Method for realizing adherent cell in-vitro amplification by utilizing stirring type bioreactor matched with three-dimensional porous hydrogel carrier

Abstract

The invention discloses a method for realizing in-vitro amplification of adherent cells by utilizing a stirring type bioreactor matched with a three-dimensional porous hydrogel carrier. The method comprises the following steps: sterilizing a hydrogel carrier and a stirred bioreactor, adding a cell culture medium, ventilating, inoculating cells, starting the stirred bioreactor for culturing, observing the color change of a culture solution in the culture process, and changing the solution when the culture solution turns yellow, thereby ensuring the nutrition required by cell culture. The hydrogel with high biocompatibility is used as a carrier, a porous three-dimensional growth environment is provided, cells can be attached to the interior of the hydrogel carrier, the cell growth space is sufficient, the cells can be cultured for more generations, the number and density are greatly increased, the amplification multiple can reach 10 times or above, the production efficiency is improved, and the production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of bioreactors, and in particular relates to a method for realizing in vitro expansion of adherent cells by utilizing a stirring bioreactor in combination with a three-dimensional porous hydrogel carrier. Background Art

[0002] Unlike artificially domesticated suspension culture cells, adherent cells must be attached to the surface of an object during the culture process and are extremely sensitive to shear force and fluctuations in temperature, pH, and dissolved oxygen. Most existing culture technologies are limited to small-scale and laboratory levels. Reference 1 explains that the adherent shake flask culture process is one of the existing adherent cell culture methods and is the main traditional adherent cell culture method. It is low-cost, but labor-intensive and has a low cell density ([1] Wen Lingyan. Preliminary study on the full suspension shake flask culture process of PK15 cells [J]. Fujian Animal Husbandry and Veterinary Medicine, 2022, 44(1):3.). Reference 2 records that stirred culture can be used for the expansion of adherent cells. The advantages are simple process and flexible operation, but the shear force generated is large, which can easily damage the cells and the adherent cells may fall off and die ([2] Luo Haiyan et al. "Research, application and development of stirred animal cell reactors." Bioprocessing (2016).).

[0003] At present, most adherent cell cultures use the shake flask process, which involves inoculating cells into shake flasks and then placing them in a constant temperature incubator, allowing the cells to grow in the shake flasks, and then washing the cells off the flask walls after a certain period of time through digestion and other methods. This production method has a low degree of automation, is highly dependent on the standardized operation of operators, and has strict requirements on the production environment. In addition, there is a lack of online sensors to detect physical and chemical properties in real time, and product uniformity cannot be effectively guaranteed. The cell yield is low, the production cost is high, and sampling cannot be performed during the culture process. These problems limit the commercial application of adherent cells.

[0004] Traditional bioreactors are used in the large-scale production of engineered cells, but many problems such as their mechanical structure, ventilation method, shear force of stirring blades, cell damage and cell separation caused by bubble breakage limit their application in large-scale in vitro culture of adherent cells.

[0005] Chinese patent application 202310121635.6 discloses a method for in vitro expansion of adherent cells, which uses a bioreactor and a paper carrier to culture adherent cells, uses a propeller to suspend the paper carrier and attach the adherent cells to the paper carrier, thereby realizing bioreactor culture of adherent cells. Although this method can utilize the automation of the bioreactor, the biocompatibility of the paper carrier is low, and the cells can only be attached to the surface of the paper carrier, and some of them will still be affected by shear force, which may cause cell damage, which still limits the expansion of adherent cells. Summary of the invention

[0006] In order to solve the many technical problems of adherent growth, oxygen and nutrient supply, seed expansion and sampling and analysis during the culture process of existing large-scale adherent cell culture in vitro, the present invention provides a method for in vitro expansion of adherent cells using a stirred bioreactor in combination with a three-dimensional porous hydrogel carrier.

