MSC culture process for improving yield of protein polymer
By optimizing the MSCs culture process, including adjusting the inoculation density, microcarrier dosage, rotation speed, and culture medium type, and using a stirred reactor for rotary culture and irradiation stimulation, the problem of low protein polymer yield in existing technologies has been solved, achieving more efficient protein polymer production and improved safety.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2026-04-02
AI Technical Summary
In existing technologies, the yield of protein polymers produced by culturing mesenchymal stem cells (MSCs) is low and the cost is high. Optimizing culture conditions to obtain more MSCs and increase the yield of protein polymers is a challenge.
By optimizing the culture process of MSCs, including adjusting the inoculation density, microcarrier dosage, rotation speed, culture container type and culture medium type, a stirred reactor was used for rotary culture, and irradiation stimulation was carried out in the culture bag. Finally, protein polymers were obtained by lysis separation and purification.
This enabled more efficient acquisition of MSCs, increased protein polymer yield, reduced costs, and improved safety and efficiency of purification procedures.
Smart Images
Figure CN2025120958_02042026_PF_FP_ABST
Abstract
Description
MSCs culture process for improving protein polymer yield
[0001] The present disclosure claims priority to the Chinese patent application No. 202411370250.4, filed on September 29, 2024, entitled "MSCs culture process for improving protein polymer yield", the whole content of the above application is incorporated herein by reference. TECHNICAL FIELD
[0002] The present application belongs to the field of biopharmaceuticals, and specifically relates to a MSCs culture process for improving protein polymer yield. BACKGROUND
[0003] The protein polymer produced by mesenchymal stem cells (MSCs) after irradiation stimulation has multiple activities, especially good nerve repair function, and has good therapeutic effect on neurodegenerative diseases. The laboratory preparation method of the protein polymer is disclosed in CN2024109443259. However, the laboratory preparation method has extremely low yield and high cost.
[0004] The protein polymer is produced by MSCs after irradiation stimulation, and the increase of the number of MSCs is conducive to obtaining more protein polymers. However, MSCs are sensitive to culture conditions, and how to optimize the culture conditions to obtain more MSCs is still a challenging work. SUMMARY
[0005] The present application aims to overcome at least one deficiency of the prior art and provide a MSCs culture process for improving protein polymer yield.
[0006] The technical solution adopted by the present application is as follows:
[0007] The MSCs culture process for improving protein polymer yield comprises the following steps:
[0008] Resuscitation and inoculation of MSCs;
[0009] The inoculated MSCs are transferred into a culture container added with microcarriers for rotation or shaking culture, and then are aseptically transferred into a culture bag after 60h-108h of culture;
[0010] Irradiation stimulation is performed on the culture bag;
[0011] After the stimulation is completed, the MSCs are lysed to obtain protein polymers.
[0012] In some examples of the MSCs culture process, the seeding density of the mesenchymal stem cells is (1.5-5.0)E+5 cells / mL or 5E+3-5E+4 cells / cm 2 .
[0013] In some examples of the MSCs culture process, the amount of the microcarrier used is 2-5 g / L.
[0014] In some examples of the MSCs culture process, the amount of the microcarrier used is 2-4 g / L.
[0015] In some examples of the MSCs culture process, the rotation speed is gradually increased from 40-60 rpm to 70-90 rpm within 20-30 h.
[0016] In some examples of the MSCs culture process, the amount of the microcarrier used is 2-4 g / L, and the rotation speed is gradually increased from 40-60 rpm to 70-82 rpm within 24 h.
[0017] In some examples of the MSCs culture process, the culture vessel used is a stirred reactor.
[0018] In some examples of the MSCs culture process, the medium used for resuscitation of the mesenchymal stem cells is a complete MSCs culture medium.
[0019] In some examples of the MSCs culture process, the medium used for irradiation stimulation culture is a serum-free MSCs culture medium.
[0020] In some examples of the MSCs culture process, the mesenchymal stem cells are selected from umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and human placenta-derived mesenchymal stem cells.
[0021] The above features can be combined arbitrarily without conflict.
[0022] The present application has the following advantages:
[0023] The MSCs culture process of some examples of the present application can obtain more MSCs and purify more protein polymers. BRIEF DESCRIPTION OF DRAWINGS
[0024] Fig. 1 is a cell growth curve under different seeding modes.
[0025] Fig. 2 is a cell growth curve under different seeding densities.
[0026] Fig. 3 is a cell growth curve under different amounts of microcarriers.
