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In response to industry challenges, Cytoniche has developed the corresponding 3D FloTrix® large-scale cell cultivation process — the 3D TableTrix® G Series three-dimensional porous degradable microcarriers. Combined with a bioreactor, it achieves suspended cultivation of adherent cells. Subsequently, through incremental amplification, large-scale, high-quality cell amplification can be realized.

This article introduces the G Series microcarriers for the large-scale cultivation and virus production of 293T cells, providing a detailed explanation of the application effects of the G Series microcarriers through a complete process and real data.

[293T Cell Cultivation Case]

Cell: 293T

Microcarrier: G02 (pre-sterilized) 2g/L

Culture Medium: DMEM+10% FBS

Medium Change: DMEM+5% FBS

Amplification Ratio: 1:6

Cell Seeding: 1×10⁸ cells seeded in a 10-layer factory, cultured for 72h, and then used for reactor seeding.

First-level Cell Seeding:

Pre-pump microcarriers and culture medium into the reactor for aseptic verification. Inoculate at a density of 4.0×10⁵ cells/mL with a carrier density of 2g/L, adjust to a constant speed of 45rpm after 8-16h.

Daily sampling and cell counting, and glucose content detection. When the glucose concentration is below 1.5g/L, replace 80% of the medium with medium change. Cell passaging is performed when the cell density reaches above 3.0×10⁶ cells/mL.

Settle the microcarriers, wash with PBS twice, digest with cell digestion enzyme for about 10-15min, and cells detach from the microcarriers. Terminate digestion using complete culture medium.

Second-level Cell Seeding:

Prepare microcarriers (target volume × 2g/L) in advance in the reactor by soaking them in the medium for aseptic verification.

Pump the liberated cells and microcarriers from the previous level into the prepared stainless steel reactor. Adjust to a constant speed of 45rpm after 8-16h.

Daily sampling and cell counting, and glucose content detection. When the glucose concentration is below 1g/L, replace 80% of the medium with medium change. Choose an appropriate cell density for plasmid transfection and virus packaging.

Cell Amplification Cultivation Results:

293T cells can be incrementally amplified, and cell proliferation is stable, reaching a maximum of 6.58×10⁶/mL (Figure 1), with a recovery rate of 96%.

Figure 1: Large-scale expansion cell proliferation curve of 293T cells

Cell Transfection and Virus Packaging Test:

Using 3D TableTrix® microcarriers to cultivate 293T cells, transfecting lentiviral plasmids. After 72h, the virus titer is tested and compared with the 2D production process. The results show that there is no significant difference in the titer of the original lentivirus packaged with 3D microcarriers compared to the 2D process (Figure 2). The traditional 2D process parameters can be easily transferred to the 3D microcarrier cultivation process, achieving large-scale virus harvest.

Figure 2: Transfection efficiency and virus titer of 2D and 3D cultivation show no difference, and the process is transferable

Figure 3: Transfection and packaging of lentivirus in 293T cells

Figure 4: Residual host DNA (HCD) and protein (HCP) in 3D harvested virus are significantly lower than in 2D

Table 1: High batch yield, effectively saving labor and space

Process Application Validation Results:

Based on the 3D TableTrix® G Series microcarriers, the large-scale cultivation process of 293T cells achieves cell proliferation of 10 times in 5 days under conditions of microcarrier density of 2g/L and cell seeding density of 300,000 cells/mL. Cell recovery rates are all above 90% through tank digestion. By cultivating 293T cells through this process, the yield of lentivirus original liquid is higher than that of 2D-cultured cells.

This process of microcarrier usage is simple, and the microcarriers can be degraded to harvest cells, meeting the production needs of gene therapy drugs.

[Conclusion]

Compared to existing cultivation methods, the 3D TableTrix® Microcarrier G Series exhibits many unique advantages in the cultivation of 293T cells:

▶ Large Surface Area: Large-scale amplification of cells can be achieved with relatively low-density G Series microcarriers.

▶ Mild Degradation: Achieves non-destructive cell harvesting, simple process, and easy amplification.

▶ 3D Biomimetic Elastic Porous Structure: Cells adhere more strongly, reducing shear forces from stirred reactors, effectively protecting host cells and reducing host cell residues.

Using 3D TableTrix® G Series microcarriers, combined with the 3D FloTrix® automated bioreactor, the desired cell quantity can be harvested in a short time, significantly reducing costs such as labor, time, and instrument consumables. This provides a better choice for gene therapy drug research and production enterprises, bringing a new driving force!

[3D TableTrix® G Series Microcarriers]

Cytoniche's core product — 3D TableTrix® Microcarriers, the related technology is derived from the scientific and technological achievements transformation of Tsinghua University, serving as a microenvironment for the growth of stem cells (Microniche), achieving high-quality in vitro expansion of stem cells.

 • Has obtained inspection reports from the China Drug Administration (CDA) and other authoritative institutions,

Report Numbers: SH202311161; Inspection<2023>012302052;

• 2 Chinese CDE pharmaceutical excipient qualifications,

Record Numbers: F20200000496, F20210000003;

• 3 US FDA DMF Type II & IV, MF Type II qualifications,

Record Numbers: 037798 & 035481, 29721.

Meet different cell cultivation needs through customized microcarriers.

Cytoniche's Full Range of 3D Microcarriers

【CytoNiche】

CytoNiche was established in 2018, led by Professor Yanan Du's research team from the School of Medicine at Tsinghua University, with Tsinghua University as a shareholder. The core technology originated from the transformation of scientific achievements at Tsinghua University and was recognized as a leading technology in "Science and Technology Innovation in China" by the China Association for Science and Technology. As a national-level high-tech enterprise, a national-level specialized and new technology "Little Giant" enterprise, a potential unicorn enterprise, it has also received key research and development special support from the Ministry of Science and Technology.
As an expert in high-quality three-dimensional cell manufacturing, CytoNiche provides a one-stop customized solution for cell scale-up based on 3D microcarriers. The company has built an original 3D cell smart manufacturing platform, achieving large-scale, automated, intelligent, and closed-cell drug and derivative production preparation. This helps global customers establish the most advanced cell drug production lines. After pioneering the production process pipeline for "billion-level" stem cells, the company is accelerating towards "hundred billion-level," dedicating efforts to empower the cell and gene therapy industry with 3D cell scale-up smart manufacturing technology to benefit more patients.