Plant source microcarrier, preparation method and application
Soy protein isolate microcarriers prepared by emulsification crosslinking method solve the problems of high cost, complex process and safety of animal-derived microcarriers in the existing technology, realize low cost and high efficiency of cell culture and recovery, and are suitable for applications of a variety of cell types.
Patent Information
- Application Number
- CN202511170766.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-20
- Publication Date
- 2025-11-07
AI Technical Summary
Existing commercial microcarriers use animal-derived materials as a matrix, which are characterized by high cost, complex processes, difficulties in cell recovery, and biosafety risks, making it difficult to meet the demand for low-cost and high-efficiency cell culture.
Using soy protein isolate as a matrix, solid microspheres with a particle size of 50–350 μm were prepared by emulsification cross-linking. Dispersant enzyme II was used to gently recover cells, simplifying the operation and reducing costs.
We provide low-cost, high-safety soy protein isolate microcarriers that support various cell growth processes, have high cell recovery rates, reduce production costs, eliminate the risk of animal-derived contamination, and are suitable for various cell culture fields.
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Figure CN120905115A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological materials, in particular to a plant-derived microcarrier, a preparation method and an application thereof. BACKGROUND
[0002] Three-dimensional (3D) cell culture technology has important value in the fields of drug screening, regenerative medicine and cell therapy due to its ability to simulate the in vivo microenvironment. Microcarriers, as the core tool for 3D culture, directly affect the cell expansion efficiency and downstream application effect. Currently, commercial microcarriers (such as Cytodex series) are mostly based on animal-derived materials such as collagen and gelatin, which have the following defects: ① high cost: animal-derived materials and complex processes increase the cost of industrial application; ② process complexity: surface modification (such as polylysine coating) is required to promote cell adhesion, increasing production costs; ③ difficulty in cell recovery: traditional enzyme digestion methods (such as trypsin) can easily damage cell activity and make it difficult to completely separate the cell-carrier complex; ④ biological safety risk: animal-derived components may carry pathogens (such as viruses and prions), leading to product contamination risk.
[0003] In recent years, plant-derived proteins have become a highly potential microcarrier preparation matrix material due to their low cost, excellent biocompatibility, degradability, low immunogenicity and rich active groups. However, how to prepare and obtain a certain plant-derived protein microcarrier preparation matrix material is a hot and painful point in the industry. SUMMARY
[0004] To solve the technical problems mentioned in the background art, the present application provides a plant-derived solid microsphere carrier, a preparation method and an application thereof, and the technical solution is as follows:
[0005] The plant-derived microcarrier is derived from soybean protein isolate, and the microcarrier is a solid microsphere with a particle size of 50-350 μm and a density of 1.03 g / cm 3 .
[0006] The application of the plant-derived microcarrier as described above is used for cell culture.
[0007] Preferably, the cells are any one of VERO cells, SHED cells or C2C12 cells.
[0008] Preferably, the cells can be recovered by lysing the microcarriers with a lysis solution.
[0009] The preparation method of the plant-derived microcarrier comprises the following steps:
[0010] S1: adding a surfactant to the oil phase to prepare a surfactant-containing oil phase solution; adding a crosslinking agent to the water phase solution and mixing uniformly;
[0011] S2: adding the aqueous phase solution into the oil phase solution containing surfactant, and emulsifying and cross-linking to prepare a water-in-oil emulsified mixed solution by stirring method;
[0012] S3: after the emulsifying and cross-linking, inactivating the cross-linking agent, and after the system is cooled to room temperature, washing to remove the oil phase to obtain the microcarriers.
[0013] Preferably, in S1, the aqueous phase is 5.0-10.0% soybean protein isolate aqueous solution, which is treated by ultrasonic defoaming before use.
[0014] Preferably, in S1, the aqueous phase is treated by ultrasonic defoaming before use.
[0015] Preferably, in S1, the cross-linking agent is transglutaminase, and the addition amount is 50-150 U / g of soybean protein isolate substrate.
