Gelatin hydrogel containing skin cell component seeds as well as preparation method and application of gelatin hydrogel

Abstract

The invention discloses gelatin hydrogel containing skin cell component seeds as well as a preparation method and application of the gelatin hydrogel, and belongs to the technical field of biomedicine. The iPSCs are used as raw materials, are differentiated into components with full-thickness skin structures and cells, then are coated with gelatin hydrogel, and are transplanted into SD rat bodies, so that inflammatory cells and inflammatory factors can be reduced, the number of epidermal stem cells is increased, fibroblast expression related to scar formation is reduced, and advanced scar formation can be inhibited. The treatment effect of the method is most obvious on the third day, the size of the wound is obviously smaller than that of a frostbite group, and the wound of a treatment group is completely healed on the seventh day, which shows that wound closure can be accelerated and infection can be reduced after treatment by the method.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a gelatin hydrogel containing skin cell component seeds and a preparation method and application thereof. Background Art

[0002] Frostbite is very common and is damage to the skin and underlying tissues caused by cold. The early stages of frostbite, known as chilblains, do not cause permanent damage to the skin and symptoms include cold and tingling sensations, followed by numbness and inflammation or discoloration of the skin. As frostbite worsens, the skin may become hard or waxy.

[0003] Mild frostbite generally has no complications. It can be cured by quickly leaving the low temperature environment, rewarming quickly, and applying frostbite cream locally. However, for full-thickness frostbite of the skin, it may cause complications such as chronic infection, non-healing wounds, and scars. The current treatment method mainly involves oral drugs that improve local blood circulation, but this type of drug improves the blood supply and oxygen supply to skin cells by improving the state of skin microvessels, so it acts on skin cells indirectly. This indirect effect takes a long time to take effect and cannot repair skin cells that have been damaged by low temperatures. Moreover, these drugs are only suitable for early frostbite before blood vessels have necrotized and spasmed. Once it is in the late stage, surgical amputation can only be performed for treatment. Summary of the invention

[0004] The present invention uses iPSCs cells as raw materials, which differentiate into components with full-thickness skin structures and cells, and then wrap them with gelatin hydrogels. Transplanting them into SD rats can reduce inflammatory cells and inflammatory factors, increase the number of epidermal stem cells, reduce the expression of fibroblasts associated with scar formation, and inhibit late scar formation. Moreover, the method of the present invention has the most obvious therapeutic effect on the third day, the wound size is significantly smaller than that of the frostbite group, and on the seventh day, the wound of the treatment group has completely healed, which shows that after treatment by the method of the present invention, wound closure can be accelerated and infection can be reduced.

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

[0006] The present invention provides a method for preparing a gelatin hydrogel containing skin cell component seeds. The preparation method comprises the following steps: immersing skin cell component seeds obtained by differentiation of iPSCs cells into a gelatin hydrogel having a mass volume fraction of 40-50% and a temperature of 30°C-40°C. After the gelatin hydrogel is restored to room temperature, the hydrogel will be evenly wrapped on the skin cell component seeds to prepare the gelatin hydrogel containing the skin cell component seeds.

[0007] Preferably, the mass volume fraction of the gelatin hydrogel is 45%.

[0008] More preferably, the temperature of the gelatin hydrogel is 35°C.

[0009] More preferably, the process of obtaining skin cell component seeds by differentiation of iPSCs cells comprises the following steps:

[0010] (1) On day -2, iPSCs were dissociated into single cells and collected by centrifugation to obtain embryoid bodies;

[0011] (2) On day 0 of differentiation, differentiation medium was added to induce ectoderm formation; the differentiation medium was Essential 6 medium + 1.5-2.5% Matrigel + 7.5-12.5M SB-431542 + 3-5ng / mL bFGF + 2-3ng / mL BMP4;

[0012] (3) On the third day of differentiation, E6LF medium was added to induce the formation of non-epithelial mesenchymal cells; the E6LF medium was Essential 6 medium + 200-300 ng / mL bFGF + 0.75-1.25 μM LDN;

