Focusing on innovative 3D cell technology products and services
Optimal Conditions for Microcarrier Cell Culture Technology | Introduction to the Seeding Process

Optimal Conditions for Microcarrier Cell Culture Technology | Introduction to the Seeding Process

  • Categories:Company News
  • Author:CytoNiche
  • Origin:CytoNiche
  • Time of issue:2023-11-30
  • Views:670

(Summary description)Not only featuring optimal process references but also offering a chance to apply for a free trial of our core products.

Optimal Conditions for Microcarrier Cell Culture Technology | Introduction to the Seeding Process

(Summary description)Not only featuring optimal process references but also offering a chance to apply for a free trial of our core products.

  • Categories:Company News
  • Author:CytoNiche
  • Origin:CytoNiche
  • Time of issue:2023-11-30
  • Views:670


Microcarriers are microbead materials with a diameter of 50 to 300 μm, slightly denser than water, providing a surface for the attachment and growth of adherent cells. Compared to traditional 2D matrix materials, they have a higher specific surface area, enabling the cultivation of a greater number of cells in equivalent culture vessels. Microcarriers can be utilized in conjunction with bioreactors for large-scale suspension culture, effectively saving labor, resources, and space.

The microcarrier cell culture technology process includes three main stages: cell seeding, cell proliferation, and cell harvesting. Among them, cell seeding is the process of introducing seed cells into a culture vessel containing microcarriers, allowing cells to attach to the surface of the microcarriers. Choosing appropriate seeding conditions enables efficient and uniform cell attachment to microcarriers (Figure 1), thereby fully utilizing the microcarrier's specific surface area—an essential prerequisite for ensuring subsequent large-scale cell expansion.


Figure 1: Cells attached to the surface of 3D microcarriers (Image source: Cytoniche, all rights reserved)

Below, we will briefly explore how different seeding process parameters in microcarrier cell culture technology impact cell seeding effectiveness, providing guidance for selecting suitable seeding conditions.


[PART 1: Cellular State]

In general, cells used for seeding microcarriers should ideally be in the exponential growth phase rather than the plateau phase. According to literature reports, seeding cells in the exponential growth phase, as opposed to the confluent phase, may increase cell yield in microcarrier culture by 2-3 times [1].

To ensure uniform attachment, single-cell suspension should be used for seeding microcarriers instead of cell aggregates. In cases where adherent cells are cultivated in 2D wall-adherent conditions, it is crucial to ensure that cells adhering to the wall are adequately digested into rounded single cells using proteolytic enzymes (e.g., trypsin), rather than merely detaching cells from the culture surface. Otherwise, cells that detach without sufficient digestion may retain cell-cell adhesion, spontaneously forming clusters, leading to uneven attachment to microcarriers.

It is worth noting that prolonged enzymatic digestion may damage cell viability or induce apoptosis in certain cell types (such as some types of stem cells). Therefore, appropriate enzyme selection and digestion duration based on different cell characteristics are crucial to preserving cell activity [2].


[PART 2: Microcarrier Concentration | Cell Density | Cell-to-Microcarrier Ratio]

The maximum yield of cells cultivated on microcarriers is directly related to the total growth area of cells, i.e., the total surface area of microcarriers, and is also constrained by the amount of culture medium used. Therefore, higher microcarrier concentration and cell density do not necessarily yield better results. Instead, the goal is to maximize the use of nutrient-rich culture media while maintaining stable concentrations or densities of environmental parameters such as dissolved oxygen and pH.

Most manufacturers recommend microcarrier concentrations of 1 to 6 grams per liter of culture medium. In addition to being influenced by culture medium usage and microcarrier concentration, seeding cell density is also affected by the cell's own attachment rate and growth characteristics, with general recommendations ranging from 104 to 106 cells per milliliter.

In the early stages of seeding, the ratio of cells to microcarriers directly affects cell attachment efficiency and uniformity.

Too low a cell-to-microcarrier ratio may result in some microcarriers being unattached, reducing the surface utilization of microcarriers and subsequent cell proliferation. Theoretically, with a cell-to-microcarrier ratio as low as 1:1, an estimated 36.5% of microcarriers may remain unattached; raising the ratio to 4:1 can reduce this percentage to 1.8% [3].

Since microcarrier surface area has its limits, the cell-to-microcarrier ratio does not need to be excessively high to avoid wasting cells. Different cell types or the same cell type on different microcarrier surfaces may have different optimal seeding ratios [2]. For example, VERO cells seeded on solid spherical microcarriers exhibit optimal cell-to-microcarrier ratios of no less than 8:1, while porous microcarriers might require ratios ranging from 30:1 to 60:1 [4] [5].

Currently, most microcarrier manufacturers provide information on the number of particles per unit mass of microcarrier products, allowing users to calculate appropriate seeding densities based on microcarrier usage.


[PART 3: Stirring Mode and Stirring Speed]

Propeller-type stirred bioreactors are the most commonly used platforms in conjunction with microcarriers for cell culture. During the seeding stage, the culture system should be stirred appropriately to ensure full contact between cells and microcarriers, aiding in efficient and uniform cell attachment to microcarriers. Stirring modes during seeding can be categorized into continuous stirring and intermittent stirring.

