Acetylated glucomannan electrospun membrane, and preparation method therefor and use thereof

Abstract

The present application discloses an acetylated glucomannan electrospun membrane, and a preparation method therefor and a use thereof. The preparation method for an electrospun membrane of the present application comprises: first adding glucomannan into a mixed solution of pyridine and acid anhydride, carrying out solid-liquid separation after the reaction, collecting a solid phase, adding ethanol for precipitation, and collecting the precipitate to obtain acetylated glucomannan; then dissolving the acetylated glucomannan in a mixed solution of chloroform and N,N-dimethylformamide, and carrying out ultrasonic treatment to obtain an electrospinning solution; and finally, using the electrospinning solution as a raw material, and carrying out electrospinning to obtain an acetylated glucomannan electrospun membrane.

Description

Acetylated glucomannan electrospinning membrane and its preparation method and application Technical Field

[0001] The present application relates to the field of biomaterial technology, and in particular to an acetylated glucomannan electrospinning membrane and a preparation method and application thereof. Background Art

[0002] In vivo, mammalian cells primarily rely on interactions within the extracellular matrix to generate connections and signal transduction. To simulate the cell culture process in vitro, 2D cell culture is a traditional and simple method, but it only produces flat cell morphology and can even alter cell gene expression. 3D cell culture, however, is becoming the mainstream method because it better simulates the in vivo cell growth environment. Cell tissues cultured using this method are often used to study cell metabolism and tissue regeneration.

[0003] In related technologies, the extracellular matrix (Matrigel) secreted by mouse chondrosarcoma cells is widely used in 3D cell culture systems. This matrix mainly includes collagen, mucin, gelatin, fibrin, hyaluronic acid, and other components, which are conducive to cell growth and adhesion. However, this type of extracellular matrix is ​​usually of animal origin and contains disease-related components. When used as a component for 3D cell culture, its disease-related components may have an adverse effect on cell culture. In addition, the uncertainty of the extracellular matrix composition leads to poor culture reproducibility and controllability.

[0004] To solve the above problems, the present application provides an acetylated glucomannan electrospinning membrane and a preparation method and application thereof.

[0005] Summary of the Invention

[0006] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes an acetylated glucomannan electrostatic spinning membrane and its preparation method and application. The acetylated glucomannan electrostatic spinning membrane preparation method of the present application is simple, and the obtained acetylated glucomannan molecular chain is rich in a large number of acetyl groups (-COCH3). The obtained acetylated glucomannan electrostatic spinning membrane fiber diameter is 905±283nm, without obvious beaded structure, and the fibers are intertwined into a network to form interconnected gaps. When applied to cell culture, it can well simulate the structure of the extracellular matrix, which is conducive to promoting cell adhesion and proliferation.

[0007] The present application also proposes an acetylated glucomannan electrospinning membrane.

[0008] The present application also provides a composition for cell culture.

[0009] The present application also proposes the use of an acetylated glucomannan electrospinning membrane or a cell culture composition in cell proliferation and / or cell differentiation culture.

[0010] In a first aspect of the present application, a method for preparing an acetylated glucomannan electrospinning membrane is provided, comprising the following steps:

[0011] Step S1: adding glucomannan to a mixed solution of pyridine and acid anhydride, performing solid-liquid separation after the reaction, collecting the solid phase and adding ethanol for precipitation, and then collecting the precipitate to obtain acetylated glucomannan;

[0012] Step S2: dissolving the acetylated glucomannan in a mixed solution of chloroform and N,N-dimethylformamide, and subjecting the solution to ultrasonic treatment to obtain an electrospinning solution;

[0013] Step S3: using the electrospinning solution as a raw material, and obtaining an acetylated glucomannan electrospinning membrane through electrospinning.

