INSECT CELL CULTURE & BACULOVIRUS INFECTIONS

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Cells, Media, Growth Characteristics, Freezing and Thawing Cells, and Glassware
 Cell Lines Used
SF-9
SF-21
High-Five

Media
For SF-9 and SF-21 cells:
TC-100 or Grace's media + 10% FBS + supplements. (Gibco; powdered).
For powdered media, dissolve in about 800 ml water.
If yeastolate and lactalbumin are not present, add: 3. 3 g/L yeastolate and 3.3 g/L lactalbumin
For TC-100 media also add 0.03 g/L sodium bicarbonate
pH to 6.2 with 1 M KOH (about 20-30 ml).
25 mg/ml gentamicin (Sigma)
Filter sterilize through a 0.22 micron filter.
Add heat inactivated FBS to 10% (Gibco)
For High-Five cells:
Ex-Cell 401 (JRH Biosciences; liquid)
25 mg/ml gentamic in (Sigma)

Growth Characteristics: The optimal temperature for insect cell growth is 27°C. If an incubator is not available, the cells will grow at room temperature but at a slower rate . CO2 incubators are not required. Cells may be grown in T-flasks or shaker flasks.

  • T-flasks: Grow cells to 80-100% confluency and then split 1 :10. Remove adherent cells from the plastic surface of the flask by pipting media with a wide bore, bent tip pipet. Overgrown cultures will have floating cells. Floating cells in a T-flask are an indicator of an unhealthy culture. It takes about one week after splitting for the cells to reach confluency. Re-use T-flasks once. It is not a good idea to use aliquots of these cells directly in experiments because 30-50% of them are trypan blue positive after being dislodged from the plastic surface.

  • Shaker flasks: Cells may be grown on orbital shaking platforms (rotating at about 100 RPM) at 27°C in sterile Erlenmeyer flasks. The flasks should be no more than 2/5 full. (We maintain 10-15 ml of cells in a 50ml flask.) SF-21 and High-Five cells double in about 24 hours. An advantage of maintaining cells in shaker flasks is that you do not need to blow the cells off T-flasks which kills many of them. We used to see d shaker flasks at 500,000 and 400,000 cells/ml for SF21 and High-Five, respectively, but more recently seed at 150,000 and 100,000 cells/ml, respectively without problems. Add 10 U/ml heparin for the first two passages of High-Five cells to prevent clumping during adaptation to suspension culturefrom T flasks.
    Split cells when they reach about 2 x 10    6 cells/ml. (High-Five cells suffer no adverse effects at 4 x 106cells/ml.) SF-21 cells should only be passaged about 10 times. We have passaged High-Five cells without problems up to 215 times.
  • Cleaning Glass Shaker Flasks: Glass shaker flasks are bleached overnight, scrubbed with a brush and soap before cleaning in a dishwasher. Flasks are then soaked in distilled water overnight to remove residual chemicals. The water is then removed, and the flasks are autoclaved.

Freezing cells
Cells sho uld be in exponential growth and more than 98% viable before freezing in liquid nitrogen.
  1. Centrifuge the cells at 1,000 g, 5 min. Discard the supernatant and resuspend the cells in fresh media at a concentration of 1-2 X 107 cells/ml.
  2. Mix equal volumes of resuspended cells and freezing medium (85% media, 15% DMSO) and place on ice. Aliquot 1 ml volumes into cryovials.
  3. Place cryovials into a Nalgene Cryo 1 C Freezi ng Container (an isopropanol bath) and place directly at -70°C overnight.
  4. Transfer cryovials into a liquid nitrogen storage tank.

Thawing Cells
  1. Remove cryovials from liquid nitrogen and thaw rapidly (in about 1 minute) at 37°C. (JRH Biosciences recommends thawing High-Five cells slowly over about 30 minutes at room temperature. We do it quickly without problems.)
  2. Wipe the outside of the vial with 70% ethanol and transfer the cells to a T25 flask containing 4 ml complete media.
  3. Wait 2-3 hours for the cells to adhere to the plastic. Remove the media and feed adherent cells with fresh media to dilute the DMSO. (JRH Biosciences indicates that High-Five cells adhere slowly to plastic. Although we have not noted this property, they suggest waiting overnight bef ore feeding the cells.) If cells are not adherent, they are probably dead. In this case, discard and thaw another vial of cells.
  4. After 24 hours, feed the cells with fresh media.
  5. For cell maintenance, follow the procedures in the Growth Characteristics/T-flask section.

Generation of Recombinant Baculovirus
Choice of Expression Vectors: We have used vectors from Clontech and Invitrogen with success. These are based on homologous recombination b etween a transfer vector (containing your gene between the 5' portion of the lacZ gene and the 3' end of a gene needed for viral replication) that is co-transfected into insect cells with a replication defective, linear, viral DNA. A double crossover event corrects the defect, restores b-galactosidase production, and transfers your gene to the virus at the same time. Without recombination or with a single cross-over, no virus particles should be produced. Gibco developed a somewhat similar system using transposition in E. coli to generate a recombinant baculovirus. This "bacmid" (a baculovirus genome that replicates in E. coli) is then used to transfect insect cells. The multiple cloning sites in the transfer vectors are downstream of the strong polyhedrin promoter. Many variations in these vectors are available, including His6 tags and a honeybee mellitin secretory signal (both from Invitrogen).

Ligation of insert to vector: Ligate the gene of interes t into a bacterial transfer vector. Your gene will typically be flanked with portions of viral sequences that permit homologous recombination with linearized viral DNA after insect cell transfection. Vectors or inserts may include sequences for protein targeting and purification. cDNA may be used, i.e. no introns are needed.
Grow recombinant DNA molecules in E. coli. Verify the direction of the insert relative the polyhedrin promoter, and purify the plasmid for transfection into insect cells .

