Expansion of insect cells
Expansion of insect cells
Cells were mechanically isolated from the cell monolayer and cultured for expansion at 27℃ and in suspension.
Operation method
Program 27.7 Expansion of insect cells
Principle
Cells were mechanically isolated from the cell monolayer and cultured for expansion at 27°C and in suspension.
Materials and Instruments
Sf9 cells Dimethyl sulfoxide Pluronic F68 Move Routine maintenance culture For more product details, please visit Aladdin Scientific website.
Growth medium
Culture Flasks Rotating Culture Flasks and Magnetic Stirrers 27°C Incubator
1. Cells are dislodged from culture flasks by the scraping method or by flushing the cells with culture medium (( ATCC # CRL-1711 ) or cell lines obtained from the kale ring Trichoplusia ni (Tn 368 or BTI-TN-5B1-4, also known as "High Five", available from Invitrogen)) method to dislodge cells from culture flasks or to utilize cells grown in suspension in rotating flasks.
2. Count the cells with a hemocyte counter plate and check the viability of the cells by staining with Tapan Blue or Naphthalene Black.
3. Cells are grown at a density of 0. 5~1×106 cells/ml in a rotary flask, and 20~100 ml of cell suspension is injected into each 500 ml rotary flask.
4. Cultivate the cells at 20~28°C, preferably at 37°C.
5. Dilute the cells to 0. 5-1×106cells/ml every 48-72 h or when the cell density is 4-5×106 cells/ml.
6. Transfer the cells into clean culture flasks every 3-4 weeks.
7. If the cell population aggregates, speed up the agitation slightly and add surfactant Pluronic F-68 [ 0.5%~1.0% (v / v ) ] to reduce the shear force.
Freeze the cells (see also protocol 20.1).
1. Count the cells and centrifuge (1000 rpm, ~200 g, 5 min ).
2. Suspend the cells with 10% DMSO (prepared in FBS) at a density of 1 × 107 cells/ml.
3. Dispense the cells into freezing tubes, and then place the freezing tubes on ice for 1 h.
4. Pack the cryotubes into styrene foam containers.
5. Slowly freeze the tubes overnight at -70°C (~1°C/min).
6. Transfer the tubes to a liquid nitrogen freezer.
7. To revive the frozen cells, thaw the tubes rapidly at 37°C. If cells are stored in the liquid phase, care should be taken to avoid the risk of injury due to rupture of the cryotube when thawing the cells in the capped container.
8. Transfer the cells into 25 cm2 culture flasks containing 5 ml of culture medium. Tap the culture flask to disperse the cells evenly.
9. Aspirate off the culture solution after 2-3 h. When most of the cells are attached to the wall, add the culture solution. When most of the cells are attached to the wall, add 5 ml of fresh culture medium.
10. Incubate the cells for 2~3 days before isolation to allow the cells to recover. Then transfer the cells into stirred culture flasks or larger plastic flasks as described above.