[Introduction]

Background:

Research on human pluripotent stem cells (hPSCs) has made significant strides in regenerative medicine, disease modeling, drug screening, and stem cell-based therapies.

Limitations:

 In relevant clinical applications, the demand for a large quantity of hPSCs hinders stable large-scale production, constrained by the limitations of traditional 2D cultivation methods. These methods fail to provide sufficient surface area for hPSC expansion, resulting in higher consumable and time costs.

Breakthrough:

hPSCs can be cultivated in a suspended state as aggregates, and 3D suspension culture supports higher-density hPSC growth, significantly reducing the amount of culture medium used. This approach is applicable to various applications for scaling up hPSC cultures.

Control over aggregates is crucial, particularly in the early stages of aggregate formation. Therefore, the generation of size-controllable aggregates that produce a uniformly mixed effect is essential for the stable growth of cells.

hPSCs can form uniform clusters and grow steadily in Cytoniche's 3D FloTrix® miniSPIN bioreactor, with cells proliferating over threefold in just four days. The pluripotency-related indicators SSEA4 and OCT4 positivity rates can be maintained at 90% or above, making it an ideal hPSC suspension culture system.

[hPSC Cultivation Case]

1.Cultivation Method

①Preparation of 125 mL Bottles:

Clean 125 mL glass bottles, adjust the paddle position near the bottom to facilitate aggregate formation. After installation, autoclave and dry for later use (not required for disposable bottles).

②Cell Resuscitation:

Resuscitate hPSCs in mTeSR medium containing a ROCK inhibitor (10 μM Y27632) at room temperature. Inoculate at a density of 5×10⁵/mL in 125 mL bottles, place on the 3D FloTrix® miniSPIN reactor, and cultivate overnight at 70 rpm in the dark with a culture volume of 30-65 mL.

③Medium Change:

Perform partial medium changes daily for hPSC suspension culture. Let the bottle stand on the clean bench for 5 minutes, aspirate half of the culture medium from the upper part into a 50 mL centrifuge tube, and gently tap the bottle to prevent aggregate fusion.

Centrifuge the tube with the medium, discard the supernatant, add an equal volume of mTeSR medium without the ROCK inhibitor, and transfer the medium back to the bottle. Place the bottle on the reactor for continued cultivation. Ensure that aggregate fusion is avoided during the process, lasting no more than 15 minutes.

④Cell Passaging:

After 4-5 days of cultivation, passage the cells when the aggregate size is in the range of 250-350 μm. Use Accutase digestion solution for 15 minutes, perform single-cell passaging at a density of 5×105/mL, and stir at 70 rpm.

2.Cultivation Results

On the 3D FloTrix® miniSPIN reactor, cells form uniform clusters in a rounded shape.

With increased cultivation time, aggregate size increases, reaching 222±37 μm on day 4, with over threefold cell proliferation and a cell viability of over 90%.

Simultaneously, the dual-positive ratio of hPSC pluripotency markers SSEA4 and OCT4 is maintained at over 95%, indicating well-preserved differentiation functionality during the cultivation process.

[Conclusion]

 The use of the 3D FloTrix® miniSPIN reactor allows for effective suspension cultivation of hPSCs, applicable to the expansion, library establishment, and differentiation of hPSCs. With the multiple control functions of the reactor, it can be employed for exploring conditions during hPSC cell differentiation. The 125 mL culture bottles equipped with the reactor can be repeatedly used after high-pressure sterilization, saving consumable equipment costs, making it an ideal hPSC suspension culture system.

[Related Products]

3D FloTrix® miniSPIN Bioreactor

▶ Compact design, saving space, suitable for placement in different-sized incubators.

▶ Separate main unit and controller, enabling real-time control of the main unit speed without frequently opening the incubator door.

▶ S1 model with a compact external structure; M2 model with diverse control modes to meet different customer needs.

▶ Culture bottles available in 125 mL/250 mL/500 mL, with various options, suitable for high-pressure sterilization and reusable.

▶ Main unit designed with ultra-low-speed stirring, speed range from 0 to 120 rpm, with a precision of 1 rpm.

▶ Smooth operation at low speed, effectively increasing cell growth rate while avoiding shear forces that may damage cells during stirring.

▶ Controller with a 4.3-inch touch screen, user-friendly interface, simple operation.

▶ Capable of constant speed/variable speed dual-mode control, equipped with programming functionality to meet various experimental needs.

【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.