Use of a dynamic injectable degradable stem cell-hydrogel construct

Abstract

The application provides application of a dynamic injectable degradable stem cell-hydrogel construct, which can conveniently store and transport stem cells in a normal temperature environment. The dynamic hydrogel construct three-dimensionally embeds stem cells in situ through N-carboxyethyl chitosan / sodium alginate hydrogel, after storage and transportation in a normal temperature environment, the stem cells proliferate to form large and dense spherical cell colonies, which can effectively maintain high survival rate and pluripotency of the cells. The application is convenient for short-term storage and transportation of stem cells in a normal temperature environment. Most importantly, the activity and phenotype of the stem cells can be maintained, and the stem cell-hydrogel construct can be directly used without additional treatment. The dynamic degradable "ready-to-use" stem cell-hydrogel construct prepared by the application can effectively support storage, transportation and instant use of stem cells, and the hydrogel construct provides a convenient, economical and reliable instant use scheme of stem cells for biomedicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical biomaterials, and particularly relates to application of a dynamic injectable degradable stem cell-hydrogel construct. BACKGROUND

[0002] Since the activity and pluripotency of stem cells are very sensitive to various stimuli, a suitable microenvironment is crucial for the storage and transportation of stem cells. Hydrogels have the characteristics of simulating the extracellular matrix microenvironment, such as high water content, good material exchange ability and similar elastic modulus to soft tissue, which can protect the embedded cells from external stimuli. In addition, the three-dimensional network structure of hydrogels can promote the transportation of oxygen and nutrients and the discharge of metabolic products. Three-dimensional embedding of stem cells in hydrogels has promoted the development of cell therapy, tissue engineering, regenerative medicine, drug screening and other fields in the biomedical field.

[0003] The storage method can significantly affect the performance of cells, especially stem cells which are sensitive to stimuli such as external force, chemicals and temperature changes. However, the currently widely used cell cryopreservation method still has many problems, such as harsh storage conditions, expensive facilities, use of toxic reagents and complex procedures. Therefore, how to safely and effectively store and transport stem cells at room temperature is a difficult problem to be solved. Efficient storage and maintenance of stem cell pluripotency are necessary conditions for three-dimensionally embedded cells in hydrogels to be effectively used. The present application has developed a "ready-to-use" stem cell-hydrogel construct, which is three-dimensionally embedded in situ in a dynamic hydrogel under physiological conditions and stored in the dynamic hydrogel, which is expected to break through the technical bottleneck of stem cell storage. At the same time, the cell-hydrogel construct prepared by embedding stem cells in a dynamic hydrogel with biocompatibility and self-biodegradability can automatically release cells and does not use toxic reagents, does not require expensive facilities and complex operation procedures. This stem cell-hydrogel construct can be directly used from the preparation site to the destination, which overcomes many problems of cryopreservation and transportation of stem cells. The self-healing hydrogel cross-linked by dynamic reversible bonds has the characteristics of restoring its structure and function after damage through the dynamic balance of dissociation, recombination and reconstruction of the hydrogel network between the polymer chains. The reversible characteristics of the dynamic hydrogel are similar to the dynamic characteristics of the extracellular matrix microenvironment and soft tissue, but current research mainly focuses on the application of dynamic hydrogels as biomaterials in the field of tissue engineering, and there is still a lack of research on the storage and transportation of stem cells embedded in dynamic hydrogels. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides an application of a dynamic injectable degradable stem cell-hydrogel construct.The stem cell-hydrogel construct can simulate the extracellular matrix microenvironment, can store and transport stem cells in a normal temperature environment, and realizes the "ready-to-use" nature of the stem cells.The stem cells are three-dimensionally embedded in a dynamic polysaccharide-based hydrogel with biocompatibility and biodegradability to obtain a stem cell-dynamic hydrogel construct.

[0005] The stem cell-hydrogel construct is not only used for storing stem cells, but also can automatically release stem cells while maintaining the proliferation activity and pluripotency of the stem cells, and the hydrogel construct has important application value in biomedical research.

[0006] To achieve the object of the application, the following technical solutions are adopted in the application.

[0007] In a first aspect, the application provides an application of a dynamic injectable degradable stem cell-hydrogel construct, wherein the stem cell-hydrogel construct comprises a hydrogel matrix and stem cells three-dimensionally embedded in the hydrogel matrix.

[0008] The hydrogel matrix comprises, by weight fraction, 0.5-1.5 parts of oxidized sodium alginate (for example, 0.5, 0.8, 1.0, 1.2 or 1.5, etc.), 1.5-2.5 parts of N-carboxyethyl chitosan (for example, 1.5, 2 or 2.5, etc.), and 96-98 parts of a cell culture medium (for example, 96, 97 or 98, etc.).

[0009] Preferably, the oxidation degree of the oxidized sodium alginate is 50-55% according to the hydroxylamine hydrochloride titration method, for example, 50%, 51%, 52%, 53%, 54% or 55%, etc.

[0010] Preferably, the amino substitution degree of the N-carboxyethyl chitosan is 48-50%, for example, 48%, 49% or 50%, etc.

[0011] Preferably, the stem cells are selected from embryonic stem cells, adipose stem cells or bone marrow mesenchymal stem cells, etc.

[0012] The application develops a "ready-to-use" stem cell-hydrogel construct, which is expected to solve the main problems of stem cell storage. The stem cells are three-dimensionally embedded in an injectable dynamic hydrogel with biocompatibility and biodegradability in situ under physiological conditions, the stem cell-hydrogel construct is conducive to safe and efficient storage of stem cells, and the stem cells still have high proliferation activity and pluripotency after storage and transportation, can solve the actual demand of "ready-to-use" stem cells, and do not need expensive facilities and complicated operation procedures.

[0013] The dynamic hydrogel in the application comprises the following components: oxidized sodium alginate, N-carboxyethyl chitosan and cell culture medium. The dynamic hydrogel of the application significantly promotes the proliferation of stem cells and retains the pluripotency of the embedded stem cells.

