A hydrogel and its application in amplifying and culturing mesenchymal stem cells

Abstract

The present invention relates to the technical field of cell culture. The present invention provides a hydrogel, the components of which include agarose, gelatin, sodium alginate, chitosan, glutathione, insulin-like growth factor 1, and distilled water. The hydrogel prepared based on the components has better performance effects such as strength and biodegradability, can effectively promote cell proliferation and differentiation in the application of amplifying mesenchymal stem cells, and plays an important role in large-scale amplification and culture of stem cells.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a hydrogel and an application thereof in the amplification and culture of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells are a type of multipotent stem cells that have all the common characteristics of stem cells, namely, self-renewal and multidirectional differentiation capabilities. They are also the most widely used in clinical applications. When used in combination with hematopoietic stem cells, they can improve the success rate of transplantation and accelerate hematopoietic reconstruction. Mesenchymal stem cells exist not only in the bone marrow, but also in skeletal muscle, periosteum, and trabecular bone. Because they can differentiate into a wide range of tissue types, their clinical applications are very valuable.

[0003] However, mesenchymal stem cells currently have limited in vitro expansion capacity, slow proliferation after passage, reduced differentiation capacity, and other problems. The effective number of passages is only 3-7 times, and there is a large gap between supply and demand. Therefore, it is very necessary to develop a method for rapid and large-scale expansion and culture of mesenchymal stem cells. Summary of the invention

[0004] The purpose of the present invention is to provide a hydrogel and its application in the expansion and culture of mesenchymal stem cells. The hydrogel prepared based on the components has good mechanical properties, biodegradability and other performance characteristics, and can better promote cell proliferation and differentiation in the application of expanding mesenchymal stem cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a hydrogel, which comprises the following components in weight percentage: 1-5% agarose, 2-8% gelatin, 1-4% sodium alginate, 2-4% chitosan, 0.2-1% glutathione, 0.1-0.5% insulin-like growth factor 1 and 82-90% distilled water.

[0007] Preferably, the composition comprises the following components in weight percentage: 1.2-4.5% agarose, 2.2-7.8% gelatin, 1.1-3% sodium alginate, 2.3-3.8% chitosan, 0.3-0.8% glutathione, 0.15-0.45% insulin-like growth factor 1 and 82.5-87% distilled water.

[0008] The present invention also provides a method for preparing the hydrogel, comprising the following steps: mixing agarose, gelatin, sodium alginate, chitosan and distilled water, adding glutathione and insulin-like growth factor 1 in sequence, stirring and then standing to obtain the hydrogel.

[0009] Preferably, the stirring speed is 500-1000 rpm, and the stirring time is 3-6 min.

[0010] Preferably, the standing temperature is 18-30° C., and the standing time is 5-10 min.

[0011] The present invention also provides application of the hydrogel in amplifying and culturing mesenchymal stem cells.

[0012] By adopting the above technical solution, the present invention has the following beneficial effects:

[0013] 1. The hydrogel prepared by using the components of the present invention has good mechanical properties, biodegradability and other performance characteristics. Among them, the addition of glutathione and insulin-like growth factor 1 can continuously provide nutrition for the culture of mesenchymal stem cells under the sustained release effect of the hydrogel; and the two components are used in combination, and the effect is stronger.

[0014] 2. The hydrogel prepared by the technical solution of the present invention can better promote the proliferation and differentiation of cells in the application of amplifying mesenchymal stem cells, and effectively ensure the continuous amplification ability of cells. DETAILED DESCRIPTION

[0015] The invention provides a hydrogel, which comprises the following components: agarose, gelatin, sodium alginate, chitosan, glutathione, insulin-like growth factor 1 and distilled water.

[0016] In the present invention, the insulin-like growth factor 1 can stimulate cell proliferation and regeneration, and enhance the cell division and regeneration capabilities.

[0017] In the present invention, the glutathione, as an important sulfhydryl compound in stem cells, not only participates in the active oxygen metabolism of stem cells, but also plays an important role in the self-renewal and proliferation of stem cells, the aging of stem cells, the maintenance of the multidirectional differentiation potential of stem cells, and the regulation of stem cell differentiation.