[0016] Preferably, in S1, the oil phase is liquid paraffin, and the surfactant is Span 80, and the addition amount of the surfactant in the oil phase is 0.5-2.5%.
[0017] Preferably, in S2, the stirring speed is 400-800 rpm, and the emulsifying and cross-linking temperature is 50℃, and in S3, the inactivation temperature of the cross-linking agent after the emulsifying and cross-linking is completed is 90℃.
[0018] Preferably, in S1, the concentration of soybean protein isolate is 7.0%, the concentration of Span 80 is 1.5%, and the amount of transglutaminase is 100 U / g; and in S2, the stirring speed is 700 rpm.
[0019] The beneficial effects of the present application mainly include:
[0020] The present application provides a soybean protein isolate-based microcarrier for cell culture, which is low in cost, high in safety, and easy to recover cells, and is suitable for large-scale culture of adherent cells.
[0021] The present application provides a solid microcarrier, wherein the microcarrier is based on soybean protein isolate, the particle size of the solid microcarrier is 50-350 μm, the density of the solid microcarrier is 1.03 g / cm 3 .
[0022] The emulsifying and cross-linking method for preparing the solid microcarrier has the characteristics of simple operation, low equipment requirement, and easy scale-up; the raw material for preparing the microcarrier, soybean protein isolate, is widely available and low in price; the microcarrier can be used for cell culture without surface modification, and can be sterilized by simple high-temperature and high-pressure treatment; and the above characteristics reduce the production and use cost of the microcarrier.
[0023] The solid soybean protein isolate microcarrier matrix material provided by the application is of plant origin, and can eliminate the risk of animal source pollution and has good safety
[0024] The solid soybean protein isolate microcarrier provided by the application can gently and conveniently harvest cells by dispersin II lysis of the microcarrier, and the harvested cells can still maintain high activity, which will simplify the downstream processing operation of the microcarrier amplified cells.
[0025] The solid soybean protein isolate microcarrier provided by the application can support the growth of various types of cells, including VERO for vaccine production, SHED for regenerative medicine and C2C12 for artificial meat research, and has the potential to be applied in different cell culture fields as a multipurpose microcarrier. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 , a schematic diagram of preparing the solid soybean protein isolate microcarrier by the emulsification crosslinking method;
[0027] Figure 2 , an apparent morphology and particle size distribution diagram of the solid soybean protein isolate microcarrier;
[0028] Figure 3 , diagrams of the morphology and particle size distribution of the microcarrier before and after high-temperature high-pressure sterilization;
[0029] Figure 4 , changes in the particle size of the microcarrier in a simulated cell culture environment within 30 days;
[0030] Figure 5 , adhesion of VERO, SHED and C2C12 cells on the microcarrier after 24 hours of inoculation;
[0031] Figure 6 , changes in the density of VERO cells on the microcarrier in dynamic culture;
[0032] Figure 7 , states of the microcarrier under different lysis times;
[0033] Figure 8 , viability of SHED cells recovered by lysis of the microcarrier using dispersin II;
[0034] Figure 9 , state of C2C12 myogenic differentiation induction on the microcarrier for 7 days;
[0035] Figure 10 , Western blot detection result of myosin heavy chain (MyHC), a myogenic differentiation marker of C2C12 on the microcarrier. DETAILED DESCRIPTION
[0036] The technical solutions of the present application will be further described below in combination with specific embodiments. The technical solutions in the embodiments of the present application will be clearly and completely described below, and obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0037] Experimental materials and reagents:
[0038] Soybean protein isolate powder (manufacturer: Shanghai Yuan Ye Biotechnology Co., Ltd., item number: S30914); crosslinking agent TGase (glutamine transaminase) (manufacturer: Shanghai Yuan Ye Biotechnology Co., Ltd., item number: S10156); DMEM cell culture medium (manufacturer: Gibco, item number: 11965092); VERO cells (from the cell library of the laboratory); SHED cells (from the cell library of the laboratory); C2C12 cells (from the cell library of the laboratory); commercial microcarrier Cytodex 1 (manufacturer: Cytiva, item number: 17044801); Calcein-AM live cell staining working solution (manufacturer: Biyun Tian Biotechnology Co., Ltd., item number: C2013S); dispersinase II (manufacturer: Beijing Solabio Biotechnology Co., Ltd., item number: D6430); BCA kit (manufacturer: Biyun Tian Biotechnology Co., Ltd., item number: P0010); MyHC antibody (manufacturer: ABclonal, item number: A25464); GAPDH internal reference primary antibody (manufacturer: Proteintech, item number: 10494-1-AP); 647-conjugated Goat anti-Rabbit IgG (H+L) secondary antibody (manufacturer: ABclonal, item number: AS060).