[0014] The preparation method according to the embodiment of the present application has at least the following beneficial effects:

[0015] The preparation method of the acetylated glucomannan electrospinning membrane of the present application is simple. First, by a mixed reaction of pyridine, anhydride and glucomannan, acetylated glucomannan (acGM) is obtained after alcohol precipitation. The analysis results of its organic functional groups show that there are a large number of acetyl groups (-COCH3) on the obtained acGM molecular chain, and its acetylation degree reaches 3.0; further, the present application mixes the obtained acetylated glucomannan with chloroform and N, N-dimethylformamide, obtains an electrospinning solution after ultrasonic treatment, and then uses the electrospinning solution as raw material to obtain an acetylated glucomannan electrospinning membrane after electrostatic spinning treatment. The preparation method is simple, does not require the addition of solubilizing substances, is low in cost, and is suitable for industrial production.

[0016] In some embodiments of the present application, in step S1, the weight-to-volume ratio of the mixed solution of glucomannan, pyridine and acid anhydride is (0.8-1.2) g:100 mL.

[0017] In some embodiments of the present application, the volume ratio of pyridine to acid anhydride in the mixed solution is 1:0.8-1.2.

[0018] In some embodiments of the present application, in step S1, the reaction temperature is 45°C to 60°C;

[0019] Preferably, the reaction time is 48 h to 96 h.

[0020] In some embodiments of the present application, in step S1, the mass volume ratio of the solid phase to ethanol is 1 g: (2-10) mL.

[0021] In some embodiments of the present application, the ethanol is analytically pure ethanol, specifically, the ethanol is pre-cooled analytically pure ethanol.

[0022] In some embodiments of the present application, the degree of acetylation of the acetylated glucomannan is 3.0.

[0023] In some embodiments of the present application, in step S2, the weight-to-volume ratio of the mixed solution of the acetylated glucomannan to chloroform and N,N-dimethylformamide is (150-250) mg:1 mL.

[0024] In some embodiments of the present application, the volume ratio of chloroform to N,N-dimethylformamide is 1.8-2.5:1.

[0025] According to some embodiments of the present application, adding a certain amount of N,N-dimethylformamide helps prevent chloroform from volatilizing too quickly.

[0026] In some embodiments of the present application, the electrospinning parameters include: an injection rate of 0.6 mL / h to 1 mL / h, a needle inner diameter of 0.33 mm to 0.51 mm, and a needle outer diameter of 0.64 mm to 0.81 mm.

[0027] Preferably, the electrospinning parameters are: injection speed of 0.8 mL / h, needle inner diameter of 0.41 mm, and needle outer diameter of 0.64 mm to 0.81 mm.

[0028] In some embodiments of the present application, the needle may be a flat needle.

[0029] According to some embodiments of the present application, since the electrospinning solution contains an organic solvent and has a certain viscosity, the use of a flat needle is beneficial to avoid clogging due to corrosion.

[0030] In some embodiments of the present application, during the electrospinning process, the voltage at the needle tip is +10 kV to +15 kV, the voltage at the receiving plate is -2 kV to -4 kV, and the distance between the needle tip and the collecting plate is 8 cm to 12 cm. Specifically, the voltage at the needle tip is +12 kV, and the voltage at the receiving plate can be -3 kV.

[0031] In some embodiments of the present application, during the electrospinning process, the temperature is 40° C. to 45° C., and the relative humidity is 8% to 12%. Specifically, during the electrospinning process, the temperature may be 40° C., and the relative humidity may be 10%.

[0032] The second aspect of the present application provides an acetylated glucomannan electrospinning membrane, which is prepared using the preparation method described in the first aspect.

[0033] The acetylated glucomannan electrospinning membrane according to the embodiment of the present application has at least the following beneficial effects:

[0034] The fiber morphology of the acetylated glucomannan electrospun membrane prepared by the preparation method of the present application is mainly thin cylindrical, and the diameter of the fibers constituting the acetylated glucomannan electrospun membrane is 905±283 nm (n=10), and there is no obvious beaded structure.

[0035] In addition, the acetylated glucomannan electrospun membrane fibers prepared by the preparation method of the present application are intertwined into a network, forming interconnected gaps, which can better simulate the structure of the extracellular matrix and are beneficial to cell adhesion and proliferation.

[0036] In some embodiments of the present application, the fiber diameter of the acetylated glucomannan electrospun membrane is 500 nm to 1300 nm;

[0037] Preferably, the fiber diameter of the acetylated glucomannan electrospinning membrane is 905±283 nm.