[0014] In the application, the cell culture medium comprises Dulbecco's Modified Eagle Medium (DMEM) medium, serum substitute (Knockout TM SR, KSR) solution, GlutaMAX TM solution, non-essential amino acid solution, beta-mercaptoethanol solution, double antibody solution and Leukemia Inhibitory Factor (LIF) solution.

[0015] The application also provides a preparation method of an injectable "ready-to-use" stem cell-hydrogel construct which can be conveniently stored and transported in a normal temperature environment. The dynamic hydrogel construct in-situ three-dimensionally embeds stem cells by N-carboxyethyl chitosan / oxidized sodium alginate hydrogel. After storage and transportation in a normal temperature environment, the stem cells proliferate to form large and dense spherical cell colonies, which can effectively maintain high survival rate and pluripotency of the cells.

[0016] Preferably, the stem cell-hydrogel construct is prepared by a preparation method comprising the following steps:

[0017] N-carboxyethyl chitosan and oxidized sodium alginate are respectively completely dissolved in the cell culture medium, the stem cells are resuspended in the cell culture medium in which N-carboxyethyl chitosan is dissolved to obtain a cell suspension, the cell suspension is mixed with the cell culture medium in which oxidized sodium alginate is dissolved to obtain a mixed solution, and the mixed solution is incubated in a 37℃ constant temperature incubator to prepare the stem cell-hydrogel construct.

[0018] In the application, the specific steps for preparing the stem cell-hydrogel construct are as follows: the digested stem cells are resuspended in the complete medium in which N-carboxyethyl chitosan is dissolved, and then uniformly mixed with the cell culture medium in which oxidized sodium alginate is dissolved and three-dimensionally incubated to obtain the stem cell-hydrogel construct.

[0019] The application provides a method for low-cost and convenient storage of stem cells in a normal temperature environment. The method can simulate the dynamic three-dimensional extracellular matrix dynamic microenvironment in vivo, store and transport stem cells in a normal temperature environment and realize the ready-to-use property of the stem cells.

[0020] The stem cell-hydrogel construct preparation method in the application is simple, and the stem cell-hydrogel construct has the following characteristics:

[0021] ①Under physiological conditions, the mixed stem cell suspension and the cell culture medium of dissolved polysaccharide can embed the stem cells in situ in three dimensions in the hydrogel, so as to realize short-term storage and transportation of the stem cells in a normal temperature environment.

[0022] ②The synergistic effect of the self-biodegradation of the polysaccharide macromolecular network and the reversible bond can provide a self-adaptive growth dynamic microenvironment for the growth of the three-dimensionally embedded stem cell colony.

[0023] ③In addition, the excellent biocompatibility and self-biodegradability of the polysaccharide and the dynamic bond can ensure that the stem cell-hydrogel construct automatically releases the stem cells without affecting the activity and phenotype, so as to realize the "ready-to-use" stem cell-biomaterial construct.

[0024] Preferably, the N-carboxyethyl chitosan is prepared by a method comprising the following steps:

[0025] The mixed solution of chitosan and acrylic acid is prepared, magnetically stirred, pH is adjusted, dialyzed in deionized water, and the mixed solution is freeze-dried to obtain N-carboxyethyl chitosan.

[0026] Preferably, the concentration of chitosan in the mixed solution is 5-10 mM, for example, it can be 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM, etc.

[0027] Preferably, the concentration of acrylic acid in the mixed solution is 20-25 mM, for example, it can be 20 mM, 21 mM, 22 mM, 23 mM, 24 mM or 25 mM, etc.

[0028] Preferably, the chitosan is acid-soluble chitosan.

[0029] Preferably, the degree of deacetylation of the chitosan is 86-88%, for example, it can be 86%, 87% or 88%, etc.

[0030] Preferably, the molecular weight of the chitosan is 200000-300000 Da, for example, it can be 200000 Da or 300000 Da, etc.

[0031] Preferably, the temperature of the magnetic stirring is 50-52℃, for example, it can be 50℃, 51℃ or 52℃, etc., and the time of the magnetic stirring is 2.5-3 days, for example, it can be 2.5 days or 3 days, etc.

[0032] Preferably, the pH is adjusted to 10-12, for example, it can be 10, 11 or 12, etc.

[0033] Preferably, the dialysis bag used for dialysis has a molecular weight cut-off of 8000 Da.

[0034] Preferably, the oxidized sodium alginate is prepared by a method comprising the following steps:

[0035] The sodium alginate, sodium periodate and deionized water are mixed to form a reaction solution, which is oxidized under light-proof conditions. Then, ethylene glycol is added to terminate the oxidation reaction. The mixture obtained by the reaction is dialyzed and then freeze-dried to obtain the oxidized sodium alginate.

[0036] Preferably, the concentration of the sodium alginate in the reaction solution is (1.0-1.2g) / 100mL, for example, 1.0g / 100mL, 1.1g / 100mL or 1.2g / 100mL, etc.

[0037] Preferably, the concentration of the sodium periodate in the reaction solution is (0.54-1.2g) / 100mL, for example, 0.54g / 100mL, 0.8g / 100mL or 1.2g / 100mL, etc.

[0038] Preferably, the viscosity of the sodium alginate is greater than 350mpa.s.

[0039] Preferably, the oxidation reaction time is 5-6h, for example, 5h or 6h, etc., and the oxidation reaction temperature is 20-25℃, for example, 20℃, 23℃ or 25℃, etc.

[0040] Preferably, the volume concentration of the ethylene glycol in the reaction system is 1.5-2%, for example, 1.5% or 2%, etc.

[0041] Preferably, the stirring reaction time is 1-2h, for example, 1h or 2h, etc.

[0042] Preferably, the dialysis bag used in the dialysis has a molecular weight cut-off of 3000Da.

[0043] Preferably, the mass percentage of the N-carboxyethyl chitosan in the mixed solution is 1.5-2.5wt%, for example, 1.5wt%, 2wt% or 2.5wt%, etc.

