A double-network amphiphilic hydrogel microcarrier capable of maintaining cell stemness and application thereof

Abstract

The application discloses a kind of double-network amphiphilic hydrogel microcarriers capable of maintaining cell stemness and application thereof, and belongs to the technical field of cell culture.The present application copolymerizes SBMA with acrylamide (AAm) monomer to form a double-network structure with acrylic acid (PAA) to prepare hydrogel microcarriers.The double-network structure hydrogel microcarriers PSAm obtained based on amphiphilic materials can effectively expand MSCs while maintaining their cell stemness for a long time, which is conducive to long-term culture of MSCs.Compared with ordinary culture, the artificial subculture process can be reduced, manpower and material resources can be saved, resource waste can be avoided, and a new experience method is provided for long-term culture, preservation and maintenance of cell stemness of MSCs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, in particular to a double-network amphiphilic hydrogel microcarrier capable of maintaining cell stemness and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are star cells in the field of regenerative medicine, which are highly valued due to their self-renewal ability and multi-directional differentiation potential. MSCs can be differentiated into various tissue cells other than hematopoietic cells in vitro under specific induction conditions, especially tissue cells derived from mesoderm and neural ectoderm. However, the differentiation mechanism of MSCs is complex, and the control of differentiation efficiency and differentiation direction is still a difficulty in current research.

[0003] In order to improve the yield and differentiation ability of MSCs, researchers are constantly exploring new culture systems and carrier materials. The commercial microcarriers for culturing cells such as Cytodex-3, SoloHill and Cultispher have been used for industrialized MSCs production. However, long-term culture may lead to aging or differentiation of MSCs, affecting their pluripotency and immunomodulatory ability. The existing microcarriers are basically developed for efficient proliferation of cells, but they cannot guarantee the long-term maintenance of stemness of stem cells. Therefore, it is particularly important to develop a microcarrier that can efficiently proliferate MSCs and maintain their stemness for a long time. SUMMARY

[0004] The purpose of the present application is to provide a double-network amphiphilic hydrogel microcarrier capable of maintaining cell stemness and application thereof, in order to solve the problems existing in the prior art. The double-network structure hydrogel microcarrier PSAm constructed based on amphiphilic materials can effectively expand MSCs, while maintaining their cell stemness for a long time, which is conducive to long-term expansion of MSCs, reduces the process of manual subculture, saves manpower and resources, avoids waste of resources, and provides a new experience method for long-term culture, preservation and maintenance of cell stemness of MSCs.

[0005] To achieve the above purpose, the present application provides the following scheme:

[0006] The present application provides a double-network amphiphilic hydrogel microcarrier capable of maintaining cell stemness, which comprises the following raw materials: oil phase, water phase, initiator, sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate and collagen.

[0007] The water phase comprises: acrylic acid, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, acrylamide, N,N'-methylenebisacrylamide and water.

[0008] In the present application, the 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl] ammonium] propane-1-sulfonic acid inner salt (SBMA) is copolymerized with acrylamide (AAm) monomer to form a first network, and the second network of the acrylic acid (PAA), after a step polymerization reaction, to form a PSAm hydrogel; wherein the hydrogen bond formed by the amino group on the acrylamide and the ionic bond formed by the anion and cation side groups on the SBMA stably connect the first network and the second network, improving the mechanical properties and stability of the hydrogel. After the amide group on the PAAm chain in the PSAm hydrogel reacts with sulfo-SANPAH, collagen can be connected, improving the adhesion of the hydrogel and facilitating the colonization and growth of cells.

[0009] Further, the oil phase includes paraffin oil, Span 80, and Tween-80.

[0010] Further, in the water phase, the mass ratio of the acrylic acid, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl] ammonium] propane-1-sulfonic acid inner salt, acrylamide, N,N'-methylene bisacrylamide, and water is (0.05-0.2):(1.0-1.5):(0.2-0.4):(0.01-0.02):(39.9-40).