[0038] The third aspect of the present application provides a composition for cell culture, comprising the acetylated glucomannan electrospinning membrane of the second aspect and gelatin.

[0039] The cell culture composition according to the embodiment of the present application has at least the following beneficial effects:

[0040] The cell culture composition of the present application includes an acetylated glucomannan electrospun membrane and gelatin, which serves as a coating material during the cell culture process, can significantly reduce cell death, promote continuous cell proliferation, and has an obvious synergistic effect.

[0041] In some embodiments of the present application, the addition ratio of the gelatin is 0.05% to 0.2%.

[0042] Specifically, the addition ratio of the gelatin can be 0.1%.

[0043] In some embodiments of the present application, the cell culture composition is prepared by soaking the acetylated glucomannan electrospun membrane in a 0.05% to 0.2% gelatin solution.

[0044] Specifically, the soaking time is 6 hours to 10 hours.

[0045] The fourth aspect of the present application provides use of the acetylated glucomannan electrospinning membrane of the second aspect or the cell culture composition of the third aspect in cell proliferation and / or cell differentiation culture.

[0046] According to the application of the embodiments of the present application, there are at least the following beneficial effects: applying the acetylated glucomannan electrospinning membrane or cell culture composition of the present application to cell proliferation and / or cell differentiation culture can significantly reduce cell death and promote continuous cell proliferation. In addition, the results of promoting the directed differentiation of NILB-hiPSCs cells show that it can significantly promote the further dispersion of NILB-hiPSCs, the continuous extension of neural synapses, the reduction of cell nuclei, and the formation of neuron-like cells.

[0047] In some embodiments of the present application, the cells include totipotent stem cells, pluripotent stem cells, or unipotent stem cells.

[0048] Specifically, the stem cells may be human induced pluripotent stem cells;

[0049] More specifically, the stem cells may be human induced pluripotent stem cells directed to differentiate into motor neurons.

[0050] In some embodiments of the present application, the cell differentiation culture includes directed differentiation culture of human induced pluripotent stem cells;

[0051] Specifically, the cell type to be differentiated is a neuron-like cell;

[0052] More specifically, the culture medium for directed differentiation culture is Neurobasal medium supplemented with 1×B27 additive (ThermoFish), 1×N2 additive (ThermoFish), 0.5×double antibody (Gibco), BDNF (10 ng / mL, Alomone Lab), GDNF (10 ng / mL, Alomone Lab) and NT3 (10 ng / mL, Alomone Lab).

[0053] Other features and advantages of the present application will be set forth in the following description, and in part will be apparent from the description, or may be learned by practicing the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present application is further described below with reference to the accompanying drawings and embodiments, wherein:

[0055] FIG1 is an infrared spectrum of acetylated glucomannan and glucomannan in Example 1 of the present application.

[0056] FIG2 is a hydrogen nuclear magnetic resonance spectrum of acetylated glucomannan and glucomannan in Example 1 of the present application.

[0057] FIG3 is a diagram showing the morphology observation results of the acetylated glucomannan electrospun membrane of the present application.

[0058] FIG4 is a fluorescence micrograph of the present invention's stem cells cultured on an acetylated glucomannan (acGM) electrospun membrane.

[0059] Figure 5 is a diagram showing the results of directed differentiation of stem cells at different growth stages on the acetylated glucomannan (acGM) electrospinning membrane of the present application, wherein Figure A in Figure 5 is a flow chart of induced differentiation, and Figure B in Figure 5 is a diagram showing the results of directed differentiation on the third and fifth days.

[0060] FIG6 is a graph showing the expression of TUJ1, a universal neuronal marker, on day 7 of differentiation culture in the present application.

[0061] FIG. 7 is a diagram showing the expression of the motor neuron-specific marker HB9 on day 7 of differentiation culture of the present application.

[0062] FIG8 is a graph showing the expression of the neuronal universal markers TUJ1 and MAP2 on day 14 of differentiation culture of the present application. DETAILED DESCRIPTION

[0063] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0064] In the description of this application, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples.