[0044] Preferably, the mass percentage of the oxidized sodium alginate in the mixed solution is 0.5-1.5wt%, for example, 0.5wt%, 1wt% or 1.5wt%, etc.

[0045] Preferably, the incubation time is 15-25 minutes, for example, 15 minutes, 18 minutes, 20 minutes, 22 minutes or 25 minutes, etc.

[0046] In the present application, the incubation is carried out in a constant-temperature incubator at a temperature of 37℃ and with 5% CO2.

[0047] Further, the construction also comprises testing the self-healing performance, rheological performance of the dynamic hydrogel construction, and the proliferation activity, alkaline phosphatase activity of the embedded stem cells, and the relative expression amount of pluripotency markers (OCT3 / 4, NANOG, SSEA-1), and the alkaline phosphatase activity of the stem cells released by the dynamic hydrogel and the relative expression amount of NANOG, Dppa5a, Sox2.

[0048] The present application facilitates short-term storage and transportation of stem cells in normal temperature environment. Most importantly, the activity and phenotype of the stem cells can be maintained, and the stem cell-hydrogel construction can be directly used without additional treatment. The dynamic degradable "ready-to-use" stem cell-hydrogel construction prepared by the present application can effectively support the storage, transportation and immediate use of stem cells, and provides a convenient, economical and reliable immediate use scheme for stem cell application in the biomedical and research fields.

[0049] In the second aspect, the present application provides an injection comprising the stem cell-hydrogel construction of the first aspect.

[0050] The numerical range of the present application includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed, and the present application does not exhaustively list the specific point values included in the range for the sake of brevity and simplicity.

[0051] Compared with the prior art, the present application has the following beneficial effects:

[0052] The N-carboxyethyl chitosan / sodium alginate hydrogel significantly promotes the proliferation of stem cells. The three-dimensionally embedded stem cells are stored in a sealed container for three days, and then transferred to fresh culture medium for culture for 3 days, and the stem cells still maintain good pluripotency. In addition, the stem cells released automatically by the dynamic hydrogel maintain pluripotency after continuous culture for 15 generations (passage every five days, culture for 25 days). The synergistic effect of biodegradation of the hydrogel and the reversible cross-linked macromolecular network provides a dynamic microenvironment and growth space for stem cell proliferation and maintenance of pluripotency. The dynamic degradable "ready-to-use" stem cell-hydrogel construction prepared by the present application can effectively support the storage, transportation and immediate use of stem cells, and provides a convenient, economical and reliable stem cell application scheme for the biomedical and research fields, and realizes short-term storage and transportation of stem cells in normal temperature environment. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1A is a self-healing performance test result diagram of N-carboxyethyl chitosan / sodium alginate hydrogel;

[0054] Figure 1Bare the amplitude sweep rheology test results of N-carboxyethyl chitosan / sodium alginate hydrogel and stem cell-hydrogel construct at a constant angular velocity of 10 rad / s;

[0055] Figure 1C are the alternating shear strain test results of N-carboxyethyl chitosan / sodium alginate hydrogel and stem cell-hydrogel construct (10 rad / s, 1% and 800%, three cycles with 200 s interval).

[0056] Figure 2A are the optical microscope photos of stem cells three-dimensionally embedded in dynamic hydrogel (scale bar: 50 μm);

[0057] Figure 2B are the live / dead cell staining photos of stem cells three-dimensionally embedded in dynamic hydrogel after continuous culture for 3 dS+72 h (scale bar: 50 μm);

[0058] Figure 2C are the cell viability statistics of stem cells three-dimensionally embedded in dynamic hydrogel after continuous culture for 3 dS+72 h;

[0059] Figure 3A are the immunofluorescence staining results of pluripotency markers (OCT3 / 4, NANOG, SSEA-1) of stem cells three-dimensionally embedded in dynamic hydrogel after continuous culture for 3 dS+72 h;

[0060] Figure 3B are the relative expression amount detection results of pluripotency markers (OCT3 / 4, NANOG, SSEA-1) of stem cells three-dimensionally embedded in dynamic hydrogel after continuous culture for 3 dS+72 h;

[0061] Figure 4A are the optical microscope photos of P7 and P15 generation stem cells which were subcultured from the released stem cells (P0) of dynamic hydrogel (scale bar: 50 μm);

[0062] Figure 4B are the alkaline phosphatase staining photos of P7 and P15 generation stem cells which were subcultured from the released stem cells (P0) of dynamic hydrogel (scale bar: 50 μm);

[0063] Figure 4C are the immunofluorescence staining photos of the released stem cells of dynamic hydrogel (scale bar: 50 μm);

[0064] Figure 4D are the cell doubling time statistics of the released stem cells of dynamic hydrogel;

[0065] Figure 4EThe alkaline phosphatase cell viability statistics results of the dynamic hydrogel released stem cells (P0) for subculture P15 generations;

[0066] Figure 4F The NANOG, Dppa5a, Sox2 relative expression detection results of the dynamic hydrogel released stem cells. DETAILED DESCRIPTION

[0067] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application, and should not be regarded as specific limitations on the present application.

[0068] The specific technologies or conditions not specified in the embodiments are carried out according to the technologies or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments not specified by the manufacturer are all conventional products that can be commercially available through regular channels.

[0069] The material sources in the following specific embodiments are shown in Table 1.

[0070] Table 1

[0071] Materials Manufacturer Specification DMEM medium Gibco 500 mL Serum replacement (KSR) Gibco 500 mL GlutaMAX Gibco 50 mL Non-essential amino acids Gibco 50 mL Beta-mercaptoethanol Sigma-Aldrich 100 mL Dual antibody Gibco 100ml Leukemia inhibitory factor (LIF) Millipore 1 mL

[0072] Example 1

[0073] The present embodiment provides a preparation method of a dynamic hydrogel for storing stem cells in a normal temperature environment, comprising the following steps:

[0074] Step one: preparing N-carboxyethyl chitosan by Michael addition reaction.