[0018] In the present invention, the weight percentage of each component is preferably 1-5% agarose, 2-8% gelatin, 1-4% sodium alginate, 2-4% chitosan, 0.2-1% glutathione, 0.1-0.5% insulin-like growth factor 1 and 82-90% distilled water; further preferably 1.2-4.5% agarose, 2.2-7.8% gelatin, 1.1-3% sodium alginate, 2.3-3.8% chitosan, 0.3-0.8% glutathione, 0.15-0.45% insulin-like growth factor 1 and 82.5-87% distilled water; further preferably 2-4% agarose, 3-7% gelatin, 1.5-2.5% sodium alginate, 2.5-3.5% chitosan, 0.5-0.7% glutathione, 0.2-0.4% insulin-like growth factor 1 and 83-86% distilled water.

[0019] The present invention also provides a method for preparing the hydrogel, comprising the following steps: mixing agarose, gelatin, sodium alginate, chitosan and distilled water, adding glutathione and insulin-like growth factor 1 in sequence, stirring and then standing to obtain the hydrogel.

[0020] In the present invention, agarose, gelatin, sodium alginate, chitosan and distilled water are mixed evenly, and then glutathione and insulin-like growth factor 1 are added in sequence, and stirring is performed while adding. The stirring of the present invention is preferably performed using a rotor stirrer, and the stirring speed is preferably 500-1000rpm, more preferably 600-900rpm, and more preferably 800rpm; the stirring time is preferably 3-6min, more preferably 3.5-5min, and more preferably 4min. After the stirring is completed, the mixture is allowed to stand, and the standing temperature is preferably 18-30°C, more preferably 20-28°C, and more preferably 25°C; the standing time is preferably 5-10min, more preferably 6-9min, and more preferably 8min.

[0021] The present invention also provides application of the hydrogel in amplifying and culturing mesenchymal stem cells.

[0022] In the present invention, the density of mesenchymal stem cell seeding is preferably (0.2-0.8)×10 5 / mL, and more preferably (0.4-0.6)×10 5 / mL, and more preferably 0.5×10 5 Pieces / mL.

[0023] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] Example 1

[0025] A hydrogel, comprising: 4% agarose, 5.5% gelatin, 2.3% sodium alginate, 2.5% chitosan, 0.5% glutathione, 0.2% insulin-like growth factor 1 and 85% distilled water;

[0026] The preparation method of the above hydrogel comprises the following steps:

[0027] The agarose, gelatin, sodium alginate, chitosan and distilled water in the weight percentages are uniformly mixed, and then glutathione and insulin-like growth factor 1 are added in sequence, and a rotor stirrer is used to stir at a speed of 800 rpm for 4 minutes, and then the mixture is allowed to stand at 25° C. for 8 minutes.

[0028] Example 2

[0029] A hydrogel, comprising: 3% agarose, 8% gelatin, 3% sodium alginate, 2.4% chitosan, 0.2% glutathione, 0.4% insulin-like growth factor 1 and 83% distilled water;

[0030] The preparation method of the above hydrogel comprises the following steps:

[0031] The agarose, gelatin, sodium alginate, chitosan and distilled water in the weight percentages are uniformly mixed, and then glutathione and insulin-like growth factor 1 are added in sequence, and a rotor stirrer is used to stir at a speed of 600 rpm for 5 minutes, and then the mixture is allowed to stand at 20° C. for 10 minutes.

[0032] Example 3

[0033] A hydrogel, comprising: 2.5% agarose, 3% gelatin, 2% sodium alginate, 4% chitosan, 0.7% glutathione, 0.3% insulin-like growth factor 1 and 87.5% distilled water;

[0034] The preparation method of the above hydrogel comprises the following steps:

[0035] The agarose, gelatin, sodium alginate, chitosan and distilled water in the weight percentages are uniformly mixed, and then glutathione and insulin-like growth factor 1 are added in sequence, and a rotor stirrer is used to stir at a speed of 500 rpm for 4 minutes, and then the mixture is allowed to stand at 28° C. for 6 minutes.

[0036] Comparative Example 1

[0037] A hydrogel, comprising: 5% agarose, 3.9% chitosan, 0.8% glutathione, 0.3% insulin-like growth factor 1 and 90% distilled water;

[0038] The preparation method of the above hydrogel is the same as that of Example 1.

[0039] Comparative Example 2

[0040] A hydrogel, comprising: 3.5% agarose, 6.5% gelatin, 3% sodium alginate, 2% chitosan and 85% distilled water;

[0041] The preparation method of the above hydrogel is the same as that of Example 1.

[0042] Comparative Example 3

[0043] A hydrogel, comprising: 1% agarose, 15% gelatin, 1% sodium alginate, 8% chitosan, 0.1% glutathione, 0.1% insulin-like growth factor 1 and 74.8% distilled water;

[0044] The preparation method of the above hydrogel is the same as that of Example 1.