[0065] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0066] Example 1

[0067] This embodiment provides a method for preparing an acetylated glucomannan (acGM) electrospinning membrane, comprising the following steps:

[0068] (1) Preparation of acetylated glucomannan (acGM)

[0069] Weigh a quantitative amount of 1 g of glucomannan (GM) and fully swell it in 200 mL of water. Use it after freeze-drying. After stirring 100 mL of a mixture of pyridine and anhydride (1:1, V / V) at 50°C for 30 minutes, slowly pour it into a round-bottom flask with GM and stir for 72 hours. After filtering the reaction solution, mix the filter residue with a mixture of 100 mL of pyridine and anhydride (1:1, V / V) and stir it in a round-bottom flask at 50°C for 72 hours. After filtering the reaction solution, collect the filter residue and add three times the volume of anhydrous ethanol to precipitate overnight. Filter, collect the filter residue, repeatedly resuspend it with ethanol, and centrifuge it 5 times. After the product is dialyzed for 2 days, freeze-dried to obtain the final reaction product, acetylated glucomannan.

[0070] (2) Preparation of electrospinning solution

[0071] 200 mg of the acetylated glucomannan prepared above was weighed and dissolved in 1 mL of a mixed reagent (chloroform:DMF=2:1), and stirred thoroughly until completely dissolved. The solution was allowed to stand and ultrasonicated to precipitate all bubbles, thereby obtaining a uniform and transparent electrospinning solution.

[0072] (3) Preparation of electrospinning membrane

[0073] The electrospinning solution was transferred into a 2.5 mL plastic syringe with a flat 22G needle, which was fixed on an electrospinning apparatus to prepare an electrospinning membrane, thereby obtaining an acetylated glucomannan electrospinning membrane. The electrospinning parameters were as follows: voltage: 15 kV, with a voltage of +12 kV at the needle tip and a voltage of -3 kV at the receiving plate; an injection rate of 0.8 mL / h; a distance of 10 cm between the needle tip and the collecting plate; a temperature of 40° C., and a relative humidity (RH) of 10%.

[0074] Example 2

[0075] This embodiment provides an acetylated glucomannan electrospinning membrane, which is prepared by the method of the above embodiment 1.

[0076] Example 3

[0077] This example provides a set of cell culture compositions, comprising the acetylated glucomannan electrospinning membrane prepared in Example 1 and 1% Gelatin (purchased from STEMCELL technologies).

[0078] Test example: Performance characterization of acetylated glucomannan electrospun membrane

[0079] 1. Experimental methods

[0080] (1) Identification of organic functional groups of acetylated glucomannan: The glucomannan (GM) raw material and the acetylated glucomannan (acGM) product in Example 1 were mixed with potassium bromide at a mass ratio of 1 / 200, ground into powder, and pressed into tablets. The tablets were detected by infrared spectrometer (FI-IR) with a scanning range of 4000-450 cm -1 The characteristic peak of carbonyl (-C=O) was observed at 1735 cm -1 The obtained product or raw material (10 mg) was fully dissolved in deuterated chloroform (600 μL), and the obtained product or raw material (10 mg) was fully dissolved in deuterated chloroform (600 μL). 1 H NMR changes at δ = 2.1 ppm (-COCH3).

[0081] (2) Scanning electron microscopy (SEM) of acetylated glucomannan electrospun membranes: A small amount of the glucomannan (GM) raw material and the acetylated glucomannan (acGM) product from Example 1 were directly adhered to a conductive adhesive. The samples were then surface-coated with gold using a sputtering coater (Quorum SC7620). Images of the electrospun fibers were captured using a scanning electron microscope (SEM, TESCAN MIRA LMS) at an accelerating voltage of 3 kV. The fiber diameter distribution was analyzed using ImageJ DiameterJ software.

[0082] 2. Experimental results and analysis

[0083] (1) Analysis of organic functional groups of acetylated glucomannan

[0084] The structure was characterized by infrared spectroscopy, wherein the infrared spectra of glucomannan and acetylated glucomannan are shown in Figure 1. It can be seen from the figure that acetylated glucomannan has a peak at 1747 cm -1 A strong absorption peak appeared near the carbonyl stretching vibration peak at 1372 cm -1 There is an obvious absorption peak near the methyl group. -1 A strong absorption peak appears near the acGM, which is the absorption peak of the saturated ester group, proving that there are a large number of acetyl groups (-COCH3) on the acGM molecular chain.

