Developing New Insect Cell Lines

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Developing New Insect Cell Lines Powered By Docstoc
					    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
                                                             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
Culture media
   “Old Standards”                     Additives
       Grace’s                             FBS and/or other
       Schneider’s                          complex additives
       Mitsuhashi and Maramorosch          Growth factors (?)
       and others                          Hormones
                                                 Ecdysone
   Commercial Serum-free                        JH
       Ex-Cell ™ 400 series                Reduced glutathione,
       Sf-900 II                            cysteine or
       Insect-XPRESS ™                      phenylthiourea
       SFX-Insect ™                        Nutrients
       Drosophila-SFM                      Conditioned medium
       and others                          Hemolymph
                                            Antibiotics
                                                                     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
Embryos – method 1
                       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.   Macerate eggs with ‘mortar and pestle’
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
Embryos – method 2
                        1.   Can use various ages of embryos
                        2.   Clean and disinfect eggs
                                (with 70% ethanol and/or other disinfectants )

                                  OR
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
                       7
Embryonic cell lines
                                                                          8
Whole neonate larvae – method 1
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
                                                                                   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
5.   Once larvae hatch, they                                    4. Wrap top of
     will crawl toward the                                         tube in foil
     light into the medium
6.   Use a pipet or glass rod
     to crush larvae
7.   Transfer medium to
                                    Melanin   inhibitor may be necessary
     flask as primary culture
                                    (Reduced glutathione, cysteine or phenylthiourea)
                                                                                      10
Older larvae -methods
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
             11
Dissection
                                                    12
Cell Source –
Larval tissues

   Successful for cell lines
       Reproductive
                            Not previously
            Ovaries
            Testes
                             used for cell lines
       Hemocytes               Malphigian
                                 tubules
       Fat body
                                Tracheoles
       Imaginal discs
                                Salivary glands
       Midguts
                                Muscles/Aorta
       Nerves
                                Endocrine glands
               13
Reproductive
organs

Ovaries
                        14
Reproductive
organs
               Testes
                                                          15
Hemolymph




            Melanin   inhibitor may be necessary
            (Reduced glutathione, cysteine or phenylthiourea)
           16
Fat body
                 17
Imaginal discs
                                        18
Digestive tract




                  Older (non-
                  feeding) larva
                  (different species)
                 19
Nervous system
                  20
Tracheal system
                  21
Salivary glands
(Silk glands)
                   22
Aorta and
Skeletal muscles
                   23
Endocrine glands
                                                            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
   Grace’s “organized neglect”
                                         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
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
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
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
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
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).
                                                        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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posted:8/7/2012
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