Transfection of Insect Cells, Screening and Viral Amplification: We use liposome-mediated transfections. Others report success with electroporation and calcium phosphate-mediated transfections.
  1. Transfer 1-2 x 106 cells into a 35 mm tissue culture dish. The cells should be >95% viable. Allow to adhere for a few hours, or seed at a lower density and grow overnight.
  2. If using Clontech's lipofec tin (1 mg/ml stock), dilute 5.5 ml of lipofectin in 49.5 ml sterile water in a polystyrene tube. In a separate polystyrene tube, mix 0.5 mg of recombinant transfer vector with 0.1 mg of linear viral DNA. QS to 50 ml with sterile water. Add the 50 ml of the diluted lipofectin mixture and mix gently. Incubate without further mixing at room temperature for 15 min.
    If using Invitrogen's InsectinPlus liposomes, combine 1     mg of the replication-defective linearized viral vector with 4 mg of recombinant transfer plasmid, 1 ml media without serum, and 20 ml InsectinPlus. Vortex for 10 seconds, then incubate at room temperature for 15 min.
  3. Remove the media from the cells and wash with media without serum or antibiotics. Co-transfect these cells by slowly adding the liposome-DNA mixture in 0.5 – 1.0 ml media. Avoid shearing the large baculoviral DNA molecules. Allow cells to incubate with this mixture for 5 hr. Qs to 1.5 ml with complete media and incubate at 27°C for about three days.
  4. Harvest the transfected cell supernatants some time after 3 days post-transfection. Wait until the cells show signs of infection - irregular shaped cells and increased volumes. In different experiments, we have collected the supernatants anywhere from day 3 to 10.
  5. Plaque Assay: Infect insect cells with this supernatant to isolate single viral plaques. Seed 35 mm tissue culture dishes with 1.5 x 106 insect cells (1.0 x 106 cells also works fine). When the cells have adhered and are at least 50% confluent, remove the medium and slowly add 200 ml of serially diluted supernatants from step 4. After 1 hour, aspirate this solution and overlay with 1.5 ml of pre-warmed (37< /FONT>°C) 1% molten agarose (made from 2% SeaPlaque agarose melted in water, and then diluted with an equal volume of pre-warmed media). Once the agarose has solidified, add 1.5 ml medium to each plate and transfer to a humidified storage box.
  6. Viral vectors now typically include markers for identification of infected cells (ex. beta-galactosidase). Assuming you are using such a vector, maintain the cells for 5 - 7 days. Aspirate the media, and stain the cells with 0.03% X-gal (diluted in medium). Remove the X-gal solution after 5 hours, invert the plates and transfer them to a dark area. Check daily for blue plaques. They typically appear after 5 days. Determine the titer of the viral stock. Select a few individual plaques by encircling them with a sterile Pasteur pipet and transferring them to 0.5 ml media.
  7. Use 20-100% of this "plaque pick" supernatant to infect 1-2 x 106 cells in 1.5 ml of media in 35 mm culture dishes. Collect the media after 3-4 days (look for signs of infection). The cells should appear grainy with irregularly shaped membranes. Centrifuge at 1,000 g, 5 min. Save this passage 1 (P-1) supernatant, and determine the titer by plaque assay as described in steps 5 - 6. Using the remaining cells, verify protein expression by Western Blot.
  8. Begin to amplify the viral stock by infecting 0.4-5.0 x 105 cells with the P-1 stock at an MOI (Multiplicity of Infection) = 0.1 (see example below). Infect these cells for 4-10 days until cells show signs of being well infected. Cells will grow for a few days and then start to die. Collect the cells and centrifuge at 1,000 g, 5 min. Save this passage 2 (P-2) supernatant. Titer by plaque assay and amplify again until a titer of >1 x 107 is obtained.


Generation of Recombinant Protein with Baculovirus Infections:
For initial production of recombinant protein, cells in exponential growth are infected at an MOI of from 0.3 to 5.0 for 3-4 days (ex. 1,030,000 cells/ml x 12 ml x MOI of 0.3 = 3,708,000 pfu; 3,708,000 pfu/62,500 pfu/ml = 59.3 ml virus to add.) After 3 days, cells are typically 30% dead. Nearly all cells are twice normal size and contain granules easily seen with a microscope. Both features are characteristics of infection. The cells are centrifuged at 1,000 g for 5 min., washed once in 0.5 x PBS, and resuspended in 0.5x PBS at a minimum of 1,000,000 cells/ml. Sonicate the cells for 10 seconds on setting 4 and pellet the debris at 12,000 g in a microcentrifuge for 3 min. Initially the pellet and supernatant are tested to determine where the protein of interest is located. In our case, we save the supernatant. This is brought to the desired concentration (in our case with 0.5 X PBS) for immediate use, or mixed with an equal volume of glycerol to produce a 50% glycerol stock for storage at -70°C. These frozen stocks last for at least a year.

Optimize the level of protein expression (MOI, time course of infection) and test for appropriate activity in a bioassay. Post-translational modifications may change with le ngth of infiection. Numbers of cells and volumes may be scaled up to produce larger amounts of protein. Reported yields vary considerably, but most are in the 1 - 100 mg /L range.

    Send comments and updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF
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Copyright 1998 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Insect Cell Culture & Baculovirus Infections. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/baculpro.html). 1998. Oklahoma City. R evision Date: October 8, 1998."