[0027] Figure 4 is a cell growth curve under different reactors.
[0028] Figure 5 is a cell growth curve under different rotation speeds of the reactor. DETAILED DESCRIPTION
[0029] A MSCs culture process for improving the yield of protein polymer, comprising the following steps:
[0030] Resuscitation and inoculation of mesenchymal stem cells;
[0031] After inoculation, the mesenchymal stem cells are transferred into a culture container added with microcarriers for rotation or shaking culture, and after 60h-108h of culture, the mesenchymal stem cells are aseptically transferred into a culture bag;
[0032] Irradiation stimulation is performed on the culture bag;
[0033] After the stimulation is completed, the mesenchymal stem cells are lysed, and protein polymer is obtained by separation and purification.
[0034] In some examples of the MSCs culture process, the inoculation density of the mesenchymal stem cells is (1.5-5.0)E+5 cells / mL or 5E+3-5E+4 cells / cm 2 . Preferably, the inoculation density is (2.0-2.5)E+5 cells / mL or (1.4-1.8)E+04 cells / cm 2 .
[0035] In some examples of the MSCs culture process, the inoculation density of the mesenchymal stem cells is (2.0-2.11)E+5 cells / mL or (1.4-1.6)E+4 cells / cm 2 , and data show that under this inoculation density, more cells can be harvested after 96h of culture.
[0036] In some examples of the MSCs culture process, the amount of the microcarriers is 2g / L-5g / L.
[0037] In some examples of the MSCs culture process, the amount of the microcarriers is 2g / L-4g / L.
[0038] Data show that under the same culture time, the highest cell amount is harvested when the amount of the microcarriers is 5g / L, the cell expansion is 2.7 times; when the amount of the microcarriers is 4g / L, the expansion is 2.9 times, and when the amount of the microcarriers is 3g / L, the cell expansion is 3.9 times. In terms of cell division, the cell state is best when the amount of the microcarriers is 3g / L.
[0039] In some examples of the MSCs culture process, the rotation speed is gradually increased from 40rpm-60rpm to 70rpm-90rpm within 20h-30h.
[0040] In some examples of the MSCs culture process, the microcarriers are used in an amount of 2-4 g / L, and the rotation speed is gradually increased from 40-60 rpm to 70-82 rpm within 24 hours.
[0041] The data show that more cells can be harvested when cultured at the preferred rotation speed.
[0042] In some examples of the MSCs culture process, a stirred reactor is used as the culture vessel. The data show that, compared with the shaker culture, the rotation and stirring culture has a better culture effect and more cell proliferation, which is unexpected.
[0043] In some examples of the MSCs culture process, the medium used for the recovery of mesenchymal stem cells is complete MSCs culture medium. This can better recover the mesenchymal stem cells.
[0044] In some examples of the MSCs culture process, the medium used for the irradiation stimulation culture is serum-free MSCs culture medium. This can avoid the introduction of animal-derived components, which is beneficial for subsequent purification operations and can improve the safety of protein polymers.
[0045] In some examples of the MSCs culture process, the mesenchymal stem cells are selected from umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and human placenta-derived mesenchymal stem cells.
[0046] The present application optimizes the MSCs culture process conditions to obtain a large number of MSCs, and further irradiates them to produce more protein polymers.
[0047] The technical solutions of the present application are further illustrated below in combination with experiments.
[0048] For convenience of comparison, some operations are unified as follows:
[0049] The cell recovery operation is unified as follows:
[0050] Start the dry heat recovery instrument and preheat it. After preheating, take one MSC cell, tear off the label, and put the cryopreserved tube containing the mesenchymal stem cells into the dry heat recovery instrument to start thawing.
[0051] Transfer the thawed cells to a 15 mL centrifuge tube containing 9 mL of 37.0℃±1.0℃ preheated complete culture medium.
[0052] Centrifuge the diluted cell suspension at room temperature (125×g) for 5 min.
[0053] Discard the supernatant, and resuspend the cell pellet in 15 mL of preheated complete medium at 37.0°C ± 1.0°C. After mixing, take a sample for counting, and according to 8.00 x 10 3 cells / cm 2 Transfer to a T flask and place the T flask in a 37.0°C ± 1.0°C, 5.0% ± 0.5% CO2 incubator for culture.