[0011] Further, in the oil phase, the volume ratio of the paraffin oil, Span 80, and Tween-80 is (4-6):(3-5):1.

[0012] Further, the mass of the initiator is 25%-35% of the mass of the acrylic acid.

[0013] Optionally, the initiator is selected from any one of ammonium persulfate (APS), azobisisobutyronitrile, azobisisoheptyl nitrile, dimethyl azobisisobutyrate, azobisisobutyl amidoxime hydrochloride, hydrogen peroxide, potassium persulfate, and tert-butyl hydroperoxide.

[0014] Further, in the double-network amphiphilic hydrogel microcarrier, the concentration of the collagen is 1.5-3 mg / mL.

[0015] Optionally, the collagen is selected from any one of type I collagen, type II collagen, type III collagen, type V collagen, and type XI collagen.

[0016] Further, the volume ratio of the water phase and the oil phase is 1:(3-5).

[0017] The present application also provides a preparation method of the above double-network amphiphilic hydrogel microcarrier, comprising the following steps:

[0018] (1) uniformly mix the acrylic acid, 3-[N,N-dimethyl-[2-(2-methylprop-2-enyl oxy) ethyl] ammonium] propane-1-sulfonic acid inner salt, acrylamide, N,N'-methylene bisacrylamide and water to obtain an aqueous phase;

[0019] (2) uniformly mix the paraffin oil, Span 80 and Tween-80 to obtain an oil phase;

[0020] (3) after mixing the aqueous phase and the oil phase, an initiator is added, and heating is carried out to 60 DEG C for 3-5 h; centrifugation is carried out to obtain microspheres;

[0021] (4) the microspheres are mixed with a solution of sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate, and after reaction under 360 nm ultraviolet light for 4-6 min, liquid is removed; after repeating the operation for 2-3 times, a collagen solution is added to the reacted microspheres to react overnight to obtain the double-network amphiphilic hydrogel microcarrier.

[0022] Further, the mixing ratio of the microspheres and the solution of sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate is 0.1 mg: 150-250 muL; the concentration of the solution of sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate is 0.5-2 mM;

[0023] The ratio of the microspheres and the collagen solution is 0.1 mg: (0.5-2) mL; the concentration of the collagen solution is 0.1-0.3 mg / mL.

[0024] The application further provides application of the double-network amphiphilic hydrogel microcarrier or the double-network amphiphilic hydrogel microcarrier prepared by the above preparation method in culturing mesenchymal stem cells; when the double-network amphiphilic hydrogel microcarrier is used to culture mesenchymal stem cells, the proliferation speed of the mesenchymal stem cells can be improved, and the cell stemness can be maintained for a long time.

[0025] The application discloses the following technical effects:

[0026] The application discloses a two-network structure hydrogel microcarrier PSAm based on amphiphilic material, which can effectively expand MSCs while maintaining cell stemness for a long time. The design principle of the hydrogel microcarrier is as follows: SBMA is copolymerized with acrylamide (AAm) monomer to form a two-network structure with acrylic acid (PAA) to prepare the microcarrier. The design can improve the mechanical properties of the PSAm, such as transparency, solubility ratio and toughness, and on the other hand, the use of zwitterionic material can resist nonspecific protein adsorption and maintain stem cell stemness. In the application, the amide group on the PAAm chain is reacted with sulfo-SANPAH to connect proteins (such as collagen), and the modification endows the PSAm with the basic ability (cell adhesion ability) of the cell culture microcarrier. Experiments show that the microcarrier PSAm has excellent biocompatibility, cell adhesion, proliferation and cell stemness maintaining ability, compared with ordinary culture, can reduce the artificial subculture process, save manpower and material resources, avoid resource waste, and provide a new experience method for long-term culture, preservation and maintenance of MSCs. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0028] Figure 1 It is a reaction principle diagram for preparation of the PSAm microspheres in Example 1; a is a gel forming process of the PSAm microspheres, and b is a polymerization process of SBMA and AAm;

