Dual-network amphiphilic hydrogel microcarrier capable of maintaining dryness of cells and application of dual-network amphiphilic hydrogel microcarrier

Abstract

The invention discloses a dual-network amphiphilic hydrogel microcarrier capable of maintaining cell dryness and application of the dual-network amphiphilic hydrogel microcarrier, and belongs to the technical field of cell culture. According to the invention, SBMA and an acrylamide (AAm) monomer are copolymerized to form a dual-network structure with acrylic acid (PAA), so that the hydrogel microcarrier is prepared. The hydrogel microcarrier PSAm with the double-network structure constructed on the basis of the amphiphilic material can effectively amplify MSCs, can keep the dryness of the cells for a long time, is beneficial to long-term amplification culture of the MSCs, and can reduce the artificial subculture process, save manpower and material resources and avoid resource waste compared with common culture; and a new experience method is provided for long-term culture, preservation and cell dryness maintenance of the MSCs.

Description

Technical Field

[0001] The invention 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 Art

[0002] Mesenchymal stem cells (MSCs), as star cells in the field of regenerative medicine, have attracted much attention due to their self-renewal ability and multidirectional differentiation potential. MSCs can differentiate into a variety of tissue cells other than hematopoiesis in vitro under specific induction conditions, especially tissue cells derived from mesoderm and neuroectoderm. However, the differentiation mechanism of MSCs is complex, and the control of its 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. Currently, commercial microcarriers for culturing cells, such as Cytodex-3, SoloHill and Cultispher, have been used for industrial MSCs production. However, long-term culture may cause MSCs to age or differentiate, affecting their multipotency and immunoregulatory ability. Existing microcarriers are basically only developed around efficient cell proliferation, but cannot guarantee that stem cells remain in an undifferentiated state for a long time. Therefore, it is particularly important to develop a microcarrier that can efficiently proliferate MSCs and maintain their cell stemness for a long time. Summary of the invention

[0004] The purpose of the present invention is to provide a double-network amphiphilic hydrogel microcarrier capable of maintaining cell stemness and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention is based on the double-network hydrogel microcarrier PSAm constructed based on amphiphilic materials, which can effectively amplify MSCs and maintain their cell stemness for a long time, which is conducive to the long-term expansion of MSCs. Compared with ordinary culture, it can reduce the artificial subculture process, save manpower and material resources, avoid waste of resources, and provide a new empirical method for the long-term culture, preservation and maintenance of cell stemness of MSCs.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a double-network amphiphilic hydrogel microcarrier capable of maintaining cell stemness, comprising the following raw materials: an oil phase, an aqueous phase, an initiator, sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate and collagen;

[0007] 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.

[0008] In the present invention, 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 acrylic acid (PAA) is subjected to a one-step polymerization reaction to form a PSAm hydrogel; wherein the hydrogen bonds formed by the amino groups on the acrylamide and the ionic bonds formed by the anionic and cationic side groups on the SBMA stably connect the first network and the second network, thereby improving the mechanical properties and stability of the hydrogel. After the amide groups on the PAAm chains in the PSAm hydrogel react with sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), collagen can be connected, thereby improving the adhesion of the hydrogel and facilitating the colonization and growth of cells.

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

[0010] Furthermore, in the aqueous phase, the mass ratio of 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).

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

[0012] Furthermore, 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, azobisisoheptanenitrile, dimethyl azobisisobutyrate, azobisisobutylamidine hydrochloride, hydrogen peroxide, potassium persulfate and tert-butyl hydroperoxide.

[0014] Furthermore, in the double-network amphiphilic hydrogel microcarrier, the concentration of 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] Furthermore, the volume ratio of the water phase to the oil phase is 1:(3-5).

