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

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Testes

Reproductive organs

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

Hemolymph

16Fat body

17Imaginal discs

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Older (non-feeding) larva (different species)

Digestive tract

19Nervous system

20Tracheal system

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(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)

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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