Intermittent stirring involves periodically activating the propeller for short durations, while keeping the propeller inactive for the rest of the time, cycling every few hours to a day, and then switching to continuous stirring mode after seeding is complete. Intermittent stirring allows cells ample time to attach to microcarriers in a static state, making it suitable for situations where cell attachment rates are slow or seeding densities are low, and has been widely used in the cultivation of mesenchymal stem cells (MSCs), pluripotent stem cells (PSCs), and embryonic stem cells (ESCs) [2].

Continuous stirring mode is more suitable for situations where cell attachment rates are faster. A study on VERO cells adhering to two different microcarrier surfaces found that, for the positively charged solid spherical microcarrier with a faster cell attachment rate, continuous stirring mode resulted in better cell attachment uniformity than intermittent stirring mode. Conversely, for another microcarrier based on ECM material with a slower cell attachment rate, the opposite conclusion was reached [5].

It can be observed that the suitable stirring mode during seeding is influenced by factors such as cell attachment rate, seeding density, and cell-microcarrier ratio. Cell attachment rate depends on principles of attachment (e.g., charge adsorption or cell-ECM interaction) and microcarrier characteristics (e.g., solid or porous). Therefore, the choice of stirring mode should be a comprehensive consideration of cell and microcarrier characteristics.

In addition to stirring mode, stirring speed is also a crucial factor affecting cell seeding effectiveness.

◆ Too low stirring speed prevents thorough mixing of microcarriers with cells, leading to spontaneous settling and clustering of cells, causing uneven attachment [6].

◆ High stirring speed increases the shear force applied to cells, potentially causing cell damage or other stress-induced changes in cellular properties, affecting cell quality [7]. Simultaneously, cells are more prone to detach from microcarrier surfaces under high shear forces [6].

 Thus, it is essential to use a gentle stirring speed that allows microcarriers to be fully and dynamically suspended while aiding in seeding.



 The effectiveness of cell seeding in microcarrier cell culture technology is influenced by various process parameters such as cellular state, microcarrier concentration, cell density, cell-to-microcarrier ratio, stirring mode, and stirring speed.

Technical personnel should have a comprehensive understanding of the basic principles of cell attachment to microcarriers, considering the characteristics of both cells and microcarriers, to select suitable seeding process conditions and ensure optimal cell culture results.


[Related Products]

 3D TableTrix® Microcarriers

More Biomimetic: Composed of tens of thousands of elastic three-dimensional porous microcarriers, with a porosity >90%, controllable particle sizes ranging from 50-500μm, and uniformity ≤100μm, forming a truly biomimetic 3D cell culture.

Full Qualifications:Holds 2 China CDE (Center for Drug Evaluation) qualifications for pharmaceutical excipients, filing numbers: F20200000496, F20210000003.

Holds 3 U.S. FDA (Food and Drug Administration) DMF (Drug Master File) Type II & IV, MF Type II qualifications, filing numbers: 037798 & 035481, 29751.

Easy Harvest: Specific degradation technology fractures microcarriers, resulting in a more efficient and gentle harvest compared to traditional methods.

Safer: Supported by authoritative institution reports on residual detection after fracture, cytotoxicity, thermal reactions, genetic toxicity, in vivo immunotoxicology, as well as safety assessment reports on hemolysis, subcutaneous injection site irritation, systemic hypersensitivity, and intraperitoneal injection drug toxicity.

Easy to Scale Up: Through 3D cultivation methods, combined with CytoNiche's 3D Cell Smart Manufacturing Platform's full product line, achieving fully automated closed large-scale cell cultivation and realizing harvests on the scale of billions.

Customized Microcarriers: Tailored to meet various cell culture needs.




Scan the QR code or call 400-012-6688 to apply for a free trial.




[1]Clark J., Hirtenstein M., Gebb C. Critical parameters in the microcarrier culture of animal cells. Dev. Biol. Stand. 46, 117-124 (1980)

[2]Derakhti S., Safiabadi-Tali S. H., Amoabediny G., Sheikhpour M. Attachment and detachment strategies in microcarrier-based cell culture technology: A comprehensive review. Mater. Sci. Eng. C 103, 109782 (2019)

[3]Panchalingam K. M., Jung S., Rosenberg L., Behie L. A. Bioprocessing strategies for the large-scale production of human mesenchymal stem cells: a review. Stem Cell Res. Ther.6, 225 (2015)

[4]Mendonca R. Z., Prado J. C. M., Pereira C. A. Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures. BioProcess Eng. 20, 565-571 (1999)

[5]Ng Y-C., Berry J. M., Butler M. Optimization of physical parameters for cell attachment and growth on microporous microcarriers. Biotechnol. Bioeng.50, 627-635 (1996)

[6]Heathman T. R. J., Nienow A. W., Rafiq Q. A., Coopman K., Kara B., Hewitt C. J. Agitation and aeration o stirred-bioreactors for the microcarrier culture of human mesenchymal stem cells and potential implications for large-scale bioprocess development. Biochem. Eng. J. 136, 9-17 (2018)

[7]Martin C., Olmos E., Collignon M-L., De Isla N., Blanchard F., Chevalot I., Marc A., Guedon E. Revisiting MSC expansion from critical quality attributes to critical culture process parameters. Process Biochem. 59, 231-243 (2017)


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.


Scan the QR code to read on your phone

Latest News

Copyright: Beijing CytoNiche Biotechnology Co., Ltd.
Copyright: Beijing CytoNiche Biotechnology Co., Ltd.