[0029] Figure 2 It is an appearance morphology diagram of the two-network amphiphilic hydrogel microcarriers PSAm and PAm in Example 1, and the scale is 100 microns;

[0030] Figure 3 It is an infrared spectrum diagram of the PSAm microspheres, PAm microspheres and P(AAm-SBMA) microspheres;

[0031] Figure 4 It is a proliferation condition of mesenchymal stem cells in different microcarrier culture systems; the scale of 3d and 7d is 100 microns, and the scale of 21d is 250 microns. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be non-limiting examples of the present application, and are understood to be a description of certain aspects, features and embodiments of the present application.

[0033] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values of the parameter are combined with a stated value of the parameter in range form. These are only examples of the various embodiments of the present application, which should only be considered to be illustrative. Accordingly, other embodiments of the present application are possible. For example, the various embodiments of the present application described above can be modified or combined as aspects of a single embodiment. Accordingly, other embodiments of the present application are possible. For example, the various embodiments of the present application described above can be modified or combined as aspects of a single embodiment. Such modifications and variations are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the present application there is a full equivalency of all the features and aspects of the present application with respect to any reference made herein, and their equivalents. It is therefore to be understood that all modifications and variations of this application, which do not depart from the scope of the present application, are considered to be within the scope of the present application.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0035] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the present application will be apparent to those of ordinary skill in the art from the description and examples that follow. The description and examples are intended to be illustrative of certain aspects of the application and are not intended to limit the scope of the application in any way.

[0036] With respect to the use of "comprising", "including", "containing", "having" and "ensing" and the like, these terms are used in the sense of "open ended" and are intended to mean including but not limited to.

[0037] The raw materials used in the present application are as follows:

[0038] Acrylic acid: PAA, CAS No. 9007-20-9;

[0039] 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1 -sulfonic acid inner salt: SBMA, CAS No. 3637-26-1 ;

[0040] Acrylamide: AAm, CAS No. 79-06-1 ;

[0041] N,N'-Methylenebisacrylamide: MBAA, CAS No. 110-26-9;

[0042] Ammonium persulfate: APS, CAS No. 7727-54-0.

[0043] Polyethyleneimine: PEI, CAS No. 9002-98-6.

[0044] Unless otherwise indicated, the reagents and materials used in the following examples are available from commercial vendors and the test methods referred to involve conventional procedures.

[0045] Example 1

[0046] I. Preparation of double-network amphiphilic hydrogel microcarriers

[0047] 1. According to the formulation shown in Table 1, PAA, SBMA, AAm, MBAA and water were placed in a conical flask, and the conical flask was shaken until each raw material was dissolved and mixed uniformly to obtain PSAm aqueous phase and PAm aqueous phase.

[0048] Table 1 Formulation of PSAm aqueous phase and PAm aqueous phase

[0049]

[0050] 2. Paraffin oil, Span 80 and Tween-80 were mixed uniformly at a volume ratio of 5:4:1, heated to 60°C and uniformly stirred for 30 min to stabilize the system to obtain an oil phase.

[0051] 3. The obtained PSAm aqueous phase and PAm aqueous phase were respectively polymerized with the oil phase at a volume ratio of 1:4 (as shown in Table 2), and the specific operation was as follows: the aqueous phase was added to a dropping funnel and slowly dropped into the oil phase, and stirred at a centrifugal force of 200 r / min during the process until a reverse microemulsion was formed. After adding initiator APS (the amount of APS added was 30% of the mass of PAA) to the reverse microemulsion, it was heated to 60°C and kept for 4 h. After cooling to 25°C, the product was centrifuged and washed, and then filtered through a 30-mesh nylon mesh to obtain PSAm microspheres and PAm microspheres. Figure 1 4. 0.1 mg of PSAm microspheres and PAm microspheres were respectively placed in 200 μL of 1 mM sulfo-SANPAH, irradiated with 360 nm ultraviolet light for 5 minutes, shaken to remove the liquid, and repeated 3 times. Then, the microspheres were soaked and washed with 50 mM HEPES (pH 8.5) for 15 minutes, and after repeating the washing 3 times, 1 mL of 0.2 mg / mL collagen type I solution was added, and the reaction was carried out at 4°C on a shaker overnight. The microspheres were washed with PBS to obtain double-network amphiphilic hydrogel microcarriers PSAm and PAm. The appearance of the two microcarriers is shown in

