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Application Note

Improved Cell Culture Productivity Using Millicell HY Multilayer Flasks


INTRODUCTION
Numerous research applications, including cell-based-assays and small-scale virus production, rely on T-flasks for cell culture. Traditionally, researchers have used multiple single-layer flasks to generate the necessary quantity of cells. This consumes valuable incubator space and results in a repetitive, slow harvesting process. However, multilayer flasks are making this process much more efficient. Millipores new Millicell HY multilayer flasks feature a unique design that increases the surface area for cell growth while maintaining a footprint similar to traditional T-flasks. Researchers can culture more cells in the same space and environment by growing cells on multiple layers the Millicell HY 3- and 5-layer flasks offer 600 and 1000 cm2 of total surface area, respectively. This is up to 13 times the area of a standard single-layer T75 flask. Because cell seeding density and media volume are linearly proportional to surface area, no reoptimization of original cell culture conditions is required, regardless of the type of flask used. Millicell HY flasks also have easy pipette access for aspirating and dispensing, and allow for pouring if desired, which improves the overall handling compared to other multilayer flasks on the market.
5. Lay the flask down to spread the cell suspension evenly across each layer. 4. Tilt the flask on its end to partition the cell suspension. 3. Turn the flask on its side to distribute the cell suspension equally among all layers. 2. Raise the end of the flask and pipette up and down to mix cells and media. 1. Fill the Millicell HY flask with growth media and add cells.

6. Alternate flasks to stack and save space in the incubator.

Figure 1. Millicell HY Flasks have easy pipette access for aspirating and dispensing, and are designed to distribute cells and media evenly across each layer by using the procedure demonstrated above.

METHODS

Flask performance evaluation


CHO-k1 cells were seeded in single-layer flasks and Millicell HY multilayer flasks at 20,000 cells/cm in 0.2 mL/cm culture
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Lentivirus production and titer determination


GFP-lentivirus was produced in both a traditional single-layer T75 flask and a 600 cm2 Millicell HY flask by transfecting HEK 293 cells with a plasmid mix. Supernatant containing infectious lentivirus was collected on the third day. Fractions were serially diluted and 100 L aliquots of each dilution were mixed with 10,000 HEK 293 cells per well in 96-well tissue culture-treated plates. On day 3, cells were fixed with 4% paraformaldehyde, washed 3 times with PBS, and analyzed by fluorescence microscopy. The total input of infectious particles (Ivp) was calculated using the formula: Input in Ivp = (# fluorescent cells) x (dilution factor) x (input volume in mL) (assay volume in mL).

media and incubated for 48 h at 37 C, 6% CO2, 95% relative humidity (RH). Cells were washed with 0.02% EDTA followed by harvest via enzyme dissociation for 10-15 min at 37 C. Cell count and viability measurements were conducted on a guava easyCyte flow cytometer using ViaCount flex

reagent for 1000 acquisition events.

Stem cell proliferation


129/S6 mouse embryonic stem cells (ESCs; Millipore Cat. No. SCR012) were seeded at 40,000 cells/cm in 0.25 mL/cm
2 2

culture medium (Millipore catalogue no. ES-101-B) and incubated for 48 h at 37 C, 6% CO2, 95% RH. Cells were washed twice with phosphate-buffered saline (PBS) followed by harvest via enzyme dissociation for 10 min at room temp. Cell count and viability measurements were performed using a guava easyCyte flow cytometer with ViaCount reagent. To confirm the stem cell profile, undifferentiated ESCs were dissociated into a single cell suspension and collected. Cells were fixed with 4% paraformaldehyde and permeabilized with 100% methanol. Oct-4 and SSEA-1 expression was determined using specific primary antibodies (Millipore catalogue numbers MAB4419 and MAB4301, respectively). Samples were analyzed using an easyCyte flow cytometer.

Influenza virus production and titer determination


Influenza A virus was produced in both Millicell HY flasks and standard T-flasks by infecting MDCK cells with an equal ratio of influenza virus stock. After incubation for 3 days, virus was harvested and virus titer determined by hemagglutination assay.