[0039] Example 1
[0040] 1. Preparation of soybean protein isolate solution: 40 mL of deionized water was added to a 100 mL beaker, and 2.8 g of soybean protein isolate powder was accurately weighed using an analytical balance. The soybean protein isolate powder was added to the beaker in small amounts and stirred with a glass rod until there was no dry powder. Then a magnetic stirrer was used to continuously stir at a speed of 800 rpm for 1 h. The obtained solution was placed in an ultrasonic cell disruptor for ultrasonic dispersion to assist in dissolving and defoaming the SPI. The power was set to 200 W, the pulse was opened for 4 s and closed for 5 s, and the cycle was repeated 80 times. A 7.0% (w / v) soybean protein isolate solution was prepared.
[0041] 2. Preparation of solid soybean protein isolate microcarriers: Figure 1The preparation of the solid soybean protein isolate microcarrier in Example 1 is shown in the schematic diagram, and the preparation process is described in detail as follows: 1.5% (v / v) Span 80 is added into 15 mL liquid paraffin, and the mixture is placed on a constant temperature magnetic stirrer, the rotation speed is set to 700 rpm, and the temperature is set to 50°C. The mixture is stirred and heated for 15 min to fully mix and heat the system. According to the enzyme dosage of 100 U / g, the crosslinking agent TGase is added into 3 mL 7.0% soybean protein isolate solution, and the solution is mixed by inversion and then added into the liquid paraffin containing Span 80. The emulsion is crosslinked for 4 h. After the reaction is completed, the temperature of the constant temperature magnetic stirrer is adjusted to 90°C, and the temperature of the reaction solution is increased to 90°C and then maintained for 5 min to inactivate the TGase. Then, the reaction solution is transferred to an ice water bath for cooling. After the microspheres are settled, the upper liquid paraffin is discarded, and the obtained microspheres are washed with deionized water for three times until no obvious oil stains are observed. Then, deionized water containing 1% (v / v) Tween 80 is added into the washed microspheres, and the mixture is stirred at a low speed for 5 min on a magnetic stirrer to dissolve the residual oil stains. Then, the microspheres are washed with deionized water for three times to remove the oil phase. The morphology of the microspheres is observed by a microscope. Finally, the transparent spherical solid soybean protein isolate microcarriers are obtained, the particle size is 50-350 μm, and the density is 1.03 g / cm 3 ( Figure 2 )。
[0042] Example 2
[0043] 1. After the microcarriers are fully swelled by PBS, the microcarriers are placed in a high-pressure steam sterilization pot and sterilized at 121°C for 15 min. The morphology of the microspheres before and after sterilization is observed by a microscope, and the average particle size of the microspheres is statistically analyzed to detect the change in the particle size of the microspheres before and after high-temperature and high-pressure treatment. Figure 3 The results show that the morphology and particle size of the microcarriers do not change significantly before and after high-temperature and high-pressure sterilization, indicating that the particle size of the microspheres can still be maintained stable after high-temperature and high-pressure treatment.
