Comparative cell growth study using CELLSTAR AutoFlask

Transcription

1 Comparative cell growth study using CELLSTAR AutoFlask Introduction With the increasing amount of cell culture work performed in today s life sciences laboratories, the need for automation is evident. Whether it is to provide cells for cell-based assays or cell-based products, the process of culturing large amounts of cells is a timeconsuming, laborious process. Over the past decade, a number of large-scale solutions for automated cell maintenance and growth have been introduced to the market. These systems have demonstrated the ability to automate the many aspects of cell culture. Greiner Bio-One, in collaboration with the Genomics Institute of the Novartis Research Foundation (GNF); San Diego (USA) has developed a unique cell culture flask, the CELLSTAR AutoFlask TM, designed for automated tissue culture. The external dimensions correspond to ANSI Standards to render the flask suitable for use on a wide range of cell culture and liquid handling systems, while also maintaining the height standard, in order to work with existing automation equipment. As with all Greiner Bio-One CELLSTAR products the AutoFlask is made of high-grade polystyrene characterized by exceptional clarity to facilitate optical control of cell proliferation and morphology. A proprietary physical surface treatment renders a hydrophilic surface, significantly increasing the attachment of adherent cells. In keeping with the CELLSTAR product line, CELLSTAR AutoFlask is guaranteed sterile, nonpyrogenic, non-cytotoxic and free of RNase and DNase. A unique feature of the AutoFlask is the centrifugation pocket which enables separation of cells from supernatant inside the flask. Upon horizontal centrifugation cells are collected inside the pocket whereas the medium remains in the bottom vessel of the flask. (For detailed information please refer to CELLSTAR AutoFlask instruction for use; chapter 3: Automation and manual handling) The robot accessible pre-slitted multiple entry septum allows sterile media and solution exchange and the removal of cell based products. Pre-slitting inhibits coring of the septum by the injection needle, permits reclosing of the septum slit and ensures sterility of the flask content throughout the whole cultivation process. The septum is located at the A1 position to ease programming and piercing with any liquid handling system. In comparison with competitive products, it is not necessary for the CELLSTAR AutoFlask to be in the upright position to access the flask. It can remain in a horizontal position during the entire cultivation process to minimize cell stress due to repeated movement of the flask. An integrated filter membrane facilitates gas exchange during the cultivation of cells while providing a sterile barrier against contaminations. The hydrophobic membrane is PTFE coated to prevent penetration of moisture from the internal liquid. The high airflow rate of the filter material, combined with an optimal vent design for oxygen supply allows cultivation of even very sensitive cells. The following provides a comparative cell growth study to display the exceptional features of CELLSTAR AutoFlask for automated and manual cell cultivation.

2 Material Item Manufacturer Cat.-No. Ham s F12 medium Biochrom AG F0815 DMEM medium Biochrom AG F0435 L-Alanyl-L-Glutamine Biochrom AG K0302 Fetal calf serum Invitrogen Life Technologies Trypsin/EDTA solution (0.05%/0.02%) Biochrom AG L2143 CELLSTAR AutoFlask Greiner Bio-One GmbH CELLSTAR Cell Culture Flask Greiner Bio-One GmbH CELLSTAR Cell Culture Flask Greiner Bio-One GmbH Methods Cell cultures were prepared and maintained according to standard cell culture procedures. Cell culture media were supplemented with 4 mm L-Alanyl-L-Glutamine. The cell line CHO was cultivated in Ham s F12 medium with 10% fetal calf serum (FCS), whereas SKNMC cells were maintained in Dulbecco s MEM with 10% FCS. Cells were cultivated in T-175 tissue culture flasks before harvest and transfer into automated cell culture flasks. Depending on the experimental approaches, cells were either cultivated for 24, 48, 72 or 96 hrs in an incubator at 37 C and 5% CO 2. Maximum cell concentrations were determined by cell harvest and counting using a CASY Cell Counter [Schärfe System; Germany]. Experimental approaches 1. Cell line specific cell growth using CELLSTAR AutoFlask L929, SKNMC, HeLa, HEK293, CHO and NH3T3 cells were cultivated in their appropriated media for five days using either a CELLSTAR AutoFlask or a CELLSTAR T-75 tissue culture flask to determine general cell growth of each individual cell line. Maximum cell numbers were analyzed after the cultivation period by electric sensing zone method [CASY Cell Counter; Schärfe System; Germany]. 2. Growth area dependent maximum cell concentration As cell growth areas of CELLSTAR AutoFlask and competitive automated flasks differ from each other (see table below) the initial seeding concentration was calculated in alignment to these values. This approach inhibits any falsification of results due to unequal space for cell propagation. Seeding of SKNMC and CHO cells facilitated cell proliferation and vitality analysis of a sensitive [SKNMC], and a common [CHO] cell culture line. The following cell concentrations were seeded in accordance with standard cell culture procedures: Product Growth Area CHO [cells/flask] CHO [cells/cm 2 ] AutoFlask 83.6 cm x x10 3 Competitor cm x x10 3 Competitor cm x x10 3