[0007] The technical solution of the present invention is as follows:

[0008] A method for achieving in vitro expansion of adherent cells using a stirred bioreactor in combination with a three-dimensional porous hydrogel carrier is specifically as follows: placing the hydrogel carrier in a stirred bioreactor, adding cell culture medium, and performing ventilation treatment, then inoculating cells, starting the stirred bioreactor for cultivation, observing the color change of the culture medium during the cultivation process, and replacing the culture medium when it turns yellow to ensure the nutrients required for cell culture.

[0009] The hydrogel carrier of the present invention can be any type of hydrogel, and the type of the hydrogel carrier is not specifically limited. In a specific embodiment of the present invention, a sulfonated bacterial cellulose / gelatin hydrogel composited with sulfonated bacterial cellulose and gelatin is taken as an example.

[0010] The hydrogel carrier and the stirred bioreactor of the present invention are both sterilized.

[0011] Preferably, the density of the hydrogel carrier of the present invention is 0.4-1 g / cm 3 The usage density of the hydrogel carrier is 3 to 10 g / L.

[0012] In the method of the present invention, the cell culture medium, aeration conditions, inoculation density, and operating parameters of the stirred bioreactor are adaptively adjusted according to the type of inoculated cells.

[0013] In one embodiment of the present invention, the seeded cells are stem cells, and the seeding density is 3 to 5×10 5 cells / ml, the operating parameters of the stirred bioreactor are: pH value is 7.0-7.5, temperature is 35-37°C, dissolved oxygen is 25%-75%, stirring speed is 50-300rpm; the culture time is 12-18 days.

[0014] In another embodiment of the present invention, the seeded cells are Vero cells, and the seeding density is 3 to 5×10 5 cells / ml, the operating parameters of the stirred bioreactor are: pH value of 7.0-7.5, temperature of 35-37°C, dissolved oxygen of 35%-75% and stirring speed of 50-300rpm; the culture time is 4-9 days.

[0015] In another embodiment of the present invention, the inoculated cells are liver cells, and the inoculation density is 3 to 5×10 5 cells / ml, the operating parameters of the stirred bioreactor are: pH value is 7.0-7.5, temperature is 35-37°C, dissolved oxygen is 25%-75%, stirring speed is 50-300rpm; the culture time is 12-18 days.

[0016] Furthermore, the method further comprises sucking out the hydrogel carrier after the culture is completed, digesting the cells adherently grown on the hydrogel carrier with pancreatic enzyme, and then eluting.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) The hydrogel carrier used has a porous three-dimensional structure and a high porosity. Adherent cells can attach to the inside of the hydrogel carrier. During the culture process, the cell lifting paddle in the stirred bioreactor can suck the hydrogel carrier out from the bottom of the tank and throw it out through the inside of the stirring paddle, so that the hydrogel carrier in the tank is completely maintained in a suspended state and does not contact any device other than the cell lifting paddle. The cell lifting paddle allows the culture medium and gas to pass through, while increasing the gas-liquid exchange area in the tank, greatly improving the oxygen utilization rate; the lower stirring speed reduces the shear force generated when the hydrogel carrier collides with the stirring paddle, and also greatly reduces the possibility of cell damage during sampling, solving the problems of insufficient attachment surface area for high-density cell growth and excessive oxygen consumption and insufficient oxygen supply in traditional methods.

[0019] (2) The hydrogel carrier has an elastic modulus suitable for the growth of adherent cells, and the hydrogel has a good affinity for cytokines, which is conducive to the adhesion of growth factors, solving the problem of high-density culture of adherent cells, improving production efficiency and reducing production costs.

[0020] (3) When using hydrogel carriers alone for adherent cell culture, even on three-dimensional carriers, cells will be restricted in growth due to the traditional two-dimensional plane environment of the carriers. Basically, they can only be cultured for three generations, and the final expansion multiple is 7-8 times. In bioreactors, there is sufficient three-dimensional space, which solves the problem of limited cell expansion space. Cells can be cultured for more generations, and the number and density are greatly increased, and the expansion multiple can reach more than 10 times.