[0054] The operation of cell passage / inoculation is as follows:
[0055] After culturing the mesenchymal stem cells for 72 h ± 2 h, discard the supernatant, wash once with the corresponding volume of DPBS (Dulbecco's Phosphate Buffered Saline), and discard the supernatant. Then, add the corresponding volume of TrypLE to infiltrate the cells. Digest the cells in a 37.0°C ± 1.0°C, 5.0% ± 0.5% CO2 incubator for 2 min to 3 min, and observe the cell shedding during the period. After the cells begin to shed, stop the digestion by adding the corresponding volume of preheated complete medium at 37.0°C ± 1.0°C, mix well by blowing, take 0.5 mL for counting, record the cell density and viability (greater than 90%), and inoculate according to the passage density of 8.00 x 10 3 cells / cm 2 Perform the calculation, transfer the corresponding cell suspension to a 50 mL centrifuge tube, centrifuge at room temperature (125 x g) for 5 min, discard the supernatant, resuspend the pellet with complete medium, mix well by blowing, and then transfer to the culture flask after the volume is adjusted to the corresponding volume with the culture medium. Then, place the flask in a 37.0°C ± 1.0°C, 5% ± 0.5% CO2 incubator for culture. Discard the excess cells. Or, after adjusting the volume, transfer the culture medium to the reactor for culture in a sterile manner.
[0056] For convenience of comparison, the MSCs used for producing protein polymers after inoculation are uniformly MSCs culture medium produced by Beijing Huagang Biological Technology Co., Ltd. The microcarriers used are Cytodex-1 microcarriers produced by Cytiva Co.
[0057] Experiment 1: Effect of different inoculation modes on MSCs culture
[0058] Inoculation mode is one of the key factors affecting growth. Long-term constant-speed culture shear force has a great impact on cells. Long-term intermittent culture results in short cell adhesion time and affects growth. The basic parameters of the inoculation mode test process are shown in Table 1, the inoculation modes of different experimental groups are shown in Table 2, and the counting results of the inoculation mode test experiment are shown in Table 3.
[0059] Table 1: Inoculation mode test process parameters
[0060] Table 2: Inoculation mode test experimental groups
[0061] Table 3: Inoculation mode test counting results (cells / mL)
[0062] The inoculation mode test growth curves are shown in Figure 1.
[0063] As can be seen from Table 3 and Figure 1, under the conditions of different inoculation modes and the same inoculation density, the growth curve results of constant speed inoculation (40 rpm) and intermittent inoculation (40 rpm, 5 min; 0 rpm, 15 min) showed no significant difference. Considering the execution of the scale-up process, constant speed inoculation was subsequently selected. The cells inoculated in the full volume expanded by 5.4 times, and the cells inoculated in the half volume expanded by 4.6 times. The amount of cells harvested by full volume inoculation was slightly higher than that by half volume inoculation.
[0064] Experiment 2: Effect of inoculation density on MSC culture
[0065] The density of the cells is also one of the key factors affecting growth. Too high or too low density will affect the growth curve of the cells, resulting in a decrease in the amount of harvested cells and thus affecting the final protein yield. The purpose of this experiment is to study the effect of inoculation density on cell growth in a spinner. The conventional inoculation density in a T flask was used as a starting point to investigate the growth of higher cell density in a spinner. The basic parameters of the inoculation density test process are shown in Table 4, the inoculation densities of different experimental groups are shown in Table 5, and the counting results of the inoculation density experiment are shown in Table 6.
[0066] Table 4: Inoculation density test process parameters
[0067] Table 5: Inoculation density experimental groups
[0068] Table 6: Inoculation density experiment counting results (cells / mL)
[0069] The cell growth curves of different experimental groups are shown in Figure 2. As can be seen from Table 6 and Figure 2, under the conditions of different inoculation densities and the same inoculation mode, the inoculation density of 2.11E+5 cells / mL (1.60E+4 cells / cm 2 ) was the experimental group with the highest cell density harvested at 96 h. This inoculation density was subsequently selected for the remaining process optimization.
[0070] Experiment 3: Effect of microcarrier dosage on MSC culture
[0071] The higher the amount of microcarriers is not better, the less amount of microcarriers will limit the growth of cells, and the excessive amount of microcarriers will reduce the utilization rate of microcarriers. This experiment investigates the difference of cell growth in spinner under different microcarrier concentrations. The basic parameters of microcarrier dosage experiment process are shown in Table 7, the microcarrier dosage of different experimental groups is shown in Table 8, and the experimental results are shown in Table 9.
