SH-SY5Y

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History

The cell line SH-SY5Y is a third generation neuroblastoma, cloned from SH-SY5, which is from SH-SY, which is from SK-N-SH. The original cell line was isolated from a woman's metastatic bone tumor in 1970. In each successive generation, the nucleus was removed and put into a new cytoplasm in the process known as cloning. The SH-SY5Y cells possess two X chromosomes (making them genetically female), and have blood type A with a positive Rh group (A+).

Morphology

The cells possess an abnormal chromosone 1, where there is an additional copy of a 1q segment and is referred to trisomy 1q. SH-SY5Y cells are known to be dopamine beta hydroxylase active, acetylcholinergic, glutamatergic and adenosinergic. The cells have very different growth phases, outlined in the surrounding pictures. The cells both propagate via mitosis and differentiate by extending neurites to the surrounding area. While dividing, the aggregated cells can look so different from the differentiated cells, that new scientists often mistake one or the another for contamination. The dividing cells can form clusters of cells which are reminders of their cancerous nature, but certain treatments such as retinoic acid and BDNF can force the cells to dendrify and differentiate.

Media and Cultivation

The most common growing cocktail used is a 1:1 mixture of DMEM and Ham's F12 medium and 10% supplemental fetal bovine serum. The DMEM usually contains 1.5g/L sodium bicarbonate, 2mM L-Glutamine, 1mM sodium pyruvate and 0.1 mM nonessential amino acids. The cells are always grown at 37 degrees Celsius with 95% air and 5% carbon dioxide. It is advised to cultivate the cells in flasks which are coated for cell culture adhesion, this will aid in differention and dendrification of the hybridoma.

Recommended culture medium: Ham's F12:EMEM (EBSS) (1:1) + 2 mM L-Glutamine + 1% Non-essential amino acids (NEAA) + 15% FBS. Subculture: Split at 70-80% confluency, approx. 1:10 to 1:100, seed at 1x103 to 1x104 viable cells/cm2. Trypsinize using 0.25% solution, with or without EDTA, 37oC and 5% CO2. Cells may reattach slowly and may remain in suspension for several days. NOTE: do not continue culture beyond 20 passages. This cell line is a thrice-cloned sub-line of bone marrow biopsy-derived SK-N-SH. SH-SY5Y has a dopamine-β-hydroxylase activity and can convert glutamate to GABA. Will form tumors in nude mice in approx. 3-4 weeks. The loss of neuronal characteristics has been described with increasing passage numbers. Therefore it is recommended not to be used after passage 20 or verify specific characteristics such as noradrenalin uptake or neuronal tumor markers.

Splitting

Splitting is the act of taking a cell rich culture and dividing it up into many less dense cultures. This is done either for preventing overcrowding, or for expanding the number of cultivated flasks. Although every lab does this differently, the general procedure is as follows.

• Aspirate off the old cell media
• Rinse twice with sterile PBS
• Add about 2 milliliters of Trypsin with 0.53mM EDTA: a protease and a metal chelator, respectively
  • This will break apart all of the cellular proteins that adhere the cells to the flask.
    • If the trypsin is left too long, the cells will fall apart, but not long enough and the cells won't seed very well.
    • For SH-SY5Y cells, the best time is around two to three minutes.
  • Tap, and rock by hand such that all of the cells are covered with trypsin
  • Be sure that the cells begin to flow with the liquid with each rocking.
• Add an approproate amount of fresh, prewarmed feeding media (5mL, 3mL).
  • Note: It doesn't matter, but just know that it must be a set amount, so that it can be further diluted to the correct ratio. The optimum ratio is around 1:30, but 1:50, 1:200, and 1:1000 have all been used.
  • Note: The DMEM has protease inhibitors, which neutralize the actions of the Trypsin.
• Take pipette into the cell-rich media and titrate gently.
  • Do not tritrate too hard, or the cells will not survive, but not enough and the cells growth will be chunky (it is better to err on the chunky side).
  • When finished, the flakes and clumps of cells should be mostly gone and the media should resemble something like cloudy grapefruit juice.
• Take one milliliter and place into a sterile centrifuge tube. Dilute as needed, (29mL works well).
• Seed or take the diluted cell media and place into a fresh, sterile flask, or dish, or plate.
• Redilute the original flask as needed (or not, newer flasks grow better)
• Screw cap back on and place flask/dish/plate into incubator

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