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Baculovirus and insect cell culture

Growing and Infecting Insect Cells


 
Media for both Hi5’s (protein production) and SF9s (virus production):
500mL Grace’s Supplemented Media, Invitrogen 11605094
50mL (10%) FBS* (current lot Atlanta Biologicals, S11150, E0012)
5mL Antibiotic/Antimycotic, Invitrogen 15240062
5mL Pluronic F-68, Invitrogen 24040032
FILTER THE MEDIA
 
*The brand, type, and lot of serum is very important.  These cells are picky.  Having the inappropriate lot of serum will result in low confluency, low infectivity, and low productivity.  I tested 4 lots of serum by growing the cells (both Hi5s and SF9s, they have different tastes, but you should be able to find one they both like) in small flasks and counting overtime.  I went with the serum they grew the fastest in, then did several test infections to make sure they would make protein.
 
Some key points about baculovirus:
  • These viruses cannot replicate in mammalian cells and are attenuated (meaning they can’t survive outside of the cell culture system).
  • They are extremely light sensitive, so the light in the cold room where they are stored should never be left on, and the viruses should be covered in foil.
  • They kill the insect cells after a day or two in culture.  Therefore all pipets used for Hi5 and Sf9 cell culture should be the plastic disposable type, and all glassware used to grow the cells should be acid washed (see the acid washing procedure on the last page).
  • Insect cells produce the most amount of protein or virus in their last 18 hours.  For highest yields infected Hi5s should be grown until most or all of the cells stain with trypan blue.
 
Starting the cells from a frozen vial:
  • Thaw cells rapidly at 37oC, immediately dilute in 10mL media.  Spin down, wash 2x with media.
  • Resuspend in 15mL, add to a T75 flask.  
  • Grow at 27oC for 2-3 days, until confluent.  Sometimes it takes longer (5-7 days).
  • Split into a larger flask, T175 or T225.  Grow until confluent. Freeze cells down in 10%DMSO/FBS at 1x107 cells/mL if needed.
  • Split into a 1L bottle (not the Bellco 1L spinner flasks).  Use a medium-sized spinner bar, grow in 100-200mL media, set at a medium spinner speed (~3), at 27oC.  
  • These cells do not like being below 2x105/mL or above 2x106/mL.  From a frozen vial it takes a couple weeks before they are growing consistently.  Do not use them for experiments or infections until then.
  • We maintain both Hi5’s and Sf9s in 1L bottles, and unless they become contaminated, they can be split twice a week for about 6 months.  After this time, we start fresh cultures (they start to become less efficient at protein production over time).
 
Co-transfections (to make a new viral construct):
  • Clone your gene into the pBacp10pH construct, which contains the homologous recombination sites necessary to make baculoviruses.
  • Plate 2x106 Sf9s in a 6-well dish, allow to adhere for 20min-1hour.
  • Remove media, carefully rinse once with Grace’s Unsupp. media (Invitrogen 11595030).
  • Add 500uL Transfection Buffer A (for our lot of serum, this is 5% FBS in Grace’s Unsupp).
  • In a 1.5mL microtube, combine 500uL Transfection Buffer B (25mM Hepes, 125mM CaCl2, 140mM NaCl, pH 7.1), 0.5ug Sapphire BV DNA (Orbigen bvd-10001), and 2-3ug plasmid (from a midi-prep, we’ve found mini-prep DNA does not work as well).  Add this to the cells/Buffer A drop-wise while gently swirling the plate.
  • Incubate for 4 hours at 27oC.
  • Remove Transfection buffers, carefully rinse 1x with Grace’s Unsupp.
  • Add 5mL of media, incubate at 27oC for 7-10 days (until the cells are mostly dead). To avoid evaporation, put plate inside a plastic baggie.  No cell death means no virus was made.
  • Harvest supernatant and sterile filter.
 
Virus Cloning (by serial dilution):
  • Dilute Hi5’s to 2x104 cells/mL, make about 100mL for each virus being cloned.
  • Transfer 9mL to 6-15mL conical tubes (104-109) and 10mL to 2-15mL conical tubes (103 and 0).
  • Add 10uL of virus to the 103 tube, mix well.  Remove 1mL from this tube and add to the 104 tube.  Continue serial diluting 1:10 through 109.  Do not add any virus to the 0 tube.
  • Using a multichannel, pipet 100uL of each dilution into 1/2 of a 96-well plate (6 columns for each dilution of virus).
  • Incubate at 27oC for 7-10 days.  To avoid evaporation, put plates inside a plastic baggie.
  • Identify infected wells.  A true clone should come from a dilution in which less than 30% of the wells were infected.  I try to use only clones from a less than 15% dilution to be safe.
  • Soluble clones should be tested by ELISA of the supernatant, transmembrane constructs should be tested by flow cytometry.
  • Choose the best clone (highest ELISA reading, or brightest staining by flow) to grow up a small viral stock (6 well dish, 2x106 SF9s, 5mL media, 20uL cloned virus). Incubate 7 days, harvest supes, and filter.
 
