Cell culture fixed bed and fixed bed bioreactor

Abstract

The invention relates to the technical field of cell culture, and discloses a cell culture fixed bed and a fixed bed bioreactor, the cell culture fixed bed comprises a cell growth layer and a liquid diversion layer; the cell growth layers and the liquid diversion layers are alternately overlapped to form a cell growth layer-liquid diversion layer-cell growth layer structure; the cell culture fixed bed structure is wound by a reel; the cell growth layer is used as a base material for cell attachment growth and provides an attachment surface for cell growth; the liquid diversion layer provides a liquid circulation channel for the culture medium, liquid of the culture medium forms turbulent flow when passing through the liquid diversion layer, cells are driven by the turbulent flow to be filtered by the cell growth layer, and the cells are intercepted and attached to the cell growth layer to grow. According to the invention, large-scale culture can be realized through a small space.

Description

Technical Field

[0001] The present application relates to the technical field of cell culture, and in particular to a fixed bed for cell culture and a fixed bed bioreactor. Background Art

[0002] The use of fixed-bed bioreactors is an inevitable trend for large-scale, industrial production of stable adherent cells. Currently, the technologies used for the amplification of adherent cells are mainly microcarrier and sheet carrier technologies.

[0003] Microcarrier technology is expensive and has complicated pre-treatment methods. The technology is essentially a suspension culture of microcarriers, where adherent cells are attached to the microcarriers, achieving 2D (two-dimensional plane) adherent culture of cells from a microscopic perspective.

[0004] Because the diameter of microcarriers is much larger than that of cells, they are subject to great shear force from the stirring blades in the suspension reactor, and a considerable portion of microcarriers are broken each time they are cultured. In addition, the cells are not evenly attached to the microcarriers, resulting in poor sampling uniformity. For primary cells or virus culture processes with contact inhibition, a large number or even all cells will fall off in the late stage of cell growth or after virus inoculation, which is very unfavorable for cell production and product preparation.

[0005] Sheet carriers fix the regular paper-like culture carriers in the reactor through fixed partitions. Because of the blocking of the partitions, the cells will not flow with stirring like microcarriers after attachment, which solves the shear force problem in the microcarrier culture process. Compared with microcarriers, sheet carriers are cheaper and easier to store. They have a 3D mesh structure. Cells are fixed in the mesh structure to achieve 3D culture. Cells are fixed by the fiber mesh, which overcomes the problem of cell shedding in the late stage of microcarrier culture.

[0006] Although sheet carriers have advantages over microcarriers in terms of price, preservation and cell culture, there is a problem that sheet carriers cannot solve. Because of the obstruction of the upper and lower partitions, sheet carriers cannot be used for cell sampling. This means that during the culture period, the growth status of the cells is a black box for the operator, and the cell growth can only be roughly estimated through indirect culture parameters.

[0007] Moreover, the distribution of the sheet carriers in the fixed bed is anisotropic, that is, randomly distributed. This leads to uneven liquid flow direction at the microscopic level. In addition, the use of bottom-layer ventilation will retain a large number of bubbles on the surface of the carrier and between the carriers, which is not conducive to gas-liquid exchange and cell growth of cells. Summary of the invention

[0008] In order to be more conducive to the growth of cultured cells and to realize large-scale culture in a smaller space, the present application provides a cell culture fixed bed and a fixed bed bioreactor.

[0009] On the one hand, the present application provides a cell culture fixed bed, which adopts the following technical solution:

[0010] A cell culture fixed bed comprises a cell growth layer and a liquid guide layer; the cell growth layer and the liquid guide layer are alternately stacked to form a cell growth layer-liquid guide layer-cell growth layer structure; the cell culture fixed bed structure is wound in a scroll shape; the cell growth layer serves as a substrate for cell attachment and growth, providing an attachment surface for cell growth; the liquid guide layer provides a channel for liquid circulation for a culture medium, turbulence is formed when the liquid of the culture medium passes through the liquid guide layer, cells are filtered through the cell growth layer driven by the turbulence, and the cells are retained and attached to the cell growth layer for growth.

[0011] By adopting the above technical solution, during inoculation, the culture medium flows from bottom to top through the gap between the cell growth layer and the liquid guide layer through the fixed bed, and the cells in the culture medium will be retained in the liquid guide layer full of pores during this period and grow attached; the culture medium flows along the pores between the cell growth layer and the liquid guide layer, and exchanges nutrients and gases with the cells on the cell growth layer during the flow. Due to the alternating superposition design of the cell growth layer and the liquid guide layer, and the reel winding structure, the fixed bed can provide a large amount of cell growth area within a limited volume. This helps to improve the cell culture efficiency and increase the cell yield. The turbulence formed by the liquid guide layer helps to promote the material exchange between the cells and the culture medium, providing a more optimized growth environment for the cells. At the same time, the filtering effect of the cell growth layer also helps to remove impurities and waste in the culture medium, further improving the cell growth environment. The reel winding structure makes the fixed bed more compact in volume, which is conducive to saving space. In addition, since the design of the fixed bed allows the culture medium to be recycled, the consumption cost of the culture medium can also be reduced.

[0012] Optionally, the width of the liquid guide layer is less than or equal to the width of the cell growth layer.

[0013] By adopting the above technical solution, when the liquid guide layer is less than , it provides a channel entrance for the culture medium to circulate, so that the culture medium flows through the fixed bed along the gap between the two cell growth layers, so that the cells adhere and provide nutritional conditions for the cells; and it helps to save consumables. When it is equal to , the cell growth layer is periodically distributed on the fixed bed through the regular winding method of the cell growth layer, and the linear speed of each part of the culture medium is uniform when it flows through the fixed bed carrier.