3 Product Growth Area SKNMC [cells/flask] SKNMC [cells/cm 2 ] AutoFlask 83.6 cm x x10 4 Competitor cm x x10 4 Competitor cm x x flasks of each supplier were cultivated with 30 ml of the corresponding media for 72 hrs. Cell attachment and morphological development was analyzed each day. On the last day of cultivation cells were harvested by Trypsin digestion and maximum cell numbers as well as vitality of cells were determined using a CASY Cell Counter [Schärfe System; Germany]. 3. Growth curve To analyse cell proliferation processes in more detail a defined starting concentration (see table below) was seeded in 25ml of the respective media. Maximum cell numbers were analysed on each day of cultivation. Cell Line Concentration [cells/ml] Concentration [cells/flask] SKNMC 8.0 x x10 6 CHO 2.0 x x10 5 After 24, 48, 72 and 96 hrs of cultivation 3 flasks of each supplier were harvested and cell concentrations were determined using a CASY Cell Counter [Schärfe System]. Results and Discussion 1. Cell line specific cell growth using CELLSTAR AutoFlask Maximum cell numbers AutoFlask Cells/ml [x10 6 ] L929 SKNMC HeLa HEK CHO NH3T3 Figure 1: Maximum cell numbers of various cell lines after 5 days of cultivation. L929, SKNMC, HeLa, HEK, CHO and NH3T3 cells were cultivated in their appropriated media for five days using either a CELLSTAR AutoFlask or a CELLSTAR T-75 tissue culture flask to determine general cell growth of each individual cell line. Maximum cell numbers were analyzed after the cultivation period by electric sensing zone method [CASY Cell Counter; Schärfe System; Germany].

4 Cultivation of L929, SKNMC, HeLa, HEK, CHO and NH3T3 cells displayed equivalent cell proliferation in both CELLSTAR AutoFlask and CELLSTAR T-75 tissue culture flask, indicating optimal cell growth conditions in these cell culture formats. 2. Growth area dependent maximum cell concentration 2.1 Vitality Vitality after 72hrs of cultivation 98,00 96,00 96,64 96,17 96,59 CHO SKNMC 94,00 Vitality [%] 92,00 90,00 88,00 90,79 88,49 89,18 86,00 84,00 AutoFlask Competitor 1 Competitor 2 Figure 2: Vitality of SKNMC and CHO cells after 72 hrs of cultivation. Cells have been seeded in 30 ml of the corresponding media and cultivated at 37 C and 5% CO 2. After 72hrs of cultivation cells have been harvested by Trypsin digestion and vitality has been determined using a CASY Cell Counter [Schärfe System; Germany]. Cultivation of SKNMC and CHO cells using CELLSTAR AutoFlask as well as competitive automated cell culture flaks under the above described settings led to comparable cell vitality values, indicating appropriate cell culture conditions for each experimental setup. 2.2 Maximum cell numbers Maximum cell numbers after 72hrs of cultivation Cells/cm 2 [x10 4 ] 9,00 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 8,84 8,43 CHO 7,76 SKNMC 4,91 3,93 4,34 AutoFlask Competitor 1 Competitor 2 Figure 3: Maximum Cell Numbers of SKNMC and CHO cells after 72 hrs of cultivation. Cells have been seeded in 30ml of the corresponding media and cultivated at 37 C and 5% CO 2. After 72hrs of cultivation cells have been harvested by Trypsin digestion and maximum cell numbers have been determined using a CASY Cell Counter [Schärfe System].