[0052] Figure 2

[0053] II. Observation of microsphere morphology

[0054] ​​The PSAm microspheres and PAm microspheres prepared in the above process were frozen at -80℃ for 4 hours. The microspheres were freeze-dried for 12 hours using a BIOCOOL vacuum freeze dryer FD-1A-BD (China). Then, the freeze-dried microspheres were ground into powder. Then, the PSAm microspheres and PAm microspheres were analyzed using an infrared spectrometer (Nicolet iS50, Thermo Scientific, USA).

[0055] Meanwhile, according to the formulations shown in Table 2, AAm, SBMA, MBAA and water were placed in a conical flask, and the conical flask was shaken until each raw material was dissolved and uniformly mixed to obtain a P(AAm-SBMA) aqueous phase; the obtained aqueous phase was subjected to a polymerization reaction with an oil phase according to the method described in “I. Preparation of double-network amphiphilic hydrogel microcarriers” to prepare P(AAm-SBMA) microspheres. The P(AAm-SBMA) microspheres were subjected to infrared spectrum detection.

[0056] Table 2 Formulations of aqueous phase of single-network hydrogel microspheres

[0057]

[0058] The infrared spectrum detection results of the hydrogel material of the microspheres are shown in Table 3. Figure 3 As can be seen from Table 3, infrared spectrum tests were performed on hydrogels with different compositions of PAAm hydrogel, PAA / P(SB-AAm) hydrogel and PAA / PAAm hydrogel. The characteristic peaks near 1450 cm -1 represent the C-H stretching on the N + (CH3)2 group on the SBMA fragment, the characteristic peaks at 1028 and 1188 cm -1 are related to the symmetric stretching vibration and asymmetric stretching vibration of the sulfonate SO 3- group in SBMA, the characteristic peak at 2930 cm -1 is related to the CH2 stretching in PAAm, and the characteristic peaks at 610 and 1416 cm -1 correspond to the symmetric and asymmetric stretching vibration of the carboxylate. The infrared spectrum results show that the three monomers of Am, SBMA and AA can form copolymer hydrogels through ultraviolet light copolymerization.

[0059] III. Function verification of hydrogel microcarriers

[0060] 1. 0.1 g of double-network amphiphilic hydrogel microcarriers PSAm and PAm were respectively placed in a flat plate with ultra-low attachment surface (purchased from Corning), 2 mL of serum-free MSC complete culture medium (purchased from OriCell) was placed in the flat plate, and the microcarriers were fully soaked for 30 min to construct a microcarrier culture system. After high-pressure sterilization of the microcarrier culture system, P5 MSCs (purchased from ATCC) were inoculated at an inoculation amount of 1.0 × 105 Cells / mL were cultured in a humidified environment at 37°C, 95% air, and 5% carbon dioxide. Cell culture in the microcarrier system was observed under a microscope on days 3, 7, and 21, and the results are as follows: Figure 4 As shown in the figure, the cell number was measured using Taipanblue, and the results are shown in Table 3.