[0075] The specific steps are as follows: first, dissolve chitosan in an acrylic acid solution to obtain a chitosan-acrylic acid mixed solution, the concentration of chitosan in the chitosan-acrylic acid mixed solution is 6.2 mM, and the concentration of acrylic acid in the chitosan-acrylic acid mixed solution is 21.3 mM; magnetically stir at 50°C for 3 days, the stirring speed is 90 rpm. The pH of the chitosan-acrylic acid mixed solution is adjusted to 11 by adding 1M sodium hydroxide solution dropwise. The solution is dialyzed in deionized water for three days (the dialysis bag has a molecular weight cut-off of 8000 Da), and the deionized water is replaced twice a day. Finally, the mixed solution after dialysis is freeze-dried to obtain N-carboxyethyl chitosan, and the amino substitution degree of the N-carboxyethyl chitosan is 48%.

[0076] Step two: synthesizing oxidized sodium alginate by sodium periodate oxidation.

[0077] The specific steps are as follows: 1.0 g of sodium alginate with a viscosity greater than 350 mpa.s is dissolved in 100 mL of deionized water, 1.08 g of sodium periodate (25°C, avoid light) is added, and magnetic stirring is carried out at 90 rpm for 5 hours. Then 1.5 mL of ethylene glycol is added, and the oxidation reaction is terminated after 1 hour of further reaction. The resulting mixed solution is dialyzed in deionized water (dialysis bag with a molecular weight cut-off of 3000 Da) for 3 days, with deionized water being replaced twice a day. The mixed solution after dialysis is freeze-dried to obtain oxidized sodium alginate, and the oxidation degree of the obtained oxidized sodium alginate is 50%.

[0078] Step three: preparation of stem cell-hydrogel construct.

[0079] The specific steps are as follows: a certain amount of oxidized sodium alginate or N-carboxyethyl chitosan is completely dissolved in cell culture medium (the formula of the cell culture medium is shown in Table 2), then the stem cells are resuspended in the N-carboxyethyl chitosan-dissolved culture medium to obtain a cell suspension, and then the cell suspension is mixed with the cell culture medium in which the oxidized sodium alginate is dissolved to obtain a mixed solution, wherein the mass percentage of N-carboxyethyl chitosan in the mixed solution is 2 wt%, the mass percentage of oxidized sodium alginate is 1.0 wt%, and the density of stem cells is 3 x 10 5 cell / mL, and the mixed solution is incubated at 37°C in a constant temperature incubator with 5% CO2 for 20 minutes to form a stem cell-hydrogel construct. The hydrogel matrix comprises, by weight fraction, 1 part of oxidized sodium alginate, 2 parts of N-carboxyethyl chitosan, and 97 parts of cell culture medium.

[0080] Table 2

[0081] Reagent name Volume DMEM medium 41 mL KSR 7.5 mL GlutaMAX 0.5 mL Non-essential amino acids 0.5 mL Beta-mercaptoethanol 0.36 μL Dual antibody 0.5 mL LIF 0.005 mL

[0082] The stem cell-hydrogel construct is placed in a sterile environment at room temperature with 5% CO2 for 72 hours to simulate cell transportation in a normal temperature environment. Then, the stem cell-hydrogel construct is cultured in complete culture medium, and the culture medium is replaced regularly.

[0083] Example 2

[0084] The present example provides a preparation method of a dynamic hydrogel for storing stem cells in a normal temperature environment. The preparation method is as in Example 1, the oxidation degree of the prepared oxidized sodium alginate is 50%, and the amino substitution degree of the prepared N-carboxyethyl chitosan is 48%; in the process of preparing the stem cell-embedded hydrogel construct, the hydrogel matrix comprises, by weight fraction, 0.5 parts of oxidized sodium alginate, 2.5 parts of N-carboxyethyl chitosan, and 97 parts of cell culture medium.

[0085] Example 3

[0086] The present example provides a preparation method of a dynamic hydrogel for storing stem cells in a normal temperature environment. The preparation method is according to Example 1. The oxidation degree of the prepared oxidized sodium alginate is 55%, and the amino substitution degree of the prepared N-carboxyethyl chitosan is 50%. In the process of preparing the stem cell-hydrogel construct, the hydrogel matrix comprises, by weight fraction: 1.5 parts of oxidized sodium alginate, 1.5 parts of N-carboxyethyl chitosan, and 97 parts of cell culture medium.

[0087] Example 4

[0088] The present example provides a preparation method of a dynamic hydrogel for storing stem cells in a normal temperature environment. The method is different from Example 1 only in that the oxidation degree of the oxidized sodium alginate is 40%. The remaining steps are according to Example 1.

[0089] Example 5

[0090] The present example provides a preparation method of a dynamic hydrogel for storing stem cells in a normal temperature environment. The method is different from Example 1 only in that the oxidation degree of the oxidized sodium alginate is 60%. The remaining steps are according to Example 1.

[0091] Example 6

[0092] The present example provides a preparation method of a dynamic hydrogel for storing stem cells in a normal temperature environment. The method is different from Example 1 only in that the amino substitution degree of the N-carboxyethyl chitosan is 45%. The remaining steps are according to Example 1.

[0093] Example 7

[0094] The present example provides a preparation method of a dynamic hydrogel for storing stem cells in a normal temperature environment. The method is different from Example 1 only in that the amino substitution degree of the N-carboxyethyl chitosan is 55%. The remaining steps are according to Example 1.

[0095] Example 8

[0096] The present example provides a preparation method of a dynamic hydrogel for storing stem cells in a normal temperature environment. The method is different from Example 1 only in that the hydrogel matrix comprises, by weight fraction: 0.2 parts of oxidized sodium alginate, 2.8 parts of N-carboxyethyl chitosan, and 97 parts of cell culture medium. The remaining steps are according to Example 1.

[0097] The degradation time comparison results of the dynamic hydrogel constructs in Examples 1-8 are shown in Table 3.