[0013] (4) On day 6 of differentiation, add Essential 6 medium; on days 8 and 10, replace half of the medium with fresh Essential 6 medium;

[0014] (5) On the 12th day of differentiation, the culture obtained in step (4) was collected, and OMM medium containing 1.5-2.5% Matrigel was added and cultured with shaking; the OMM medium was prepared by mixing Advanced DMEM-F12 medium, Neurobasal medium, B27, Glutamax, N2, 2-mercaptoethanol, and Normocin, wherein the volume ratio of Advanced DMEM-F12 medium to Neurobasal medium was 1:1, so that the final concentrations of B27, Glutamax, N2, 2-mercaptoethanol, and Normocin were (0.4-0.6)×, 1×, (0.4-0.6)×, 0.09-0.11 mM, and 90-110 μg / mL, respectively;

[0015] (6) On the 15th day of differentiation, replace half of the medium with OMM medium containing 0.8-1.2% Matrigel; starting from the 18th day, replace half of the medium with fresh OMM medium every 2-3 days until the skin cell component seeds mature.

[0016] More preferably, the differentiation medium in step (2) is Essential 6 medium + 2% Matrigel + 10MSB-431542 + 4ng / mL bFGF + 2.5ng / mL BMP4.

[0017] More preferably, the E6LF medium in step (3) is Essential 6 medium + 250 ng / mL bFGF + 1 μM LDN.

[0018] More preferably, the amount of Matrigel added in step (5) is 2%; the final concentrations of B27, Glutamax, N2, 2-mercaptoethanol and Normocin in the OMM culture medium are 0.5×, 1×, 0.5×, 0.1 mM and 100 μg / mL respectively.

[0019] More preferably, the concentration of Matrigel in the OMM culture medium in step (6) is 1%.

[0020] The present invention also provides gelatin hydrogel containing skin cell component seeds prepared by the above preparation method.

[0021] The present invention also provides the use of the gelatin hydrogel containing skin cell component seeds in the preparation of a product for treating frostbite.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention uses iPSCs cells as raw materials, which can effectively differentiate into cell components with full-thickness skin structure and provide high-quality skin repair materials.

[0024] (2) After transplantation, the levels of inflammatory cells and inflammatory factors can be significantly reduced, which helps to improve the wound healing environment.

[0025] (3) The present invention can increase the number of epidermal stem cells and promote skin regeneration.

[0026] (4) Reduce the expression of fibroblasts associated with scar formation, thereby inhibiting late scar formation and improving the therapeutic effect. The treatment group showed obvious effects on the third day, and the wound size was significantly smaller than that of the frostbite group, proving that it quickly played a role in treating frostbitten skin. On the seventh day, the wounds in the treatment group were completely healed, indicating that this method can accelerate wound closure and reduce the risk of infection.

[0027] The reasons for the above advantages all come from using skin cell component seeds to treat frostbitten skin after frostbite. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1a It is a culture flow chart of the skin cell component seeds in Example 1, which is a schematic diagram showing the key time nodes of the skin cell component seed culture. Correspondingly, the bright field photographs on the right represent the microscopic morphology of the skin cell component seeds on the 16th, 35th, 55th, 75th and 90th days (scale: 400μm).

[0029] Figure 1b The immunofluorescence staining diagram of the skin cell component seeds at different time points (15 days, 35 days, 55 days, and 75 days) in Example 1. The skin cell component seeds expressed epithelial cell markers (TFAP2A, ECAD, and KRT17) from day 15 to 35, and from day 55 onwards, the skin cell component seeds expressed mesenchymal-related markers (PDGFRα), glial cell-related markers (SOX10 and P75), proliferation markers (PCNA), and epidermal stem cell markers (KRT15 and KRT5). From day 75 onwards, the skin cell component seeds expressed epidermal germ cell markers (LHX2, PCAD, and EDAR) and dermal layer condensates (PDGFRα and P75).