[0021] (4) When using paper carriers for adherent cell culture in bioreactors, the paper carriers have low biocompatibility, and because the paper is also a two-dimensional plane, cells can only adhere to the surface of the paper carrier, and the cells are still affected by the growth space and partial shear force. Other commercial microcarriers also have such problems. When the present invention uses a hydrogel carrier and a bioreactor, the hydrogel carrier has better biocompatibility and provides a porous three-dimensional growth environment. Cells can attach to the inside of the hydrogel carrier, and the cell growth space is sufficient, which also solves the effect of shear force on cells in traditional stirred bioreactors.

[0022] (5) The problem of the carrier being easily entangled with the pipes in the equipment is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are field emission scanning electron micrographs of sulfonated bacterial cellulose / gelatin hydrogel at different magnifications.

[0024] Figure 2 The figure is a morphological diagram of mouse liver cells expanded and cultured by the method of the present invention under an optical microscope.

[0025] Figure 3 This is a morphological image of mouse liver cells cultured using only hydrogel carriers under an optical microscope.

[0026] Figure 4 It is a confocal microscope image of mouse liver cells amplified and cultured by the method of the present invention.

[0027] Figure 5 This is a confocal micrograph of mouse liver cells cultured using only the hydrogel carrier.

[0028] Figure 6 The figure is a density change diagram of mouse liver cells expanded and cultured using the method of the present invention within one month.

[0029] Figure 7 This is a light microscope image of Vero cells grown using commercial microcarriers and bioreactors. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments.

[0031] The hydrogel carrier used in the following examples is a sulfonated bacterial cellulose / gelatin hydrogel, which was prepared with reference to [Xinmeng L, Weixiao D, Shujun W, et al. Three-Dimensional Sulfated Bacterial Cellulose / Gelatin Composite Scaffolds for Culturing Hepatocytes. [J]. Cyborg and bionic systems (Washington, DC), 2023, 40021-0021], and its field emission scanning electron microscopy images at different magnifications are shown in FIG. Figure 1 As shown. Figure 1 As can be seen from Figure (a) in the figure, the hydrogel carrier has a porous three-dimensional structure with high porosity and uniform pores, which is suitable for gas-liquid exchange of cells. Figure 1 As can be seen from Figure (b), there is sufficient three-dimensional growth space inside the hydrogel carrier, which can provide a large surface area for high-density cell growth and is suitable for cell adherent growth.

[0032] In the following examples, the stirred bioreactor used was manufactured by Nanjing Jinwei Bioengineering Equipment Co., Ltd. The Lab / D animal cell culture bioreactor has online monitoring facilities for pH, dissolved oxygen, temperature, and ventilation during the culture process.

[0033] Example 1

[0034] The in vitro expansion of stem cells is achieved by using a stirred bioreactor in combination with a three-dimensional porous hydrogel carrier, which specifically includes the following steps:

[0035] 1) Cleaning and sterilizing the hydrogel carrier and the stirred bioreactor;

[0036] 2) growing the stem cells to the logarithmic phase and then dispersing them by trypsin digestion to obtain a stem cell suspension;

[0037] 3) Place the hydrogel carrier in a stirred bioreactor, and the density of the hydrogel carrier is 0.4-1 g / cm 3 The density of the hydrogel carrier is 3-10 g / L. Then add stem cell culture medium ( The medium was then aerated to introduce the gases (oxygen, air and carbon dioxide) required for cell growth. 5 Inoculate stem cells at an inoculum of 10 cells / ml, start the stirred bioreactor, set the parameters as follows: pH 7.0-7.5, temperature 35-37°C, dissolved oxygen 25%-75%, stirring speed 50-300 rpm, and culture for 12-18 days.

[0038] 4) Observe the color change of the culture medium during the culture process. When the culture medium turns yellow, replace the medium. Draw the culture medium in the tank from the extraction tube and inject new culture medium to maintain the nutrients required for cell culture.

[0039] 5) After the culture is completed, the culture medium and the hydrogel carrier are directly aspirated from the extraction tube. The exosomes are in the culture medium; the stem cells are located on the hydrogel carrier and need to be digested with pancreatic enzymes before elution.

