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Nick Kohlstrom, George Wang, Linette Philip and Ma Sha, Eppendorf Inc., Enfield, CT, USA
Summary
The feasibility of culturing mammalian cells in the New Brunswick S41i CO2 constant temperature shaker in this study was first demonstrated by culturing Chinese hamster ovary cells (CHO). And compare it to the performance of two mainstream CO2 constant temperature shakers. Hybridoma cells cultured with New Brunswick S41i performed as well as other similar products in terms of cell growth rate and viable cell rate, while also comparing CO2 gas consumption with high-end eco-design and advanced parameter control, New Brunswick S41i The gas consumption of the CO2 constant temperature shaker is 10 times lower than that of similar products, showing superior performance.
Review
Cars are not just the only source of CO2 emissions, and laboratory equipment such as CO2 incubators can produce more than 20,000 liters of CO2 per year. Eppendorf proposes environmental initiatives to reduce the environmental impact of its products. The CO2 consumed in most incubators is released into the atmosphere, while Eppendorf's new constant temperature shaker, New Brunswick S41i, releases very small amounts of CO2 under normal cell culture conditions without affecting the performance of the shaker. In this study, the performance of the New Brunswick S41i constant temperature shaker was evaluated by culturing hybridoma cells and Chinese hamster ovary cells (CHO). This study also compares the CO2 consumption of the New Brunswick S41i constant temperature shaker with other similar products. The data reveals that the CO2 consumption of the New Brunswick S41i constant temperature shaker is 5 to 10 times lower than that of the same type of product, and its carbon footprint is also 5 to 10 times lower than its competitors.
The high-end design reduces gas leakage, the tightly sealed glass inner door is protected by an external door, and the tightly sealed drive structure isolates it from the culture chamber. Comparison of growth rate, cell density and viable cell rate based on cell culture The performance of the New Brunswick S41i constant temperature shaker is the industry leader.
This new CO2 incubator is equipped with four eccentric drives to provide precise and stable environmental control for the growth of suspended cells. The shaker drive is optimized for high performance in humid and carbon dioxide enriched environments.
Materials and Method device
> New Brunswick S41i CO2 constant temperature shaker with high temperature disinfection
> Similar products 1
> Similar products 2
> Vi-CELL® Analyzer (Beckman Coulter, Germany)
> YSI 2700® Analyzer (YSI Life Sciences, USA)
> New Brunswick Galaxy® Gas Analyzer
> Omega® FMA-1608A Thermal Mass Flowmeter (Omega Engineering, USA)
> Eppendorf supplies
- Research® plus single channel pipette
- epT.IPS®
- Easypet® 
Media and cells:
> DG44 Chinese Hamster Ovary Cells (Invitrogen)
> EX-CELL® Chemically Defined Chinese Hamster Ovary Cell Serum Free Medium (Sigma)
> Hybridoma Cell DA4-4; American Type Culture Collection: HB57
> DMEM Medium (American Type Culture Collection, ATCC)
> 5% fetal bovine serum (Gibco)
> 100x penicillin (Gibco)
Chinese hamster ovary cell (CHO) culture method
CHO cells were grown in serum-free medium of Chinese hamster ovary cells supplemented with 1% antibiotic penicillin (EX-CELL®). Six 250 mL conical flasks were each inoculated with 60 mL of medium at a seeding density of 3 x 105 cells/mL. And each conical flask was prepared from the same batch of medium. The Erlenmeyer flasks were placed in six different positions on the shaker plate and the results were uniform. The Erlenmeyer flask was incubated at 37 ° C with a CO2 concentration of 5%, an air content of 95%, and a rotational speed of 130 RPM (4.69 rcf).
CHO cells were grown for 14 days and analyzed for glucose content, cell density and viable cells on days 3, 5, 7, 10, 12 and 14 using a YSI 2700 analyzer and a Beckman Coulter Vi-CELL analyzer. rate.
Hybridoma cell culture method
DA4-4 hybridoma cells were grown in DMEM medium supplemented with 5% fetal bovine serum and 1% antibiotic penicillin. Each 250 mL Erlenmeyer flask was inoculated with 45 mL of medium, and the media in all Erlenmeyer flasks were prepared in the same batch, placed in six different New Brunswick S41i CO2 constant temperature shakers, similar products 1 and similar products 2 position. The Erlenmeyer flask was incubated at 37 ° C with a CO2 concentration of 5%, an air concentration of 95%, and a rotational speed of 95 RPM (2.52 rcf). Hybridoma cells were subcultured at 2×105 cells/mL on day 2 and day 4, respectively. Each conical flask was sampled daily and analyzed for glucose content, cell density and cell viability using a YSI 2700 analyzer and a Beckman Coulter Vi-CELL analyzer.
Gas consumption
The New Brunswick S41i CO2 constant temperature shaker and similar products 1 are set at 37 °C, 95 RPM, 5% CO2, and ensure that the environment has been running for 12 hours. The CO2 inlet pressure is set at the lowest recommended by the manufacturer. Verify the CO2 concentration in each shaker using an off-line CO2 analyzer. A mass flow meter was used to record the volume of gas consumed by each shaker after 48 hours. Repeat the test three times and the average result is shown on the right:
result
1) Growth evaluation of CHO cells and hybridoma cells 
2) Determination of gas consumption <br> When the CO2 concentration is set at 5%, the measurement of CO2 consumption shows that at the same time, other similar products consume more CO2 gas than the New Brunswick S41i CO2 constant temperature shaker. (As shown in Figure 4) 
Discussion and conclusion
During the development of the New Brunswick S41i CO2 constant temperature shaker, we have considered the increasing demand for environmentally friendly products and the pursuit of better performance. This study validated the performance of a new CO2 incubator with a New Brunswick shaker inside by culturing two cell lines that are common in research and production. The experiment did not optimize the culture process and media.
The experimental results show that the New Brunswick S41i CO2 constant temperature shaker can reduce environmental impact to a minimum and effectively culture mammalian cells. The New Brunswick S41i CO2 Shaker has four eccentric drives that provide a precise and stable environment for the growth of suspended cells.
Eco-friendly, low-consumption New Brunswick S41i CO2 constant temperature shaker for culture of hybridoma cells and Chinese hamster ovary (CHO) cells
Figure 1: Average viable cell density and viable cell rate of cultured CHO cells in a New Brunswick S41i CO2 shaker
The maximum viable cell density of CHO live cells reached 4.72×10 6 cells/mL on the seventh day. The cell rate was maintained at 98% for the first five days and then slowly decreased (as shown in Figure 1).
Figure 2: Comparison of average viable cell density of hybridoma cells in the New Brunswick S41i CO2 constant temperature shaker, homogeneous product 1 and similar products 2
Compared with CHO cells, hybridoma cells were passaged on the second and fourth day of exponential growth phase, so that the cultured cells maintained a 95% survival rate in the first six days. The maximum viable cell density reached 1.81×106 cells/mL on the sixth day of culture (as shown in Figures 2 and 3).
Figure 3: Comparison of average viable cell rates of hybridoma cells in the New Brunswick S41i CO2 constant temperature shaker, homogeneous product 1 and similar products 2
Figure 4: Average CO2 gas consumption per shaker in 24 hours (standard liter)