[0030] Figure 1c Bright field and immunofluorescence images (DAPI: cell nucleus) of hair follicles seeded with skin cell components on day 90 in Example 1 (scale bars: 200 and 50 μm).

[0031] Figure 1d Immunofluorescence of seed epithelial cell markers (ECAD), epidermal basal cell markers (KRT15, ​​KRT5, KRT14 and P63), mature epithelial markers (KRT1 and KRT10), proliferation marker (PCNA), desmosomes (DSG1) and adhesion receptors (ITGB1) of mature skin cell components on day 120 in Example 1 (scale bars: 200 and 50 μm).

[0032] Figure 2a , Figure 2b These are the screening results of different hydrogels in Example 1.

[0033] Figure 3 The time points for treatment and skin tissue collection in Example 2.

[0034] Figure 4 These are wound photos of the treatment group and frostbite group in Example 2 (upper row: frostbite group, lower row: treatment group) and quantitative broken lines of wound area, where Fro: frostbite group, Fro+SO: treatment group.

[0035] Figure 5 The KRT14 immunofluorescence photos of the skin tissues of the treatment group and the frostbite group in Example 2 (upper row frostbite group, lower row treatment group), one day after frostbite (Day 0) the skin cell component seeds were used for treatment, wherein Figure 5The first row shows the expression of KRT14 in nude mouse skin 1 day after treatment (Day 1), 3 days after treatment (Day 3), 7 days after treatment (Day 7), and 14 days after treatment (Day 14). The second row shows the expression of TRK14 in the corresponding frostbite group (only frostbite, no treatment). Bar = 200u.

[0036] Figure 6 This is the immunofluorescence staining (MYH2) of myofibroblasts in the skin tissue of nude mice in the treatment group and the corresponding normal control group and frostbite group 7 days after treatment in Example 2 (top, normal control; middle, frostbite group; bottom, treatment group) bar=200u. DETAILED DESCRIPTION

[0037] Example 1

[0038] 1. Preparation of skin cell component seeds (for details, see Figure 1a ):

[0039] (1) iPSCs were cultured in the mTeSR culture system to a density of 70%.

[0040] (2) On day (-2), iPSCs were dissociated into single cells and aggregated in a 96-well plate at a concentration of 3000 viable cells / well to form embryoid bodies.

[0041] (3) On day 0 of differentiation, the embryoid bodies were transferred to a new 96-well plate and differentiation medium (Essential 6 medium (manufacturer: Thermo Fisher, catalog number: A1516401) supplemented with 2% Matrigel (manufacturer: Corning, catalog number: 354230), 10 μM SB-431542 (manufacturer: Selleck, catalog number: S1067), 4 ng / mL bFGF (manufacturer: Peprotech, catalog number: 100-18B) and 2.5 ng / mL BMP4 (manufacturer: Peprotech, catalog number: 120-05)) was added to induce ectoderm formation.

[0042] (4) On the third day of differentiation, 25 μl of E6LF medium (Essential 6 medium supplemented with 250 ng / mL bFGF and 1 μM LDN (manufacturer: Selleck, catalog number: S7507)) was added to induce the formation of non-epithelial mesenchymal cells.

[0043] (5) On day 6, add 75 μl of Essential 6 medium to each well. On days 8 and 10, replace half of the medium with fresh Essential 6 medium.

[0044] (6) On day 12, the embryoid bodies were transferred to a 24-well plate and 500 μl of OMM medium supplemented with 2% Matrigel (50 ml volume containing 24.5 ml Advanced DMEM-F12 medium (manufacturer: Gibco, catalog number: 12634028), 24.5 ml Neurobasal medium (manufacturer: Gibco, catalog number: 21103049), 500 μl B27 (manufacturer: Thermo Fisher, catalog number: 17504044), 500 μl Glutamax (manufacturer: Gibco, catalog number: 35050), 250 μl N2 (manufacturer: Gibco, catalog number: 17502048), 91 μl 2-mercaptoethanol (manufacturer: 21985023, catalog number: 21985023) and 100 μl Normocin (manufacturer: ant-nr-1, catalog number: InvivoGen)) was added and placed on a shaker at 70 rpm for continued culture to induce further development of the skin cell component seeds.

