Editing 293E Protein Production (section)

== Optimized Condition for Gag-VLP<br> ==

<br>[[Image:Culture_medium2.jpg]]<br> 

[[Image:Culture_medium.jpg]]<br>

<br>

[[Image:Insulin_transferrin_lipidmix.jpg]]<br>

<br>

<br>

<br>

<br>

1. Transfection<br>

Grow cells in mid-log phase (2~3 X10^ cells/ml), spin down, use new medium and do transfection with 25 kDa L-PEI (1 ug DNA + 2 ug PEI), Dilute DNA and PEI in culture medium. 1 ug DNA per ml cells. Incubate DNA-PEI for 15 min then add to cells.<br>

Best cell density for transfection: 10^6 cells/ml for freestyle medium (mid-log phase 2.9 X 10^6 cells/ml), and 2X10^6 for supplemented freestyle (5.4 X10^6 cells/ml)

2. Harvest: <br>

2 or 3 days after transfection, spin down at 500 g for 15 min at 4 oC, collect supernatant.<br>

3. Spin down at 25000 rpm (115,254 xg) for 3 hours at 4 oC using a SW32 rotor.<br>

5. Resuspend in cool PBS (250 ul per tube), incubate at 4 oC for 2 hour.<br>

6. Add Benzonase, 200 U/ml,&nbsp; treat for 1 hour at room temperature.<br>

7. Concentrate VLP by spin ultrafiltration column. Load to 130 ml Sepharose 6FF (XK 16/70 column ), using PBS. Flow rate is 2 ml/min. (60 cm/h), 2.5 ml/tube.&nbsp;<br>

8. Filter through 0.2 uM . Add Trehalose to 15%. Filter with 0.2 uM filter and store at 4 oC.<br>

Should be ~7000 copies of Cre/FLP per Gag VLP<br>



Supplements:

Lipids: 100X, 100X Synthecol, Sigma. Fatty acids, 100X F7050, Sigma. Tocopherol acetate, 0.2 mg/ml, T1157 Sigma. Emulsifying agent polysorbate 80 (2.5 mg/ml, P4780, Sigma



=== Equipment  ===

Rinse glassware three times using Milli-Q H2O (or equivalent), followed by steam sterilization. 

Boxes, Styrofoam (large and 20-position small) or programmable cooler (see Steps 12 and 16) 

Centrifuge 

Cryovials 

Flasks, Erlenmeyer, glass, reusable (Corning) 

Flasks, Erlenmeyer, plastic, 125-mL disposable shake (Corning) 

Freezer at -80°C 

Freezer storage, liquid nitrogen, vapor phase 

Hemocytometer 

Hood, laminar flow 

Incubator preset to 37°C, humidified, 5% CO2 

Microscope 

Shaker, orbital 

Ensure that the orbital shaker can operate continuously under humidified conditions. Do not shut off the orbital shaker in the humidified incubator or it will seize upon subsequent use. When not in use for cultures, leave the shaker agitating or remove it from the incubator until needed. Avoid shakers with digital controls, as they are susceptible to humidity. The shaker must contain holders for shake flask sizes ranging from 50 to 2000 mL. If no holders are supplied, equip the platform with an anti-slip adhesive mat. Shake flasks must stay on the platform up to '''120 rpm'''. 

Tubes, conical, 15-mL 

Water bath preset to 37°C<br>Previous SectionNext Section<br><br> 

=== METHOD  ===

Conduct all operations aseptically in a laminar flow hood. Cells growing in serum-free media are highly sensitive to chemical contamination; thorough rinsing of all equipment that contacts the cells with highly pure water is compulsory.<br> 

'''Cell Thawing and Maintenance''' 

1. Prepare a 15-mL conical tube containing 10 mL of prewarmed cell culture medium appropriate for the cell line to be used. 

2. Thaw the cells rapidly in a 37°C water bath. 

3. Add the cells to the 15-mL tube. Invert to mix. 

4. Dilute a 100-μL aliquot of cells with 100 μL of PBS containing 25 mg/mL '''erythrosin B'''. Determine the cell density and viability using a hemocytometer. 