[0098] Table 3

[0099] Sample Degradation time Example 1 72h Example 2 96h Example 3 74h Example 4 68h Example 5 64h Example 6 69h Example 7 68h Example 8 64h

[0100] From the comparison of Example 1 and Examples 4-5, when the oxidation degree of oxidized sodium alginate is lower or higher than 50%, the reaction between 1 part of aldehyde groups on oxidized sodium alginate and 2 parts of amino groups on N-carboxyethyl chitosan is incomplete, thus accelerating the self-degradation of stem cell-hydrogel construct; from the comparison of Example 1 and Examples 6-7, when the amino substitution degree of N-carboxyethyl chitosan is lower or higher than 48%, the reaction between 1 part of aldehyde groups on oxidized sodium alginate and 2 parts of amino groups on N-carboxyethyl chitosan is incomplete, thus accelerating the self-degradation of stem cell-hydrogel construct; from the comparison of Example 1 and Example 8, oxidized sodium alginate is easy to degrade, and the degradation time of stem cell-hydrogel construct can be adjusted by adjusting the concentration and oxidation degree of oxidized sodium alginate.

[0101] Example 1 for performance testing of stem cell-hydrogel construct

[0102] (1) Self-healing test

[0103] The hydrogel prepared in Example 1 is a N-carboxyethyl chitosan / oxidized sodium alginate hydrogel. The self-healing test of the hydrogel prepared in Example 1 is as follows: Figure 1A Figure 1 is a photograph of the self-healing performance test of N-carboxyethyl chitosan / oxidized sodium alginate hydrogel.

[0104] Firstly, the self-healing performance is evaluated by macroscopic test. As shown in Figure 1A (i), a round piece of hydrogel is cut into two halves from the middle, one half is dyed blue with methylene blue and the other half is dyed pink with rhodamine B. As shown in Figure 1A (ii), two pieces of hydrogel with different colors are combined together, and the interface healing process of N-carboxyethyl chitosan / oxidized sodium alginate hydrogel is observed under a microscope at 37°C for 5 minutes. As shown in Figure 1A (iii), the boundary between the two kinds of hydrogel is very clear in the initial stage, Figure 1A (iii), with the passage of time, the interface gradually becomes blurred, and the pigments diffuse into the phosphate buffer solution after 30 minutes Figure 1A (iv), and the contact interface almost disappears after 120 minutes Figure 1A (v). The above test results show that N-carboxyethyl chitosan / oxidized sodium alginate hydrogel has good self-healing ability.

[0105] (2) Rheological property test

[0106] The influence of three-dimensionally embedded stem cells on the dynamic behavior and self-healing performance of hydrogel is studied by rheological test, and the rheological properties of original N-carboxyethyl chitosan / oxidized sodium alginate hydrogel are compared.

[0107] The N-carboxyethyl chitosan / sodium alginate hydrogel and stem cell-hydrogel constructs were cut into 15 mm in diameter and 2 mm in thickness discs and then placed between the lower and upper plates of a rheometer for testing. In the amplitude sweep strain experiment, the angular velocity was fixed at 10 rad / s and the strain range was 1-1000%. After reaching the maximum strain of 1000%, the strain was adjusted to 1% and maintained for 10 minutes. The storage modulus (G') and loss modulus (G") of the sample at different strains were recorded during the test. In the alternating shear strain test, the angular velocity was 10 rad / s and the strain of each experimental cycle was from 1.0% to 800%. The storage modulus (G') of the material at different strains was recorded during the test.

[0108] Figure 1B Figure 3 is the results of the rheological property test of the stem cell-hydrogel construct and N-carboxyethyl chitosan / sodium alginate hydrogel at 10 rad / s. When the strain increased from 1% to 1000%, the storage modulus (G') of the prepared stem cell-hydrogel construct decreased sharply from 430.83 Pa to 3.28 Pa, while the corresponding loss modulus (G") increased from 2.64 Pa to 27.98 Pa. Similarly, the storage modulus of the N-carboxyethyl chitosan / sodium alginate hydrogel decreased from 226.73 Pa to 1.46 Pa, while the corresponding loss modulus increased from 1.07 Pa to 15.47 Pa. The results showed that the mechanical properties of the stem cell-hydrogel construct were higher than those of the original N-carboxyethyl chitosan / sodium alginate hydrogel, and the change trends of the storage modulus and loss modulus of the N-carboxyethyl chitosan / sodium alginate hydrogel and the stem cell-hydrogel construct were similar during the strain increase (1%-1000%). The intersection of the values of the storage modulus (G') and loss modulus (G") of the two samples occurred when the strain was 40%, at which time the mechanical properties of the samples were in the gel-solution transition state.

[0109] Figure 1CThe self-healing property of the stem cell-hydrogel construct was further confirmed by the alternate shear strain test (alternating at 1% and 800% shear strain every 200s), and compared with the original N-carboxyethyl chitosan / sodium alginate hydrogel. The hydrogels experienced the transition between gel-sol state under the alternate shear strain. When the shear strain increased from 1% to 800%, the original N-carboxyethyl chitosan / sodium alginate hydrogel and the stem cell-hydrogel construct showed the same trend, i.e. the storage modulus (G') value of the two samples decreased rapidly by about two orders of magnitude due to shear thinning, and when the shear strain decreased from 800% to 1%, the storage modulus of the two samples recovered to the original value. This phenomenon indicates that the network structure of the dynamic hydrogel is destroyed under large strain, and the sample experiences gel-sol transition, and the storage modulus decreases sharply; under small strain, the storage modulus of the N-carboxyethyl chitosan / sodium alginate hydrogel and the stem cell-hydrogel construct can recover to the original value, indicating that the embedded stem cells have no significant effect on the self-healing property of the N-carboxyethyl chitosan / sodium alginate hydrogel.