[0014] Optionally, a thickness ratio of the cell growth layer and the liquid guide layer after winding is in a range of 1:4 to 1:2.

[0015] By adopting the above technical solution, if the thickness ratio exceeds 1:4 to 1:2, the culture area will be reduced or the liquid flow in the guide layer will not be smooth.

[0016] Optionally, the thickness of the cell growth layer is less than 0.22 mm.

[0017] Optionally, the mass per unit area of ​​the cell growth layer is in the range of 0.1 oz / yd 2 ~3.0oz / yd 2 .

[0018] By adopting the above technical solution, the mass per unit area of ​​the cell growth layer is between 0.1oz / yd 2 Up to 3.0oz / yd 2 There is a reasonable range between 2 and 3, which can meet the culture requirements of different types of cells and provide good system stability and cost-effectiveness.

[0019] Optionally, the cell growth layer has an air permeability range of 50 cm 3 / s / cm 2 ~300cm 3 / s / cm 2 .

[0020] By adopting the above technical solution, the air permeability of the cell growth layer is between 50cm 3 / cm 2 Up to 300cm 3 / cm 2 The range between 2 and 3 is a reasonable one and can meet the needs of most cell cultures.

[0021] Optionally, the cell growth layer has a porous mesh structure with a porosity ranging from 5% to 90%.

[0022] By adopting the above technical solution, when the porosity is in a lower range (such as 5%-30%), although a certain attachment area and material exchange channel can be provided, it may limit the cell growth space and the penetration efficiency of nutrients. When the porosity is moderate (such as 30%-70%), the cell attachment growth and the supply of nutrients can be balanced, which is conducive to the healthy growth and proliferation of cells. When the porosity is too high (such as 70%-90%), although more growth space and better material exchange efficiency can be provided, the mechanical strength and stability of the material may be sacrificed, increasing the risk in the cell culture process.

[0023] Optionally, the cell growth layer is a mesh fluffy filter membrane structure.

[0024] By adopting the above technical solution, the net-like fluffy filter membrane structure is conducive to the culture medium passing through the membrane pores, providing nutrition for the cells and facilitating cell growth.

[0025] Optionally, the light transmittance of the cell growth layer ranges from 10% to 90%.

[0026] Optionally, the fiber diameter of the cell growth layer ranges from 10 μm to 300 μm.

[0027] By adopting the above technical solutions, the cell growth layer with a smaller fiber diameter (such as 10μm to 50μm) usually has a larger specific surface area, providing more attachment points for cells, which is conducive to the initial attachment and stable growth of cells. The cell growth layer with a larger fiber diameter (such as close to 300μm) may reduce the cell attachment area, but the larger fiber gap may be conducive to the penetration of nutrients and the discharge of metabolites. In practical applications, reasonable selection and optimization should be made based on factors such as cell type, culture conditions, material properties and cost-effectiveness. By selecting an appropriate fiber diameter range, the healthy growth of cells and the accumulation of metabolites can be promoted, and the performance and efficiency of the cell culture system can be improved.

[0028] Optionally, the breaking strength of the cell growth layer ranges from 20N to 300N, and the breaking elongation ranges from 10% to 80%.

[0029] By adopting the above technical solution, a cell growth layer with a breaking strength between 20N-300N can usually meet the needs of most cell cultures. A higher breaking elongation means that the cell growth layer can undergo a larger deformation without breaking when subjected to external force, which is beneficial to cell growth and differentiation. However, too high a breaking elongation may also cause the cell growth layer to deform or relax during long-term use, affecting its stability and reliability. A breaking elongation between 10%-80% can meet the needs of most cell cultures.

[0030] Optionally, the cell growth layer is extremely hydrophilic, and a water drop angle is less than 120°.

[0031] Optionally, the cell growth layer is made of a material that is resistant to oil, diluted alkali, and high temperature and high pressure sterilization at 120-130°C.

[0032] Optionally, the material of the cell growth layer is PBT (polybutylene terephthalate), PTT (polytrimethylene terephthalate) or PET (polyethylene terephthalate).

[0033] By adopting the above technical scheme, PBT (polybutylene terephthalate) is milky white, translucent to opaque, semi-crystalline solid; it has high heat resistance, toughness, fatigue resistance, but is not resistant to strong acids and alkalis, can resist organic solvents, is flammable, and decomposes at high temperatures. The surface morphology and structure of PTT (polypropylene terephthalate fiber) is in the shape of a smooth strip, and the light reflection and refraction are strong, and the fiber luster is strong. There are gaps on the surface, which have certain moisture conduction, breathability and warmth retention. It can be made into fiber products with various cross-sectional shapes, and can also be directly processed into colored PTT fibers during the manufacturing process. The "Z"-shaped spiral structure makes it have high elasticity and good resilience that are superior to other fibers. It has a low glass transition temperature and can be dyed at normal pressure. The dye is easier to enter the fiber, and the dyeing performance is better than PET fiber. It combines the anti-fouling properties of polyester, the softness of nylon, and the bulkiness of acrylic. PET (polyethylene terephthalate) is a transparent, tough, low-cost and widely used thermoplastic polyester resin.

[0034] Optionally, the thickness of the liquid guiding layer is greater than 0.9 mm.

[0035] Optionally, the width of the liquid guiding layer ranges from 0.5 to 20 cm.

[0036] Optionally, the material of the liquid guide layer is PP (polypropylene).

[0037] Optionally, the liquid guiding layer has a mesh structure.