[0061] Table 3. Cell count (cells / mL) in different microcarrier culture systems

[0062] Microcarrier culture system 3d 7d 21d PSAm 6.0 x 10 5 ]] 3.4 x 10 6 ]] 8.4 x 10 9 ]] PAm 6.3 x 10 5 ]] 2.8 x 10 6 ]]> 2.4 x 10 9 ]]>

[0063] from Figure 4 As shown in Table 3, the PSAm microcarrier culture system is more conducive to the proliferation of MSCs than the PAm microcarrier culture system, indicating that the addition of SBMA to the hydrogel microcarrier system is more beneficial for cell adhesion to the microcarrier and efficient proliferation. Therefore, PSAm microcarriers were chosen for subsequent functional validation.

[0064] 2. Place 0.1 g of the dual-network amphiphilic hydrogel microcarrier PSAm in a plate with an ultra-low adhesion surface (purchased from Corning). Add 2 mL of serum-free MSC complete culture medium (purchased from OriCell) to the plate and soak the microcarrier for 30 min to construct the PSAm microcarrier culture system. After autoclaving the microcarrier culture system, inoculate it with P5 generation MSCs (purchased from ATCC) at an inoculation density of 1.0 × 10⁻⁶. 5 Cells / mL were cultured at 37°C in a humidified environment with 95% air and 5% carbon dioxide. A monolayer culture dish was also set up as a blank control group.

[0065] MSCs were digested from the microcarrier culture system and placed in centrifuge tubes on days 5, 14, and 21 of culture. Cells were incubated at 4°C with anti-CD105-FITC, anti-CD90-FITC, and anti-CD45-FITC (abcam, 1:100) for 0.5 hours, followed by fixation with 4% paraformaldehyde for 10 minutes. The cell suspension was collected and filtered through a 300-mesh nylon screen. Phenotypic analysis was performed using an Accuri C6-Plus cell sorter (BD) and FlowJo software (Tree Star). The results are shown in Table 4.

[0066] Table 4. Results of cell phenotypic antibody detection by flow cytometry

[0067]

[0068] As shown in Table 4, in the ordinary culture medium culture process (Blank group), with the prolongation of the culture time, the positive rates of CD105 and CD90 decrease rapidly, on the 21st day, the positive rate of CD105 is only 46.7%, and the positive rate of CD90 is only 63.3%, and the positive rate of CD45 shows an upward trend, indicating that the MSCs culture time is too long, and the cell stemness and MSCs differentiation ability decrease; compared with the Blank group, the MSCs cultured by the hydrogel microcarrier PSAm decrease slowly with the prolongation of the culture time, and the positive rate of CD45 even shows a downward trend, indicating that the hydrogel microcarrier PSAm of the application can maintain the cell stemness of mesenchymal stem cells in a long culture process.

[0069] Comparative Example 1

[0070] I. The microcarriers were prepared according to the method of "I. Preparation of double-network amphiphilic hydrogel microcarriers" in Example 1, except that step 4 was omitted to obtain the hydrogel microcarriers.

[0071] II. Function verification

[0072] 1. 0.1 g of the hydrogel microcarriers was placed in a flat plate with an ultra-low attachment surface (purchased from Corning), 2 mL of serum-free MSC complete culture medium (purchased from OriCell) was placed in the flat plate, and the microcarriers were fully soaked for 30 min to construct a microcarrier culture system. After high-pressure sterilization of the microcarrier culture system, P5 MSCs (purchased from ATCC) were inoculated at an inoculation amount of 1.0 × 10 5 cells / mL, and cultured in a humidified environment of 37°C, 95% air and 5% carbon dioxide. On the 3rd, 7th and 21st days, the cell number was measured using Taipan blue, and the results are shown in Table 5.

[0073] Table 5 Cell number (cells / mL)

[0074] - 3d 7d 21d Cell number 0.3 x 10 2 ]] 0.6 x 10 2 ]] 1.3 x 10 3 ]]>

[0075] As shown in Table 5, compared with the hydrogel microcarriers PSAm in Example 1, the adhesion of the hydrogel microcarriers after omitting the collagen modification to the cells is significantly reduced, and the cell proliferation rate decreases.