5 Analysis of maximum cell numbers revealed only minor differences for CHO cells when compared to competitor 1 (5%) or competitor 2 (9%). The sensitive SKNMC cells, whose cell proliferation rate can be affected by inappropriate cell culture conditions such as inefficient tissue culture treatment and reduced ventilation, exhibited a significant difference in comparison to competitive automated tissue culture flasks. Expansion of SKNMC cells using CELLSTAR AutoFlask led to 25% more cells compared to competitor 1 and 13% increased cell numbers in comparison to competitor 2. As total cell numbers are standardized to the respective cell growth areas (see experimental approaches), falsification of results due to unequal space for cell propagation can be excluded. 3. Growth curve 3.1 CHO growth curve 140,00 Growth curve CHO Cells/flask [x10 5 ] 120,00 100,00 80,00 60,00 40,00 AutoFlask Competitor 1 Competitor 2 20,00 0, hrs 48hrs 72hrs 96hrs Figure 4: Growth Curve CHO. CHO cells have been seeded with a starting concentration of 2.0 x10 4 cells/ml and cells have been cultivated at 37 C and 5% CO 2 for 96 hrs. Each 24 hrs cells have been harvested by Trypsin digestion and maximum cell number has been determined using a CASY Cell Counter [Schärfe System]. Detailed analysis of CHO cell propagation revealed accelerated cell proliferation after the second day in vitro in CELLSTAR AutoFlask when compared to competitor 1 and 2. Maximum cell numbers dependent to the provided cell growth area were achieved between 72 and 96 hours of cultivation, whereas comparable cell numbers were not achieved in the same timeframe using automation friendly tissue culture flasks originated from competitor 1 or 2. This indicates cell splitting and expansion can be initiated 24 hours earlier than with competitive automated tissue culture flasks, enabling a significant time saving throughout the whole cell preparation process.

6 3.2 SKNMC growth curve 7,00 Growth curve SKNMC 6,00 Cells/flask [x10 5 ] 5,00 4,00 3,00 2,00 1,00 0,00 AutoFlask Competitor 1 Competitor hrs 48hrs 72hrs 96hrs Figure 5: Growth Curve SKNMC. SKNMC cells have been seeded with a starting concentration of 8.0 x10 4 cells/ml and cells have been cultivated at 37 C and 5% CO 2 for 96 hrs. Each 24 hrs cells have been harvested by Trypsin digestion and maximum cell number has been determined using a CASY Cell Counter [Schärfe System]. SKNMC cells cultivated in CELLSTAR AutoFlask demonstrated a similar effect as CHO cells, although less prominent due to a slower proliferation rate compared to CHO cells. Despite this inhibition in cell division, CELLSTAR AutoFlask provided exceptional cultivation conditions leading to increased cell numbers after only four days in vitro. Conclusion Compared to competitive automated cell culture flasks, Greiner Bio-One s CELLSTAR AutoFlask provides optimal cultivation conditions for both common and very sensitive cell lines. The high airflow filter membrane, optimized vent design for maximum oxygen supply, and specific hydrophilic tissue culture treatment of Greiner Bio-One s CELLSTAR surfaces enhanced cell attachment and proliferation to significantly accelerate cell expansion and time saving throughout the entire cultivation process. 08/