MSCs culture process for improving yield of protein polymer

Abstract

The invention belongs to the field of biological pharmacy, and discloses an MSCs (mesenchymal stem cells) culture process for improving the yield of protein polymers, which comprises the following steps: resuscitating and inoculating mesenchymal stem cells, transferring the mesenchymal stem cells into a culture container added with a microcarrier for rotating or shaking culture, and aseptically transferring the cultured mesenchymal stem cells into a culture bag; performing irradiation stimulation on the culture bag; after stimulation is completed, the mesenchymal stem cells are split, separated and purified, and the protein polymer is obtained. According to the MSCs culture process of some examples, more MSCs can be obtained, and more protein polymers can be obtained through purification.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a MSCs culture process for improving the yield of protein polymers. Background Art

[0002] The protein polymers produced by mesenchymal stem cells (MSCs) after irradiation stimulation have multiple activities, especially good nerve repair function, and have good therapeutic effects on neurodegenerative diseases. CN2024109443259 discloses a laboratory preparation method of protein polymers. However, the laboratory preparation method has extremely low yield and high cost.

[0003] Protein polymers are produced by MSCs after irradiation stimulation and culture. Increasing the number of MSCs is conducive to obtaining more protein polymers. However, MSCs are sensitive to culture conditions, and how to optimize culture conditions to obtain more MSCs is still a challenging task. Summary of the invention

[0004] The object of the present invention is to overcome at least one disadvantage of the prior art and provide a MSCs culture process for improving the yield of protein polymers.

[0005] The technical solution adopted by the present invention is:

[0006] A MSCs culture process for increasing the yield of protein polymers comprises the following steps:

[0007] Mesenchymal stem cell resuscitation and inoculation;

[0008] The inoculated mesenchymal stem cells are transferred into a culture container with microcarriers and cultured by rotation or shaking. After 60 to 108 hours of culture, the cells are aseptically transferred into a culture bag.

[0009] The culture bag was stimulated by irradiation;

[0010] After the stimulation is completed, the mesenchymal stem cells are lysed, separated and purified to obtain protein polymers.

[0011] In some examples of 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 .

[0012] In some examples of MSCs culture process, the dosage of the microcarrier is 2-5 g / L.

[0013] In some examples of MSCs culture process, the dosage of the microcarrier is 2-4 g / L.

[0014] In some examples of MSCs culture processes, the rotation speed is gradually increased from 40 to 60 rpm to 70 to 90 rpm within 20 to 30 hours.

[0015] In some examples of MSCs culture process, the dosage of the microcarrier is 2-4 g / L, and the rotation speed is gradually increased from 40-60 rpm to 70-82 rpm within 24 hours.

[0016] In some examples of MSCs culture processes, the culture vessel used is a stirred reactor.

[0017] In some examples of MSCs culture processes, the culture medium used for resuscitation of mesenchymal stem cells is a complete MSCs culture medium.

[0018] In some examples of MSCs culture processes, the culture medium used during irradiation stimulation culture is a serum-free MSCs culture medium.

[0019] In some examples of 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.

[0020] The above features can be combined arbitrarily unless they conflict with each other.

[0021] The beneficial effects of the present invention are:

[0022] The MSCs culture process of some examples of the present invention can obtain more MSCs and purify more protein polymers. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 are the cell growth curves under different inoculation modes.

[0024] Figure 2 This is the cell growth curve at different seeding densities.

[0025] Figure 3 This is the cell growth curve with different microcarrier dosages.

[0026] Figure 4 These are the cell growth curves under different reactors.

[0027] Figure 5 This is the cell growth curve under different reactor speeds. DETAILED DESCRIPTION

[0028] A MSCs culture process for increasing the yield of protein polymers comprises the following steps:

[0029] Mesenchymal stem cell resuscitation and inoculation;

[0030] The inoculated mesenchymal stem cells are transferred into a culture container with microcarriers and cultured by rotation or shaking. After 60 to 108 hours of culture, the cells are aseptically transferred into a culture bag.

[0031] The culture bag was stimulated by irradiation;

[0032] After the stimulation is completed, the mesenchymal stem cells are lysed, separated and purified to obtain protein polymers.

[0033] In some examples of 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 Preferably, the seeding density is (2.0-2.5)E+5 cells / ml or (1.4-1.8)E+04 cells / cm 2 The data showed that at this seeding density, more cells could be harvested after 96 h of culture.