[0076] 2. 0.1 g of the hydrogel microcarriers was placed in a flat plate with an ultra-low attachment surface (purchased from Corning), 2 mL of serum-free MSC complete culture medium (purchased from OriCell) was placed in the flat plate, and the microcarriers were fully soaked for 30 min to construct a microcarrier culture system PSAm. After high-pressure sterilization of the microcarrier culture system, P5 MSCs (purchased from ATCC) were inoculated at an inoculation amount of 1.0 × 105 The MSCs were cultured at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide at a density of 1.0 x 105cells / mL.

[0077] At the 5th, 14th and 21st day of culture, the MSCs were digested from the microcarrier culture system and placed in centrifuge tubes. The cells were incubated with anti-CD105-FITC, anti-CD90-FITC and anti-CD45-FITC (abcam, 1:100) at 4°C for 0.5 hours, and then the cells were fixed with 4% paraformaldehyde for 10 minutes. The cell suspension was collected and filtered through a 300-mesh nylon net. Phenotype analysis was performed using an Accuri C6-Plus cell sorter (BD) and FlowJo software (Tree Star). The results of the analysis are shown in Table 6.

[0078] Table 6 Results of cell phenotype antibody detection by flow cytometry

[0079]

[0080]

[0081] As can be seen from Table 6, compared with the hydrogel microcarriers PSAm of Example 1, the positive rates of CD105 and CD90 decreased with the extension of the culture time, and at the 21st day, the positive rate of CD105 was only 56.7%, and the positive rate of CD90 was only 66.2%, and the positive rate of CD45 showed an upward trend, indicating that after long-term culture of MSCs, the phenomenon of decreased cell stemness and decreased differentiation ability of MSCs occurred.

[0082] Comparative Example 2

[0083] I. The microcarriers were prepared according to the method of “I. Preparation of double-network amphiphilic hydrogel microcarriers” in Example 1, except that the mass ratio of SBMA to AAm was adjusted to 1:1, and the total mass was still 1.50 g, to obtain the hydrogel microcarriers.

[0084] II. Function verification

[0085] 1. 0.1 g of hydrogel microcarriers were placed in a flat plate with an ultra-low attachment surface (purchased from Corning), 2 mL of serum-free MSC complete culture medium (purchased from OriCell) was placed in the flat plate, and the microcarriers were fully soaked for 30 minutes to construct a microcarrier culture system. After high-pressure sterilization of the microcarrier culture system, P5 MSCs (purchased from ATCC) were inoculated at a concentration of 1.0 x 105cells / mL, and cultured at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide. At the 3rd, 7th and 21st day, the cell number was measured using Taipan blue, and the results are shown in Table 7. 5 The MSCs were cultured at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide at a density of 1.0 x 105cells / mL.

[0086] Table 7 Cell number (cells / mL)

[0087] - 3d 7d 21d Cell number 5.9 x 10 5 ]]> 2.9 x 10 6 ]]> 4.3 x 10 9 ]]>

[0088] As can be seen from Table 7, compared with the hydrogel microcarrier PSAm in Example 1, after adjusting the mass ratio of SBMA to AAm, the cell proliferation rate decreased.

[0089] 2. 0.1 g of hydrogel microcarriers were placed in a flat plate with an ultra-low attachment surface (purchased from Corning), 2 mL of serum-free MSCs complete culture medium (purchased from OriCell) was put into the flat plate, and the microcarriers were fully soaked for 30 min to construct a microcarrier culture system PSAm. After high-pressure sterilization of the microcarrier culture system, P5 MSCs (purchased from ATCC) were inoculated at a concentration of 1.0 x 10 5 cells / mL, and cultured at 37°C in a humidified environment with 95% air and 5% carbon dioxide.