[0038] Optionally, the mesh of the liquid guiding layer is in a rhombus shape, and the side length of the rhombus ranges from 4 mm to 10 mm.

[0039] By adopting the above technical solution, the design of diamond mesh facilitates the uniform distribution and flow of liquid in the guide layer. Compared with meshes of other shapes, diamond mesh can more effectively reduce the flow resistance of liquid, allowing liquid to pass through the guide layer more smoothly. Within the range of 4MM-10MM, it ensures that the mesh is neither too large to cause poor filtration effect, nor too small to increase flow resistance; the mesh can effectively balance the filtration effect and fluidity.

[0040] Optionally, the cell growth layer is soaked in 0.5M NaOH for 4 to 148 hours at a temperature range of 20 to 45°C before being placed in the bioreactor for cell attachment.

[0041] By adopting the above technical solution, NaOH (sodium hydroxide) is a strong alkali with strong corrosiveness and bactericidal ability. In the field of cell culture, NaOH is often used to disinfect cell growth layers, culture vessels, etc. Through immersion treatment, NaOH can optimize the hydrophilicity of the cell growth layer, and can also effectively kill bacteria, fungi and other microorganisms attached to the surface of the cell growth layer, ensuring a sterile environment for cell culture. The concentration of 0.5M NaOH is moderate, which can achieve a good disinfection effect without causing excessive corrosion to the cell growth layer. Soaking for 4 hours can ensure that NaOH fully penetrates into every corner of the cell growth layer and completely kills potential microorganisms. During the production or storage process, the cell growth layer may be attached with some dust, grease or other impurities. These impurities may affect the attachment and growth of cells. Through NaOH immersion treatment, these impurities can be effectively removed, making the surface of the cell growth layer cleaner and smoother. At the same time, NaOH can effectively remove toxins in the cell growth layer, avoiding the situation where the cell culture layer may introduce pyrogens during subsequent culture. Provide a purer growth environment for cells.

[0042] Optionally, the wound fixed bed is sterilized under high temperature and high pressure, with a temperature range of 121° C. to 123° C. and a time range of 30 min to 60 min.

[0043] By adopting the above technical solution, the sterilizer uses electricity to provide heat energy to heat the inside of the pot, which can raise the temperature to a suitable temperature for sterilization. In addition, during the heating process, the water inside the sterilizer will produce a certain amount of steam, and the pressure in the pot is adjusted by controlling the pressure valve to form a high-pressure steam sterilization state. The length of the sterilization time depends on the temperature, pressure, and the material and structure of the items to be sterilized. 121℃ is a commonly used sterilization temperature that can ensure that most bacteria and their spores are completely inactivated. A sterilization time of 45 minutes can ensure that the bacteria and their spores on the fixed bed are fully inactivated.

[0044] Optionally, the cell growth layer and liquid conduction layer may be sterilized by gamma irradiation.

[0045] By adopting the above technical solutions, the rays have strong penetrating power and can directly act on the biological macromolecules such as DNA, RNA and protein of microorganisms, causing the microorganisms to lose their reproductive ability or die by breaking chemical bonds and destroying molecular structures. At the same time, gamma rays can also further destroy the cell structure of microorganisms and improve the sterilization effect through indirect effects, such as stimulating water molecules to produce free radicals. By strictly controlling the irradiation dose, ensuring the uniformity of irradiation and performing appropriate post-irradiation treatment, the sterilization effect can be ensured without negatively affecting the performance of the cell growth layer and the liquid guide layer.

[0046] Optionally, the cell growth layer is sealed with a chitosan solution to form a chitosan thin layer with a nanometer thickness on the surface of the cell growth layer.

[0047] By adopting the above technical solution, the hydrophilicity of the cell growth layer is further maintained and not destroyed. The material is sealed with a 30,000 KD 0.2% (w / v) chitosan solution. In this way, the shelf life of the PET film is extended from 1 year to 3 years. At the same time, the cell growth layer treated in this way has a strong antibacterial ability.

[0048] The setting of chitosan solution changes the characteristics of the interface between the cell growth layer and the liquid guide layer. Chitosan is highly hydrophilic, providing more attachment sites for cells, enriching the environment for cell attachment, and helping cells to attach more stably to the cell growth layer. It also increases the elastic properties of the fixed bed. At the same time, chitosan is relatively thin (about 2 nanometers) and is mainly distributed on the surface of the cell growth layer. It can play a certain adsorption role on the culture medium flowing through it, thereby further optimizing the material exchange environment between cells and culture medium, enabling cells to better obtain nutrients from the culture medium, discharge metabolic waste, and promote cell growth and metabolism.

[0049] The presence of chitosan will form a more complex and diverse nano-microenvironment on the surface of the cell growth layer, which will affect the liquid flow state in the cell growth layer. It will promote the formation of turbulence and further enhance the efficiency of gas-liquid exchange. The enhancement of turbulence is conducive to the more complete dissolution and transfer of oxygen in the culture medium to the cells. At the same time, the carbon dioxide and other gases produced by the cells can be discharged more quickly, providing a good gas environment for the respiratory metabolism of the cells, which is more conducive to the growth and proliferation of cells.

[0050] On the other hand, the present application provides a fixed bed bioreactor, which adopts the following technical solution:

[0051] A fixed bed bioreactor comprises a tank body, in which a cell culture fixed bed as described in any one of the above items is arranged; the cell culture fixed bed accounts for 40% to 98% of the volume of the bioreactor tank.

[0052] By adopting the above technical solution, the cell culture fixed bed occupies between 40% and 98% of the volume of the bioreactor tank. This design enables the reactor to provide as much cell growth area as possible within a limited volume, thereby improving the efficiency and yield of cell culture.