[0017] The present invention also provides a method for preparing the above-mentioned double-network amphiphilic hydrogel microcarrier, comprising the following steps:

[0018] (1) 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 uniformly to obtain an aqueous phase;

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

[0020] (3) After mixing the aqueous phase and the oil phase, adding an initiator, heating to 60° C. and maintaining for 3-5 hours; and centrifuging to obtain microspheres;

[0021] (4) mixing the microspheres with a solution of sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate, reacting under 360 nm ultraviolet light for 4-6 minutes, and then removing the liquid; repeating the operation 2-3 times, adding the collagen solution to the reacted microspheres and reacting 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 μL; the concentration of the solution of sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate is 0.5-2 mM;

[0023] 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.

[0024] The present invention also provides the use of the above-mentioned double-network amphiphilic hydrogel microcarrier or the double-network amphiphilic hydrogel microcarrier prepared by the above-mentioned preparation method in culturing mesenchymal stem cells. When the double-network amphiphilic hydrogel microcarrier is used to culture mesenchymal stem cells, the proliferation rate of mesenchymal stem cells can be increased and the stemness of the cells can be maintained for a long time.

[0025] The present invention discloses the following technical effects:

[0026] The present invention discloses a hydrogel microcarrier PSAm with a double network structure based on amphiphilic materials, which can effectively amplify MSCs while maintaining its cell stemness for a long time. The design principle of the hydrogel microcarrier of the present invention is to use SBMA and acrylamide (AAm) monomers to form a double network structure with acrylic acid (PAA) to prepare the microcarrier. On the one hand, this design can improve the mechanical properties of PSAm, such as transparency, solubility ratio and toughness, and on the other hand, the use of zwitterionic materials can resist the adsorption of nonspecific proteins and maintain the stemness of stem cells. In the present invention, the amide group on the PAAm chain reacts with sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate (sulfo-SANPAH) to connect proteins (such as collagen), and this modification gives PSAm the basic ability (cell adhesion ability) as a cell culture microcarrier. Experiments have shown that the microcarrier PSAm of the present invention has excellent biocompatibility, cell adhesion, proliferation and ability to maintain cell stemness. Compared with ordinary culture, it can reduce the artificial subculture process, save manpower and material resources, avoid waste of resources, and provide a new empirical method for the long-term culture, preservation and maintenance of cell stemness of MSCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The schematic diagram of the preparation reaction of PSAm microspheres in Example 1; a is the gelation process of PSAm microspheres, and b is the polymerization process of SBMA and AAm;

[0029] Figure 2 This is the appearance morphology of the double-network amphiphilic hydrogel microcarriers PSAm and PAm in Example 1, with a scale of 100 μm;

[0030] Figure 3 The infrared spectra of PSAm microspheres, PAm microspheres and P(AAm-SBMA) microspheres;

[0031] Figure 4 The proliferation of mesenchymal stem cells in different microcarrier culture systems; the scale bar for 3d and 7d is 100 μm, and the scale bar for 21d is 250 μm. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

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

[0038] Acrylic acid: PAA, CAS number is 9007-20-9;

[0039] 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt: SBMA, CAS number is 3637-26-1;

[0040] Acrylamide: AAm, CAS number is 79-06-1;

[0041] N, N'-methylenebisacrylamide: MBAA, CAS number is 110-26-9;

[0042] Ammonium persulfate: APS, CAS number is 7727-54-0.

[0043] Polyethyleneimine: PEI, CAS number is 9002-98-6.

[0044] Unless otherwise specified, the reagents and materials used in the following examples can be purchased through conventional channels, and the detection methods involved are all conventional methods in the art.

[0045] Example 1

[0046] 1. Preparation of double network amphiphilic hydrogel microcarriers

[0047] 1. According to the formula shown in Table 1, PAA, SBMA, AAm, MBAA and water are placed in a conical flask, and the conical flask is shaken until the raw materials are dissolved and mixed evenly to obtain PSAm aqueous phase and PAm aqueous phase.

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

[0049]

[0050] 2. Mix paraffin oil, Span 80 and Tween-80 in a volume ratio of 5:4:1, heat to 60°C and stir evenly for 30 minutes to stabilize the system to obtain an oil phase.