Growing a viral stock:
  • Transfer 3x107 SF9s into 5 large flasks.
  • Add 150uL of the small viral stock and bring the volume up to 50mL with media.
  • Incubate 10 days, or until the cells are mostly dead.
  • Harvest the supes by spinning down the cells, sterile filter.
  • Store viral stocks at 4oC in the dark, these viruses are light sensitive so they should be covered with foil.
  • Titer your virus using the same method as cloning.  Calculate the approx. viral titer using the dilution where less than 30% of the wells are infected: #of wells infected X dilution of the plate X 0.1mL/well = Units/mL.  Most viral stocks grown up in 50mL with 3x107 cells are around 2-6x108U/mL.
 
Sequencing a viral construct:
  • Transfer 3x105 SF9s into a 24-well plate.  Add 20uL virus and bring volume up to 1mL with media.
  • Incubate for 3 days at 27oC.
  • Harvest cells by spinning (in a 1.5mL conical tube) at 1200rpm for 5 minutes.  Wash 1x with PBS. Pellets can be stored at –80oC.
  • Resuspend cells in 50uL Extraction Buffer (10mM Tris, pH 8.5, 0.01% gelatin, 0.45% Triton-X 100, 0.45% Tween-20, and 50mM KCl), add 1uL 10mg/mL proteinase K, incubate at 56oC for 2 hours.
  • Heat inactivate at 95oC for 20 min.
  • Use 1.5uL as template per 25uL PCR reaction, use primers outside your gene to amplify.  Use an internal primer to sequence.
 
Infecting Hi5’s for soluble protein production:
  • Expand Hi5’s in Bellco 1L spinner flasks (use the ones with stir bars, not paddles).  Keep the cells within their viable range (2x105-2x106 cells/mL) in a total volume of 400-500mL.  They will grow slower if put in larger volumes because of reduced oxygen at the bottom of the flask.
  •  Grow at 27oC, at a medium spinner speed (~3-4).  Continue splitting and expanding until there are enough cells for an infection (109 cells for every liter of protein supes).
  • For four liters of supes: Spin 4x109 cells down at 1200rpm for 15-20 minutes.  Resuspend in 40mL Grace’s Unsupp. and divide into 4 Bellco 1L spinner flasks (you can use the same flasks that you expanded the cells in, if they are still sterile).
  • Bring the volume of each flask up to 300mL with Grace’s Unsupp. media (+5mL F-68, +5mL antibiotic/antimycotic per 500mL).  Add 20mL of viral stock (at ~2-6x108U/mL).  Spin at a low spinner speed (~2) for 1 hour at 27oC.
  • To each flask add 700mL Grace’s Supplemented media (+2% FBS, +5mL F-68, +5mL antibiotic/antimycotic).  Incubate for 7-10 days at 19oC at a low spinner speed (~2).
  • Harvest supes by spinning out cells and debris (2500rpm, 20min).  Sterile filter, add 10% azide, and store at 4oC.
 
Infecting Sf9s for flow:
  • Count Sf9s and put 2x106 cells in a 6-well dish.  Bring up to 5mL with media.
  • Add 2U per cell of virus (procedure for titering virus is above).  This is usually between 5-20uL.  If you use too much virus, you will not see any negative cells and won’t get the nice 1 to 1 ratio between MHC and TCR positive cells.
  • Incubate for 3 days.  On day 3, harvest cells (dislodge from the plate by pipetting), usually only 200uL is enough for flow, and stain with the appropriate antibodies or TCR.  Harvesting on day 2 will result in less protein production (and less positive cells by flow), harvesting on day 4 will result in more dead cells and fewer uninfected cells (reducing the number of negative cells by flow and giving you the same problem as if you used too much virus).  
  • Remember this only works for membrane-bound proteins, secreted and soluble (BirA tagged) proteins must be checked by ELISA of infected supernatants.
 
Infecting Sf9s for vaccination:
  • Count Sf9s and put 3x107 cells in a T175 flask.  Cells should be healthy prior to infection.  Bring up to 35-40mL with media. This is enough for 5 mice, with extra void volume.  You can squeeze out 6 mice if you’re really careful.  If you need more, I’ve tried infecting with 5x107 (enough for 9 mice) cells in a T175 flask (and used a little more virus), and had good results.  If you need less, you can scale back proportionally, but use a smaller flask and less volume.
  • Add 2U per cell of virus (usually 100-150uL, procedure for titering virus is above).  Using too much or too little of the virus will affect antigen dose.
  • Incubate 3 days.  There’s no need to count the cells again.  If you do count them, you’ll notice that some of them are dead.  Harvest cells on day 3 (dislodge them from the flask by banging it a few times) into a 50mL conical tube.  Spin down at 1200rpm for 5 minutes.
  • Resuspend in HBSS (25mL), spin and repeat 2x.
  • Resuspend at 2.5x107 cells/mL in HBSS.  For a flask of 3x107 cells, resuspend in 1.2mL (add about 1mL, there is usually 200uL of cells and remaining volume in the tube). Transfer to a 1.5mL conical tube.  I wait to load syringes until I get to the mouse house.
  • Mix the tube well right before loading the syringe (I usually load 800uL at a time, enough for 4 mice).  Carefully inject I.P., if you miss and go S.Q. the response is much poorer.   Mix the tube again before reloading the syringe.  This is day 0.
  • Boost the mice on day 7 (infect cells as before on day 4).  Tetramer-positive T cells are not detectable in most mice prior to the boost.  They are detectable in the blood on day 10 (3 days after the boost), but peak at day 14 (7 days after the boost).  They contract by day 17 (10 days after the boost).  
  • For best results, challenge mice with tumor on day 14, waiting until day 17 is detrimental.  
 
