What you need to know about large scale cultivation (Issue 5)-Q&A on Practical Operation of BioFactory
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What you need to know about large scale cultivation (Issue 5)-Q&A on Practical Operation of BioFactory

In the previous four issues, we have acquired an in-depth comprehension of the application, operation procedures, related accessories, and serial use of the BioFactoriesTM. But how do we choose the right BioFactoryTM? What are the differences between the old and new version BioFactoryTM? What are the advantages of different choices? This issue provides the answers to these queries. 

Q: BioFactoryTM with how many layers should I choose before innoculation and seeding?

A : Based on the number of cells needed for a single batch, you can roughly calculate the cultivation area required and further determine the proper layers of BioFactory.

Step 1: Calculate the maximum number of cells that each layer of BioFactoryTM can harvest

(Maximum number of cells per square centimeter) x (632 square centimeters) = Maximum number of cells harvested from each layer of BioFactoryTM

E.g.: 10^4 x 632 square centimeters = 6.3 x 10^6

Step 2: Calculate the number of layers of BioFactoryTM required for each batch operation

(Number of cells required for each batch operation) / (Maximum number of cells harvested from each layer of BioFactoryTM ) = Number of layers of BioFactoryTM required

E.g.: (6 x 10^8) / (6.3 x 10^6) = 9.5 layers, so you can choose a 10-layer BioFactoryTM.

Q: I want a 15 or 25-layer BioFactoryTM, but I can't find the corresponding product. What should I do?


A: NEST currently offers a range of BioFactoryTM options, including single-layer, 2-layer, 5-layer, 10-layer, and 40-layer BioFactoryTM. However, BioFactoryTM with specific layer numbers can also be customized, due to NEST's continuous commitment to customized and personalized services. These customized product can be delivered in approximately one week, meeting various needs of customers.

Q: What are the respective advantages of BioFactoriesTM with 1 wide + 1 narrow mouth, 2 wide mouths, and 2 narrow mouths? And what are the differences?


A: The wide mouth facilitates the direct pouring of medium, while the narrow mouth design is intended for tube installment for aseptic operation subsequently. Please choose the proper BioFactoryTM according to your actual needs.

Q: I used another company's product before and found the surface not very flat and smooth. Does your product have this problem?

A : No matter before and after product upgrades, NEST BioFactories TM are well validated by the welding tests and mid-layer testing of the eighth layer flatness. The test results are as follows:

(Test points)

Before the upgrade, the maximum flatness was 1.3900, and after the upgrade, the maximum was 1.1894. The flatness is relatively stable, which meets the technical requirements.

Q: What cells can be cultured in BioFactoryTM?

A: NEST BioFactoryTM is intended for industrial-scale production, such as vaccines, monoclonal antibodies, or biopharmaceuticals. It is suitable for adherent cell culture. The whole process is linear amplification and the kinetic growth conditions of cells including VERO, MRC-5, 2BS, 293T, L-929, etc. won’t be altered.

Q: I used manual operation for small-scale testing, and now I am about to conduct pilot-scale amplification. Is there any way to improve efficiency?

A: To facilitate large-scale cell culture, multiple BioFactoriesTM can be connected in series through the use of sepcialized accessories that connect their tubes. As production processes vary, different connection methods and biological process accessories may be used for large-scale cell culture in BioFactoryTM. NEST provides tailored customization of tubes to meet the unique requirements of customers across different industries.

Q: How can we observe the status of cells when cultured in BioFactoryTM? And how to operate it?

A: In general, inverted microscopes are used to monitor the survival rate and growth status of cells in the BioFactoryTM of 1, 2, 4, and 10 layers. Since the culture environment between layers or within the same layer is basically the same,the overall status of cells can be learned through the observation of edge cells in the layers at the bottom or in one specific layer. If the inverted microscope is too small for a 10-layer or 40-layer BioFactoryTM, you can seed the cultured cells to another 1-layer BioFactoryTM for comparison during the cultivation and estimate the growth status of the 10-layer and 40-layer BioFactoryTM based on the growth status of the 1-layer BioFactory.

Q: How is the cell culture performance of the BioFactoryTM?

A: We made a cell suspension of VERO cells in logarithmic phase and seeded it into a 2-layer BioFactory at a concentration of 5x104/mL with a seeding volume of 400 mL.

(40x observed after 24-hour culture)

(40x observed after 48-hour culture)

(40x observed after 72-hour culture)

The findings show that the cells are robust in a normal shape and adhere evenly during growth. We compared the cell count after three days of cell culture with  2-layer chambers for cell culture of other brands, and NEST BioFactoryTM slightly surpassed them in terms of total cell count. 

Cell Culture Comparision Experiement Between 2-layer BioFactoryTM


Seeding Density(/mL)

Seeding Volume(mL)

Seeding Cell Count

Cell Count after 3 days

Expansion Fold







Brand C






Q: What types of validation documents does BioFactoryTM offer that are essential for obtaining authorization for industrial production?

A: BioFactoryTM developed by NEST has successfully passed various safety performance verification and applicability verifications, including the verification of raw material information, physical and chemical testing(such as incineration residue, metal elements, detection of eluates, etc.), biological testing (such as in vitro cytotoxicity test, skin sensitization test, skin irritation test, intradermal reaction test, acute systemic toxicity test, hemolysis test, etc.), eluate testing, cell growth testing (such as cell growth uniformity test, comparison of different brands of cell culture, etc.), sterile protection, and process information. All these verifications confirm the safety and effectiveness of the BioFactoryTM.

For any other questions about using the BioFactory, please feel free to contact us by info@nest-wuxi.com.