[0053] Optionally, the number of layers of the cell growth layer and the liquid guide layer after winding ranges from 1 layer to 1000 layers.

[0054] By adopting the above technical solution, the design of the number of layers of the cell growth layer and the liquid guide layer after winding between 1 and 1000 layers provides great flexibility and room for efficiency improvement. However, in actual applications, the optimal number of layers needs to be determined based on specific application requirements, cost-benefit analysis, and system complexity.

[0055] In summary, the present application includes at least one of the following beneficial technical effects:

[0056] The present invention absorbs the advantages of sheet carriers and solves the disadvantages of sheet carriers in a targeted manner. Through the regular winding method of the cell growth layer, the cell growth layer is periodically distributed on the fixed bed, and the linear speed of each part of the culture medium when flowing through the fixed bed carrier is completely uniform;

[0057] The through-type sampling strips are axially symmetrically distributed at equal distances, which can truly reflect the actual growth status of cells on the cell growth layer, making it convenient for users to make real-time adjustments to process parameters based on cell growth information;

[0058] While the rigid liquid guide layer supports the cell growth layer, its internal regular reticular structure also plays a guiding role, such as separating the PET film into relatively stable culture units, promoting faster cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is a schematic diagram of the structure of a fixed bed for cell culture.

[0060] Figure 2 This is another structural schematic diagram of a cell culture fixed bed.

[0061] Figure 3 is a schematic diagram used to demonstrate the porosity of the cell growth layer.

[0062] Figure 4 This is a schematic diagram of the fiber diameter of the cell growth layer under electron microscopy.

[0063] Figure 5 It is a diagram used to show that the mesh of the liquid diversion layer is in a diamond-shaped structure.

[0064] Figure 6 This is a comparison chart of cell density in the cell culture comparison experiment between the suspension reactor and the fixed bed reactor in Application Example 1.

[0065] Figure 7 This is a comparison chart of the total cell number in the cell culture comparison experiment between the suspension reactor and the fixed bed reactor in Application Example 1.

[0066] Figure 8This is a comparison chart of the cell culture experiment between the suspension reactor and the fixed bed reactor in Application Example 1, and the NTA detection of exosome particle concentration.

[0067] Fig. 9 This is a diagram of fixed bed cell amplification in Application Example 2, showing a comparative experiment between using a fixed bed bioreactor to culture retrovirus and a cell factory.

[0068] Fig.10 This is the comparative experiment of using fixed bed bioreactor to culture retrovirus and cell factory in Application Example 2, and the glucose metabolism diagram of fixed bed cells.

[0069] Fig.11 This is a comparative experiment between using a fixed-bed bioreactor to culture retroviruses and a cell factory in Application Example 2, and a graph of the virus yield of each batch received in the fixed bed.

[0070] Fig.12 This is a comparative experiment using fixed-bed bioreactors to culture retroviruses and cell factories in Application Example 2, and a graph of virus yield per unit area for fixed-bed and cell factories.

[0071] Figure numerals: 1. Cell growth layer; 2. Liquid guide layer. DETAILED DESCRIPTION

[0072] The following is combined with Figure 1-Figure 12 It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0073] Example 1

[0074] A cell culture fixed bed, referring to Figure 1 , including a cell growth layer 1 and a liquid guide layer 2; the cell growth layer 1 and the liquid guide layer 2 are alternately stacked to form a structure of a cell growth layer 1-liquid guide layer 2-cell growth layer 1. The cell culture fixed bed structure is wound in a reel; the cell growth layer 1 serves as a substrate for cell attachment and growth, providing an attachment surface for cell growth. The liquid guide layer 2 provides a channel for liquid circulation for the culture medium. When the liquid of the culture medium passes through the liquid guide layer 2, turbulence is formed. The cells are filtered through the cell growth layer 1 driven by the turbulence, and the cells are retained and attached to the cell growth layer 1 to grow.

[0075] During inoculation, the culture medium flows from bottom to top through the gap between the cell growth layer 1 and the liquid guide layer 2, and the cells in the culture medium will be retained in the liquid guide layer 2 full of pores during this period, and grow attached. The culture medium flows along the pores between the cell growth layer 1 and the liquid guide layer 2, and exchanges nutrients and gases with the cells on the cell growth layer 1 during the flow. Due to the alternating superposition design of the cell growth layer 1 and the liquid guide layer 2, and the reel winding structure, the fixed bed can provide a large amount of cell growth area within a limited volume. This helps to improve the cell culture efficiency and increase the cell yield. The turbulence formed by the liquid guide layer 2 helps to promote the material exchange between the cells and the culture medium, providing a more optimized growth environment for the cells. At the same time, the filtering effect of the cell growth layer 1 also helps to remove impurities and waste in the culture medium, further improving the cell growth environment. The reel winding structure makes the fixed bed more compact in volume, which is conducive to saving space. In addition, since the design of the fixed bed allows the culture medium to be recycled, the consumption cost of the culture medium can also be reduced.

[0076] The width of the cell growth layer 1 is greater than the width of the liquid guide layer 2 .

[0077] Reference Figure 2 When the width of the liquid guide layer 2 is smaller than the width of the cell growth layer 1, the liquid guide layer 2 provides a channel entrance for the culture medium to circulate, so that the culture medium flows through the fixed bed along the gap between the two cell growth layers 1, so that the cells adhere and provide nutritional conditions for the cells; and it helps to save consumables.

