Techniques for the Development of New Insect Cell Lines Workshop at 2005 Annual Meeting – SIVB...
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Transcript of Techniques for the Development of New Insect Cell Lines Workshop at 2005 Annual Meeting – SIVB...
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
2Equipment 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.)
3Culture media “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
Commercial Serum-free Ex-Cell ™ 400 series Sf-900 II Insect-XPRESS ™ SFX-Insect ™ Drosophila-SFM and others
4Source 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)
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3. After disinfection, transfer eggs to culture medium
4. Macerate eggs with ‘mortar and pestle’
Embryos – method 1
1. Can use various ages of embryos2. 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.
6Embryos – method 2
1. Can use various ages of embryos2. 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
7Embryonic cell lines
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1. Disinfect eggs– same procedure as for embryo cultures
2. Place in petri dish on dampened filter paper
3. Wait for hatch4. Place 1.0 ml, 0.25% trypsin on
Maximov slide5. Place 30+ newly hatched larvae in slide6. Cut / mince larvae to very fine pieces7. Transfer minced larvae to cent. tube / add 4.0 ml additional trypsin8. Incubate 37°C / 10 min9. Add 1.0 ml FBS to stop action of trypsin10. Triturate vigorously 11. Spin / low speed / 5 min12. Resuspend pellet / 3.0 ml growth medium + antibiotics
Whole neonate larvae – method 1
9Whole 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)
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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
11Dissection
12Cell 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
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Ovaries
Reproductive organs
14
Testes
Reproductive organs
15
Melanin inhibitor may be necessary(Reduced glutathione, cysteine or phenylthiourea)
Hemolymph
16Fat body
17Imaginal discs
18
Older (non-feeding) larva (different species)
Digestive tract
19Nervous system
20Tracheal system
21
(Silk glands)
Salivary glands
22andSkeletal musclesAorta
23Endocrine glands
24Primary Culture Techniques:Tricks of the Trade Keep a high “tissue-to-media volume” ratio
Combine explants from many individualsand/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”
25Primary 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
tearingor
Trypsin, Collagenase, Hyluronidase, etc. for ezymatic disruption
26Primary Culture Techniques:Tricks of the Trade (Cont.) Selection of colonies based on morphologyAll of these cells were present in a single early passage embryo culture
27Primary 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
28Primary 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
29Primary 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
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)
31Long 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