This protocol describes how to maintain and subculture Spodoptera frugiperda (Sf 9) cells in both monolayer and suspension (spinner) cultures, in either serum-containing or serum-free medium. A culture of insect cells is begun using frozen Sf 9 cells. Cultures are maintained by subculturing and their viability is checked periodically. Aliquots of these cultures can be frozen in a liquid nitrogen freezer for long-term storage.

Materials

TNM-FH insect medium (see recipe) containing 10% fetal bovine serum (FBS),with and without 20% (v/v) DMSO	Spodoptera frugiperda (Sf 9) cells (ATCC #CRL 1711) derived from fall armyworm ovaries
70% ethanol	0.4% trypan blue stain (Life Technologies)
Serum-free insect cell culture medium (BaculoGold Protein-Free Insect Medium from Pharmingen; Sf-900 II from Life Technologies; HyQ-CCM3 and HyQSFX-Insect from Hyclone; or ExCell 401 from JRH Biosciences)	60-mm tissue culture plates or 25-cm2flasks
27°C incubator (humidification optional)	Spinner culture flasks (for suspension cultures) and stir plate for multiple spinner flasks (all available from Techne or Bellco) or disposable (plastic) shaker flasks (Corning) and shaking incubator set to 27°C, 90 to 150 rpm
Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent)	Screw-top cryostat freezing vials
Liquid nitrogen freezer	Additional reagents and equipment for counting cells with a hemacytometer (APPENDIX 3F)

Begin culture of Sf9 cells

1. Place 3 ml TNM-FH medium containing 10% FBS in a 60-mm tissue culture plate or 25-cm² flask.

2. Thaw a frozen ampule of Sf9 cells rapidly in a 37°C water bath by moving it back and forth by hand. When ampule contents are almost completely thawed, immerse ampule in 70% ethanol to sterilize the outside.

3. Break the neck of the ampule and transfer contents to 60-mm tissue culture plate or 25-cm² flask from step 1. Rock plate gently by hand to distribute the cells evenly and incubate 2 to 3 hr at 27°C until cells have attached.

4. Remove old medium and replace with 3 ml fresh TNM-FH/10% FBS. Continue incubation, feeding culture every 3 days (by removing old medium and replacing with fresh) until cells reach confluency (form a packed monolayer). It is important to hold the plates at an angle and remove and add medium at one corner so as not to dislodge the cells from the monolayer.

Maintain and subculture monolayer cultures

5. Remove the old medium from a confluent plate or flask of Sf9 cells and resuspend cells by gently spritzing them with medium from a pipet.

6. Count the cells using a hemacytometer designed for tissue culture cells (APPENDIX 3F).

7. Seed 1–2 × 10⁶ cells from step 5 in new 60-mm plates or 25-cm² flasks and rock to evenly distribute the cells (or use a larger plate or flask with more medium if preparing a larger culture of cells). Add fresh TNM-FH/10% FBS to bring the volume to 3 ml.

8. Incubate at 27°C, feeding the culture every 3 days with TNM-FH/10% FBS, until the cells reach confluency.

Maintain and subculture suspension cultures

9. Remove medium and resuspend cells from confluent monolayer culture as described in step 5. Count the cells using a hemacytometer (APPENDIX 3F).

10a. Seed cells in a spinner culture flask at 4–5 × 10⁵ cells/ml. Incubate at 27°C with constant stirring on a stir plate set at 60 to 80 rpm. Leave the side-arm caps slightly loosened to ensure adequate aeration.

10b. Alternatively, seed cells in a shaker flask and incubate at 27°C in a shaking incubator.
When initially adapting cells, shake at 80 to 90 rpm. Increase by 10 rpm each time cells are split until cultures are shaking at 150 rpm. The volume of cell suspension in the shaker flasks should not exceed 40% of the total capacity.

11. Count cells every 2 to 3 days using a hemacytometer (APPENDIX 3F). Subculture when cells reach a concentration of 2–2. 5 × 10⁶ cells/ml by transferring the appropriate number of cells to a new flask containing fresh TNM-FH/10% FBS to achieve a final density of 4–5 × 10⁵ cells/ml. Alternatively, pour out the appropriate volume of cell suspension and replace it with fresh medium.

12. Determine cell viability by adding 0. 1 ml of 0. 4% and examining the cells under a microscope at low that take up trypan blue (dead cells) and count the total number of cells, Then calculate the percentages of dead cells and viable cells.

Adapt cells to serum-free mediu

13. Subculture monolayer cells (from step 5) or suspension cells (from step 11) intmedium composed of one part complete TNM-FH/10% FBS and one part serum-free medium (BaculoGold, Sf-900 II, or ExCell 401). Allow cells to grow to confluency (monolayer cultures) or to a density of 2–3 × 10⁶ cells/ml (suspension cultures).
Other commercially available serum- or protein-free media besides BaculoGold, Sf-900 II, and ExCell 401 may be used. The final choice of serum-free medium should be based on a comparison of cell growth curves and production of recombinant protein in different media.

14. Repeat the subculture and growth procedure as in step 13 using a medium composeof one part FBS-containing complete medium and three parts serum-free medium.

15. Repeat the subculture and growth procedure as in step 13 using a medium composeof one part FBS-containing complete medium and between 7 and 9 parts serum-free medium.

16. Subculture the cells into serum-free medium. Cells may adapt slowly to serum-free medium and may require several passages before growth rates and viability return to normal.

Freeze cells

17. Count cells to be frozen from an exponentially growing culture using a hemacytometer (APPENDIX 3F).

18. Centrifuge cells 10 min at 1000 × g (2000 rpm in a GH-3. 7 rotor), room temperatureand discard supernatant.

19. Resuspend cell pellet at 1–2 × 10⁷ cells/ml in TNM-FH/10% FBS. Add an equal volume of TNM-FH/10% FBS containing 20% DMSO and place cells on ice. Dispense 1-ml aliquots of this cell suspension into screw-top cryostat freezing vialand incubate 1 hr at −20°C, then overnight at −70°C.
Alternatively, cells can be frozen using serum-free medium (with and without DMSO) if the cells have been adapted to serum-free medium as described in steps 13 to 16.

20. Transfer frozen cells to a liquid nitrogen freezer for long-term storage.