Editing 293F Protein Production (section)

== '''Cell maintenance and Culture'''<br>  ==

<br> Both 293F and 293FT can be maintained in adhesion and suspension culture, and transfered back and forth freely. <br> For suspension subculturing, both of them use FreeStyle 293 expression media, but 500ug/ml G-418 should be supplemented to maintain the existance of T-antigen in 293FT. 

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1. Remove the cryovial of cells from the liquid nitrogen and thaw quickly in a 37°C water bath.<br><br> 

2. Just before the cells are completely thawed, decontaminate the outside of the vial with 70% ethanol. Gently break up clumps and cell pellet if present and transfer the entire contents of the cryovial into a 125 ml polycarbonate, disposable, sterile Erlenmeyer shaker flask containing 30 ml of '''pre-warmed''' FreeStyle 293 Expression Medium.<br> 

<br>3. Incubate cells in a 37°C incubator containing a humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 135 rpm.<br> 

<br>4. Next day, determine viable and total cell counts. Generally, viability is &gt; 70%; a bit lower is no reason for concern, but if viability is less than 60% thaw a new batch of cells.<br> 

<br>5. Subculture the FreeStyle 293-F cells when they reach a density between 1 x 10^6 and 3 x 10^6 viable cells/ml, usually 24 -48 hours, seed shaker flasks at 0.3 x 10^6 viable cells/ml in pre-warmed FreeStyle 293 Expression Medium. Use 125 or 250 ml polycarbonate, disposable, sterile, Erlenmeyer flasks containing 40 or 80 ml total working volume of cell suspension, respectively.<br> 

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Freeze FreeStyle 293-F cell line at a density of 1 x 10^7 viable cells/ml with a freezing medium composed of 90% fresh FreeStyle 293 Expression Medium and 10% DMSO. The freezing rate should be a decrease of 1°C per minute. Transfer frozen vials to liquid nitrogen for long-term storage.<br><br><br><br><br> 

=== Suspension Culture<br>  ===

Use polycarbonate, disposable, sterile Erlenmeyer flask with vented cap for suspension culture<br>For culture volumes above 400 ml, lower the speed of the orbital shaker if foam&nbsp; is generated. Use 90 rpm for 1 L culture.<br> 

At high stirring speeds (i.e. greater than 130 rpm) and/or depending on the impeller design, Add Pluronic® F-68 (2.5-5 ml/L of 10% Pluronic® F-68, Invitrogen Catalog no. 24040) to FreeStyleTM 293 Expression Medium.<br><br> 

Do not add antibiotics to media as this may negatively impact cell growth. <br><br> 

=== Flasks<br>  ===

{| cellspacing="1" cellpadding="1" border="1" style="width: 455px; height: 123px;"
|-
| Flask <br>Volume<br><br> 
| Culture Volume 
| Manufacturer <br><br> 
| Catalog no<br>
|-
| 125 ml 
| 25 - 40 ml 
| VWR<br> 
| 30180-036<br>
|-
| 250 ml 
| 50 - 80 ml 
| VWR<br> 
| 30180-044
|-
| 500 ml 
| 100 – 200 m 
| VWR<br> 
| 30180-052
|-
| 1 liter 
| &nbsp;200 – 400 ml 
| VWR<br> 
| 82013-164
|-
| 3 liter 
| 600 – 1000 ml 
| Corning<br> 
| 431252
|}

<br> 

=== Spinner Flask<br>  ===

<br>the optimum spinner speed was 100-130 rpm and 70-100 rpm impeller speed in Celligen stirred tank bioreactors. Seed cells at 0.3-0.5 x 10^6 viable cells/ml.<br><br> 

'''Suspension Subculture method (Mingqian Feng):''' 

1. For T-175 vented flask, put 30 ml prewarmed FreeStyle 293 expression media.<br> 2. Collect the seed cells by gently patting the flask, such that ~80-90 of the cells are single. <br> 3. Spin down, resuspend the seed cells at a final density of 3 x 10^6/ml.<br> 4. Add 3ml of seed cells to the T-175 flask, place it to the incubator at 8% CO2.<br> 5. 48h later, the cell density should be 0.8-1.2x10^6/ml (generally 1.0x10^6/ml). <br> 6. Split the cells exactly every 2 days no matter what the cell density at that time. 

=== <br> Adhesion Culture <br>  ===

<br>For adhesion subculture, both of them use D-MEM, but 500 ug/ml G418 is needed for 293FT. Both 293F and 293FT are recommended to be cultured at '''8'''% CO2. <br> 

'''Adhension Subculture method (Mingqian Feng):'''<br> Same as Suspension Subculture Method, except for using D-MEM media + 10% FBS&nbsp; instead of FreeStyle 293 Expression Media. 

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