[0051] 3. The obtained PSAm aqueous phase and PAm aqueous phase were polymerized with the oil phase at a volume ratio of 1:4 (eg Figure 1 The specific operation is to add the aqueous phase into the dropping funnel and slowly drip into the oil phase, stirring at a centrifugal force of 200r / min until a reverse microemulsion is formed. After adding the initiator APS (the amount of APS added is 30% of the mass of PAA) to the reverse microemulsion, heat it to 60°C and keep it for 4h. After cooling to 25°C, the product is washed by centrifugation and then filtered through a 30-mesh nylon mesh to obtain PSAm microspheres and PAm microspheres.

[0052] 4. Place 0.1 mg PSAm microspheres and PAm microspheres in 200 μL 1 mM sulfo-SANPAH, irradiate with 360 nm UV light for 5 minutes, shake to remove the liquid, and repeat 3 times. After that, soak and wash the microspheres with 50 mM HEPES (pH 8.5) for 15 minutes, repeat the washing 3 times, add 1 mL of 0.2 mg / mL type I collagen solution, and react on a shaker at 4 ° C overnight. Wash the microspheres with PBS to obtain double-network amphiphilic hydrogel microcarriers PSAm and PAm. The appearance of the two microcarriers is as follows Figure 2 shown.

[0053] 2. Observation of microsphere morphology

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

[0055] At the same time, according to the formula shown in Table 2, AAm, SBMA, MBAA and water were placed in a conical flask, and the conical flask was shaken until the raw materials were dissolved and mixed evenly to obtain a P(AAm-SBMA) aqueous phase; the obtained aqueous phase was polymerized with the 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 detected by infrared spectroscopy.

[0056] Table 2 Aqueous phase formula of single network hydrogel microspheres

[0057]

[0058] The infrared spectrum test results of the hydrogel material of the microspheres are as follows Figure 3 As shown in the figure, it can be seen that the infrared spectra of PAAm hydrogel, PAA / P(SB-AAm) hydrogel and PAA / PAAm hydrogel with different compositions were tested, and the 1450cm -1 The characteristic peaks nearby represent the N + CH stretching on (CH3)2 groups, 1028 and 1188 cm -1 The characteristic peaks are related to the sulfonate SO in SBMA. 3- The symmetric stretching vibration of the group is related to the asymmetric stretching vibration, 2930cm -1 The characteristic peaks are related to the CH2 stretching in PAAm, 610 and 1416 cm -1 The characteristic peaks correspond to the symmetric and asymmetric stretching vibrations of carboxylates. Infrared spectroscopy results show that the three monomers Am, SBMA and AA can form copolymer hydrogels through UV light curing.

[0059] 3. Functional verification of hydrogel microcarriers

[0060] 1. 0.1 g of double-network amphiphilic hydrogel microcarriers PSAm and PAm were placed in a plate with an ultra-low attachment surface (purchased from Corning), and 2 mL of serum-free MSCs complete culture medium (purchased from OriCell) was placed in the plate to fully soak the microcarriers for 30 min to construct a microcarrier culture system. After the microcarrier culture system was sterilized by high pressure, P5 generation MSCs (purchased from ATCC) were inoculated at an inoculation size of 1.0×105 The cells were cultured at 37°C in a humidified environment with 95% air and 5% carbon dioxide. The cell culture conditions in the microcarrier culture system were observed under a microscope on the 3rd, 7th, and 21st days. Figure 4 The cell number was measured using Taipanblue, and the results are shown in Table 3.

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

[0062] Microcarrier culture system 3d 7d 21d PSAm <![CDATA[6.0×10 5 ]]> <![CDATA[3.4×10 6 ]]> <![CDATA[8.4×10 9 ]]> PAM <![CDATA[6.3×10 5 ]]> <![CDATA[2.8×10 6 ]]> <![CDATA[2.4×10 9 ]]>

[0063] from Figure 4 As can be seen from Table 3, compared with the PAm microcarrier culture system, the PSAm microcarrier culture system is more conducive to the proliferation of MSCs, indicating that adding SBMA to the hydrogel microcarrier system is more conducive to cell adhesion to the microcarrier and efficient proliferation. Therefore, the PSAm microcarrier was selected for subsequent functional verification.