[0072] Table 7: Microcarrier dosage experiment process parameters
[0073] Table 8: Microcarrier dosage experimental groups
[0074] Table 9: Microcarrier dosage experiment count results (cells / mL)
[0075] The growth curves of cells in different experimental groups are shown in Figure 3. As shown in Table 9 and Figure 3, at the same culture time, the highest cell amount is harvested at 5g / L, the cell expansion is 2.7 times; the expansion is 2.9 times at 4g / L, and the expansion is 3.9 times at 3g / L. In terms of cell division doubling, the cell state at 3g / L is the best.
[0076] Experiment 4: Effect of reactor on MSCs culture
[0077] During cell culture, different culture containers are one of the key factors affecting cell growth. This experiment tests the effect of different reactors on cell growth. The basic process parameters of reactor test experiment are shown in Table 10, the reactor types of different experimental groups are shown in Table 11, and the experimental results are shown in Table 12.
[0078] Table 10: Reactor process parameters
[0079] Table 11: Reactor process experimental groups
[0080] Table 12: Reactor count results (cells / mL)
[0081] The growth curves of cells in different reactors are shown in Figure 4. As shown in Table 12 and Figure 4, the WAVE reactor does not double as expected, and the growth curve of 3L reactor is consistent with the previous spinner trend. The 3L reactor is used for subsequent testing.
[0082] Experiment 5: Effect of culture speed on MSCs culture
[0083] The rotation speed is one of the key factors affecting cell growth during the reactor cultivation. Too high rotation speed will result in too large shear force affecting cell growth, and too low rotation speed will result in microcarrier settlement limiting cell growth. Therefore, it is crucial to screen the appropriate rotation speed of the reactor. The present experiment tests the influence of different rotation speeds on cell growth in the reactor. The basic process parameters of the reactor cultivation rotation speed experiment are shown in Table 13, the rotation speed settings of different experimental groups are shown in Table 14, and the experimental results are shown in Table 15.
[0084] Table 13: Process parameters of reactor cultivation rotation speed
[0085] Table 14: Experimental groups of reactor cultivation rotation speed
[0086] Table 15: Counting results of reactor cultivation rotation speed (cells / mL)
[0087] The cell growth curves under different cultivation rotation speeds are shown in Figure 5. As shown in Figure 5 and Table 15, increasing the rotation speed to 80 rpm at 24 h has the best effect.
[0088] The above is a further detailed description of the present application, which cannot be regarded as a specific implementation of the present application. For ordinary skilled persons in the technical field to which the present application belongs, simple deductions or replacements without departing from the concept of the present application are within the protection scope of the present application.
Claims
1. A culture process of MSCs for increasing the production of protein polymer, characterized in that, It comprises the following steps: Mesenchymal stem cell resuscitation and inoculation; After inoculation, the mesenchymal stem cells are transferred into a culture container added with microcarriers for rotation or shaking culture, and then are aseptically transferred into a culture bag after 60-108 hours of culture; Irradiation stimulation is performed on the culture bag; After the stimulation is completed, the mesenchymal stem cells are lysed to obtain protein polymers through separation and purification.
2. The MSCs culture process according to claim 1, wherein, The mesenchymal stem cells are seeded at a density of (1.5-5.0)E+5 cells / mL or 5E+3-5E+4 cells / cm2 2 ; Preferably, the mesenchymal stem cells are seeded at a density of (2.0-2.11)E+5 cells / mL or (1.4-1.6)E+4 cells / cm2. 2 .
3. The MSCs culture process of claim 1, wherein, The amount of the microcarriers is 2-5 g / L.
4. The MSCs culture process according to claim 3, wherein, The amount of the microcarriers is 2-4 g / L.
5. The MSCs culture process of claim 1, wherein, The rotation speed is gradually increased from 40-60 rpm to 70-90 rpm within 20-30 hours.
6. The MSCs culture process according to any one of claims 1-5, wherein, The amount of the microcarriers is 2-4 g / L, and the rotation speed is gradually increased from 40-60 rpm to 70-82 rpm within 24 hours.
7. The MSCs culture process according to any one of claims 1-5, wherein, The culture container used is a stirring reactor.
8. The MSCs culture process according to any one of claims 1-5, wherein, The culture medium used when the mesenchymal stem cells are resuscitated is complete MSCs culture medium.
9. The MSCs culture process according to any one of claims 1-5, wherein, The culture medium used when the culture is irradiated and stimulated is serum-free MSCs culture medium.
10. The MSCs culture process according to any one of claims 1-5, wherein, The mesenchymal stem cells are selected from umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells and human placenta-derived mesenchymal stem cells.