RESULTS

Yield is proportional to surface area


Results indicate that cell yields from Millicell HY flasks are linearly proportional to total surface area (Figure 2). For example, the three-layer Millicell HY flask has eight times as much surface area as a standard T75 flask, and, correspondingly, yields eight times as many cells. The HY flask design also allows for uniform cell growth from layer to layer.

Adenovirus production
HEK 293 cells were grown to 80% confluence in either T150 culture flasks or 3-layer Millicell HY flasks. GFP adenovirus was added at 100:1 multiplicity of infection (MOI), and cells were incubated until they detached from the culture surface (2-4 days). Cells were harvested and lysed by three rapid freeze-thaw cycles in dry ice ethanol bath and a 37 C water bath. Lysates were clarified by centrifugation at 2500 x g for

14 12 Cell Count (T75 Flask Equivalents) 10 8 6 4 2 0 75 cm2 Millicell 600 cm2 Cell Culture Flask Type T75 Actual CHO-k1 Cell Yield Expected Cell Yield 3-Layer

5-Layer % Cell Yield Compared to Theoretical Millicell 1000 cm2

1.

15 min and used for titer determination.

1.

Adenovirus titer determination


HEK 293 cells were seeded in 96-well tissue culture-treated plates at a density of 10,000 cells per well in 100 L of cell culture medium containing 2% FBS and incubated at 37 C, 95% relative humidity, 6% CO2. Serial dilutions of adenovirus samples were prepared and 100 L of each dilution sample was added per well, with 10 replicate wells per dilution point. Evaluation of GFP-positive infection foci by fluorescence microscopy yielded 50% tissue culture infectious dose (TCID50) for each sample.

0.

0.

0.

0.

Figure 2. Actual and expected yields of CHO-k1 cells grown in single layer T75 or multilayer Millicell HY flasks.

Stem cell culture


Stem cell evaluations demonstrated very similar percent viability for cells cultured in both standard and multilayer flasks (Figure 4). The cells also demonstrated nearly identical stem cell marker expression (Figure 5). flasks (Figure 6). The small standard deviations reflect high reproducibility of virus production between flask replicates. Note that 1 T600 HY flask has a surface area equal to that of 8 T75 flasks or 4 T150 flasks. Finally, influenza virus production in the 5-layer 1000 cm2 Millicell HY flask was compared with influenza virus production in standard T150 cm2 tissue culture flasks. A single 5-layer Millicell HY flask provides approximately as much surface area for cell growth as do 7 single-layer T150 flasks, so the yields were equivalent (Figure 7).

Virus production
Next, the HY flasks were tested for performance in small scale virus production. The titer in viral particles/mL of both adenovirus and lentivirus produced in Millicell HY flasks is similar to titers obtained from traditional single layer

Average Infectious Adenovirus Average Infectious Adenovirus Average Infectious Adenovirus Titer (TCID50/mL) Titer (TCID50/mL) 50/mL) Titer (TCID

100 80 % Viability 60 40 20 0

T75 cm2 vs. T600 cm2 Average mESC Viability

1.00E+11 1.00E+10 1.00E+09 1.00E+11 1.00E+08 1.00E+10 1.00E+07 1.00E+09 1.00E+06 1.00E+08 1.00E+05 1.00E+07 1.00E+11 1.00E+04 1.00E+06 1.00E+10 1.00E+03 1.00E+05 1.00E+09 1.00E+02 1.00E+04 1.00E+08 1.00E+01 1.00E+03 1.00E+07 1.00E+02 1.00E+06 1.00E+01 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+07 Average Lentivirus Titer Average Lentivirus Titer Titer Average Lentivirus (ivp/mL) (ivp/mL) (ivp/mL) 1.00E+06 1.00E+07 1.00E+05 1.00E+06 1.00E+04 1.00E+05 1.00E+03 1.00E+07 1.00E+04 1.00E+02 1.00E+06 1.00E+03 1.00E+01 1.00E+05 1.00E+02 1.00E+04 1.00E+01 1.00E+03 1.12E+06 3-layer (600 cm2) Millicell HY Flask 3-layer (600 cm2) Millicell HY Flask Single-layer T150 Flask 8.45E+05 8.45E+05 3-layer (600 cm2) Millicell HY Flask 1.12E+06 1.12E+06 3.22E+10 3.32E+10 3.22E+10 3.32E+10

% Viable % Dead

3.22E+10 Single-layer T150 Flask Single-layer T150 Flask

3.32E+10 3-layer (600 cm2) Millicell HY Flask 3-layer (600 cm2) Millicell HY Flask

mESC in T75 (n=3) Device Type

mESC in T600 (n=3)

Figure 4. Stem cell viability in standard single-layer T75 flask versus 3-layer Millicell HY T600 flask.