[0110] Test Example 2: Performance detection of stem cells embedded in dynamic hydrogel

[0111] (1) Evaluation of cell activity

[0112] In this test example, the cell activity of the stem cells embedded in the hydrogel in Example 1 was detected. The growth state of the cells was observed and recorded by an inverted fluorescence microscope during the culture process, and after 96h of culture, the embedded cells were detected by live / dead cell staining.

[0113] The components of the live / dead staining kit were Calcein AM and EthD-1. After the stem cell-hydrogel construct was cultured for a certain time, it was transferred to a detection solution containing 2μM Calcein AM and 4μM EthD-1 for 25 minutes, and then washed with a phosphate buffer solution. The treated sample was observed under a fluorescence microscope to detect the activity of the stem cells.

[0114] Figure 2A is an optical microscope image of the stem cells embedded in the hydrogel (scale: 50μm); from Figure 2A It can be seen that the diameter of the stem cell colony gradually increased from about 5μm (3dS+24h) to 60μm after transportation, forming a dense spherical cell mass, which confirmed that the dynamic hydrogel can promote cell proliferation during storage and transportation.

[0115] Figure 2BFigure 6 is a live / dead cell staining image of stem cells after continuous culture of stem cell-hydrogel construct for 3dS+72h (scale bar: 50 pm). The results show that most stem cells can adapt to the culture environment in dynamic hydrogel and maintain a cell survival rate of more than 90% (acridine orange labels live cells as green, and iodinated propyl iodide labels dead cells as red). Figure 2C Figure 7 is a statistical result of cell viability of stem cells after continuous culture of stem cell-hydrogel construct for 3dS+72h, as shown in Figure 2C Figure 7, the stem cell viability at the 3dS+24h, 3dS+48h and 3dS+72h transport time points is 96.92%, 95.02% and 90.67% respectively, which confirms that the N-carboxyethyl chitosan / sodium alginate hydrogel has good cell compatibility.

[0116] (2) Evaluation of totipotency of three-dimensionally embedded stem cells

[0117] Maintaining the pluripotency of stem cells is an important characteristic for stem cell-hydrogel construct to be widely used. In order to prove whether the embedding, storage and transport process will affect the totipotency of stem cells, the present test example uses immunofluorescence staining method to detect the pluripotency markers (OCT3 / 4, NAONG, SSEA-1) of stem cells embedded in dynamic hydrogel in Example 1. After culturing the stem cells three-dimensionally embedded in dynamic hydrogel for 96 hours, the culture solution is aspirated, and the preheated phosphate buffer solution is used to rinse three times. Then, 1 mL of 4% paraformaldehyde is added for fixation for 20 minutes, and the preheated phosphate buffer solution is used to rinse three times. After washing, 0.5 mL of 0.2% Triton X-100 is added to make the stem cells permeable for 10 minutes, and the preheated phosphate buffer solution is used to rinse three times. Then, 1 mL of 5% bovine serum albumin solution is added to block the stem cells at room temperature for 6 hours. After completion of the blocking, the blocking solution is aspirated, and 0.5 mL of primary antibody diluted with 1% bovine serum albumin solution (for different antibodies, the dilution ratio is diluted according to the usage instruction) is added to the stem cells, which are placed in a wet box at 4°C overnight. The next day, the stem cells are taken out and placed at room temperature for 30 minutes to balance the temperature, and then washed with 1% bovine serum albumin solution for 1 hour each time. After washing, the subsequent operation is carried out in the dark, and 0.5 mL of secondary antibody diluted with 1% bovine serum albumin solution (dilution ratio 1:500) is added to the stem cells, which are placed at room temperature for 6 hours. Then, the stem cells are washed with 1% bovine serum albumin solution for three times, and 0.5 mL of DAPI staining solution is added. After 5 minutes, the laser confocal microscope is used to observe and record the staining results.

[0118] Figure 3A and Figure 3Bimmunofluorescence staining results of pluripotency markers (OCT3 / 4, NANOG, SSEA-1) of stem cells embedded in dynamic hydrogels after continuous culture for 3dS+72h and relative expression. The above immunofluorescence staining results are as follows: after continuous culture for 3dS+72h, the pluripotency markers OCT3 / 4, NANOG and SSEA-1 of stem cell colonies growing in the dynamic hydrogel are highly expressed, indicating that the transported stem cells retain the expression of stem cell markers at the protein level, i.e. after embedding, storage and transportation, stem cells can normally express pluripotency markers, proving that three-dimensional culture of embedded stem cells in dynamic hydrogels can well maintain the pluripotency of stem cells. The above results show that the dynamic characteristics of macromolecular networks and sufficient nutrient and oxygen transport in biodegradable hydrogels can ensure the cell colony expansion and pluripotency of three-dimensionally embedded stem cells during storage and transportation.

[0119] Test Example 3 Performance testing of stem cells released from dynamic hydrogels

[0120] (1) Growth status characterization of released stem cells

[0121] In order to further evaluate the proliferation ability and pluripotency of stem cells released from dynamic hydrogels after long-term culture, the cell morphology and doubling time of stem cells just released from the hydrogel (denoted as S+T+P0) and cells cultured for 7 generations, 15 generations (denoted as S+T+P7, S+T+P15) were characterized.

[0122] Figure 4A Optical microscope photos of P0, P7 and P15 of stem cells released from dynamic hydrogels for subculture, the results show that whether it is S+T+P0 stem cells (ii) just released from dynamic hydrogels, or S+T+P7 (iii) and S+T+P15 (iv) stem cells continued to culture, they can form dense cell colonies, and there is no obvious morphological difference compared with conventional culture stem cells (control group).

[0123] (2) Alkaline phosphatase activity characterization of dynamically released stem cells

[0124] In order to determine whether the stem cells released from the hydrogel automatically still retain pluripotency, alkaline phosphatase (Alkaline phosphatase, ALP) staining method was used to preliminarily detect the pluripotency of recovered stem cells released.

[0125] 2.1 Alkaline phosphatase qualitative detection kit (1 kt, Sigma-Aldrich) was used to detect the pluripotency marker expression of stem cells.