[0034] In some examples of MSCs culture process, the dosage of the microcarrier is 2-5 g / L.

[0035] In some examples of MSCs culture process, the dosage of the microcarrier is 2-4 g / L.

[0036] The data showed that at the same culture time, when the dosage of microcarriers was 5g / L, the highest cell quantity was harvested, and the cells expanded by 2.7 times; when the dosage of microcarriers was 4g / L, the cells expanded by 2.9 times; when the dosage of microcarriers was 3g / L, the cells expanded by 3.9 times. In terms of cell division and doubling, the cell state at 3g / L was the best.

[0037] In some examples of MSCs culture processes, the rotation speed is gradually increased from 40 to 60 rpm to 70 to 90 rpm within 20 to 30 hours.

[0038] In some examples of MSCs culture process, the dosage of the microcarrier is 2-4 g / L, and the rotation speed is gradually increased from 40-60 rpm to 70-82 rpm within 24 hours.

[0039] The data showed that more cells could be harvested when cultured at the optimal rotation speed.

[0040] In some examples of MSCs culture processes, the culture vessel used is a stirred reactor. Data show that compared with shaker culture, rotary stirring culture has better culture effects, more cell proliferation, and unexpected effects.

[0041] In some examples of MSCs culture processes, the culture medium used for resuscitation of mesenchymal stem cells is a complete MSCs culture medium, which can better resuscitate the mesenchymal stem cells.

[0042] In some examples of MSCs culture processes, the medium used in irradiation stimulation culture is serum-free MSCs medium, which can avoid the introduction of animal-derived components, which is beneficial for subsequent purification operations and can also improve the safety of protein polymers.

[0043] In some examples of 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.

[0044] The technical solution of the present invention is further explained below in conjunction with experiments.

[0045] For the convenience of comparison, some operations are unified as follows:

[0046] The cell recovery procedures are as follows:

[0047] Turn on the dry heat resuscitation instrument to preheat. After the preheating is completed, take out a piece of MSC cells, tear off the label, put the cryopreservation tube containing mesenchymal stem cells into the dry heat resuscitation instrument, and start thawing;

[0048] Transfer and dilute the thawed cells into a 15 ml centrifuge tube containing 9 ml of complete medium preheated at 37.0℃±1.0℃;

[0049] The diluted cell suspension was centrifuged at room temperature (125 × g) for 5 min;

[0050] Discard the supernatant and resuspend the cell pellets in 15 ml of complete medium preheated at 37.0°C ± 1.0°C. Mix well and take samples for counting. 3 cells / cm 2 Transfer to a T bottle and place the T bottle in a 37.0℃±1.0℃, 5.0%±0.5% CO2 incubator for culture.

[0051] The cell passaging / seeding procedures are unified as follows:

[0052] After 72h±2h of mesenchymal stem cell recovery culture, the supernatant was discarded, and the corresponding volume of DPBS was added to wash once, and the supernatant was discarded; then the corresponding volume of TrypLE was added to infiltrate the cells; the cells were digested in a 37.0℃±1.0℃, 5.0%±0.5% CO2 incubator for 2min~3min, and the cell shedding was observed. After the cells began to shedding, the corresponding volume of 37℃±1.0℃ preheated complete medium was taken to terminate the digestion, and the mixture was mixed by blowing, and 0.5ml was taken for counting, and the cell density and viability (greater than 90%) were recorded. According to the subculture density of 8.00×103 cells / cm 2 Calculate, transfer the corresponding cell suspension to a 50ml centrifuge tube, centrifuge at room temperature (125×g) for 5 minutes, discard the supernatant, resuspend the precipitate in complete medium, mix well by blowing, dilute to the corresponding volume with culture medium, transfer to a culture bottle, and then culture in a 37.0℃±1.0℃, 5%±0.5%CO2 incubator. Discard excess cells. Or transfer to a reactor aseptically for culture after diluting to the same volume.

[0053] For the sake of comparison, the MSCs culture medium used for the production of protein polymers after inoculation was uniformly 3D FloTrix mesenchymal stem cell serum-free culture medium produced by Beijing Huakan Biotechnology Co., Ltd. The microcarrier used was Cytodex-1 microcarrier produced by Cytiva.

[0054] Experiment 1: Effects of different seeding modes on MSCs culture

[0055] The inoculation mode is one of the key factors affecting growth. Long-term constant-speed culture shear force will have a greater impact on cells. Long-term intermittent culture will result in a short cell attachment time, affecting 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.