5. Centrifuge the cells at '''200g''' for 5 min at room temperature. 

6. Decant the supernatant. Loosen the pellet by gently tapping the tube. Dilute the cells to 2.5 × 10<sup>5</sup> cells/mL in a 125-mL disposable shake flask using cell culture medium. 

Operating culture volumes must be in the range of 16%-33% of the nominal shake flask volume for sufficient oxygenation. 

7. Place the shake flask on the orbital shaker (~120 rpm) in the incubator. 

Center the shaker in the incubator. Make sure that the walls, door, and wire are not contacted during agitation. 

8. Count the cells at 48 h post-thawing. If necessary, dilute to 2.5 × 105 cells/mL using cell culture medium. 

9. When the doubling time is 24 h for two consecutive passages, subculture every 2 or 3 d to maintain cell densities between 2.5 × 105 and 1.2 × 106 cells/mL using medium supplemented with G418 (25 μg/mL or 50 μg/mL, for 293-6E or 293E cells, respectively). 

See Troubleshooting. 

10. Dilute the cultures to 1.5 × 10<sup>5</sup> or 7.5 × 10<sup>4 </sup>cells/mL for weekends or long weekends (3 or 4 d, respectively). 

'''Cell Freezing''' 

11. Prepare the freezing mixture by adding DMSO to fresh cell culture medium (10:90, v/v). 

12. Label the cryovials. Place in a small Styrofoam box. Prechill the large Styrofoam box in a -80°C freezer. 

13. Count the cells. Determine the volume needed for cryopreservation. 

Cells must be in the exponential phase (between 8 × 10<sup>5</sup> and 1.2 × 10<sup>6</sup> cells/mL). 

14. Centrifuge the cells at 200g for 5 min. Decant the supernatant. Dissociate the pellet by gently tapping the tube. 

15. Add the freezing medium drop-wise to the cells while swirling the tube to obtain the desired density (i.e., 5 × 10<sup>6</sup> to 5 × 10<sup>7</sup> cells/mL per vial). 

16. Quickly aliquot the cells into the labeled vials. Immediately transfer to a -80°C freezer. Do not open the freezer door for at least 2 h. 

Alternatively, freeze cells at 1°C/min in a programmable cooler. 

17. Transfer the vials to liquid nitrogen freezer storage (vapor phase) the following day. 

Store for a maximum of 4 d. 

'''''<br>TROUBLESHOOTING''''' 

Problem: The cells do not grow well. 

[Step 9] 

Solution: If cells do not grow with the expected doubling time, consider the following: 

1. The cultureware is not sufficiently clean. Glassware for all culture manipulations and reagent preparations must be rinsed at least three times with high-purity H2O. 

2. The H2O used may not be sufficiently pure. Check the source of the ultrapure H2O. 

3. Check that the filtration system (if applicable) used to make the ultrapure H2O is functioning properly. 

4. Cell aggregation can give a false impression of slower growth rates. Check regularly that the orbital shaker is at the right speed and that the incubator is at the correct temperature and is supplying the prescribed amount of CO2. 

5. The cells may be too old. The number of cell passages indicates the age of the cells in service. A new cryovial can be thawed every 3 mo, on average. 

6. Verify expiration dates, culture medium lots, and batches of additives used (e.g., Pluronic F-68 and antibiotics). 

7. Check for mycoplasma contamination; if positive, thaw new cells. 

<br> 

=== Culture  ===

=== <br>  ===

1. One day before transfection pass HEK-293E cells&nbsp; in Ex-cell 293 serum-free medium with the final cell density of 1x106 cells/ml. <br> 

2. Before transfection determine the cell number in the suspension culture using PCV <br> 

3. Spin down 20x10^6 cells and resuspend in 0.89 ml of RPMI 1640 (Invitrogen, Carlsbad, CA).<br> 

4. Transfect cells with DNA (1.5 μg/1x10^6 cells) + PEI (3 to 4.5 μg/10^6 cells). Incubate cells with DNA-PEI for 3 hours.<br> 

5. Dilute cells with fresh Ex-Cell 293 serum-free media (Sigma, cat. no. 14571C) to the final cell density of 1x10^6 cells/ml.<br> 

6. Add VPA to final conc of 3.75 mM 24 hours post transfection. 

7. Havest cells at 3 to 7 days. 

ESF serum free medium (Expression Systems cat. no. 98-001) can also be used?