[0090] On the 5th day, 14th day and 21st day of culture, the MSCs were digested from the microcarrier culture system and placed in a centrifuge tube. The cells were incubated with anti-CD105-FITC, anti-CD90-FITC and anti-CD45-FITC (abcam, 1:100) at 4°C for 0.5 hours, and then the cells were fixed with 4% paraformaldehyde for 10 minutes. The cell suspension was collected and filtered through a 300-mesh nylon mesh. Phenotype analysis was performed using an Accuri C6-Plus cell sorter (BD) and FlowJo software (Tree Star). The analysis results are shown in Table 8.

[0091] Table 8 Flow cytometry cell phenotype antibody detection results

[0092]

[0093]

[0094] As can be seen from Table 8, compared with the hydrogel microcarrier PSAm of Example 1, as the culture time increased, the positive rates of CD105 and CD90 decreased, and on the 21st day, the positive rate of CD105 was only 80.2%, and the positive rate of CD90 was only 89.3%, and the positive rate of CD45 showed an upward trend, indicating that after long-term culture of MSCs, the cell stemness decreased, and the differentiation ability of MSCs decreased.

[0095] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A bi-networked amphiphilic hydrogel microcarrier capable of maintaining cell stemness, characterized in that, The raw materials include: an oil phase, an aqueous phase, an initiator, sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate and collagen; The aqueous phase comprises: acrylic acid, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, acrylamide, N,N'-methylenebisacrylamide and water; The oil phase comprises paraffin oil, Span 80 and Tween-80; In the aqueous phase, the mass ratio of the acrylic acid, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, acrylamide, N,N'-methylenebisacrylamide and water is (0.05-0.2):(1.0-1.5):(0.2-0.4):(0.01-0.02):(39.9-40).

2. The dual-network amphiphilic hydrogel microcarriers of claim 1, wherein, In the oil phase, the volume ratio of the paraffin oil, Span 80 and Tween-80 is (4-6):(3-5):

1.

3. The dual-network amphiphilic hydrogel microcarriers of claim 1, wherein, The mass of the initiator is 25%-35% of the mass of the acrylic acid.

4. The dual-network amphiphilic hydrogel microcarriers of claim 1, wherein, In the double-network amphiphilic hydrogel microcarrier, the concentration of collagen is 1.5-3 mg / mL.

5. The dual-network amphiphilic hydrogel microcarriers of claim 1, wherein, The volume ratio of the aqueous phase to the oil phase is 1:(3-5).

6. A method for the preparation of the double-network amphiphilic hydrogel microcarriers according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) uniformly mixing the acrylic acid, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, acrylamide, N,N'-methylenebisacrylamide and water to obtain an aqueous phase; (2) uniformly mixing the paraffin oil, Span 80 and Tween-80 to obtain an oil phase; (3) after mixing the aqueous phase and the oil phase, adding an initiator and heating to 60℃ for 3-5 h; Centrifuging to obtain microspheres; (4) mixing the microspheres with a sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate solution, reacting under 360 nm ultraviolet light for 4-6 min, removing the liquid, repeating the operation 2-3 times, adding a collagen solution to the reacted microspheres and reacting overnight to obtain the double-network amphiphilic hydrogel microcarrier.

7. The preparation method according to claim 6, characterized in that, The mixing ratio of the microspheres to the sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate solution is 0.1 mg:150-250 μL; the concentration of the sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate solution is 0.5-2 mM; The ratio of the microspheres to the collagen solution is 0.1 mg:(0.5-2) mL; the concentration of the collagen solution is 0.1-0.3 mg / mL.

8. Use of the double-network amphiphilic hydrogel microcarriers according to any one of claims 1 to 5 or of the double-network amphiphilic hydrogel microcarriers prepared according to the method of claim 6 or 7 for culturing mesenchymal stem cells, characterized in that, When mesenchymal stem cells are cultured by using the double-network amphiphilic hydrogel microcarrier, the proliferation speed of the mesenchymal stem cells can be improved, and the stemness of the cells can be maintained for a long time.

Images