[0056] Table 1: Inoculation mode test process parameters

[0057]

[0058] Table 2: Inoculation mode test experimental groups

[0059] Experimental Group Culture mode 24h after inoculation Speed ​​24h after inoculation 1 Constant speed 40rpm 2 Interval 40rpm5min; 0rpm15min 3 Constant speed Half volume 4 Constant speed Full volume

[0060] Table 3: Inoculation mode test count results (cells / ml)

[0061] Cultivation time 0h 24h 48h 72h 96h Constant speed 1.06E+05 8.96E+04 3.34E+05 6.20E+05 NA Interval 1.06E+05 1.59E+05 3.16E+05 5.82E+05 5.51E+05 Half volume 2.10E+05 3.46E+05 3.27E+05 5.92E+05 9.59E+05 Full volume 2.10E+05 2.55E+05 4.52E+05 7.17E+05 1.14E+06

[0062] Inoculation mode test growth curve Figure 1 shown.

[0063] From Table 3 and Figure 1 It can be seen that under different inoculation modes and the same inoculation density, there is no significant difference in the growth curve results of constant speed inoculation (40rpm) and intermittent inoculation (40rpm, 5min; 0rpm, 25min). Considering the execution of the amplification process, constant speed inoculation was used in the follow-up. The cells were amplified 5.4 times by full volume inoculation and 4.6 times by half volume inoculation. The amount of cells harvested by full volume inoculation was slightly higher than that by half volume inoculation.

[0064] Experiment 2: Effect of seeding density on MSCs culture:

[0065] Cell density is also one of the key factors affecting growth. Too high density or too low density will affect the growth curve of cells, resulting in a reduction 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 on the Spinner. Starting from the conventional inoculation density of the T bottle, the growth of higher cell density on the Spinner was investigated. 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 inoculation density experimental counting results are shown in Table 6.

[0066] Table 4: Inoculation density test process parameters

[0067]

[0068] Table 5: Inoculation density experimental groups

[0069] Experimental Group <![CDATA[Inoculation density (cells / cm 2 )]]> Seeding density (cells / ml) 1 8.00E+03 1.05E+05 2 1.60E+04 2.11E+05 3 2.00E+04 2.64E+05

[0070] Table 6: Seeding density test counting results (cells / ml)

[0071] Experimental Group 0h 4h 24h 48h 72h 96h 1 1.05E+05 1.01E+05 1.59E+05 3.16E+05 5.82E+05 5.51E+05 2 2.11E+05 1.73E+05 2.58E+05 4.95E+05 9.55E+05 NA 3 2.64E+05 2.35E+05 3.95E+05 7.08E+05 7.77E+05 7.59E+05

[0072] Cell growth curves of different experimental groups are shown in Figure 2. Figure 2 As shown in Table 6 and Figure 2 It can be seen that under the conditions of different inoculation densities and the same inoculation mode, the inoculation density is 2.11E+5 cells / ml (1.60E+4 cells / cm 2 ) is the experimental group with the highest cell density at 96h harvest. This inoculation density was selected for subsequent process optimization.

[0073] Experiment 3: Effect of microcarrier dosage on MSCs culture

[0074] The higher the amount of microcarriers used, the better. A smaller amount of microcarriers will limit cell growth, and an excessive amount of microcarriers will reduce the utilization rate of microcarriers. This experiment investigated the difference in cell growth on the Spinner with different microcarrier concentrations. The basic process parameters of the microcarrier dosage experiment are shown in Table 7, the microcarrier dosages of different experimental groups are shown in Table 8, and the experimental results are shown in Table 9.

[0075] Table 7: Microcarrier dosage experimental process parameters

[0076]

[0077] Table 8: Microcarrier dosage experimental groups

[0078] Experimental Group Microcarrier dosage (g / L) 1 3.0 2 4.0 3 5.0

[0079] Table 9: Microcarrier dosage experiment counting results (cells / ml)

[0080] Experimental Group 0h 24h 48h 72h 96h 1 2.00E+05 3.64E+05 7.80E+05 6.43E+05 4.70E+05 2 2.80E+05 3.44E+05 8.12E+05 6.56E+05 5.02E+05 3 3.50E+05 4.94E+05 9.45E+05 7.30E+05 6.88E+05

[0081] The growth curves of cells in different experimental groups are shown in Figure 2. Figure 3 As shown in Table 9 and Figure 3 It can be seen that at the same culture time, 5g / L harvested the highest cell mass, and the cells expanded 2.7 times; 4g / L expanded 2.9 times, and 3g / L expanded 3.9 times. In terms of cell division and doubling, 3g / L had the best cell state.

