Microcarrier cell culture technology and application principle

Microcarrier cell culture technology is a common cell culture technique in cell culture. Regarding microcarrier cell culture technology, taking animal cells as an example, the details are as follows:

First, the application of microcarrier culture technology

Microcarrier culture technology was used in large-scale culture of animal cells in 1967. After more than 30 years of development, the technology has gradually become more sophisticated and mature, and is widely used in the production of vaccines, genetic engineering products.

Microcarrier culture is currently recognized as the most promising large-scale culture technique of animal cells, which has the advantages of both suspension culture and adherent culture, and is easy to enlarge. At present, microcarrier culture is widely used to culture various types of cells to produce vaccines and protein products such as 293 cells, myoblasts, Vero cells, and CHO cells.

There are two types of reactors that are used more: Braun's BioStatB reactor, a double-blade bubble-free aeration mixing system; NBS's CelliGen, CelliGen PlusTM and Bioflo 3000 reactors, using the Cell-lift double screen mixing system. Both systems enable efficient separation of cultured cells and harvested products.

Second, microcarrier introduction

Microcarriers refer to microbeads having a diameter of 60-250 μm which can be applied to the growth of adherent cells. It is generally composed of natural glucan or various synthetic polymers. Since the first microcarriers developed by Van Wezel with DEAE-Sephadex A 50, more than a dozen types of microcarriers have been sold on the international market, including liquid microcarriers, macroporous gelatin microcarriers, and polystyrene. Microcarriers, PHEMA microcarriers, chitin microcarriers, polyurethane foam microcarriers, alginate gel microcarriers, magnetic microcarriers, and the like. There are three commonly used commercial microcarriers: Cytodex 1, 2, 3, Cytopore and Cytoline.

• Size of the microcarrier: Increasing the surface area per unit volume (S/F) is very beneficial for cell growth. The microcarrier diameter is made as small as possible, preferably between 100 and 200 μm.

• The density of the microcarriers: generally 1.03-1.05g/cm2, the density can be gradually increased with the attachment and growth of the cells.

• Surface charge of microcarriers: According to research, the basic factor controlling cell attachment is charge density rather than charge properties. If the charge density is too low, the cell attachment is insufficient, but the charge density is too large, which may have a "toxic" effect.

Third, the principle and operation of microcarrier culture

1. Principle:

The principle is that the particle-microcarriers which are harmless to the cells are added to the culture solution of the culture vessel, and the cells are allowed to adhere to the surface of the microcarriers as a carrier, while the microcarriers are kept in a suspended state by continuous agitation.

The proliferation of anchorage-dependent cells on the surface of microcarriers undergoes three stages of adhesion, adherence, growth and expansion into a single layer. Cells can only proliferate if they are attached to the surface of a solid substrate, so the attachment of cells to the surface of the microcarriers is the key to further spreading and growth. Adhesion is mainly based on electrostatic attraction and van der Waals force. Whether the cells adhere to the surface of the microcarrier depends mainly on the probability of contact and compatibility of the cells with the microcarriers.

2. Stirring speed:

Since animal cells have no cell walls and are sensitive to shear forces, it is not possible to increase the contact probability by increasing the stirring speed. The usual mode of operation is to use a low agitation speed during the adherence period, and to stop when stirring; after a few hours, when the cells adhere to the surface of the microcarrier, the set low rotation speed is maintained and the culture phase is entered. The agitation of the microcarrier culture was very slow with a maximum speed of 75 r/min.

3. The compatibility of cells with microcarriers is related to the physical and chemical properties of the microcarrier surface. Generally, when the cells enter physiological pH, the surface is negatively charged. If the microcarriers are positively charged, the electrostatic attraction can accelerate the cell attachment speed. If the microcarriers are negatively charged, the cells are difficult to adhere to the adherend due to electrostatic repulsion, but when the culture solution is dissolved or the surface of the microcarriers adsorbs divalent cations as a medium, the negatively charged cells can also be attached.

4. The growth of cells on the surface of microcarriers has many factors affecting the growth of cells on the surface of microcarriers. There are three main aspects:

• On the cellular side, such as cell population, status, and type.

• In terms of microcarriers, such as the surface state of microcarriers, adsorbed macromolecules and ions; when the surface of the microcarrier is smooth, the cell expands rapidly, and the surface is porous and expands slowly.