Infection for ELISA or protein gels:
  • Infect Sf9s as you would for flow (2x106 cells in a 6-well dish).  Make lysis buffer containing detergent [(10mM NaH2PO4), 10% glycerol, 0.6% CHAPs, 0.5mM DTT], filter, and add protease inhibitors as you would for purified protein (1:1000 of PMSF, and 1:100 of leu/pep).  Resuspend cells at 1x107 cells/mL (200uL) in lysis buffer containing detergent.
  • Vortex and incubate while rotating at 4oC for 4 hours (or overnight).  
  • For ELISA, dilute supes 1:10, 1:100, and 1:1000.  For the Ld ELISA, the 1:100 dilution falls within the linear range of my standard curve.  If you don’t dilute the supes, you’re ELISA results will be ridiculous.
  • For protein gels, run about 15uL per lane (you can run denaturing gels, and dilute with BME-containing running buffer).  
  • To get a clearer band, freeze/thaw the cells several times in HBSS (at the same concentration).  Spin down at 3000rpm for 20 minutes.  Remove the HBSS, soluble protein (BirA or secreted) should be in the HBSS, and run about 15uL per lane as above.  Resuspend the cell pellet in lysis buffer as before, membrane-bound proteins should be in this part.
  • These gels can also be used for western blots, although western blot MHC antibodies are rare.
  • The infected cell lysates can also be used for immunoprecipitations.
 
Common problems with Hi5s and SF9s:
 1)    They aren’t growing to confluency or they die:
  • They are old- you need to start a new batch from a frozen vial.
  • The glassware they are growing in wasn’t properly cleaned (detergents or acid left on the glass).
  • The serum isn’t their favorite.
  • They are infected with a baculovirus- always use plastic disposable pipets and make sure you acid wash any glassware that comes in contact with baculovirus.
  • The antibiotic is old- always store in frozen aliquots.
  • They overgrew- keep them below 2x106/mL.
  • They are contaminated- take an aliquot of media and put it at 37oC, bacteria, yeast, or fungus should grow out to a visible amount if they are.
 
2)    They aren’t making protein:
  • Something is wrong with the viral construct- double check/sequence the plasmid DNA you used to make the baculovirus (is it the right construct, are there mutations, etc), and sequence the viral construct (see above).
  • The cells are old- start a new batch from a frozen vial.
  • Your viral titer is either too high (killing the cells too quickly) or too low (not enough to have a good infection)- titer your virus to find out (see above).
  • You are using the wrong cell type- Hi5s are better at making large amounts of protein because they stay alive longer after infection.
  • Check to see if there is protein in the supes (by ELISA).  If there is, then something is wrong with the purification, not with protein production.
  • If all else fails, start over.  Make a new viral construct by co-infection.  I’ve never had a baculovirus not work without a reason- usually something was wrong with the original plasmid vector.
 
Acid washing glassware:
  • Even though it’s a huge pain, we acid wash our own glassware because Fran in the K/M lab has had problems growing these cells in glassware washed through the facility (they don’t rinse them well enough to remove all detergents and acid).
  • Any glassware that contacts insect cells, insect cell media, or baculovirus should be acid washed.
  • Rinse the dishes with water to remove cells, bacteria, and media, and fill part way with diluted bleach (~10%). Sit for 5-15 min in the bleach water.
  • Rinse once with DI water and disassemble (take off lids and remove the stir bars from the spinner flasks).  The stir bar shafts are really fragile, so be careful not to drop them and don’t drop anything on them.
  • Fill the sink and dishes with DI water, add some 7x soap (enough to see bubbles in the water, if you add too much you’ll have rinse extra times).  Sit for at least 2 hours, overnight is also okay.
  • Drain the sink and rinse the dishes 6x with DI water (remember to rinse lids and stir bars also).
  • Fill the sink and dishes with DI water, add 250-300mL of HCl.  Sit for at least 2 hours, overnight is also okay.
  • Drain the sink and rinse the dishes 8x with DI water, being careful to rinse the outside, edges, and spouts of the dishes, stir bars, and lids.
  • Dry on paper towels overnight.
  • Reassemble (wearing gloves), cover the lids with foil, and autoclave.  Vent caps to avoid explosions.