[0082] Experiment 4: Effects of the reactor on MSCs culture

[0083] During cell culture, different culture containers are one of the key factors affecting cell growth. This experiment tests the effects of different reactors on cell growth. The basic process parameters of the 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.

[0084] Table 10: Reactor process parameters

[0085]

[0086] Table 11: Reactor process experimental group

[0087] Experimental Group Reactor Type 1 Wave arm reactor (WAVE) 2 Stirred reactor (3L reactor)

[0088] Table 12: Reactor counting results (cells / ml)

[0089] Experimental Group 0h 5h 24h 48h 72h 96h 1 2.11E+05 3.03E+05 2.74E+05 1.88E+05 2.40E+05 2.44E+05 2 2.11E+05 1.54E+05 4.23E+05 5.00E+05 6.00E+05 6.01E+05

[0090] Growth curves of cells in different reactors Figure 4 As shown in Table 12 and Figure 4 The data showed that the WAVE reactor did not double as expected, and the growth curve of the 3L reactor was consistent with the previous spinner trend. The 3L reactor was used for subsequent tests.

[0091] Experiment 5: Effect of culture speed on MSCs culture

[0092] During the reactor culture process, the rotation speed is one of the key factors affecting cell growth. Too fast a rotation speed will cause excessive shear force to affect cell growth, and too low a rotation speed will cause microcarrier sedimentation, resulting in restricted cell growth. Therefore, it is very important to screen a suitable reactor speed. This experiment tested the effect of different rotation speeds on the reactor on cell growth. The basic process parameters of the reactor culture speed experiment are shown in Table 13, the speed settings of different experimental groups are shown in Table 14, and the experimental results are shown in Table 15.

[0093] Table 13: Process parameters of reactor culture speed

[0094]

[0095] Table 14: Experimental groups of reactor culture speed

[0096] Experimental Group Start training speed Increase the speed 1 55rpm 24h increase to 80rpm 2 55rpm Increase to 100rpm in 24h 3 55rpm 48h increase to 80rpm

[0097] Table 15: Reactor culture speed counting results (cells / ml)

[0098] Experimental Group 4h 24h 48h 72h 96h 1 1.51E+05 2.61E+05 4.45E+05 5.59E+05 2.40E+05 2 1.92E+05 2.11E+05 2.72E+05 3.17E+05 2.92E+05 3 1.43E+05 1.86E+05 3.86E+05 4.10E+05 2.37E+05

[0099] The growth curves of cells at different culture speeds are as follows Figure 5 As shown. Figure 5 As shown in Table 15, increasing the rotation speed to 80 rpm for 24 h has the best effect.

[0100] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A MSCs culture process for improving protein polymer production, characterized in that: The steps include: Mesenchymal stem cell resuscitation and inoculation; The inoculated mesenchymal stem cells are transferred into a culture container with microcarriers and cultured by rotation or shaking. After 60 to 108 hours of culture, the cells are aseptically transferred into a culture bag. The culture bag was stimulated by irradiation; After the stimulation is completed, the mesenchymal stem cells are lysed, separated and purified to obtain protein polymers.

2. The MSCs culture process according to claim 1, characterized in that: 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 .

3. The MSCs culture process according to claim 1, characterized in that: The dosage of the microcarrier is 2-5 g / L.

4. The MSCs culture process according to claim 3, characterized in that: The dosage of the microcarrier is 2-4 g / L.

5. The MSCs culture process according to claim 1, characterized in that: The rotation speed is gradually increased from 40 to 60 rpm to 70 to 90 rpm within 20 to 30 hours.

6. The MSCs culture process according to any one of claims 1 to 5, characterized in that: The dosage of the microcarrier 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 to 5, characterized in that: The culture vessel used was a stirred reactor.

8. The MSCs culture process according to any one of claims 1 to 5, characterized in that: The culture medium used for mesenchymal stem cell recovery is complete MSCs culture medium.

9. The MSCs culture process according to any one of claims 1 to 5, characterized in that: The culture medium used for irradiation stimulation culture was serum-free MSCs culture medium.

10. The MSCs culture process according to any one of claims 1 to 5, characterized in that: 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.

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