[0078] When the width of the liquid guide layer 2 is equal to the width of the cell growth layer 1, Figure 1 As shown, the cell growth layer 1 is regularly wound so that the cell growth layer 1 is periodically distributed on the fixed bed, and the linear velocity of each part of the culture medium is uniform when flowing through the fixed bed carrier.

[0079] The thickness ratio of the cell growth layer 1 and the liquid guide layer 2 after winding is in the range of 1:4 to 1:2. In this embodiment, the thickness ratio of the cell growth layer 1 and the liquid guide layer 2 after winding is 11:25. In other embodiments, the thickness ratio of the cell growth layer 1 and the liquid guide layer 2 after winding can also be 1:3.5 or 1:4. If the thickness ratio exceeds 1:4 to 1:2, the culture area will be reduced or the liquid in the guide layer will not flow smoothly.

[0080] The thickness of the cell growth layer 1 is not greater than 0.22 mm, and is set to 0.22 mm in this embodiment. In other embodiments, the thickness of the cell growth layer 1 may also be 0.2 mm or 0.1 mm. In this embodiment, a vernier caliper is used for measurement.

[0081] The mass per unit area of ​​the cell growth layer 1 is in the range of 0.1 oz / yd 2 ~3.0oz / yd 2 , in this embodiment, it is set to 0.8 oz / yd 2 In other embodiments, the mass per unit area of ​​the cell growth layer 1 may also be 0.1 oz / yd 2 、0.5oz / yd 2 、1.0oz / yd 2 、2.0oz / yd 2 or 3.0oz / yd 2 .

[0082] The mass per unit area of ​​the cell growth layer 1 is between 0.1 oz / yd 2 Up to 3.0oz / yd 2 There is a reasonable range between 2 and 3, which can meet the culture requirements of different types of cells and provide good system stability and cost-effectiveness.

[0083] The range of air permeability of cell growth layer 1 is 50cm 3 / s / cm 2 ~300cm 3 / s / cm 2 , in this embodiment, it is set to 161cm 3 / s / cm 2 In other embodiments, the air permeability of the cell growth layer 1 can also be 50cm 3 / s / cm 2 , 100cm 3 / s / cm 2 、150cm 3 / s / cm 2 , 200cm 3 / s / cm 2 、250cm 3 / s / cm 2 or 300cm 3 / s / cm 2 .

[0084] The air permeability of the cell growth layer 1 is between 50cm 3 / cm 2 Up to 300cm 3 / cm 2 The range between 2 and 3 is a reasonable one and can meet the needs of most cell cultures.

[0085] Reference Figure 3 , the cell growth layer 1 presents a porous network structure.

[0086] The porosity of the cell growth layer 1 ranges from 5% to 90%, and is set to 35.22% in this embodiment. In other embodiments, the porosity may also be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0087] When the porosity is in a lower range (such as 5% to 30%), although it can provide a certain attachment area and material exchange channel, it may limit the growth space of cells and the penetration efficiency of nutrients.

[0088] When the porosity is moderate (eg, 30% to 70%), the attachment growth of cells and the supply of nutrients can be balanced, which is beneficial to the healthy growth and proliferation of cells.

[0089] When the porosity is too high (eg, 70% to 90%), more growth space and better material exchange efficiency can be provided.

[0090] The cell growth layer 1 is a net-like fluffy filter membrane structure, which is conducive to the culture medium passing through the membrane pores, providing nutrition for the cells and facilitating cell growth.

[0091] The light transmittance of the cell growth layer 1 ranges from 10% to 90%, and the measured average value in this embodiment is 54%. In other embodiments, the light transmittance can also be 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0092] The fiber diameter of the cell growth layer 1 ranges from 10 μm to 300 μm, and is measured to be 28.35 μm in this embodiment. In other embodiments, the fiber diameter of the cell growth layer 1 can also be 26.576 μm, 27.375 μm, 28.084 μm, 32.07 μm, 32.303 μm, 35.534 μm, 50 μm, 100 μm, 200 μm or 300 μm.

[0093] A cell growth layer 1 with a smaller fiber diameter (such as 10 μm to 50 μm) generally has a larger specific surface area, provides more attachment points for cells, and is beneficial to the initial attachment and stable growth of cells. A cell growth layer 1 with a larger fiber diameter (such as close to 300 μm) may reduce the cell attachment area, but a larger fiber gap may be beneficial to the penetration of nutrients and the discharge of metabolites. In practical applications, reasonable selection and optimization should be made based on factors such as cell type, culture conditions, material properties, and cost-effectiveness. By selecting an appropriate fiber diameter range, the healthy growth of cells and the accumulation of metabolites can be promoted, and the performance and efficiency of the cell culture system can be improved.

[0094] The breaking strength of the cell growth layer 1 ranges from 20N to 300N, and in this embodiment the measured average value is 88.9N. In other embodiments, it may be 25N, 50N, 76.9N, 77.5N, 89.5N, 97.8N, 100N, 103N, 150N, 200N or 300N.

[0095] The elongation at break of the cell growth layer 1 ranges from 10% to 80%, and the measured average value in this embodiment is 39%. In other embodiments, it may be 20%, 29%, 30%, 35%, 40%, 41%, 43%, 47%, 50%, 60%, 70% or 80%.

[0096] A cell growth layer 1 with a breaking strength between 20N and 300N can usually meet the needs of most cell cultures. A higher breaking elongation means that the cell growth layer 1 can undergo a larger deformation without breaking when subjected to external force, which is beneficial to cell growth and differentiation. However, an excessively high breaking elongation may also cause the cell growth layer 1 to deform or relax during long-term use, affecting its stability and reliability. A breaking elongation between 10% and 80% can meet the needs of most cell cultures.

