What does Adherent Cell Culture Mean? The adherent cell culture is a form of cell culture in which cells grow in a flask until they cover the entire surface of the flask or until the medium becomes depleted of nutrients. The cells then need to be passaged or subculture to prevent the culture from dying. To do this, a protease is used to release cells from the flask. However, proteases can be damaging to some cell lines because they break the membrane markers.
Cell Culture Techniques?
Cell culture technology is widely used in biotechnology, with gene and vaccine manufacturers leveraging it for manufacturing advanced therapies. It has the potential to break down barriers and industrialize therapies. Moreover, cell culture media is highly versatile, allowing biotech companies to develop novel therapies and vaccines without using animal models.
Types of Cell Culture
`Cell subculture can be divided into two categories, adherent and suspension cultures. Anchorage-dependent cell lines are most common in vertebrates, while suspension-dependent cell lines are popular among insect cells. Most insect cell lines are adaptable to both suspension and adherent cultures.
There are several factors that drive cell adhesion. Understanding the attachment process is a key part of developing new manufacturing formats and therapeutic approaches. The academic sector is working to understand these processes. The key drivers of cell attachment include the transglutaminase integrin, fibronectin, and temperature-sensitive chemistry.
How to Promote Growth of adhered cell culture?
BioNOC(tm) II adhered cell culture media are carriers designed to promote cell growth. They are ideal for most types of anchorage-dependent cell culture, and are available in serum-free or serum-containing media. These nonwoven fabric strips are manufactured in strict cGMP guidelines.
Before starting the culture, ensure that the cells have been adequately neutralized. This can be done by adding a soybean trypsin inhibitor at 1 mg/mL to the cell culture medium. This step is especially important when using a serum-free medium. Finally, make sure that the culture medium is neutralized by adding 5% CO2 to 95% air filtered through a 0.2 mm filter.
TC-treated polystyrene plates
A major advantage of TC-treated polystyrena (TC-TP) plates for adherent cell culture is their perfect surface that enables the most adherent cells to grow in a healthful environment. These plates have a proprietary vacuum plasma treatment that guarantees uniform surface behavior. In contrast, the surface of an untreated TCP is not suitable for cell attachment. Moreover, the TC-TPs offer an enhanced surface chemistry, which increases cell attachment.
The TC-treated PT plates are transparent polystyrene microplates. The surface of these microplates is hydrophilic, and serum components are easily bound. They are particularly useful for adhesion and expansion of human pluripotent stem cells. This unique coating also facilitates the formation of spheroid cultures.
TC-treated PS plates provide a cost-effective means of growing a range of cells. Moreover, these TPPS plates are a good choice for cell culture because of their optical clarity. They are also compatible with device holders.
TC-treated polystyrena plates are also available with tissue culture treatment (TC-TP) for anchorage-dependent cell cultures. They are available in different sizes and shapes. These plates are available with chimney wells, which reduce the chances of cross-contamination.
In this study, researchers evaluated the performance of TCT plates for adherent cell culture. They found that plasma-deposited coatings were comparable to those produced by commercial collagen-coated microplates. Furthermore, a TC-treated polystyrenе plate had an angle of contact with the cells of the Vero cell line.
The results of the TCPs showed that PBMCs with higher levels of CD14 positive cells tended to adhere more than those in suspension. Further, post hoc tests also revealed significant differences between adhesion and suspension-cell cultures in cell-repellent and temperature-sensitive cultures.
There are several aspects to consider when selecting a particle-based carrier for adherent cell culture. The goal is to maximize cell yield while minimizing costs without sacrificing cell quality. It is also important to consider downstream processing steps and technology limitations. These considerations can help design a process workflow that is both effective and efficient. For instance, the carrier type should be compatible with the cell line that will be cultured.
The crystallinity of particle-based carriers is critical in controlling cell-substrate interactions. High-crystalline particles have larger spreading areas and aspect ratios than low-crystalline particles, and they exhibit reduced negative surface potential.
These features result in enhanced cell infiltration.
Microcarriers are small particles that allow adherent cells to attach to them and grow. Microcarriers have the potential to provide surface matrices for large-scale cell culture. However, these carriers are not without disadvantages. For example, they may cause uneven oxygen distribution and shear stress, which can impact the cell quality. Particle-based carriers can be made of glass or fibers. Prior to choosing a microcarrier, the topography should be carefully examined.
Microcarriers with higher charge densities promote cell adhesion and are suitable for weak cell lines. However, they also pose a challenge in terms of harvesting. Cells may detach from the carrier at the end of culture. A study on L929 mouse fibroblast cells found that these cells tended to be adherent to sulfuric acid particles.
Particle-based carriers can facilitate the isolation of specific subpopulations. The ability to isolate a cell’s secretion is crucial to the quantitative analysis of the cell secretion. Furthermore, particle-based carriers are compatible with commercially available flow sorters.
Particle-based carriers are also useful for preventing cell clumping. The process of seeding onto these carriers is very similar to traditional methods. The cells are seeded on the particle and allowed to settle with a majority of the cells in the cavities of the particles. The cells adhere to the particles through integrin binding sites.
Manual selection of pigmented clusters of cells
Using a manual selection method, pigmented clusters of cells can be selected from a cultured cell suspension by manually selecting the pigmented clusters. This method eliminates centrifugation and reduces the amount of cellular stress. The process also results in a higher TEER value, and these values increase with the pigmentation level of the cells.
One of the major challenges associated with iPSC adherent culture is the time-consuming and labor-intensive nature of this procedure. To overcome this, the protocol presented here offers methods for culture iPSCs in a time-efficient manner and provides a reliable platform for cGMP manufacturing. The optimized cell culture techniques described here will make iPSC production more reproducible and increase the quality.
Another key factor to obtaining high-quality iPSCs is the optimum cell culture conditions. When creating iPSCs, it is essential to sub-culture cells according to their density. Excessive cell density may result in 3D-like aggregates and decreased viability of the cells.
Once a culture has reached the induction phase, large numbers of contaminant cells are present. This can prevent smooth dissociation, which is why it is essential to perform trituration. Using a pipette tip, carefully break up the cell layers, ensuring a single-cell suspension.
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