[0064] 2. Place 0.1 g of double-network amphiphilic hydrogel microcarrier PSAm in a plate with ultra-low attachment surface (purchased from Corning), and put 2 mL of serum-free MSCs complete culture medium (purchased from OriCell) in the plate to fully soak the microcarrier for 30 min to construct the microcarrier culture system PSAm. After the microcarrier culture system was sterilized by high pressure, P5 generation MSCs (purchased from ATCC) were inoculated at an inoculation volume of 1.0×10 5 The cells were cultured at 37°C in a humidified environment containing 95% air and 5% carbon dioxide. A single-layer culture dish was set up as a blank control group.

[0065] On the 5th, 14th and 21st day of culture, 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 fixed with 4% paraformaldehyde for 10 minutes. The cell suspension was collected and filtered through a 300-mesh nylon mesh. Phenotypic analysis was performed using an Accuri C6-Plus cell sorter (BD) and FlowJo software (Tree Star). The analysis results are shown in Table 4.

[0066] Table 4 Cell phenotype antibody detection results by flow cytometry

[0067]

[0068] As can be seen from Table 4, in the ordinary culture medium culture process (Blank group), with the extension of culture time, the positive rates of CD105 and CD90 decreased rapidly. On the 21st day, the positive rate of CD105 was only 46.7%, CD90 was only 63.3%, and the positive rate of CD45 showed an upward trend, indicating that if the MSCs culture time is too long, the cell stemness will decrease and the differentiation ability of MSCs will decrease. Compared with the Blank group, the MSCs cultured with the hydrogel microcarrier PSAm, with the extension of culture time, the positive rates of CD105 and CD90 decreased slowly, and the positive rate of CD45 even showed a downward trend, indicating that the hydrogel microcarrier PSAm of the present invention can maintain the cell stemness of mesenchymal stem cells in a longer culture process.

[0069] Comparative Example 1

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

[0071] 2. Functional Verification

[0072] 1. Place 0.1 g of hydrogel microcarriers on a plate with ultra-low attachment surface (purchased from Corning), add 2 mL of serum-free MSCs complete culture medium (purchased from OriCell) to the plate, and fully soak the microcarriers for 30 min to construct a microcarrier culture system. After high-pressure sterilization of the microcarrier culture system, inoculate P5 MSCs (purchased from ATCC) at an inoculation volume of 1.0 × 10 5 The cells were cultured at 37°C in a humidified environment containing 95% air and 5% carbon dioxide. The cell numbers were measured using Taipanblue on days 3, 7, and 21. The results are shown in Table 5.

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

[0074] - 3d 7d 21d Cell number <![CDATA[0.3×10 2 ]]> <![CDATA[0.6×10 2 ]]> <![CDATA[1.3×10 3 ]]>

[0075] It can be seen from Table 5 that compared with the hydrogel microcarrier PSAm in Example 1, the hydrogel microcarrier after omitting collagen modification has significantly reduced adhesion to cells and decreased cell proliferation rate.

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

[0077] On the 5th, 14th and 21st day of culture, 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 fixed with 4% paraformaldehyde for 10 minutes. The cell suspension was collected and filtered through a 300-mesh nylon mesh. Phenotypic analysis was performed using an Accuri C6-Plus cell sorter (BD) and FlowJo software (Tree Star). The analysis results are shown in Table 6.

[0078] Table 6 Cell phenotype antibody detection results by flow cytometry

[0079]

[0080]

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

[0082] Comparative Example 2

[0083] 1. Microcarriers were prepared according to the method of "1. 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 hydrogel microcarriers.