[0097] The cell growth layer 1 is extremely hydrophilic, and the water drop angle is less than 120°.

[0098] The material of the cell growth layer 1 is PBT (polybutylene terephthalate), and in other embodiments, PTT (polypropylene terephthalate fiber) or PET (polyethylene terephthalate) is selected. The main purpose is to be oil-resistant, alkali-resistant, and able to withstand high-temperature and high-pressure sterilization at 120-130°C.

[0099] PBT (polybutylene terephthalate) is a milky white, translucent to opaque, semi-crystalline solid; it has high heat resistance, toughness, and fatigue resistance, but is not resistant to strong acids and alkalis, can resist organic solvents, is flammable, and decomposes at high temperatures. The surface morphology and structure of PTT (polypropylene terephthalate fiber) is in the shape of a smooth strip, and the light reflection and refraction are strong, and the fiber luster is strong. There are gaps on the surface, which have certain moisture conduction, breathability and warmth retention. It can be made into fiber products with various cross-sectional shapes, and can also be directly processed into colored PTT fibers during the manufacturing process. The "Z"-shaped spiral structure makes it have high elasticity and good resilience that are superior to other fibers. It has a low glass transition temperature and can be dyed at normal pressure. The dye is easier to enter the fiber, and the dyeing performance is better than PET fiber. It combines the anti-fouling properties of polyester, the softness of nylon, and the bulkiness of acrylic. PET (polyethylene terephthalate) is a transparent, tough, low-cost and widely used thermoplastic polyester resin.

[0100] The thickness of the liquid guiding layer 2 is greater than or equal to 0.9 mm, and the average value measured by a vernier caliper in this embodiment is 0.9 mm. In other embodiments, it can also be set to 1.0 mm, 2.0 mm or 3.0 mm.

[0101] The width of the liquid guiding layer 2 ranges from 0.5 to 20 cm, and is set to 5 cm in this embodiment. In other embodiments, the width of the liquid guiding layer 2 can also be 1 cm, 7.5 cm, 10 cm, 15 cm or 20 cm.

[0102] The material of the liquid guiding layer 2 is PP (polypropylene) and is designed in a mesh structure. The mesh of the liquid guiding layer 2 is rhombus-shaped, and the side length of the rhombus ranges from 4 mm to 10 mm, and is set to 4 mm in this embodiment. In other embodiments, the side length of the rhombus can also be 5 mm, 6 mm, 7 mm, 8 mm, 9 mm or 10 mm.

[0103] The diamond mesh design helps the liquid to be evenly distributed and flow in the diversion layer. Compared with meshes of other shapes, diamond mesh can more effectively reduce the flow resistance of the liquid, allowing the liquid to pass through the diversion layer more smoothly. Within the range of 4mm to 10mm, it ensures that the mesh is neither too large to cause poor filtration effect, nor too small to increase flow resistance; the mesh can effectively balance the filtration effect and fluidity.

[0104] The cell growth layer 1 is soaked in 0.5M NaOH for 4 to 148 hours before being placed in the bioreactor for cell attachment, which is set to 4 hours in this embodiment; the temperature range is 20 to 45°C, which is set to 25°C in this embodiment; wherein M is mol / L.

[0105] NaOH (sodium hydroxide) is a strong alkali with strong corrosive and bactericidal abilities. In the field of cell culture, NaOH is often used to disinfect the cell growth layer 1, culture vessels, etc. Through soaking treatment, NaOH can optimize the hydrophilicity of the cell growth layer 1, and can also effectively kill bacteria, fungi and other microorganisms attached to the surface of the cell growth layer 1, ensuring a sterile environment for cell culture. The 0.5M NaOH concentration is moderate, which can achieve a good disinfection effect without causing excessive corrosion to the cell growth layer 1. Soaking for 4 hours can ensure that NaOH fully penetrates into every corner of the cell growth layer 1 and completely kills potential microorganisms. During the production or storage process, the cell growth layer 1 may be attached with some dust, grease or other impurities. These impurities may affect the attachment and growth of cells. Through NaOH soaking treatment, these impurities can be effectively removed, making the surface of the cell growth layer 1 cleaner and smoother. At the same time, NaOH can effectively remove endotoxins from the cell growth layer 1, avoiding the situation where the cell culture layer may introduce pyrogens during subsequent culture. Provide a purer growth environment for cells.

[0106] The wound fixed bed is sterilized at high temperature and high pressure, the temperature range is 121°C to 123°C, and in this embodiment, it is set to 121°C, and the sterilization time range is 30 to 60 minutes, and in this embodiment, the sterilization time is set to 45 minutes. In other embodiments, the sterilization time can also be 30 minutes, 40 minutes, 50 minutes or 60 minutes, depending on the size of the reactor.

[0107] Preferably, under the condition of a pressure of 100Kpa, the boiling point of water will increase accordingly, thereby ensuring that the temperature in the sterilizer can reach the required sterilization temperature. When the temperature exceeds the temperature range of the optimal physiological activity of cells, cell metabolism will slow down, and the cell's proteins, enzymes and nucleic acids will be permanently damaged, leading to irreversible cell death. 121°C is a commonly used sterilization temperature that can ensure that most bacteria and their spores are completely inactivated. The length of the sterilization time depends on the temperature, pressure, and the material and structure of the items to be sterilized. A sterilization time of 45 minutes can ensure that the bacteria and their spores on the fixed bed are fully inactivated.

[0108] The cell growth layer 1 and the liquid guide layer 2 can be sterilized by γ-ray irradiation.