[0085] The results of nuclear magnetic resonance detection are shown in Figure 2. 1 The H NMR spectrum shows a peak in the range of δ = 2.1 ppm (-COCH3), which also indicates that the acetylation is successful.

[0086] (2) Morphology of acetylated glucomannan electrospun membrane

[0087] Scanning electron microscopy (SEM) was used to observe the morphology of the electrospun membrane. The results are shown in Figure 3. It can be observed that the obtained fiber morphology is mainly thin cylindrical. The fiber diameter of the acetylated dextran electrospun membrane is 905±283nm (n=10), and there is no obvious beaded structure. The fibers are intertwined into a network, forming interconnected gaps, which can better simulate the structure of the extracellular matrix and promote cell adhesion and proliferation.

[0088] Application Example 1: Application of acetylated glucomannan (acGM) electrospinning membrane in promoting stem cell growth

[0089] 1. Experimental methods

[0090] (1) The acGM electrospun membrane prepared in Example 1 was cut into 10*10 mm squares, placed in a 24-well cell culture plate, and soaked in 75% alcohol for 2 h. After discarding the 75% alcohol, the culture plate was placed in a biosafety cabinet and irradiated with ultraviolet light for 30 min for further disinfection.

[0091] (2) After the sterilized 24-well cell culture plates were dried, they were divided into three groups and coated with 0.1% Gelatin, acGM electrospun membrane (prepared in Example 2), and Gelatin + acGM electrospun membrane (prepared in Example 3), respectively. The Gelatin + acGM electrospun membrane was obtained by soaking the acGM electrospun membrane in 0.1% Gelatin. The culture well plates were placed in a cell culture incubator (37°C, 5% CO2) and coated overnight.

[0092] (3) After discarding the coating solution, human induced pluripotent stem cells (NILB-hiPSCs) that have been successfully constructed in the laboratory for motor neuron differentiation (this cell line conditionally expresses motor neuron-specific transcription factors Ngn2, Isl1, and Lhx3 under the induction of the drug doxycycline dox; and overexpresses the anti-apoptotic gene Bcl-xl and green fluorescent GFP) were seeded into culture wells, and stem cell culture medium mTeSR (purchased from STEMCELL technologies) containing the anti-apoptotic small molecule Y27632 (MCE, 10 μM) was added and cultured overnight in a cell culture incubator (37°C, 5% CO2).

[0093] (4) The next day, discard the culture medium and replace it with fresh stem cell culture medium mTeSR without Y27632. Continue culturing, changing the medium every other day, and observe cell morphology under a microscope.

[0094] (5) Culture for 6 days and take photos using a fluorescence microscope.

[0095] 2. Experimental results

[0096] The above experimental results are shown in Figure 4. The NILB-hiPSCs in the Gelatin group were in a semi-adherent state and were very easy to fall off and die during the culture process. The number of cells was very small and no clones were formed. The number of NILB-hiPSCs cultured in the acGM electrospinning membrane was slightly more than that in the Gelatin group, but still relatively small, and a large number of rounded cells indicated that the cells died. The NILB-hiPSCs inoculated in the Gelatin+acGM electrospinning membrane had less cell death during the culture process, could continue to proliferate, and formed a stem cell clone state, indicating that the cells were in good condition. The cell number was significantly greater than that in the Gelatin group and the acGM electrospinning membrane group.

[0097] The above results show that acGM electrospinning membrane is better than gelatin coating, among which the co-coating effect of gelatin and acGM electrospinning membrane is the best and can be used for stem cell culture.

[0098] Application Example 2: Application of Acetylated Glucomannan Electrospinning Membrane in Promoting Directed Differentiation of Stem Cells

[0099] 1. Experimental methods

[0100] (1) According to the method in the above application example 1, on day 0, NILB-hiPSCs were seeded in the Gelatin+acGM electrospinning membrane at a seeding density of 5*10 4 / well (24-well plate).