• In culture environments, such as medium composition, temperature, pH, DC, and metabolic waste, the growth of cells on microcarriers is significantly affected. If the conditions are optimal, the cells grow fast; otherwise, the growth rate is slow.

5. Microcarrier culture operation points

• In the early stage of culture: ensure that the medium and microspheres are at a stable pH and temperature level, and inoculate the cells (logarithmic growth phase, not stationary phase) to a final volume of 1/3 of the culture medium to increase the contact between the cells and the microcarriers. opportunity. The concentration of different microcarriers and the density of seeded cells are different. A microcarrier content of 2-3 g/L is often used, and a higher microcarrier concentration requires a controlled environment or frequent liquid exchange.

• After the adherent stage (3-8d), slowly add the culture solution to the working volume and increase the agitation speed to ensure complete homogenization.

• Culture maintenance period: cell count (nucleus count), glucose measurement, and cell morphology microscopy. As the cells proliferate, the microspheres become heavier and heavier, and the stirring rate needs to be increased. After about 3 days, the culture solution began to be acidic. It was necessary to change the solution: stop stirring, let the beads be precipitated for 5 min, discard the appropriate volume of the culture solution, slowly add fresh culture solution (37 ° C), and start stirring again.

• Harvest the cells: first drain the culture, rinse at least once with buffer, then add the appropriate enzyme and stir quickly (75-125 r/min) for 20-30 min. The cells and their products are then dissociated.

• Magnification of microcarrier culture: It can be amplified by increasing the content of microcarriers or the culture volume. The production of vaccines and interferons using aneuploid or primary cell culture has been amplified to over 4000L.

Bioreactor system for large-scale cell culture of microcarriers

The large-scale cultivation of this technology, the efficiency of cell expansion is affected and limited by many factors, the main limiting factors include: cell sensitivity to shear, oxygen transmission and passage and expansion culture. The various types of bioreactor systems developed can provide suitable external environment such as low shear force, high oxygen transmission efficiency and easy cell passage for microcarrier cell culture and amplification in response to the above-mentioned limiting factors.

The microreactor culture system bioreactor that has been used more often can be computer controlled, culture stirring speed and suspension uniformity, temperature change, pH stability and dissolved oxygen supply (O ₂ , N ₂ , CO ₂ , air purification) Parameters such as gas proportional adjustment, tank pressure, culture volume and aeration are all automatically controlled by the computer. Therefore, the application of bioreactor system for large-scale amplification of microcarrier cells has obvious advantages. At present, several bioreactor systems suitable for large-scale cell culture of microcarriers have been developed abroad, such as agitated bioreactor system, rotary type. Bioreactor systems and perfusion bioreactor systems, etc.

1. Stirring bioreactor system

The agitated bioreactor system has a long history of research on the large-scale amplification of microcarrier cells, but the cell culture system is prone to excessive shear force, which limits its application range. Despite this, due to the simplicity, practicality and low price of the system, there are still many reports on the successful large-scale cell expansion of the system. For example, Werner A (2000) successfully conducted large-scale expansion of hepatocytes in this system.

2. Perfusion bioreactor system

Perfusion culture is one of the current research hotspots. It is characterized by the continuous addition of fresh medium and the continuous withdrawal of the spent medium containing cellular metabolic waste, allowing the cells to proliferate in a relatively stable growth environment, saving time and effort and reducing the chance of cell contamination. And can increase the cell density by more than 10 times.

3. Rotating bioreactor

In recent years, Rotating Bioreactor Systems (RCCS) have become a more common cell culture system for large-scale cell expansion using microcarrier technology. The system is based on a bioreactor designed by NASA to simulate spatial microgravity effects. RCCS can be used for both large-scale cell culture of microcarriers and a three-dimensional complex formed by cells and scaffolds. To date, nearly one hundred tissue cells have been successfully expanded in large scale within the system.

Five, microcarrier culture advantages

• The surface area/volume (S/V) is large, so the cell yield per unit volume of culture medium is high;

• Combine suspension culture with adherent culture, which combines the advantages of both;

• The growth of cells on the surface of the beads can be observed with a simple microscope;

• Simplifies the detection and control of various environmental factors of cell growth, and has good reproducibility;

• High media utilization;

• Easy to zoom in;

• The cell harvesting process is not complicated;

• Low labor intensity;

• The system footprint and space are small.