This method is very advanced. It has the divisions of the biotechnology field such as tissue engineering, free generative therapy and drug screening. The main advantage of this technique is that uniform size aggregates can be produced in simple, suspension culture.
This board represents the possibility of the novel O-shaped vessels for the production of uniform aggregates of mammalian cells in orbital shaking suspension culture. In orbital shaking culture with conventional vessels, the circular flow occurs because of the centrifugal force of the medium surface, and this medium flow transfers cells towards center-bottom of the vessels. This effect is well known as Einsteins tea leaf paradox.
This effect causes excess accumulation of cells in center-bottom of the cells and the inhomogeneous aggregation. On the other hand, O-shaped vessels eliminate the center region which prevents medium flow sweeping cells into the center-bottom region. This limits the accumulation of cells that can realize homogenous aggregation.
Begin with a 25-square centimeter flask of HEK-293 cells for each vessel to be seated. Dissociate the cells using 0.25%Trypsin-EDTA solution and collect cells via centrifugation. Resuspend the resulting pellet in one milliliter of culture medium.
Filter the cell suspension through a 40 micron cell strainer to collect a single-cell suspension. Add Trypan blue to an aliquot of the cell suspension and perform a cell count. Then prepare 20 milliliters of cell suspension at 2 times 10 to the 5th cells per milliliter.
Connect the inlet of the O-shaped bag to a clamped 50 milliliter syringe and then detach the plunger. Lower the prepared cell suspension into the O-shaped bag through the clamped syringe. Replace the first clamp syringe with a clean syringe.
Add 55 milliliters of air through the clean syringe to expand the bag completely. Flap the inlet tube and then close the inlet. Finally, remove the clamp from the tube.
Incubate the cells in the O-shaped bag with shaking at 45 rpm in conditions of 35 degrees Celsius and 5%carbon dioxide. To change the medium in the cells, first transfer the cell suspension into a 50 milliliter tube, via the inlet. Then centrifuge for two minutes at 200G at room temperature.
After aspirating the supernatant, add 20 milliliters of culture medium and resuspend the cells. Add the resuspended cells to the O-shaped bag as before and return the sealed bag to the shaking incubator. To collect the cells, transfer the cell suspension into a 50 milliliter tube through the inlet, as before.
Wash the inside of the bag with 20 milliliters of calcium, magnesium free phosphate-buffered saline and then drain the contents into a tube to collect the remaining cells from the bag. Centrifuge the collected cell suspension for three minutes at 200 times G at room temperature and then aspirate the supernate. Add 10 milliliters of calcium, magnesium free PBS and watch the aggregates.
After centrifuging as before, remove the supernate and to leave the pellet containing the aggregates. If a cell-count is required, add four milliliters of PBS and one milliliter of Trypsin to the aggregates and incubate for 10 minutes at 37 degrees Celsius to dissociate the cells for counting. Experimental measurements show that aggregates grown in the conventional dish, had varied diameters after five days in orbital shaking.
In contrast, aggregates grown in O-shaped dishes or bags for five days, had much more uniform diameters. According to the image-based size measurement, the conventional dish culture showed two different peaks, indicating a wide deviation of aggregate size. On the other hand, aggregates in O-shaped dishes and O-shaped bags showed a single peak and less deviation in diameter than those in the conventional dish, suggesting that such aggregates may be of more uniform quality.
Haematoxylin and eosin-staining of aggregate cross-sections, showed that aggregates grown in the conventional dish had some denucleated cells and necrotic course;however aggregates grown in O-shaped vessels did not show any necrotic course and aggregates grown in the O-shaped bag were as large as those in the conventional dish. Cell counts showed that more than 85%of the cells survived for five days of suspension culture in each vessel. This video demonstrates how to handle novel O-shaped vessels for production of uniform site aggregates of mammalian cells in simple suspension culture.