[0040] Example 2

[0041] The in vitro expansion of Vero cells is achieved by using a stirred bioreactor in combination with a three-dimensional porous hydrogel carrier, which specifically includes the following steps:

[0042] 1) Cleaning and sterilizing the hydrogel carrier and the stirred bioreactor;

[0043] 2) After the Vero cells are grown to the logarithmic phase, they are digested and dispersed by trypsin to obtain a Vero cell suspension;

[0044] 3) Place the hydrogel carrier in a stirred bioreactor, and the density of the hydrogel carrier is 0.4-1 g / cm 3 The density of the hydrogel carrier is 3-10 g / L. Then, vero cell culture medium is added, and the culture medium is HyCloneTM VaccineXpress serum-free culture medium. Three kinds of supplements are added at the same time as the above culture medium: F-68, soypeptone (Fluka), soybean-derived trypsin inhibitor (Sigma-Aldrich; 1 mg / mL in PBS), and ventilation treatment was performed to introduce the gases required for cell growth (oxygen, air, and carbon dioxide). 5 Vero cells were inoculated at an inoculum of 10 cells / ml, and the stirred bioreactor was started with the following parameters: pH value of 7.0-7.5, temperature of 35-37°C, dissolved oxygen of 35%-75%, and stirring speed of 50-300 rpm. The culture was carried out for 4-9 days.

[0045] 4) Observe the color change of the culture medium during the culture process. When the culture medium turns yellow, replace the medium. Draw the culture medium in the tank from the extraction tube and inject new culture medium to maintain the nutrients required for cell culture;

[0046] 5) After the culture is completed, the hydrogel carrier is directly sucked out from the liquid extraction tube, digested with trypsin, and then eluted.

[0047] Example 3

[0048] The in vitro expansion of liver cells is achieved by using a stirred bioreactor in combination with a three-dimensional porous hydrogel carrier, which specifically includes the following steps:

[0049] 1) Cleaning and sterilizing the hydrogel carrier and the stirred bioreactor;

[0050] 2) After the liver cells are grown to the logarithmic phase, they are digested and dispersed with trypsin to obtain a liver cell suspension;

[0051] 3) Place the hydrogel carrier in a stirred bioreactor, and the density of the hydrogel carrier is 0.4-1 g / cm 3 The density of the hydrogel carrier is 3-10 g / L. Then add the liver cell culture medium and perform ventilation to introduce the gases (oxygen, air and carbon dioxide) required for cell growth. 5 Inoculate liver cells at an inoculum of 10 cells / ml, start the stirred bioreactor, set the parameters as follows: pH 7.0-7.5, temperature 35-37°C, dissolved oxygen 25%-75%, stirring speed 50-300 rpm, and culture for 12-18 days.

[0052] 4) Observe the color change of the culture medium during the culture process. When the culture medium turns yellow, replace the medium. Draw the culture medium in the tank from the extraction tube and inject new culture medium to maintain the nutrients required for cell culture;

[0053] 5) After the culture is completed, the hydrogel carrier is directly sucked out from the liquid extraction tube, and then digested with trypsin and eluted.

[0054] Figure 2 The figure is a morphological diagram of mouse liver cells expanded and cultured by the method of the present invention under an optical microscope. Figure 3 This is a morphological image of mouse liver cells cultured using only hydrogel carriers under an optical microscope. Figure 4 It is a confocal microscope image of mouse liver cells amplified and cultured by the method of the present invention. Figure 5 This is a confocal micrograph of mouse liver cells cultured using only hydrogel carriers. Figures 2 to 5 It can be observed that: the cells cultured using only the hydrogel carrier are unevenly distributed, and due to the limitation of the growth space, agglomeration and squeezing occur, which affects the further expansion of the cells; while the cells cultured by the method of the present invention are more evenly distributed on the hydrogel carrier, the cells are round, the number of cells is larger, and the expansion effect is better.

