Cell suspension culture is the use of biological reactor core technology of production of biological products large-scale culture of animal cells, is the mainstream of the current international biological products production mode, its biggest advantage is that by process control, more precise and effective at the time of maximum output can steadily improve the quality of the products.
However, the technology has not been widely used in China, and the production of biological products still mainly adopts the method of the culture of transfer cell culture with low rate of virus production, high production cost and large labor intensity.
With the development of modern biotechnology, the use of cell suspension culture to produce biological products is an inevitable trend of the development of bio-pharmaceutical industry.
The application history and present situation of suspension culture technology in biological medicine
In 1962, Capstick and other pioneers developed suspension culture for BHK21 cell domestication and used for the production of veterinary vaccine.
In 1967, Van Wezel developed microcarriers and made large scale cell wall cells in bioreactors.
In the 1980 s, CHO cell suspension culture, the development of therapeutic antibodies production technology greatly promotes the bioreactor in the application of biological pharmaceutical industry, to the end of the 20th century has entered the upgrade scale.
After 2000, with the flow and cultivation, perfusion, personalized development of technology, such as cell culture medium, as main equipment of mass culture bioreactor size tend to be more and more simple.
Now, the world more than 10000 l and volume of the reactor is more than one hundred units, has amounted to 25000 l, largest and almost mechanical stirring reactor can be amplified, mainly to Genetech, Amgen, BoehringerIngelheim and Lonza owned company.
Five of the world's top six biotech drugs in 2007 were produced by mammalian cells.
Throughout the country, there have been more than 110 vaccine-producing enterprises for human and veterinary use, and only four have been used in the suspension culture of the reactor.
The common animal vaccine companies use the domestically developed 650 L and 1200 L reactors for the production of foot-and-mouth disease vaccine.
Key techniques in suspension culture technology
The technology of suspension culture mainly includes four aspects: the acquisition of high expression cell lines, the development of personalized media, the improvement of animal cell reactor and supporting facilities, and optimization of production process.
1.The domestication of cells and strains
To increase the rate of production of biological products and security effectiveness, reproduction of the virus in the bioreactor and the choice of cells to adapt to each other, according to different strain and its expression of mass screening for the host cell to cell mass production is of great significance.
In order to achieve domestication stability, high density cell culture is usually turned into low-density cell culture, which gradually decreases serum concentration.
In the case of cell domestication, it should be noted that the cell vitality should be more than 90%, and the cell proliferation ability and the expression of secretion ability should be determined to avoid the change of the expression characteristics of the cells after domestication.
The proliferation ability, vitality and production capacity of the domesticated cells can meet the needs of large-scale industrial production.
Due to the nutritional needs of different specific cells, to achieve its domestication is different also, to the difficulty of the need to choose a suitable cell culture medium during domestication, some nutrition supplements targeted to meet the specific needs of cell, make some tamed cell can maintain its suspension or serum-free growth characteristics.
2.Personalization of cell culture medium
In large-scale culture, the nutrient content of cell culture is critical to the proliferation and maintenance of cells.
In suspension culture, the different specificity of cell nutrition metabolism, cell and virus culture process, shearing force and meet the required functions, still need to improve the stability of the target biological products, expression, all these need the support of personalized culture medium.
Sales of cell culture medium in our country is mostly in the 1950 s invented MEM, DMEM, 199, RPMI1640 cell culture medium and so on, these directories product is difficult to meet the technical requirements of bioreactor large-scale culture of animal cells, a direct impact on the biological pharmaceutical industry technology upgrades.
From long-term consideration, about the safety of human and animal biological products production are increasingly no animal components in serum-free medium, because of the serum free medium to eliminate the serum exogenous pollution and the cytotoxic effect, make the product easy to purification, recovery rate is high, the production of biopharmaceuticals, already has more than 50% cells in serum-free medium.
3.Research and development of cell culture technology
Suspension culture development and application of the process technology is mainly the development of production technology and process optimization, maintain the cell growth and virus propagation environment control of optimization, also vital role in the technology of suspension culture.
（1）Culture of suspension culture and microcarrier culture
The suspension culture technique is divided into the suspension cell culture and the microcarrier suspension culture of the wall cell.
The floating cells can be directly produced and proliferated in the reactor, the cell is free to grow, the cultivation environment is uniform, the sampling is simple, the cultivation operation is simple and controllable, the amplification is convenient, the pollution rate and the cost are low;
However, the suspension culture of the wall cells in the reactor requires the use of microcarriers, the poor nutrition environment of the cell and sphere contact parts, and the cultivation, sampling observation and amplification process are complex and costly.
Although relatively in suspension culture, microcarrier culture complex, but compared to turn flask culture still has great advantages, can increase the production scale, product quality and labor efficiency, and therefore is a major means of adherent cell suspension culture.
Common microcarriers have solid Cytodex microcarriers, flaky DISK microcarriers and porous Cytopore microcarriers.
When using DISK or Cytopore cells attached to the carrier internal growth, on the surface of cells is less, hard to use carrier cell digestion method and cell separation, and the reactor amplification process is difficult, is not fit for the release of the virus.
And more adapt to pour Cytodex tank reactor amplification process, the current coverage of the adherent cell production technology is the best form of mechanical stirring reactor entity microcarrier suspension culture system, which can realize the most effective optimization process and the optimum process design, the optimum design of continuous perfusion culture embodied in high density.
（2）Suspension culture
The selection of cell and virus, the product stability, the selection of cell culture, the size of cell culture, etc.
It is divided into batch culture, flow and infusion culture.
The batch culture can directly reflect the growth and metabolic changes of the cells in bioreactors, and the operation is simple, but the early metabolic waste products are more and inhibit the growth of cells, and the cell growth density is not high.
The flow plus culture is simple, high yield, easy to enlarge and widely used, but it needs to be designed with flow and medium.
Perfusion culture cultivation of small volume, recovery volume is big, the product of the tank retention time is short, can in time to the low temperature preservation, to keep the activity of the product, but the operation is multifarious, cell culture medium low use efficiency, such as the rotating filter easy to block.
Different viruses adopt different ways of culture, such as the production of biological products that are secreted and the products are less active, suitable for the use of perfusion culture, and the culture of perfusion is more suitable for microcarrier culture.
The same biological products because of its different nutritional environment, its production technology will change too, such as BHK21 cells produce foot-and-mouth disease virus, the batch culture in low serum culture, take poison before the need for replacement of serum free maintain fluid;
In the absence of serum culture, it may be better to cooperate with the flow.
Application of cell suspension culture in livestock and poultry vaccines
Vaccine immunization is one of the important measures to control animal infectious diseases.
How to improve the quality of vaccine has become the focus of animal vaccine manufacturers.
Industrial cell culture production vaccine is an effective means to improve the quality of vaccine and reduce cost.
（1）Swine virus vaccine
Rabies virus in 2 L of bioreactor breeding, the maximum titer of virus can reach 1.38 x 108pfu/mL, and the production of vaccine quality meets WHO requirements.
The cell culture of non-serum microcarrier is beneficial to the reproduction and production of enteritis virus.
There are also reports on the mass production of pig pseudo rabies, pig blue ear disease, pig disease, pig disease, pig disease and other diseases.
（2）Avian influenza vaccine
Poultry vaccines are produced mainly by SPF, but the vaccine requires a large amount of SPF chicken, which is long and prone to contamination and can only be injected with one virus strain at a time.