FAQs for EmCD CHO-S 203 Medium Series

The EmCD CHO-S 203 medium series from Eminence is specifically developed for the ExpiCHO-S cell line to support high-density suspension culture and efficient transient expression. EmCD CHO-S 203 Basal Medium is chemically defined and free from animal-derived components, allowing for rapid production of milligram to gram quantities of antibodies or recombinant proteins with stable quality and manageable safe. Currently, the 203 Platform independently developed by Eminence has been widely utilized in preclinical drug research and therapeutic recombinant protein production by biopharmaceutical companies, for which there is an increasing number of inquiries in this regard. Eminence has prepared a series of FAQs specifically for the Eminence 203 platform for everyone.

I. FAQs (Resuscitation, Passage and Culture):

Q1: What is the appropriate passage density for CHO-S cells in EmCD CHO-S 203, and what should be done if such density is exceeded?

A: The appropriate passage density for CHO-S cells is 4-6 × 10⁶/mL. If the density goes beyond this range, cells should be passaged at a density of 0.5 × 10⁶/mL for 2-3 generations until cell resuscitation.

Q2: Is it necessary to acclimate when switching from the original medium to 203 Basal Medium?  

A: In general, cells can be directly switched from the original medium to EmCD CHO-S 203 Basal Medium for passage culture. If CHO-S cell status declines, it is advisable to acclimate the cells, stabilize them for three generations, and then proceed with experiments.

Q3: Is it necessary to add glutamine during resuscitation of CHO-S cells in 203 Medium? How much and how often should it be added? Is supplementation required after transfection?

A: Yes, glutamine should be added at a concentration of 4-6 mM/L. During resuscitation or passage, it is not required after transfection.

Q4: If there is a little clumping when CHO-S cells are cultured in EmCD CHO-S 203, can it be adjusted by adding an anti-clumping agent?

A: If there is a little clumping, it is not necessary to add any anti-clumping agent, and it is enough to adjust the cell status through passage. If clumping is much, two options can be considered: ① When harvesting cells for passage, wait for 5-10 seconds for cells to settle, and then harvest the upper-layer cells without clumping for passage. ② Pass cells through a sieve before counting and passage.

Q5: CHO-S cells exhibit slow growth during passage, with a prolonged doubling time, or they show wall attachment after transfection. What could be the cause?

A: The doubling time for CHO-S cells in EmCD CHO-S 203 Basal Medium is approximately 17 ± 1 h. First, confirm if glutamine (at a concentration of 6 mM) has been added. Second, ensure that shaker parameters (37℃, 8% CO₂, 100 ± 5 rpm, amplitude) are consistent. Finally, confirm the cell source and whether the original medium contains insulin or growth factors.

II. FAQs (Cell Transfection):

Q1: How many days can CHO-S cells be cultured after transfection without 203 Feed added? How many days can they be cultured with the Feed added?

A: Based on testing results the platform from Eminence, CHO-S cells can be cultured for 10 days after transfection without 203 Feed, and the viability is maintained around 70%. With the 203 Feed added, cells can generally be cultured for 10-13 days.

Q2: What are the effects of the 203 Feed, and how much difference in expression titer is there compared to that without the Feed added?

A: According to the testing results with the Eminence’s platform, addition of feeds increases the expression titer by 30% compared to that without feeds. However, there may be differences in the results among different platforms, so it is recommended to set up multiple test groups, including (1) no feed, (2) with feeds, and (3) with the platform feed, for testing.

Q3: How should the temperature and time point for cooling down after transfection be chosen?

A: Based on the testing results with the Eminence's platform, cooling to 32℃ at 24 h after transfection is conducive to maintaining cell status and increasing the expression titer. It is also recommended preparing a 32℃ shaker in advance rather than gradually cooling cells from 37℃ to 32℃ inside the incubator.

Q4: How are the sample collection time points after transfection determined?

A: The collection time points are generally determined based on individual company’s platform processes. Eminence recommends collection on days 10-12 or when the viability drops below 60%.

Q5: What is the initial volume for dilution of plasmid and PEI in the medium during transfection?

A: Both plasmid and PEI are diluted in the medium at an initial volume of 5%, so the final volume of the prepared transfection reagent should be 10% of the initial volume.

Q6: Using the 203 medium series, liposomes can express, but PEI cannot. What could be the cause?

A: The possibility of PEI transfection method, the most common method in transient transfection processes, not resulting in expression is very low. However, transfection efficiency often depends heavily on the concentration of plasmid DNA and PEI, so it is recommended to optimize their concentrations for the best transfection efficiency.

III. FAQs (Medium Parameters):

Q1: What product specifications are currently available for the 203 medium series?

A: EmCD CHO-S 203 Basal Medium is currently available in liquid with the specification of 1000 mL and dry powder with the specifications of 10, 20, and 100 L. EmACF CHO 203 Feed is currently available in liquid with the specification of 500 mL.

Q2: Are EmCD CHO-S 203 medium series chemically defined?

A: The EmCD CHO-S 203 media are chemically defined and devoid of any growth factors, hydrolysates, serum, serum substitutes, proteins, or any animal-derived components, ensuring the safety and stability of the cell culture process. EmACF CHO 203 Feed is free from animal-derived components, growth factors or L-glutamine. When used together with EmCD CHO-S 203 Basal Medium, it is particularly suitable for the transient transfection of ExpiCHO-S cells, ensuring high-density culture and maximizing expression. With this medium, milligram to gram quantities of antibodies or recombinant protein samples with stable quality and favorable safety can be rapidly produced.

Q3: What is the osmolality of the 203 Basal Medium/203 feed ?

A: The osmolality of EmCD CHO-S 203 Basal Medium is 300 mOsm/kg. The osmolality of EmACF CHO 203 Feed is 1000 mOsm/kg.

Q4: What are the glucose contents in the 203 Basal Medium/203 Feed?

A: The glucose content in EmCD CHO-S 203 Basal Medium is 6 g/L. The glucose content in EmACF CHO 203 Feed is 33 g/L.