[0126] After culturing the stem cells in a six-well plate for 48 hours, the original culture medium was aspirated, 0.5 mL of fixative was added, and after soaking for 20 minutes, the fixative was aspirated and the plate was washed three times with phosphate buffer solution for 5 minutes each. All subsequent steps must be performed under light-proof conditions. 0.5 mL of alkaline phosphatase staining solution was added to the culture plate and stored in the dark at room temperature for 30 minutes. Then, the plate was rinsed three times with phosphate buffer solution for 5 minutes each. After the washing is completed, an appropriate amount of hematoxylin solution was added for counterstaining for 2 minutes. Finally, the counterstain was aspirated and the plate was rinsed once with phosphate buffer solution. The plate was observed under a microscope and the experimental results were recorded.

[0127] 2.2 An alkaline phosphatase quantitative detection kit (150 mL, Nanjing Jiancheng Bioengineering Research) was used to detect cell pluripotency expression markers.

[0128] After culturing stem cells in six-well plates for 48 hours, the original culture medium was aspirated, the cells were rinsed three times with phosphate-buffered saline, and 250 μL of RIPA lysis buffer was added and incubated overnight at 4°C. The supernatant was collected the next day and assayed for stem cell pluripotency markers. The supernatant was mixed with the working solution at a ratio of 1:50 and incubated at 37°C. The change in sample absorbance was monitored at a wavelength of 410 nm for 1-3 minutes using a microplate reader to determine ΔA / min. Deionized water was used as a control group during the monitoring process. Finally, the alkaline phosphatase activity (U / L) of the sample was calculated. Simultaneously, the protein content (g / L) in the supernatant was determined using a BCA protein quantification kit. Finally, the alkaline phosphatase activity (U / g) of the final sample was calculated.

[0129] like Figure 4B As shown in the figure, under the action of alkaline phosphatase reagent, the stem cells just released and recovered (S+T+P0) and the stem cells after continued culture for S+T+P7 and S+T+P15 generations were stained brown-red like conventionally cultured stem cells, further indicating that there was no significant difference in the pluripotency between the collected stem cells and conventionally cultured stem cells ( Figure 4E These results preliminarily demonstrate that the released stem cells can form normal colonies and maintain pluripotency.

[0130] (3) Evaluation of the totipotency of released stem cells

[0131] Analysis of stem cell protein and gene (mRNA) level markers further demonstrated that the released and recovered stem cells can maintain pluripotency after long-term subculture. The experimental steps are as follows:

[0132] 3.1 Extraction of total cellular RNA

[0133] RNA was extracted using the Simply P total RNA extraction kit. The specific operation process was as follows: Under normal culture conditions, P15 stem cells and unembedded stem cells (control group) were co-cultured for 72 hours, and 2×10 6 Place 100 μL of Solution R1 in a 1.5 mL centrifuge tube. Vortex and mix for 30 seconds, then let stand at room temperature for 1 minute. Add 600 μL of Solution R2 to the mixture, mix thoroughly, and let stand at room temperature for 3-5 minutes. After standing, pipette the supernatant onto a purification column. Place the column in a centrifuge tube and centrifuge for 30 seconds. Discard the liquid in the outer tube. Add 600 μL of wash solution to the column and centrifuge for 30 seconds. Discard the liquid in the centrifuge tube and repeat the wash. Centrifuge the column again for 1 minute. Then transfer the column to a new 1.5 mL centrifuge tube, add 20-50 μL of lysis solution to the center of the membrane, let stand at room temperature for 1 minute, and centrifuge for 30 seconds to obtain total RNA.

[0134] 3.2 cDNA Synthesis

[0135] Using PrimeScript TM Synthesize cDNA using the RT Master Mix kit. To prepare cDNA, add 2 μL of 5× PrimeScript RT Master Mix to a centrifuge tube. Then, add the RNA obtained in step 1.1. Finally, add RNase-free dH2O to a total volume of 10 μL. Perform the entire preparation on ice. Gently mix the solution and incubate at 37°C for 15 minutes to generate the cDNA solution.

[0136] 3.3 RT-PCR reaction

[0137] After obtaining the cDNA solution, prepare the PCR reaction mixture in an ice bath according to Table 4. The primers for detecting each gene are listed in Table 5. Transfer the prepared PCR reaction mixture to an eight-tube tube and place it in a PCR instrument. Perform the reaction according to the pre-set two-step protocol (Table 6). After the reaction is completed, record the ct value and process the data.

[0138] Table 4

[0139] Reagent name Volume (μL) SYBR Premix Ex Taq II (Tli RNaseH Plus) (2x) 12.5 Upstream primer F (10 μM) 1.0 Downstream primer R (10 μM) 1.0 RT reaction solution (cDNA solution) 2.0 [dH2O (sterilized distilled water)] 8.5 Total 25

[0140] Table 5

[0141]

[0142] Table 6

[0143] Program process Program content Step one 95℃ for 30 s Step two 95℃ for 10 s, then 60℃ for 30 s, repeat the process 40 times Step three 95℃ for 15 s, then 60℃ for 1 min, finally 95℃ for 15 s

[0144] The stem cells of S+T+P0, S+T+P7 and S+T+P15 generations form dense cell colonies after being cultured for 48 hours, and show significant immunofluorescent staining effects on Oct3 / 4, NANOG and SSEA-1 Figure 4C ), and there is no significant difference compared with the stem cells of conventional passages. In order to quantitatively detect the pluripotency of the stem cells released by long-term passages, the stem cells of S+T+P15 are detected by RT-PCR. According to the immunofluorescent staining results Figure 4D ) and the RT-PCR analysis results, it is shown that the expression of NANOG, Dppa5a and Sox2 of the stem cells of S+T+P15 has no significant difference compared with the stem cells of conventional passages, which indicates that the released stem cells still maintain pluripotency after being continuously cultured for 25 days Figure 4F ). The above results collectively show that the N-carboxyethyl chitosan / sodium alginate hydrogel is a dynamic culture platform of stem cell-hydrogel construct, and can maintain the pluripotency of stem cells.