40 35 30 Count 25 20 15 10 5 0 0 100

T75 cm2 vs. T600 cm2 mESC Pluripotency with OCT4


Oct4 Oct4 + Control - No OCT4 T75 (n=3) T600 (n=3)

8.45E+05 Single-layer T75 Flask Single-layer T75 Flask

200

300

400 500 600 700 Green Fluorescence

800

900

1000

Influenza Virus Titer Influenza Virus Titer Titer Influenza Virus (HAU/mL) (HAU/mL) (HAU/mL)

Figure 1.00E+02 in viral particles/mL of adenovirus prepared from 6. Titer single-layer T150 flask (A) and lentivirus prepared from single-layer 1.00E+01 T75 flasks (B), each compared to 3-layer T600 Millicell HY flask. Data 9000 Single-layer 3-layer (600 cm2) represent the average tite. Error bars represent standard deviation Millicell HY Flask T75 Flask 8000 of the titer determination assay.
9000 7000 8000 6000 7000 5000 6000 4000 5000 3000 9000 4000 2000 8000 3000 1000 7000 2000 0 6000 1000 5000 0 4000 3000 2000 1000

40 35 30 Count 25 20 15 10 5 0 0 100

T75 cm2 vs. T600 cm2 mESC Pluripotency with SSEA1


SSEA1 Control - No SSEA1 T75 (n=3) T600 (n=3) SSEA1 +

5-layer (1000 cm2) Millicell HY Flask 5-layer (1000 cm2) Millicell HY Flask

7 x T150 Single-layer Flasks 7 x T150 Single-layer Flasks

200

300

400 500 600 700 Green Fluorescence

800

900

1000

0 5-layer (1000 cm2) Millicell HY Flask 7 x T150 Single-layer Flasks

Figure 5. Expression of stem cell markers Oct-4 (A) and SSEA-1 (B) in stem cells cultured in T75 or T600 flasks.

Figure 7. Total infectious influenza virus particles harvested from 7 single-layer T150 flasks and from one 5-layer Millicell HY flask.

Collectively, these data show that the expected amount of virus is produced based on the surface area used. Using Millicell HY flasks allows one to work with fewer flasks to produce an equivalent amount of virus.

mL. For each comparison, the same protocols, media concentrations, and cell seeding density were used, demonstrating the scalability and reproducibility of the flasks for various applications.

DISCUSSION
T-flasks are useful for cell culture and virus production but the number of flasks required in some cases can become burdensome and challenging to manage. Multilayer flasks like Millipores Millicell HY flasks meet a growing need. As demonstrated above, the HY flasks offer greater surface area and proportionally larger yields within a footprint similar to that of a single-layer flask. This saves both time and space. The unique design of the flask ensures that cell growth is equivalent on each layer, so standard cell culture protocols are easily scaled up. The HY flasks are appropriate for diverse applications, including stem cell culture and virus production. Stem cells cultured in the HY flask have similar viability and pluripotent marker expression levels to cells cultured in standard T flasks. Likewise, virus production assays in both the HY flasks and regular T flasks yielded equivalent titers in viral particles per

CONCLUSION
The advantages of using Millicell HY flasks over using traditional single-layer cultureware include less time required when harvesting from a single flask rather than numerous culture vessels, less incubator space required, and uniform cell health and culture conditions. Use of the Millicell HY flasks results in robust production similar to traditional single-layer flasks. Millicell HY flasks have been demonstrated to work for diverse types of cell culture, including stem cell expansion and virus production. Millicell HY flasks are also designed for easy use, with access for aspirating, dispensing, and pouring. This combination of ergonomic flask design along with performance comparable to traditional flask designs can minimize labor intensive activities in your lab today.

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