[0101] (2) Then, differentiation was induced according to the experimental flow chart A in Figure 5. Specifically, on day 1, Dox (1 μg / mL, 5 days) was added to the motor neuron induction medium (the medium formula is: neurobasal medium + B27 additive + double antibody + glutamax + NEAA + DAPT 5 μM + SU5402 5 μM) to induce NILB-hiPSCs to undergo directed differentiation into motor neurons.

[0102] (3) During the 5-day induction period, change the medium by half every day.

[0103] (4) Starting from the 6th day, the medium was changed to motor neuron maturation medium (neurobasal medium + B27 supplement + N2 supplement + double antibody + BDNF + GDNF + NT3) and culture was continued for 14 days.

[0104] 2. Experimental results

[0105] The results of the above cell differentiation are shown in Figure 5B, which show that after 3 days of Dox treatment, NILB-hiPSCs dispersed from the clone state, and synapses appeared at the edge of the clone; after 5 days of Dox treatment, NILB-hiPSCs further dispersed, the synapses continued to extend, the cell nuclei became smaller, and neuron-like cells were formed.

[0106] The above results indicate that acGM electrospun membranes co-coated with Gelatin can be used for the directed differentiation of motor neurons.

[0107] Application Example 3: Application of Acetylated Glucomannan Electrospun Membrane in Identifying Motor Neurons

[0108] 1. Experimental methods

[0109] According to the method in Application Example 2, the motor neurons in the Gelatin+acGM electrospun membrane were identified by cell immunofluorescence staining on the 7th and 14th days, respectively. The experimental steps of cell immunofluorescence include:

[0110] (1) In a 24-well plate containing Gelatin+acGM electrospun membrane, cells were washed three times with 500 μL DPBS in each well, and then 300 μL 4% PFA was added to each well and fixed at room temperature for 15 to 20 minutes.

[0111] (2) Aspirate 4% PFA and wash each well three times with 500 μL DPBS, each wash for 15 minutes.

[0112] (3) Blocking and permeabilization: Add 500 μL of tissue blocking solution to each well and block at room temperature for 1 to 2 hours.

[0113] (4) Primary antibody incubation: Discard the blocking solution, add 400 μL of primary antibody diluted in blocking solution, and incubate at 4°C overnight.

[0114] (5) The next day, discard the primary antibody and wash the sections with PBS three times, 15 min each time.

[0115] (6) Secondary antibody incubation: After washing, select the corresponding fluorescent secondary antibody according to the properties of the primary antibody, dilute it with 3% BSA, add 300 μL of the diluted secondary antibody to each slide, and incubate at room temperature in the dark for 1 to 2 hours.

[0116] (7) Remove the secondary antibody and wash with PBS three times, 15 min each time.

[0117] (8) Add 400 μL of DAPI working solution to each well, incubate at room temperature in the dark for 5 min, and then wash with PBS for 5 min.

[0118] (9) Aspirate the PBS, transfer the membrane to a glass slide, add 50% glycerol (50 μL / slide), cover the slide with a coverslip from one end of the glycerol to prevent bubbles from forming, squeeze out excess glycerol, and wipe the slide dry.

[0119] (10) After sealing, the slides were stored in the dark and placed under a Zeiss710 NLO confocal microscope for 3D multi-layer scanning.

[0120] 2. Experimental results

[0121] Figures 6 to 8 show the results of cell immunofluorescence experiments, demonstrating that motor neurons differentiated from NILB-hiPSCs were dispersed within the Gelatin+acGM electrospun membrane. By day 7 of differentiation, they expressed abundantly the universal neuronal marker TUJ1 (Figure 6) and the motor neuron-specific marker HB9 (Figure 7). After continued culture for 14 days, the neurons further matured and expressed the maturation marker MAP2 (Figure 8).

[0122] These results demonstrate that genetically modified NILB-hiPSCs can be cultured in 3D on Gelatin+acGM electrospun membranes and undergo directed differentiation into motor neurons under drug induction. These induced neurons express abundant motor neuron-related markers.

