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Techniques for the Development of New Insect Cell Lines Workshop at 2005 Annual Meeting – SIVB Saturday 4 June 3:00-4:30PM D. E. Lynn, C. Goodman, G. Caputo http://www.ars.usda.gov/SP2UserFiles/Place/12752100/InsectTechniques.pps
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.)
“Old Standards” Grace’s Schneider’s 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 Culture media • Commercial Serum-free • Ex-Cell ™ 400 series • Sf-900 II • Insect-XPRESS ™ • SFX-Insect ™ • Drosophila-SFM • and others
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)
Embryos – method 1 • Can use various ages of embryos • Clean and disinfect eggs (with 70% ethanol and/or other disinfectants ) • After disinfection, transfer eggs to culture medium • Macerate eggs with ‘mortar and pestle’ • Centrifuge to separate tissues from yolk and debris • Transfer to culture flask • If culture contains a lot of debris, replace medium at 24 hr.
Embryos – method 2 • Can use various ages of embryos • Clean and disinfect eggs (with 70% ethanol and/or other disinfectants ) OR • After disinfection, transfer eggs to culture medium • Cut or tear open chorion • Separate embryos from yolk material • Transfer embryos to standing drop of tissue culture medium • Cut/tear embryos into 3 to 8 pieces
Whole neonate larvae – method 1 • Disinfect eggs– same procedure as for embryo cultures • Place in petri dish on dampened filter paper • Wait for hatch • Place 1.0 ml, 0.25% trypsin on Maximov slide • Place 30+ newly hatched larvae in slide • Cut / mince larvae to very fine pieces • Transfer minced larvae to cent. tube / add 4.0 ml additional trypsin • Incubate 37°C / 10 min • Add 1.0 ml FBS to stop action of trypsin • Triturate vigorously • Spin / low speed / 5 min • Resuspend pellet / 3.0 ml growth medium + antibiotics
Whole neonate larvae – method 2 • Disinfect eggs– same procedure as for embryo cultures • Once larvae hatch, they will crawl toward the light into the medium • Use a pipet or glass rod to crush larvae • Transfer medium to flask as primary culture • Add 4 ml culture medium to sterile centrifuge tube • Place eggs near top • Wrap top of tube in foil • Melanin inhibitor may be necessary (Reduced glutathione, cysteine or phenylthiourea)
Older larvae -methods • Disinfect with 70% ethanol 5-10 minutes • (may also need a sodium hypochlorite pretreatment: 1 to 2 minutes with 50% household bleach plus 1% triton X-100 or other detergent) • Rinse at least twice with sterile distilled water • Transfer to culture medium
Successful for cell lines Reproductive Ovaries Testes Hemocytes Fat body Imaginal discs Midguts Nerves Cell Source –Larval tissues • Not previously used for cell lines • Malphigian tubules • Tracheoles • Salivary glands • Muscles/Aorta • Endocrine glands
Reproductive organs Ovaries
Reproductive organs Testes
Hemolymph • Melanin inhibitor may be necessary (Reduced glutathione, cysteine or phenylthiourea)
Digestive tract Older (non-feeding) larva (different species)
Salivary glands (Silk glands)
Aorta andSkeletal muscles
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 • Grace’s “organized neglect”
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
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
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
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
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)
Backup cultures (short term storage) • 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).
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