Editing 293F Protein Production (section)

== <u></u>'''Suspension Transfection Method'''<u></u>  ==

<br> 1. Collect the cells. <br> 

Approximately 24 hrs before transfection, pass FreeStyle 293-F cells at 6~7 x 10^5 cells/ml. Place the flask(s) on an orbital shaker platform rotating at 135 rpm at 37°C, 8% CO2. Do not add antibiotics during transfection.<br><br><br> 

On the day of transfection, the cell density should be about 1.2-1.5 x 10^6/ml.<br> 

<br> For one standard transfection, 30 million (3 x 10^7) cells are needed (from one T75 flask). Resuspend the cell in 30 ml of prewarmed FreeStyle 293 expression media in a 125ml SpinFlask (250ml, 500ml Cell SpinFlask may&nbsp; be needed for large scale transfection). 

<br> 

For 100 ml culture, 100 million (10X10^7) cells from one T175 flask plus one T75 flask are required. Use 1 liter VWR flask for culture.<br> 

Put the cells at 37 <sup>o</sup>C while preparing DNA/GeneExpressoMax complex (next step). 

<br><br> 2. Prepare DNA/<strike>293Fectin</strike> GeneExpressoMax complex. <br> Dilute 30 ug plasmid into 1 ml of prewarmed <strike>OptiMem</strike> DMEM serum-free medium;&nbsp; <br> 

<br> 

Dilute 60 ul <strike>293Fectin</strike> GeneExpressoMax into 1 ml of prewarmed <strike>OptiMem</strike> DMEM serum-free medium, immediately mix them, incubate at hood exactly <strike>5 min</strike>, 2 min.<br> 

<br>'''To transfect cells from one T175 flask+ one T75 flask''': dilute 100 ug DNA in 3 ml serum-free DMEM. Dilute 300 ul GeneExpresso in 3 ml serum-free DMEM. <br> 

<br> 

Mix DNA solution and <strike>293Fectin</strike> GeneExpresso solution, incubate inside the hood for <strike>25</strike> 20 min.<br> 

<br> 

3. Transfection<br> 

After incubate for 20 min, gradually drop the complex into cell suspension while whirling the cell suspension.<br> 

<br> 

4. Culture and harvest<br> 

Put the transfected cell at spin platform at a rotation speed of 75 rpm, '''8'''% CO<sub>2</sub>. The expression can be detected next day by sandwitch ELISA (generally 100-600 ng/ml at D1). 

'''Yield per ml at D1: ''' 

30 ml: 3 to 18 ug/30 ml<br> 

'''Yield per ml at D3''': <br> 

5~20 ug/ml for most proteins<br> 

100 ug/ml for short peptide Fc fusion. <br> 

1 ug/ml or less for difficult protein.<br> 

'''Expected Total Yield at D3''': <br> 

30 ml: 150 ug to&nbsp; 600 ug<br> 

100 ml: 500 ug to 2 mg<br> 

Very difficult protein: ~ 100 ug or less from 100 ml.<br> 

<br> 

For low expression protein: Culture the cells up to 7 days. There is no need to change or supplement the culture medium during the first 6 to 7 days. <br><br> It's optional but recommended that collect the supenatant and refresh the media At D3, by doing this more protein will be achieved. 

<br> If the no changing of the media, the peak of Fc fusion concentration generally appears at D6 (seldomly at D5), thus harvest at D6. <br> 

<br> 

5. Harvest <br> Collect the supernatant by spin down at 1000-3000rpm, filtered with 0.22um sterile membrane, Store at 4 <sup>o</sup>C (cold room) until purification (at least stable for several weeks, no sodium azide needed after filtration).<br> 

<br> 

<br> 

{| cellspacing="1" cellpadding="1" border="1" style="width: 661px; height: 114px;"
|-
| <br> 
| Cells from Adhesion Culture Flasks 
| DNA 
| Transfection Reagent 
| Dilution in DMEM 
| 0.5 M VPA (ul) 
| Freestyle Medium (ml)
|-
| 30 ml Transfection 
| Three&nbsp; T75 flasks 
| 30 ug 
| 90 ul of 1 mg/ml 
| 2 X 1 ml 
| 225 
| 30
|-
| 100 ml Transfection 
| One T175 + One T75 
| 125 ug 
| 375 ul of 1 mg/ml 
| 2 X 3 ml 
| 750 
| 100
|}

<br> 

Further Optimization needed:<br> 

DNA amount, cell density, and volume of Freestyle medium needed for sustain growth and protein production<br> 

<br>