[0123] In summary, the present application provides an acetylated glucomannan electrospinning membrane and its preparation method and application, the preparation method comprising: first, by a mixed reaction of pyridine, anhydride and glucomannan, acetylated glucomannan (acGM) is obtained after alcohol precipitation, and the analysis results of its organic functional groups show that there are a large number of acetyl groups (-COCH3) on the obtained acGM molecular chain, and its acetylation degree reaches 3.0; further, the acetylated glucomannan prepared in the present application is mixed with chloroform and N, N-dimethylformamide for reaction, and an electrospinning solution is obtained after ultrasonic treatment, and then the electrospinning solution is used as a raw material, and an acetylated glucomannan electrospinning membrane is obtained after electrostatic spinning treatment. The preparation method is simple, does not require the addition of solubilizing substances, has low cost, and is suitable for industrial production.

[0124] Furthermore, experiments show that the fiber morphology of the acetylated glucomannan electrospun membrane prepared by the preparation method of the present application is mainly thin cylindrical, and the fiber diameter of the acetylated glucomannan electrospun membrane is 905±283nm (n=10), and there is no obvious beaded structure. The fibers are intertwined into a network, forming interconnected gaps, which can relatively better simulate the structure of the extracellular matrix and play an important role in promoting cell adhesion and proliferation.

[0125] In addition, the acetylated glucomannan electrospun membrane prepared in this application was applied to stem cell culture, and the results showed that it could reduce cell death and promote cell proliferation and directed differentiation. Furthermore, the acetylated glucomannan electrospun membrane prepared in this application was applied to directed differentiation culture of neurons, and the results showed that it could achieve directed differentiation.

[0126] The embodiments of the present application have been described in detail above, but the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the protection of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a conflict.

Claims

1. A method for preparing an acetylated glucomannan electrospinning membrane, characterized in that: The following steps are involved: Step S1: adding glucomannan to a mixed solution of pyridine and acid anhydride, separating the solid and the liquid after the reaction, collecting the solid phase and adding ethanol for precipitation, and then collecting the precipitate to obtain acetylated glucomannan; Step S2: dissolving the acetylated glucomannan in a mixed solution of chloroform and N,N-dimethylformamide, and subjecting the mixture to ultrasonic treatment to obtain an electrospinning solution; Step S3: using the electrospinning solution as a raw material, and obtaining an acetylated glucomannan electrospinning membrane through electrospinning.

2. The preparation method according to claim 1, characterized in that: In step S1, the weight volume ratio of the mixed solution of glucomannan, pyridine and acid anhydride is (0.8-1.2) g:100 mL; the volume ratio of pyridine and acid anhydride in the mixed solution is 1:0.8-1.

2.

3. The preparation method according to claim 1, characterized in that: In step S1, the reaction temperature is 45°C to 60°C; Preferably, the reaction time is 48 h to 96 h.

4. The preparation method according to claim 1, characterized in that: In step S2, the weight-to-volume ratio of the acetylated glucomannan to the mixed solution of chloroform and N,N-dimethylformamide is (150-250) mg:1 mL.

5. The preparation method according to claim 4, characterized in that: In step S2, the volume ratio of chloroform to N,N-dimethylformamide is 1.8-2.5:

1.

6. The preparation method according to claim 1, characterized in that: In step S3, the electrospinning parameters include: an injection speed of 0.6 mL / h to 1 mL / h, a needle inner diameter of 0.33 mm to 0.51 mm, and a needle outer diameter of 0.64 mm to 0.81 mm.

7. An acetylated glucomannan electrospinning membrane, characterized in that: The method is prepared according to any one of claims 1 to 6.

8. A composition for cell culture, characterized in that The invention comprises the acetylated glucomannan electrospinning membrane according to claim 7 and gelatin.

9. The composition for cell culture according to claim 8, characterized in that The added amount of gelatin is 0.05% to 0.2%.

10. Use of the acetylated glucomannan electrospinning membrane according to claim 7 or the cell culture composition according to any one of claims 8 to 9 in cell proliferation and / or cell differentiation culture.

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