[0044] 2. After the sterilized microcarriers are transferred to a 96-well plate, DMEM cell culture medium is added, and the plate is placed in a 37°C incubator to simulate a cell culture environment. The medium is changed every 3 days, and the particle size of the microspheres is recorded. The observation is continuously recorded for 30 days. Figure 4 The results show that the particle size of the microcarriers only fluctuates slightly within 30 days, and no significant change is observed, indicating that the soybean protein isolate microcarriers provided by the application can maintain stable morphology for a long time under cell culture conditions.
[0045] Example 3
[0046] VERO, SHED and C2C12 cells are inoculated on the sterilized microcarriers, and the state of the cells after adhesion for 24 h is shown in Figure 5 .
[0047] As can be seen from the figure, the three kinds of cells can be well attached and stretched on the soybean protein isolate microcarrier, and the cell adhesion rate is close to 100%, which is comparable to the commercial microcarrier Cytodex 1. It is shown that the soybean protein isolate microcarrier can well support the adhesion of cells, and the high adhesion rate means higher culture efficiency, more stable cell state and product quality, which is one of the core goals of the microcarrier process development.
[0048] Example 4
[0049] 1. VERO cells were dynamically cultured in a suspension culture flask, and commercial microcarrier Cytodex 1 was used as a control.
[0050] 2. Cell inoculation: Cytodex 1 and self-made solid soybean protein isolate microcarriers were soaked and swollen in pH 7.4 PBS buffer for 4 h, and the use concentration of the wet microcarriers after swelling equilibrium was 20 mg / mL. The pretreated microcarriers were transferred to the culture flask, and 10 mL of DMEM complete medium (90% DMEM + 9% FBS + 1% three-antibody) was added. Then 4.5 x 10 6 cells of VERO cells were added, and the medium was supplemented to 30 mL, and the initial density of cells in the culture system was 1.5 x 10 5 cells / mL. In the early stage of inoculation, the system was shaken at a speed of 50 rpm every 2 h for 2 min, and after 12 h of inoculation, the system was continuously rotated at a speed of 50 rpm for 9 d.
[0051] 3. The medium was replaced every 1-3 days according to the real-time density of cell culture. During the period, the sample was taken every 24 h, and the cell density was counted by citric acid-crystal violet staining solution. The specific operation was as follows: 1 mL of microcarrier suspension was taken, and the unattached cells in the suspension were filtered by a cell filter, and the precipitate on the filter was transferred to a centrifuge tube, which was washed twice with PBS, and then 1 mL of 0.1% citric acid-crystal violet staining solution was added to each tube, which was vortexed after 30 min of 37°C water bath, and the cell nucleus was counted by a cell counting plate, and the cell density was recorded.
[0052] 4、 Figure 6 The density change of cells on the two kinds of microcarriers during 9 d of continuous culture was reflected, and as can be seen from the figure, during the 9 d of dynamic culture, the cell density on the solid soybean protein isolate microcarrier was comparable to that on the commercial product Cytodex 1, which showed that the soybean protein isolate microcarrier provided by the application had excellent cell expansion performance.
[0053] Example 5
[0054] 1. Lysis of microcarriers and recovery of SHED cells: Soy protein isolate microcarriers containing SHED cells were washed twice with PBS, and then 250 μL of Calcein-AM live cell staining working solution was added. The microcarriers were incubated at 37°C in the dark for 1 hour. After incubation, the staining solution was aspirated, and the microcarriers were washed twice with PBS. Then, 500 μL of 2.5 U / mL dispersin II working solution was added to each well, and the microcarriers were incubated at 37°C to lyse them. The lysis was observed and photographed every 5 minutes under a fluorescence inverted microscope. The lysis status of the microcarriers is shown in the figure below. Figure 7 As shown in the figure, the solid soy protein isolate microcarriers can be completely lysed after 30 minutes.