[0109] Gamma rays have strong penetrating power and can directly act on microbial biomacromolecules such as DNA, RNA and proteins, causing microorganisms to lose their reproductive ability or die by breaking chemical bonds and destroying molecular structures. At the same time, gamma rays can also further destroy the cell structure of microorganisms and improve the sterilization effect through indirect effects, such as stimulating water molecules to produce free radicals. By strictly controlling the irradiation dose, ensuring the uniformity of irradiation and performing appropriate post-irradiation treatment, the sterilization effect can be ensured without negatively affecting the performance of the cell growth layer 1 and the liquid guide layer 2.

[0110] In order to further maintain the hydrophilicity of the cell growth layer, the cell growth layer 1 is sealed with a 30,000 KD 0.2% (w / v) chitosan solution to form a nanometer-thick chitosan layer on the surface of the cell growth layer 1 .

[0111] Among them, 30,000 KD 0.2% (w / v) chitosan solution was used to seal the material, which extended the shelf life of the PET film from 1 year to 3 years. At the same time, the cell growth layer treated in this way has a strong antibacterial ability.

[0112] The setting of chitosan solution changes the characteristics of the interface between the cell growth layer and the liquid guide layer. Chitosan is highly hydrophilic, providing more attachment sites for cells, enriching the environment for cell attachment, and helping cells to attach more stably to the cell growth layer. It also increases the elastic properties of the fixed bed. At the same time, chitosan is relatively thin (about 2 nanometers) and is mainly distributed on the surface of the cell growth layer. It can play a certain adsorption role on the culture medium flowing through it, thereby further optimizing the material exchange environment between cells and culture medium, enabling cells to better obtain nutrients from the culture medium, discharge metabolic waste, and promote cell growth and metabolism.

[0113] The presence of chitosan will form a more complex and diverse nano-microenvironment on the surface of the cell growth layer, which will affect the liquid flow state in the cell growth layer. It will promote the formation of turbulence and further enhance the efficiency of gas-liquid exchange. The enhancement of turbulence is conducive to the more complete dissolution and transfer of oxygen in the culture medium to the cells. At the same time, the carbon dioxide and other gases produced by the cells can be discharged more quickly, providing a good gas environment for the respiratory metabolism of the cells, which is more conducive to the growth and proliferation of cells.

[0114] Example 2

[0115] Embodiment 2 of the present application discloses a fixed bed bioreactor, including a tank body, in which a cell culture fixed bed as described above is arranged; the cell culture fixed bed accounts for 40% to 98% of the volume of the bioreactor tank, for example, in this embodiment, it is set to 40% according to the specifications of the tank body. In other embodiments, it can also be set to 50%, 60%, 70%, 80%, 90% or 98%.

[0116] The cell culture fixed bed occupies between 40% and 98% of the volume of the bioreactor tank. This design enables the reactor to provide as much cell growth area as possible within a limited volume, thereby improving the efficiency and yield of cell culture.

[0117] The number of layers of the wound cell growth layer 1 and the liquid guide layer 2 ranges from 1 layer to 1000 layers, and in this embodiment the number of layers is set to 100 layers. In other embodiments, the number of layers can also be set to 1 layer, 200 layers, 300 layers, 400 layers, 500 layers, 600 layers, 700 layers, 800 layers, 900 layers or 1000 layers, depending on the size of the reactor.

[0118] The design of the number of layers of the wound cell growth layer 1 and the liquid guide layer 2 between 1 and 1000 provides great flexibility and room for efficiency improvement. However, in actual applications, the optimal number of layers needs to be determined based on specific application requirements, cost-benefit analysis, system complexity and other factors.

[0119] Application Example 1

[0120] Use our products Comparison of the immobilized culture of 293F suspension cells in the Culture Ad60 series fixed-bed bioreactor and the traditional Applikon airlift suspension reactor:

[0121] Experimental plan: According to the principle of equal initial seeding density (volume calculation), 293F suspension cells were seeded into Cell culture was performed in Culture Ad60 series fixed bed bioreactors, BT-2 tanks, FB-4 fixed beds, and 1.5 L suspended airlift bioreactors according to the conditions in Table 1.

[0122] Table 1 Comparison of culture conditions for inoculating 293F suspension cells into fixed bed bioreactors and 1.5L suspension airlift bioreactors

[0123] Airlift Suspension Reactor Fixed bed bioreactor Culture volume (L) 1.5 0.9 <![CDATA[Inoculation density (×10 6 / ml)]]> 1.18 1.00 <![CDATA[Inoculation cell amount (×10 9 )]]> 1.77 0.9 Cultivation period (days) 7 7 Training model Batch Mode Cycle Mode

[0124] Experimental results:

[0125] During the cell culture period, the cell density was tested every day, and the total number of cells in the two reactors was calculated every day. The supernatant of the airlift suspension reactor was taken on day 7, and the supernatant of the fixed-bed bioreactor was taken on day 4 and day 7, respectively. The number of exosome particles (NTA) in the supernatant was detected after centrifugation. The exosome yield of the two reactors was compared. With the same number of inoculated cells and the same culture time, the exosome yield of the fixed-bed bioreactor was 15 times that of the suspension reactor; the results are shown in Figure 2. Figure 6-Figure 8 And as shown in Table 2.

[0126] Table 2 Cultivation results of airlift suspension reactor and fixed bed reactor

[0127]

[0128] Application Example 2

[0129] Use our products Comparison between Culture Ad60 series fixed bed bioreactor for retrovirus culture and BIOFI's cell factory:

[0130] Experimental plan: Retrovirus production using a stable retroviral cell line. CultureAd60 series fixed bed bioreactor, BT-2 tank, FB-1 fixed bed and 10-layer cell factory as culture carriers, respectively, fixed bed and 10-layer cell factory at 2×104 / cm 2 Cells were plated and viral supernatants were harvested during cell expansion.

