). Transfection experiments can be modified by scaling volumes up or down as needed. The following conditions described are used for 200ml transfections. Changing culture media is not required after transfection and all reagents should be room temperature prior to use.
Transfection:
1. Determine cell density and viability using Trypan Blue exclusion Method (see page 4)
2. If you have enough cells collect 293E culture in an appropriate vessel and centrifuge at 1000 RPM for 5 minutes
3. Aspirate media (Be careful not to disturb pellet) and resuspend cells in fresh Pro293S media (by gentle pippeting) to cell density of 1x106 cells/ml.
a. For optimal results recount cells, viability (should be greater than 90%), and make sure you have single cell suspension. Return culture to incubator.
4. 30 minutes prior to tranfection add 5mL Enhancer LS (Bottle A)
5. Dilute 200µg DNA in total 10 ml volume Diluent LS (Bottle B), mix gently
6. Add 1ml Transfectol LS (Bottle D) , mix gently
7. Incubate Room Temp for 15-20 minutes
8. Add total volume of DNATransfectol-LS Complex to cell culture flask. Swirl flasks.
9. Replace cell culture to 125 RPM, 37oC, 8% CO2 incubator.
10. Collect after 6-8 days.
a. You may want to add some fresh media at about the half way point

Media Collection:
1. Collect cell culture media by centrifugation at 5000 RPM, for 15minutes.
2. Sterile filter media over 0.22 ?m GP Express Membrane filter
3. Store media 4oC prior to antibody purification

VI. ASSOCIATED PROCEDURES
A.Trypan blue viability test
1. Transfer 0.9 ml of 0.4% Trypan Blue + 0.1 ml of cell suspension in an Eppendorf Tube
2. Gently mix to avoid cell clusters by by pipetting up and down
3. Allow to stand for 2 minutes (viable cells will take up dye if incubation goes too long)
4. Pipet enough of mixture into cover slipped chambers of hemacytometer to fill
5. Count viable and non-viable cells in 5 squares. For optimal results, adjust cell density to between 20-50 cells per square 
Calculations: 
Viable cells/ml: the number of viable cells per quadrant × dilution factor × 104 cells / ml (e.g. 1:10 dilution, 200 cells counted in 5 squares = 200/5 = 40 cells per quadrant × 10 × 104 cells / ml = 4 × 106 cells / ml). cells / ml × original volume = Total cells (e.g. 40 million cells in 10 ml) 
cell viability (%): total viable cells (unstained) / total cells (stained and unstained) × 100 (e.g. 20 stained cells per quadrant: 50% viability)