[0055] 2. Viability assay of SHED cells recovered from microcarriers: Soy protein isolate microcarriers containing SHED cells were cultured for 3 days. The culture medium was discarded, and the cells were washed twice with PBS. 500 μL of 2.5 U / mL dispersin II working solution was added to each well, and the microcarriers were lysed in a 37℃ incubator. SPI MCs were incubated for 30 min, and SPI PMCs for 45 min. After lysis, the cell suspension was aspirated and added to centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in fresh culture medium. Trypan blue staining was used to calculate the viability of the recovered cells. SHED cells recovered from conventional 2D planar culture and trypsin digestion were used as a control. The viability of all recovered cells was >97%. Figure 8 The lack of significant difference indicates that the cells recovered from the soybean protein isolate microcarriers provided by this invention by lysing the microcarriers with dispersant enzyme II can still maintain a high cell viability.
[0056] Example 6
[0057] 1. Induction of myogenic differentiation of C2C12 cells on microcarriers: C2C12 cells were inoculated at a rate of 1×10⁻⁶ cells / cells. 5 Cells per well were seeded into 24-well ultra-low adsorption plates containing Cytodex 1 and solid soy protein isolate microcarriers, and co-cultured for 48 h. The proliferation medium was then replaced with myoblast differentiation medium, and the medium was changed every 2 days for 7 days of differentiation induction. Results are as follows: Figure 9 As shown in the figure, after 7 days of differentiation induction, the cells on the surface of the commercially available Cytodex 1 microcarrier detached, with only a small number of cells adhering to the surface. In contrast, on the soy protein isolate microcarrier, C2C12 cells proliferated and differentiated to form a very dense cell cluster. The cells connected multiple microcarriers together, and the surface and gaps of the carriers were filled with cells. Under the microscope, only a blurry outline of the microcarriers could be seen. This indicates that the soy protein isolate microcarrier can effectively support the growth of C2C12 cells, and its performance is far superior to that of the commercially available Cytodex 1 microcarrier, showing potential for application in artificial meat research.
[0058] 2. Western blot detection of MyHC expression in C2C12 cells differentiated on different carriers:
[0059] 1) After 7 days of myogenic differentiation induction, the cells were collected by digestion and washed twice with PBS, then added with cell lysis solution containing 1% PMSF, mixed and placed on ice for lysis for 30 min. The lysed sample was centrifuged at 4°C for 20 min, and the supernatant protein concentration was determined using a BCA kit. After determination, the three samples were diluted to the same protein concentration with PBS.
[0060] 2) SDS-PAGE electrophoresis was performed on the samples, and 5% concentrated gel and 8% separation gel were prepared. After the gel was prepared, 1x electrophoresis buffer was added. The protein sample was mixed with 4x loading buffer at a ratio of 3:1, and the mixture was boiled in boiling water for 15 min. After boiling, the sample was centrifuged at 5000 rpm for a short time.
[0061] 3) Marker 5 μL per well, sample 40 μg per well. Turn on the electrophoresis instrument, electrophoresis at 90V for 30 min, then switch to 130V for 1h, so that the protein bands are separated.
[0062] 4) After protein electrophoresis, transfer the membrane according to the order of sponge → filter paper → protein gel → PVDF membrane → filter paper → sponge, clamp them in order, and place them in the transfer slot for 2.5h at a current of 300mA.
[0063] 5) After transfer, wash the PVDF membrane with TBST three times, then add 5% skim milk powder and incubate at room temperature for 2h. After blocking, discard the blocking solution, wash the PVDF membrane with TBST three times, add the diluted MyHC and GAPDH primary antibody, and incubate on a slow shaker at 4°C overnight. After incubation, discard the primary antibody, wash with TBST three times, add the diluted secondary antibody, and incubate on a slow shaker at room temperature for 1h. After incubation, discard the secondary antibody, wash with TBST three times, add ECL developing solution, and observe under a gel imager.
