1. The freshness of all LM1 components will be the decisive factor for generating healthy rapidly growing T2ECS cultures. Almost all of the components used can suffer from excessive aging. This includes:

a. a-MEM: do not use a bottle of a-MEM that has been opened for too long (more than about 2 wk), as indicated by its strongly pink color.

b. FBS: after being decomplemented, aliquot, and store at -20°C. The exact nature of the serum batch to use seems not to be as critical as in previous culture systems. Nevertheless, differences in between batches are still apparent, and we strongly recommend some tests to be performed for obtaining the proper FBS batch. We also strongly advice that a few vials be stored at -80°C, so as to generate long-term stores.

c. In our experience, PS and HEPES can be stored at 4°C with no loss.

d. P-Mercaptoethanol is stable for at least 2 yr when stored as a 14 M solution in the dark. The working 10-1 M solutions will be stored at 4°C under aluminium foil and will be freshly prepared every week.

e. Dexamethasone, TGF-a, and TGF-P will be stored as an aliquot at -20°C. Do not refreeze a thawed aliquot, but store it at 4°C for no longer than 2 wk. Avoid preparing large amounts of TGF-a and TGF-P solutions, as those factors will be much more stable as powder.

2. At least two types of selection can be envisioned:

a. A G418 selection when the retrovirus expresses a NeoR gene. In that case, apply the selection by adding 30 ||L of a 100 mg/mL G418 solution/mL of culture medium (the final concentration of G418 being 3 mg/mL) for at least 4 d. In the case where an excessive death occurs, the living cells can be separated from the dead cells and debris through a centrifugation1050g for 15 min, no brake) on top of a Lymphocyte Separation Medium (LSM)(Eurobio; density = 1.077 g/mL). The interface will contain mostly living cells.

b. A selection procedure relying on the factor independence that is induced by some transforming oncogenes like the v-erbB oncogene (53). In that case, 48 h after infection, simply grow the cells in a LM1 medium devoid of dexamethasone, TGF-a, and TGF-P for a few days. No normal cells will grow in those conditions.

3. The viral suspensions stability should be a permanent concern. Even at -80°C, the viral titer drops with time. Fresh stock solutions of the most used virus should be prepared by a new round of infection every year. In order to minimize the impact of mutations arising through this reiterative infection protocol, generate new viral batches through transfection with the original plasmids every 5 yr or so.

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