Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

The overall goal of this procedure is to demonstrate how to grow homogenous cells with increased productivity using the three or five layer BD Falcon Multi flasks. This is accomplished by first adding media to the flask and inoculating it with a cell suspension. The second step is to mix and equilibrate the contents to evenly distribute them.

The third step is to partition transport and place the vessel flat in the incubator. The final step is to harvest the cells grown in the multi flask. Ultimately three and five times the cell yield can be harvested compared to T 1 75 flasks with homogenous cell growth across all layers of the multi flask Because it utilizes a new cell culture device.

Visual demonstration of this method is strongly recommended as the handling steps are difficult to conceptualize. Multi flask cell culture vessels are available in a three or five layer stackable format for scale up of cells, providing 525 and 875 square centimeter growth surface areas respectively, pipette access, facilitates addition, and removal of cells and reagents into and out of the vessel. The presence of the mixed port allows for rapid in vessels, mixing and equalization of media across all layers of the multi flask.

To use a multi flask for cell culture begin by placing the vessel vertically on its side in a sterile laminar flow hood. With the cap end facing up, label the multi flask while it is in this position before any contents have been added, loosen and remove the cap. Insert a 50 to 100 milliliter pipette.

Immediately pass the logo to add 35 milliliters of prewarm cell growth media per layer directly to the multi flask. To avoid bubbling of the medium, allow the liquid stream to enter along the inner logo side wall of the multi flask. Finally, using a less than or equal to 10 milliliter pipette.

Add the experimental cell suspension from a concentrated cell stock into the growth medium through the top layer and cap the flask to facilitate inves mixing of cells with media and to eliminate the need to make large volumes of cell suspensions externally. Lift and hold the multi flask upright with the logo facing towards you, and turn the vessel counterclockwise to a 45 degree angle. Then holding the vessel at the same angle.

Gently tilt the multi flask from front to back with the neck pointing away until the liquid in the layer on top drains fully downwards through the mix port. Pivot the vessel on the mix port and repeat this action to allow adequate mixing of the cells with media. Then return the multi flask back to the mixed position.

After mixing the cell suspension, place the multi flask vertically onto a flat work surface to equalize the liquid volume uniformly throughout all the layers. Finally, to partition the liquid into each layer. Hold the multi flask with the logo facing toward you as before and turn the vessel clockwise to a 45 degree angle.

Taking care to keep fluid away from the mixed port. Once the cell suspension has been partitioned, transport the multi flask at the same 45 degree clockwise angle with the media away from the mixed port to the incubator while holding the vessel at this angle, gently rotate the multi flask horizontally down onto the incubator surface with the logo facing up, using the corner away from the mixed port as a pivot. Then gently rock the vessel back and forth and side to side to distribute the cells evenly onto the culture surfaces.

Taking care not to spill liquid from each layer to stack vessels on top of each other. Once they've been placed in the incubator, ensure that the media remains partitioned and away from the mixed port when laying it flat onto the other vessel. First, hold the multi flask upright with the logo facing inward.

Then insert a five milliliter aspirating tip and empty the exhausted media to complete the aspiration. Hold the multi flask in the 45 degree counterclockwise direction while inverting the vessel inward and continue the aspiration for maximum recovery. While the vessel is inverted inward, tilt it clockwise and aspirate the residual volume in the corner opposite the mixed port prior to dissociation.

If necessary, cell monolayers can be rinsed with PBS and aspirated into a waste container. Now add tripsin EDTA dissociating reagent and bring the vessel back to the mixed position. Then after equilibrating partitioning and distributing the reagent across all the layers as just demonstrated, incubate the flask at 37 degrees Celsius for desired time.

After neutralizing the solution within inactivating reagent and repeating the mixing equilibrating and partitioning steps, pour the cell suspension into a receiving tube. Finally, tilt the multi flask clockwise to a 45 degree angle away from the mixing port. Keeping the vessel inverted.

Use a pipette to collect any remaining reagents. Take an aliquot of cells for counting in order to determine cell concentration. When using growth media in a three layer multi flask, a 75 to 150 milliliter cell culture media volume is recommended.

When using a five layer multi flask, it is possible to incubate up to 125 to 250 milliliters per vessel. For dissociation agents, 15 milliliters or more can be added per three layer multi flask, 25 milliliters or greater for five layer vessels using three layer and five layer multi flasks. Three to five times the number of baby hamster kidney cells were grown and recovered from the multi flasks compared to T 1 75 flasks as represented in this bar graph.

In this figure, it can be seen that the cell yield per unit surface area is equivalent in three and five layer multi flasks and T 1 75 flasks for baby hamster kidney cells. A human prostate adenocarcinoma cell line, a human hepatic carcinoma cell line, and a human kidney cell line eco pack 2 2 9 3, all cultured in the appropriate growth mediums for these data. Cell culture.

Medium was added to five layer multi flasks and partitioned into layers. According to the just demonstrated protocol, holes were then drilled into the individual layers and the media from each was pumped out and measured. The weight of fluid recovered from each layer was relatively uniform from layer to layer confirming equal distribution of the media throughout the vessel.

After partitioning in this figure, a simulated distribution of cells between layers of a multi flask using beads similar in size to cells was performed as evidenced by the data represented here of beads enumerated per layer. The mixed port enabled homogenous distribution of the beads and reagents between multi flask layers. Here, representative images of staining patterns of cells grown to greater than 80%confluence on three layer multi flasks.

In supplemented growth media are shown cell monolayers were fixed and stained with crystal violet. The multi flask layers were cut and the images were scanned. Note the homogenous cell patterning on all layers of the multi flask.

This final figure shows data from an analysis of spent media isolated from human embryonic kidney cell cultures after 96 hours using a bio profile flex analyzer. No difference in air saturation in the media isolated from the individual layers of five layer multi flasks versus T 1 75 flasks was detected. After watching this video, you should have a good understanding of how to use the multi flash to increase productivity.