[0055] Figure 6This is a graph showing the density changes of mouse liver cells amplified and cultured using the method of the present invention within one month. It can be seen that the number of cells has been increasing, and the growth rate first accelerates and then slows down. This is due to the cells consuming nutrients in the culture medium. In short, it can be clearly found that the growth state of the cells on the hydrogel carrier is very good and the survival rate is very high. Under this cell density, the number of cells in a 500mL bioreactor can reach 10 within a month. 8 Order of magnitude indicators, good results.

[0056] Figure 7 This is a picture of the growth of Vero cells cultured in commercial microcarriers and bioreactors under an optical microscope in the literature [Wu Yinjie, Zhang Ming, Zhou Jian, et al. Effects of microcarrier pretreatment methods on sterilization temperature and Vero cell adhesion rate [J]. Journal of Yunnan University (Natural Science Edition), 2017, 39(05): 876-882]. It can be seen that the cells are not evenly attached to the microcarriers. Some microcarriers have a lot of cells while some have no cells. The growth space provided by the microcarriers is also a circular two-dimensional plane, which limits the gas-liquid exchange and further expansion of the cells.

[0057] pass Figure 2 and Figure 7 By comparison, the distribution of cells on the microcarriers is uneven when using commercial microcarriers and bioreactor culture methods. Some microcarriers gather a lot of cells while some microcarriers have no cells. It is observed that the growth space provided by the microcarriers is a two-dimensional plane, and cell growth will be squeezed, which limits the gas-liquid exchange and further expansion of the cells; while under the method of the present invention, the cells are distributed more evenly on the hydrogel carrier, the cells are rounded, the hydrogel carrier provides a spherical three-dimensional growth space, the number of cells is larger, and the expansion effect is better. Liver cells are the most sensitive cells to shear force among adherent cells. The better growth of liver cells is enough to show that the method of the present invention can be used for in vitro expansion of adherent cells with better results.

Claims

1. A method for in vitro expansion of adherent cells using a stirred bioreactor in combination with a three-dimensional porous hydrogel carrier, characterized in that: Specifically, the hydrogel carrier is placed in a stirred bioreactor, cell culture medium is added, and ventilation is performed. Then the cells are inoculated and the stirred bioreactor is started for culture. During the culture process, the color change of the culture medium is observed. When the culture medium turns yellow, the medium is replaced to ensure the nutrients required for cell culture.

2. The method according to claim 1, characterized in that The hydrogel carrier and stirred tank bioreactor were sterilized.

3. The method according to claim 1, characterized in that The hydrogel carrier is a sulfonated bacterial cellulose / gelatin hydrogel composited with sulfonated bacterial cellulose and gelatin.

4. The method according to claim 1, characterized in that: The density of the hydrogel carrier is 0.4~1g / cm 3 The usage density of the hydrogel carrier is 3~10g / L.

5. The method according to claim 1, characterized in that The inoculated cells are stem cells, and the inoculation density is 3~5×10 5 cells / ml, the operating parameters of the stirred bioreactor are: pH 7.0~7.5, temperature 35~37℃, dissolved oxygen 25%~75%, stirring speed 50~300rpm; the culture time is 12~18 days.

6. The method according to claim 1, characterized in that The inoculated cells are Vero cells, and the inoculation density is 3~5×10 5 cells / ml, the operating parameters of the stirred tank bioreactor are: pH 7.0~7.5, temperature 35~37℃, dissolved oxygen 35%~75% and stirring speed 50~300rpm; the culture time is 4~9 days.

7. The method according to claim 1, characterized in that The inoculated cells are liver cells, and the inoculation density is 3~5×10 5 cells / ml, the operating parameters of the stirred bioreactor are: pH 7.0~7.5, temperature 35~37℃, dissolved oxygen 25%~75%, stirring speed 50~300rpm; the culture time is 12~18 days.

8. The method according to claim 1, characterized in that After the culture is completed, the hydrogel carrier is sucked out, and the cells adherently grown on the hydrogel carrier are digested with trypsin and then eluted.

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