[0064] The Western blot detection results of myogenic differentiation marker myosin heavy chain (MyHC) are as follows Figure 10As shown, the expression amount of MyHC of the cells on the soybean protein isolate microcarrier is significantly higher than that of Cytodex 1, which is 1.86 times of the control group, which indicates that C2C12 cells have a more excellent myogenic differentiation capacity on the solid soybean protein isolate microcarrier than Cytodex 1. The solid soybean protein isolate microcarrier is self-assembled with the cells to form a micro-tissue block under the connection of the myoblasts, and has a potential for constructing artificial meat tissue containing high-density myoblasts.
[0065] Experimental conclusion:
[0066] 1. The emulsification cross-linking method for preparing the solid soybean protein isolate microcarrier has the characteristics of simple operation, low equipment requirement and easy scale preparation; and the raw material of the microcarrier, soybean protein isolate, is widely available and cheap; the microcarrier can be used for cell culture without surface modification, and can be sterilized by simple high-temperature and high-pressure treatment, and the above characteristics reduce the production and use cost of the microcarrier.
[0067] 2. The solid soybean protein isolate microcarrier matrix material provided by the application is of plant origin, which eliminates the risk of animal source pollution and is safe
[0068] The solid soybean protein isolate microcarrier provided by the application can gently and conveniently harvest cells by dispersinase II lysis of the microcarrier, and the harvested cells can still maintain high activity, which will simplify the downstream processing operation of the microcarrier after cell expansion.
[0069] 3. The solid soybean protein isolate microcarrier provided by the application can support the growth of various types of cells, including VERO cells for vaccine production, SHED cells for regenerative medicine and C2C12 cells for artificial meat research, and has the potential to be used as a multipurpose microcarrier in different cell culture fields.
[0070] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.
Claims
1. Plant-derived microcarriers, characterized in that, It is derived from soybean protein isolate, and is a solid microsphere with a particle size of 50-350 μm and a density of 1.03 g / cm 3 .
2. Use of plant-derived microcarriers according to claim 1, characterized in that, For cell culture.
3. Use of plant-derived microcarriers according to claim 2, characterized in that, The cell is any one of VERO cell, SHED cell or C2C12 cell.
4. A method for the preparation of plant-derived microcarriers, characterized in that, The method comprises the following steps: S1: adding a surfactant into an oil phase to prepare a surfactant-containing oil phase solution; adding a crosslinking agent into an aqueous phase solution and mixing uniformly; S2: adding the aqueous phase solution into the surfactant-containing oil phase solution and preparing a water-in-oil emulsion mixed solution by emulsion crosslinking through stirring; S3: after the emulsion crosslinking, inactivating the crosslinking agent, and washing to remove the oil phase after the system is cooled to room temperature to separate the microcarrier.
5. The method for preparing plant-derived microcarriers as described in claim 4, characterized in that, In the S1, the aqueous phase is a 5.0-10.0% aqueous solution of soybean protein isolate, which is subjected to ultrasonic defoaming treatment before use.
6. The method for preparing plant-derived microcarriers according to claim 5, wherein, In the S1, the aqueous phase is subjected to ultrasonic defoaming treatment before use.
7. The method for preparing plant-derived microcarriers as described in claim 5, characterized in that, In the S1, the crosslinking agent is transglutaminase, and the addition amount is 50-150 U / g of soybean protein isolate substrate.
8. The method for preparing plant-derived microcarriers as described in claim 7, characterized in that, In the S1, the oil phase is liquid paraffin, and the surfactant is Span 80, and the addition amount of the surfactant in the oil phase is 0.5-2.5%.
9. The method for preparing plant-derived microcarriers according to claim 8, wherein, In the S2, the stirring speed is 400-800 rpm, and the emulsion crosslinking temperature is 50°C; in the S3, the inactivation temperature of the crosslinking agent after the emulsion crosslinking is completed is 90°C.
10. The method for preparing plant-derived microcarriers according to claim 9, wherein, In the S1, the concentration of soybean protein isolate is 7.0%, the concentration of Span 80 is 1.5%, and the amount of transglutaminase is 100 U / g; in the S2, the stirring speed is 700 rpm.