Shreffler:Culturing Het1A

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Materials

  • BEGM Medium (Lonza CC-3170), a complete medium prepared by adding supplements (CC-4175) to basal medium (BEBM, CC-3171). Buying together is cheaper.
    • Use all Lonza supplements except GA (gentamycin/amphotericin), which is toxic to Het-1A cells. Instead, use Shrefflab penicillin/streptomycin in 1:100 dilution of stock.
    • Do not sterile-filter this medium.
  • Coating solution - this is complete medium containing 0.01 mg/ml fibronectin, 0.03 mg/ml bovine collagen type 1, and 0.01 mg/ml bovine serum albumin (BSA).
    • Prepare 1 mg/ml stocks of fibronectin (Sigma F4759) and BSA (Sigma A9418) in complete medium. Prepare 1mg/ml stock of bovine collagen type 1 (Sigma C9791) in 0.1M acetic acid. Collagen will not dissolve, but will form a suspension after ~3h on a shaker. Do not sterile-filter stock solutions. Use aliquots of these stocks to prepare coating solution (after thawing aliquots, thorough mixing may be necessary to redissolve/resuspend fibronectin and collagen).

Procedure

  • Coat culture flask (surface 75 cm squared) w/ 5 ml coating solution. Rock gently to coat entire surface (quick circular motions work well). Incubate overnight at 37°C.
  • Suction off coating solution before adding Het-1A cells in ~20 ml complete medium.
  • Gro cells until confluent (covering intire surface of flask). This will take ~5 days. Add or refresh medium when it turns yellow. If there are many non-adherent (dead) cells or debris, suction off medium and refresh completely.
  • To harvest cells, suction off medium and incubate cells w/ 2-3 ml 0.05% trypsin in 0.53mM EDTA (Cellgro 25-052-CI) at room temperature. Cells will detach after ~5 min (check under microscope). Rock as little as possible to avoid cell clumps. Add 20 ml com[plete medium to dilute trypsin-EDTA and wash the flask. Put cells in 50 ml tube and centrifuge @125g for 7 min. Remove supernatant, resuspent celll pellet in complete medium, and count with trypan blue. Cells are ready for use in experiment or to be split into new coated flasks (~2x10^6 cells/flask).
    • An alternative method to stopping trypsin activity is by using soybean trypsin inhibitor (STI), as described in ATCC's protocol. This may enhance cell survival. Add 3 ml 0.1% STI in PBS after trypsinization instead of 20 ml medium. Wash flask, put cells in tube, etc.
  • Cryopreserve cells if necessary in fetal bovine serum + 10% DMSO. Put ~4x10^6 cells in 1 ml per vial. Spin cells down, resuspend in half the needed volume of FBS, and add an equal volume of FBS + 20% DMSO. Then, aliquot in vials and put in pre-cooled (4°C) Mr. Frosty cryopreservation container. Put container in -80°C freezer over night. Place vials into liquid nitrogen storage.
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