[0045] Comparative Example 4

[0046] A hydrogel, the components of which are the same as those in Example 1;

[0047] The preparation method of the above hydrogel comprises the following steps:

[0048] After the agarose, gelatin, sodium alginate, chitosan, glutathione and insulin-like growth factor 1 in the weight percentage are mixed evenly, distilled water is added, and a rotor stirrer is used to stir at a speed of 100 rpm for 2 minutes, and then the mixture is allowed to stand at 60° C. for 8 minutes to obtain the mixture.

[0049] Test Example 1

[0050] The performance of the hydrogels obtained based on Examples 1-3 and Comparative Examples 1-4 was tested.

[0051] (I) Porosity

[0052] The hydrogels obtained in Examples 1-3 and Comparative Examples 1-4 were freeze-dried and weighed, which was recorded as W0. The mass of the pycnometer filled with ethanol was W1, the mass after ethanol fully filled the gaps in the hydrogel and was filled with ethanol was W2, and the mass of the remaining ethanol and the pycnometer after taking out the hydrogel was W3, which were weighed separately using an analytical balance.

[0053] Porosity (%) = [(W2-W3-W0) / (W1-W3)] × 100

[0054] Table 1 Porosity of hydrogels in different groups

[0055] Group Porosity (%) Example 1 88.34 Example 2 86.92 Example 3 87.55 Comparative Example 1 97.13 Comparative Example 2 92.49 Comparative Example 3 91.32 Comparative Example 4 96.65

[0056] It can be seen from Table 1 that the porosity of the hydrogel prepared in Comparative Examples 1-4 is higher than that in Examples 1-3. The higher the porosity of the hydrogel, the faster its water absorption and dehydration speed, and the corresponding mechanical properties and sensitivity to surface liquid changes are reduced, which is not conducive to the culture of mesenchymal stem cells.

[0057] (ii) Swelling rate

[0058] The hydrogels obtained in Examples 1-3 and Comparative Examples 1-4 were placed in PBS solutions, respectively, and balanced at 37° C. for 24 hours. The hydrogels were then taken out to absorb the surface moisture, and the weights after swelling were respectively measured as M1. The hydrogels were then freeze-dried to obtain the weights M2.

[0059] Swelling rate (%) = (M1-M2) / M1×100

[0060] Table 2 Swelling rate of hydrogels in different groups

[0061] Group Swelling rate (%) Example 1 19.13 Example 2 18.87 Example 3 19.46 Comparative Example 1 10.32 Comparative Example 2 17.25 Comparative Example 3 11.54 Comparative Example 4 12.18

[0062] It can be seen from Table 2 that the swelling ratio of the hydrogels prepared in Examples 1-3 of the present invention is relatively high.

[0063] (III) Degradability

[0064] The hydrogels obtained in Examples 1-3 and Comparative Examples 1-4 were freeze-dried and weighed, recorded as M3, and then placed in a PBS solution for swelling. The swollen samples were placed in 15 mL centrifuge tubes, and 5 mL of PBS solution containing 2U / mL type I collagenase was added to each tube. The centrifuge tubes were then placed in a constant temperature water bath shaker at 37°C, and the enzyme solution was replaced every 2 days to maintain enzyme activity. The sample was taken out on the 20th day, rinsed twice with deionized water, and then freeze-dried and weighed, recorded as M4.

[0065] Degradability (%) = (M3-M4) / M3×100

[0066] Table 3 Degradability of hydrogels in different groups

[0067] Group Degradability (%) Example 1 83.75 Example 2 82.84 Example 3 83.12 Comparative Example 1 50.67 Comparative Example 2 71.35 Comparative Example 3 59.24 Comparative Example 4 63.58

[0068] It can be seen from Table 3 that the hydrogels prepared in Examples 1-3 of the present invention have better degradability.

[0069] In summary, it can be seen that the hydrogel prepared by the technical solution of the present invention has better comprehensive performance characteristics and is more suitable for the culture of mesenchymal stem cells.