[0084] 2. Functional Verification

[0085] 1. Place 0.1 g of hydrogel microcarriers on a plate with ultra-low attachment surface (purchased from Corning), add 2 mL of serum-free MSCs complete culture medium (purchased from OriCell) to the plate, and fully soak the microcarriers for 30 min to construct a microcarrier culture system. After high-pressure sterilization of the microcarrier culture system, inoculate P5 MSCs (purchased from ATCC) at an inoculation volume of 1.0 × 10 5 The cells were cultured at 37°C in a humidified environment containing 95% air and 5% carbon dioxide. The cell numbers were measured using Taipanblue on the 3rd, 7th and 21st days, and the results are shown in Table 7.

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

[0087] - 3d 7d 21d Cell number <![CDATA[5.9×10 5 ]]> <![CDATA[2.9×10 6 ]]> <![CDATA[4.3×10 9 ]]>

[0088] It can be seen from Table 7 that, 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. Place 0.1 g of hydrogel microcarriers in a plate with ultra-low attachment surface (purchased from Corning), add 2 mL of serum-free MSCs complete culture medium (purchased from OriCell) in the plate, and fully soak the microcarriers for 30 min to construct the microcarrier culture system PSAm. After the microcarrier culture system was sterilized by high pressure, P5 generation MSCs (purchased from ATCC) were inoculated at an inoculation volume of 1.0 × 10 5 The cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide.

[0090] On the 5th, 14th and 21st day of culture, 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 fixed with 4% paraformaldehyde for 10 minutes. The cell suspension was collected and filtered through a 300-mesh nylon mesh. Phenotypic 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 Cell phenotype antibody detection results by flow cytometry

[0092]

[0093]

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

[0095] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A double-network amphiphilic hydrogel microcarrier capable of maintaining cell stemness, characterized in that: The method comprises the following raw materials: 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.

2. The double-network amphiphilic hydrogel microcarrier according to claim 1, characterized in that: The oil phase includes paraffin oil, Span 80 and Tween-80.

3. The double-network amphiphilic hydrogel microcarrier according to claim 1, characterized in that: In the aqueous phase, the mass ratio of 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).

4. The double-network amphiphilic hydrogel microcarrier according to claim 2, characterized in that: In the oil phase, the volume ratio of the paraffin oil, Span 80 and Tween-80 is (4-6):(3-5):

1.

5. The double-network amphiphilic hydrogel microcarrier according to claim 1, characterized in that: The mass of the initiator is 25%-35% of the mass of the acrylic acid.

6. The double-network amphiphilic hydrogel microcarrier according to claim 1, characterized in that: In the double-network amphiphilic hydrogel microcarrier, the concentration of collagen is 1.5-3 mg / mL.

7. The double-network amphiphilic hydrogel microcarrier according to claim 1, characterized in that: The volume ratio of the water phase to the oil phase is 1:(3-5).

8. A method for preparing the double-network amphiphilic hydrogel microcarrier according to any one of claims 1 to 7, characterized in that: The steps include: (1) 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 uniformly to obtain an aqueous phase; (2) mixing the paraffin oil, Span 80 and Tween-80 uniformly to obtain an oil phase; (3) After mixing the water phase and the oil phase, add an initiator, heat to 60° C. and maintain for 3-5 hours; After centrifugation, microspheres were obtained; (4) mixing the microspheres with a solution of sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate, reacting under 360 nm ultraviolet light for 4-6 minutes, and then removing the liquid; repeating the operation 2-3 times, adding the collagen solution to the reacted microspheres and reacting overnight to obtain the double-network amphiphilic hydrogel microcarrier.

9. The preparation method according to claim 8, characterized in that: The mixing ratio of the microspheres and the solution of sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate is 0.1 mg:150-250 μL; the concentration of the solution of sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate 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.

10. Use of the double-network amphiphilic hydrogel microcarrier according to any one of claims 1 to 7 or the double-network amphiphilic hydrogel microcarrier prepared by the preparation method according to claim 8 or 9 in culturing mesenchymal stem cells, characterized in that: When the double-network amphiphilic hydrogel microcarrier is used to culture mesenchymal stem cells, the proliferation rate of the mesenchymal stem cells can be increased and the stemness of the cells can be maintained for a long time.

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