[0131] Experimental results:

[0132] The fixed-bed bioreactor was used to harvest viruses on day 2, day 5, day 6 and day 7, and the cell factory was used to harvest viruses in batches on day 7. After the virus was harvested, the supernatant was taken and the number of retrovirus copies was tested as the basis for the virus yield. The experimental results showed that the retrovirus yield per unit area of ​​the fixed-bed bioreactor was 2.6 times that of the cell factory; the experimental results are as follows Figure 9-12 shown.

[0133] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A cell culture fixed bed, characterized in that: The invention comprises a cell growth layer (1) and a liquid guide layer (2); the cell growth layer (1) and the liquid guide layer (2) are alternately stacked to form a structure of a cell growth layer (1)-liquid guide layer (2)-cell growth layer (1); the cell culture fixed bed structure is wound in a reel; the cell growth layer (1) serves as a substrate for cell attachment and growth, providing an attachment surface for cell growth; the liquid guide layer (2) provides a channel for liquid circulation for the culture medium; turbulence is formed when the liquid of the culture medium passes through the liquid guide layer (2); cells are filtered through the cell growth layer (1) under the drive of the turbulence, and the cells are retained and attached to the cell growth layer (1) for growth.

2. The cell culture fixed bed according to claim 1, characterized in that: The width dimension of the liquid guiding layer (2) is less than or equal to the width dimension of the cell growth layer (1).

3. The cell culture fixed bed according to claim 1, characterized in that: The thickness ratio of the cell growth layer (1) and the liquid guide layer (2) after winding is in the range of 1:4 to 1:

2.

4. The cell culture fixed bed according to claim 1, characterized in that: The thickness of the cell growth layer (1) is less than 0.22 mm.

5. The cell culture fixed bed according to claim 1, characterized in that: The mass per unit area of ​​the cell growth layer (1) is in the range of 0.1 oz / yd 2 ~3.0oz / yd 2 .

6. The cell culture fixed bed according to claim 1, characterized in that: The cell growth layer (1) has an air permeability range of 50 cm 3 / s / cm 2 ~300cm 3 / s / cm 2 .

7. The cell culture fixed bed according to claim 1, characterized in that: The cell growth layer (1) is in a porous mesh structure, and the porosity ranges from 5% to 90%.

8. The cell culture fixed bed according to claim 1, characterized in that: The cell growth layer (1) is a net-like fluffy filter membrane structure.

9. The cell culture fixed bed according to claim 1, characterized in that: The light transmittance of the cell growth layer (1) ranges from 10% to 90%.

10. The cell culture fixed bed according to claim 1, characterized in that: The fiber diameter of the cell growth layer (1) ranges from 10 μm to 300 μm.

11. The cell culture fixed bed according to claim 1, characterized in that: The breaking strength of the cell growth layer (1) is in the range of 20N-300N, and the breaking elongation is in the range of 10%-80%.

12. The cell culture fixed bed according to claim 1, characterized in that: The cell growth layer (1) is extremely hydrophilic, and the water drop angle is less than 120°.

13. The cell culture fixed bed according to claim 1, characterized in that: The cell growth layer (1) is made of a material that is resistant to oil, resistant to diluted alkali, and resistant to high temperature and high pressure sterilization at 120-130°C.

14. The cell culture fixed bed according to claim 13, characterized in that: The material of the cell growth layer (1) is PBT (polybutylene terephthalate), PTT (polytrimethylene terephthalate fiber) or PET (polyethylene terephthalate).

15. The cell culture fixed bed according to claim 1, characterized in that: The thickness of the liquid guiding layer (2) is greater than 0.9 mm.

16. The cell culture fixed bed according to claim 1, characterized in that: The width of the liquid guiding layer (2) is in the range of 0.5 to 20 cm.

17. The cell culture fixed bed according to claim 1, characterized in that: The material of the liquid guiding layer (2) is PP (polypropylene).

18. The cell culture fixed bed according to claim 1, characterized in that: The liquid guiding layer (2) has a mesh structure.

19. The cell culture fixed bed according to claim 18, characterized in that: The mesh of the liquid guiding layer (2) is in a rhombus shape, and the side length of the rhombus ranges from 4 mm to 10 mm.

20. The cell culture fixed bed according to claim 1, characterized in that: The cell growth layer (1) is soaked in 0.5M NaOH for 4 to 148 hours at a temperature of 20 to 45°C before being placed in a bioreactor for cell attachment.

21. The cell culture fixed bed according to claim 1, characterized in that: The wound fixed bed is sterilized at high temperature and high pressure, with a temperature range of 121° C. to 123° C. and a time range of 30 min to 60 min.

22. The cell culture fixed bed according to claim 1, characterized in that: The cell growth layer (1) and the liquid guide layer (2) are sterilized by gamma irradiation.

23. The cell culture fixed bed according to claim 1, characterized in that: The cell growth layer (1) uses a chitosan solution to seal the material, thereby forming a chitosan thin layer with a nanometer thickness on the surface of the cell growth layer (1).

24. A fixed bed bioreactor, characterized in that: It comprises a tank body, in which a cell culture fixed bed as claimed in any one of claims 1 to 23 is arranged; the cell culture fixed bed accounts for 40% to 98% of the volume of the bioreactor tank.

25. The fixed bed bioreactor according to claim 24, characterized in that The number of layers of the cell growth layer (1) and the liquid guide layer (2) after winding ranges from 1 layer to 1000 layers.

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