[0070] Test Example 2

[0071] (I) Cell viability

[0072] UCMSCs cultured to the fourth generation (purchased from ATCC official website) were taken and 0.5×10 5 The cells were inoculated at a density of 100 μg / mL in the wells of a 96-well culture plate. Seven test groups and one control group were set up, with 5 wells in each group and 200 μL in each well; the blank group was cultured with the same volume of culture medium for culturing UCMSCs. Then the cells were cultured at 37°C and 50 mL / L CO2 saturated humidity. After 24 hours of complete cell attachment, the hydrogels prepared in Examples 1-3 and Comparative Examples 1-4 were added to the wells of the test group, respectively, and the wells of the control group and the blank group were not treated. After incubation in a CO2 incubator for 72 hours, the supernatant was removed, 100 μL (5 g / L) of MTT reagent and 500 μL of serum-free culture medium were added to each well, and the culture was terminated after incubation for 4 hours. The supernatant was removed, 500 μL of dimethyl sulfoxide was added to each well, and the absorbance value (OD) was read on a UV spectrophotometer at a wavelength of 595 nm.

[0073] Cell viability (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100

[0074] Table 4 Cell viability of UCMSCs in different groups

[0075] Group Cell viability (%) Example 1 96.64 Example 2 96.03 Example 3 96.25 Comparative Example 1 77.31 Comparative Example 2 78.50 Comparative Example 3 83.72 Comparative Example 4 85.24

[0076] It can be seen from Table 4 that, compared with the comparative examples 1-4, the cell viability of UCMSCs in the examples 1-3 of the present invention is higher, which indicates that culturing UCMSCs with the hydrogels prepared in the examples 1-3 of the present invention can improve the cell viability.

[0077] (II) Cell proliferation

[0078] UCMSCs cultured to the fourth generation (purchased from ATCC official website) were taken and 0.5×10 5 The cells were inoculated at a density of 100 μL / mL in the wells of a 96-well culture plate, and 8 groups of wells were selected, with 5 wells in each group and 200 μL in each well. The hydrogels prepared in Examples 1-3 and Comparative Examples 1-4 were added to 7 groups of wells, respectively, and the other group was not treated (as a control group). The proliferation rate of UCMSCs in the hydrogel after 10 days of culture was measured using a CCK-8 kit. 10 μL of CCK-8 solution was added to each well and incubated in a constant temperature incubator at 37°C for 2.5 hours. The liquid was aspirated and placed in another 96-well plate, and the absorbance values ​​(OD) of the experimental group and the control group were measured using a fully automatic microplate reader.

[0079] Cell proliferation rate (%) = OD experimental group / OD control group × 100

[0080] Table 5 Cell proliferation of UCMSCs in different groups

[0081]

[0082]

[0083] It can be seen from Table 5 that the cell proliferation rate of UCMSCs in Examples 1-3 of the present invention is higher than that in Comparative Examples 1-4, indicating that culturing UCMSCs with the hydrogel prepared in Examples 1-3 of the present invention can promote cell proliferation.

[0084] It can be seen from the above embodiments and test examples that the hydrogel prepared by the technical solution of the present invention has good mechanical properties, biodegradability and other performance characteristics; and the use of the hydrogel of the present invention to expand mesenchymal stem cells can effectively promote cell proliferation and significantly improve cell viability and cell proliferation rate.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A hydrogel, characterized in that: The invention is composed of the following components in weight percentage: 1.2-4.5% agarose, 2.2-7.8% gelatin, 1.1-3% sodium alginate, 2.3-3.8% chitosan, 0.3-0.8% glutathione, 0.15-0.45% insulin-like growth factor 1 and 82.5-87% distilled water; The preparation method of the hydrogel comprises the following steps: mixing agarose, gelatin, sodium alginate, chitosan and distilled water, adding glutathione and insulin-like growth factor 1 in sequence, stirring and then standing to obtain the hydrogel; The stirring speed is 500-1000 rpm, and the stirring time is 3-6 min; The standing temperature is 18-30° C., and the standing time is 5-10 min.

2. The method for preparing the hydrogel according to claim 1, characterized in that: The method comprises the following steps: mixing agarose, gelatin, sodium alginate, chitosan and distilled water, adding glutathione and insulin-like growth factor 1 in sequence, stirring and then standing to obtain the hydrogel.

3. The method for preparing the hydrogel according to claim 2, characterized in that: The stirring speed is 500-1000 rpm, and the stirring time is 3-6 min.

4. The method for preparing the hydrogel according to claim 2, characterized in that: The standing temperature is 18-30° C., and the standing time is 5-10 min.

5. Use of the hydrogel according to claim 1 or the hydrogel prepared by the hydrogel preparation method according to claims 2 to 4 in the amplification and culture of mesenchymal stem cells.

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