[0145] Test Example 4

[0146] The stem cell-hydrogel constructs are prepared by using adipose-derived stem cells or bone marrow mesenchymal stem cells respectively, and the cell activity performance is detected.

[0147] The adipose-derived stem cell-hydrogel construct and the bone marrow mesenchymal stem cell-hydrogel construct are constructed by referring to the method of Example 1 respectively, and the survival rate of the stored stem cells is detected, and 3 parallel experiments are set in each group. Table 7 is the survival rate statistics of the adipose-derived stem cells and the bone marrow mesenchymal stem cells.

[0148] Table 7

[0149]

[0150] From the results in Table 7, it can be seen that the stem cell-hydrogel construct is suitable for the storage and transportation of various types of stem cells, and can still maintain a high survival rate after being stored at room temperature for 72 hours. The hydrogel can realize the convenient storage of stem cells in a room temperature environment.

[0151] In summary, the present application provides a preparation method of an injectable "ready-to-use" stem cell-hydrogel construct which can conveniently store and transport stem cells in a room temperature environment, i.e. by embedding cells in three dimensions in situ and storing them in a dynamic hydrogel under physiological conditions, which is expected to break through the technical bottleneck of stem cell storage. The biodegradable and reversible cross-linked macromolecular network of the hydrogel has a synergistic effect, which provides an adaptive growth space and a dynamic microenvironment for adapting to the proliferation and pluripotency maintenance of stem cells. The present application is convenient for realizing the short-term storage and transportation of stem cells in a room temperature environment, and provides a convenient, economical and reliable stem cell application scheme for the field of biomedicine and research.

[0152] The applicant states that the above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and it should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought out by those skilled in the art, and all of them fall within the protection scope and disclosure scope of the present application.

Claims

1. Application of a dynamic injectable and degradable stem cell-hydrogel construct, characterized in that: The stem cell-hydrogel construct includes a hydrogel matrix and stem cells three-dimensionally embedded in the hydrogel matrix; The hydrogel matrix comprises, by weight, 0.5-1.5 parts of oxidized sodium alginate, 1.5-2.5 parts of N-carboxyethyl chitosan, and 96-98 parts of cell culture medium; The cell culture medium includes: DMEM culture medium, serum replacement solution, GlutaMAX solution, non-essential amino acid solution, β-mercaptoethanol solution, double antibody solution and leukemia inhibitory factor solution; The amino substitution degree of the N-carboxyethyl chitosan is 48-50%; the oxidation degree of the oxidized sodium alginate is 50-55% according to the hydroxylamine hydrochloride titration method; The stem cell-hydrogel construct is prepared by a preparation method comprising the following steps: Completely dissolving N-carboxyethyl chitosan and oxidized sodium alginate in a cell culture medium, respectively, resuspending stem cells in the cell culture medium containing the N-carboxyethyl chitosan to obtain a cell suspension, mixing the cell suspension with the cell culture medium containing the oxidized sodium alginate to obtain a mixed solution, and incubating the mixed solution in a 37° C. constant temperature incubator to prepare the stem cell-hydrogel construct; The mass percentage of N-carboxyethyl chitosan in the mixed solution is 1.5-2.5wt%; the mass percentage of oxidized sodium alginate in the mixed solution is 0.5-1.5wt%; and the incubation time is 15-25 minutes.

2. The use according to claim 1, characterized in that The N-carboxyethyl chitosan is prepared by a method comprising the following steps: A mixed solution of chitosan and acrylic acid was prepared, magnetically stirred, pH was adjusted, dialyzed in deionized water, and the mixed solution was freeze-dried to obtain N-carboxyethyl chitosan.

3. The use according to claim 2, characterized in that The concentration of chitosan in the mixed solution is 5-10 mM.

4. The use according to claim 2, characterized in that The concentration of acrylic acid in the mixed solution is 20-25 mM.

5. The use according to claim 2, characterized in that The chitosan is acid-soluble chitosan.

6. The use according to claim 2, characterized in that The deacetylation degree of the chitosan is 86-88%.

7. The use according to claim 2, characterized in that The molecular weight of the chitosan is 200,000-300,000 Da.

8. The use according to claim 2, characterized in that The temperature of the magnetic stirring is 50-52° C., and the time of the magnetic stirring is 2.5-3 days.

9. The use according to claim 2, characterized in that The pH was adjusted to 10-12.

10. The use according to claim 2, characterized in that The molecular weight cut-off of the dialysis bag used in the dialysis is 8000Da.

11. The use according to claim 1, characterized in that The oxidized sodium alginate is prepared by a method comprising the following steps: Sodium alginate, sodium periodate and deionized water are prepared into a reaction solution, which is oxidized under light-proof conditions, and then ethylene glycol is added with stirring to terminate the oxidation reaction. The resulting mixed solution is dialyzed and then freeze-dried to obtain oxidized sodium alginate.

12. The use according to claim 11, characterized in that The concentration of sodium alginate in the reaction solution is (1.0-1.2 g) / 100 mL.

13. The use according to claim 11, characterized in that The concentration of sodium periodate in the reaction solution is (0.54-1.2 g) / 100 mL.

14. The use according to claim 11, characterized in that The viscosity of the sodium alginate is greater than 350 mPa.s.

15. The use according to claim 11, characterized in that The oxidation reaction time is 5-6 hours, and the oxidation reaction temperature is 20-25°C.

16. The use according to claim 11, characterized in that The volume concentration of the ethylene glycol in the reaction system is 1.5-2%.

17. The use according to claim 11, characterized in that The stirring time is 1-2h.

18. The use according to claim 11, characterized in that The molecular weight cut-off of the dialysis bag used in the dialysis is 3000 Da.

19. An injection, characterized in that: The injection comprises the stem cell-hydrogel construct according to any one of claims 1 to 18.

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