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Techniques for the Development of New Insect Cell Lines Workshop at 2005 Annual Meeting – SIVB Saturday 4 June 3:00-4:30 PM D. E. Lynn, C. Goodman, G.

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Presentation on theme: "Techniques for the Development of New Insect Cell Lines Workshop at 2005 Annual Meeting – SIVB Saturday 4 June 3:00-4:30 PM D. E. Lynn, C. Goodman, G."— Presentation transcript:

1 Techniques for the Development of New Insect Cell Lines Workshop at 2005 Annual Meeting – SIVB Saturday 4 June 3:00-4:30 PM D. E. Lynn, C. Goodman, G. Caputo http://www.ars.usda.gov/SP2UserFiles/Place/12752100/InsectTechniques.pps

2 2 Equipment Laminar flow hood (clean bench) Dissecting microscope Alcohol lamp/Alcohol jar or Clorox/water rinse Fine surgical instruments Plus the usual tissue culture equipment and supplies (26-28°C incubator, pipettor, pipets, culture dishes, flasks, etc.)

3 3 Culture media Old Standards Graces Schneiders Mitsuhashi and Maramorosch and others Additives FBS and/or other complex additives Growth factors (?) Hormones Ecdysone JH Reduced glutathione, cysteine or phenylthiourea Nutrients Conditioned medium Hemolymph Antibiotics Commercial Serum-free Ex-Cell 400 series Sf-900 II Insect-XPRESS SFX-Insect Drosophila-SFM and others

4 4 Source of Cells Eggs (embryos) Many cell types are actively dividing and undifferentiated Whole larvae (neonate) All cell types Some (most?) are already terminally differentiated Larval tissues (from older larvae) Specific cell types Many terminally differentiated Adult tissues Reproductive tissues (esp. ovaries)

5 5 3.After disinfection, transfer eggs to culture medium 4.Macerate eggs with mortar and pestle Embryos – method 1 1.Can use various ages of embryos 2.Clean and disinfect eggs (with 70% ethanol and/or other disinfectants ) 5.Centrifuge to separate tissues from yolk and debris 6.Transfer to culture flask 7.If culture contains a lot of debris, replace medium at 24 hr.

6 6 Embryos – method 2 1.Can use various ages of embryos 2.Clean and disinfect eggs (with 70% ethanol and/or other disinfectants ) 3.After disinfection, transfer eggs to culture medium 4.Cut or tear open chorion 5.Separate embryos from yolk material 6.Transfer embryos to standing drop of tissue culture medium 7.Cut/tear embryos into 3 to 8 pieces OR

7 7 Embryonic cell lines

8 8 1.Disinfect eggs– same procedure as for embryo cultures 2.Place in petri dish on dampened filter paper 3.Wait for hatch 4.Place 1.0 ml, 0.25% trypsin on Maximov slide 5.Place 30+ newly hatched larvae in slide 6.Cut / mince larvae to very fine pieces 7.Transfer minced larvae to cent. tube / add 4.0 ml additional trypsin 8.Incubate 37°C / 10 min 9.Add 1.0 ml FBS to stop action of trypsin 10.Triturate vigorously 11.Spin / low speed / 5 min 12.Resuspend pellet / 3.0 ml growth medium + antibiotics Whole neonate larvae – method 1

9 9 Whole neonate larvae – method 2 1.Disinfect eggs– same procedure as for embryo cultures 2.Add 4 ml culture medium to sterile centrifuge tube 3.Place eggs near top 4.Wrap top of tube in foil 5.Once larvae hatch, they will crawl toward the light into the medium 6.Use a pipet or glass rod to crush larvae 7.Transfer medium to flask as primary culture Melanin inhibitor may be necessary (Reduced glutathione, cysteine or phenylthiourea)

10 10 1. Disinfect with 70% ethanol 5-10 minutes a. (may also need a sodium hypochlorite pretreatment: 1 to 2 minutes with 50% household bleach plus 1% triton X-100 or other detergent) 2. Rinse at least twice with sterile distilled water 3. Transfer to culture medium Older larvae -methods

11 11 Dissection

12 12 Cell Source – Larval tissues Successful for cell lines Reproductive Ovaries Testes Hemocytes Fat body Imaginal discs Midguts Nerves Not previously used for cell lines Malphigian tubules Tracheoles Salivary glands Muscles/Aorta Endocrine glands

13 13 Ovaries Reproductive organs

14 14 Testes Reproductive organs

15 15 Melanin inhibitor may be necessary (Reduced glutathione, cysteine or phenylthiourea) Hemolymph

16 16 Fat body

17 17 Imaginal discs

18 18 Older (non- feeding) larva (different species) Digestive tract

19 19 Nervous system

20 20 Tracheal system

21 21 (Silk glands) Salivary glands

22 22 and Skeletal muscles Aorta

23 23 Endocrine glands

24 24 Primary Culture Techniques: Tricks of the Trade Keep a high tissue-to-media volume ratio Combine explants from many individuals and/or Use a standing drop of medium for the first 24-48 hours Supply fresh medium on a fairly regular (7-10 day) interval Graces organized neglect

25 25 Primary Culture Techniques: Tricks of the Trade (Cont.) Mechanical vs. enzymatic disruption of tissues No single right method Microscalpel, microscissors or mortar/pestle for mincing/macerating or Two fine-tipped forceps for tearing or Trypsin, Collagenase, Hyluronidase, etc. for ezymatic disruption

26 26 Primary Culture Techniques: Tricks of the Trade (Cont.) Selection of colonies based on morphology All of these cells were present in a single early passage embryo culture

27 27 Primary Culture Techniques: Tricks of the Trade (Cont.) Selection of colonies based on morphology Make a cell scraper from a pipet tip Use flattened edge to scrape off cells, then suction into tip with pipettor Transfer to a new dish or multiwell plate

28 28 Primary Culture Techniques: Tricks of the Trade (Cont.) Suspended vs. attached cell selection Gentle rinse and transfer for suspended Flushing, scraping, and/or enzymes for attached

29 29 Primary Culture Techniques: Tricks of the Trade (Cont.) Temperature 26-28°C for most temperate insect species 16-18° may improve maintenance of preferred traits (virus susceptibility, for example)

30 30 Once a normal subculture routine is established, leftover cells from each passage can be stored for a few weeks at a lower temperature. Add fresh medium to the leftover cells in the parent culture Leave the parent at room temperature or use a cool incubator (such as the 16-18°C incubator used for low temperature cells). Backup cultures (short term storage)

31 31 Long term storage New cell lines should be stored in liquid nitrogen at the lowest possible passage. Record cell identity, passage level, medium, supplements, cryoprotectant used, number of ampoules prepared, name of person doing the freeze, location in the freezer (Freezer no., Canister, Cane) in log book Multiple storage locations is recommended


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