[0045] (7) On day 15, replace half of the medium with fresh OMM medium supplemented with 1% Matrigel. Starting from day 18, replace half of the medium with fresh OMM medium every 2-3 days until the skin cell component seeds are mature (i.e., staining with immunofluorescence, expression of Figure 1d marker shown).

[0046] 2. Preparation of Gelatin Hydrogel Loaded with Therapeutic Components:

[0047] (1) Prepare 25%, 30%, 35%, 40%, and 45% hydrogels. Accurately weigh gelatin (3.75g, 4.5g, 5.25g, 6.00g, 6.75g, manufacturer: Sigma, product number: V900863) and add it to 15mL phosphate buffered saline (PBS). The pH value of the PBS solution is 7.4 to ensure suitability for biocompatible applications. Subsequently, the mixture is placed in an 80°C water bath and stirred evenly to promote complete dissolution of the gelatin until the solution becomes transparent, which usually takes about 15 to 30 minutes. After the solution has completely cooled to room temperature, slowly pour it into a pre-prepared 50 mL centrifuge tube to avoid the generation of bubbles and ensure the uniformity of the hydrogel. Finally, the fluidity of hydrogels of different concentrations was tested at 25°C, 37°C, and 80°C, respectively. Results ( Figure 2b ) showed that at 25°C and 37°C, the viscosity of the hydrogel was high and the fluidity was poor, which was suitable for maintaining shape and structure; while at 80°C, the viscosity of the hydrogel was significantly reduced and the fluidity was enhanced, which was suitable for applications requiring fluidity. These data are helpful to further optimize the formulation of hydrogels to meet different biomedical needs.

[0048] During the preparation of the hydrogel, SBF (simulated biological fluid) absorption test was performed to evaluate its biocompatibility. Figure 2a ) shows the absorption capacity of gelatin hydrogels of different concentrations in SBF, with the horizontal axis being time (min) and the vertical axis being the absorption amount (g / g). As can be seen from the figure, the absorption amount of the hydrogel gradually increases over time, indicating that its adsorption capacity for SBF gradually increases. At different concentrations, the gelatin hydrogel with a concentration of 45% showed the best absorption performance and reached the peak of absorption, which indicates its potential in biological applications. This figure can be used to deeply analyze the characteristics of the hydrogel and reveal its application prospects in fields such as biocompatibility and drug delivery.

[0049] (2) Immerse the skin cell component seeds in a hydrogel with a concentration of 45% and cool to room temperature.

[0050] (3) Wash twice with PBS.

[0051] 3. Transplantation of skin cell components loaded with gelatin hydrogel into frostbitten skin:

[0052] (1) Disinfect the affected skin three times and apply local anesthesia.

[0053] (2) A 0.4 cm opening was made in the skin below the frostbite (to the depth of the dermis) and the skin cell component seeds loaded with gelatin hydrogel were transplanted into the skin. The incision was closed and sutured, disinfected again, and the wound was covered with 3M dressing and changed daily.

[0054] Example 2

[0055] 1. Preparation of nude mice with full-thickness skin frostbite:

[0056] (1) Male nude mice (Vital River) aged 8 to 12 weeks were used in this example, and the procedures were approved by the Ethics Review Committee of the Beijing National Center for Protein Science and were performed in accordance with the guidelines of the Animal Experimentation and Use Committee of the Beijing Center for Protein Science. Before and after surgery, the animals were housed in a temperature-controlled facility with a 12-h light / dark cycle, with 4 mice per cage.

[0057] (2) On day -1, mice were individually anesthetized with isoflurane, the skin was cleaned with a 70% isopropyl alcohol cotton ball, and a circle with a diameter of 0.8 cm was gently drawn on the skin using a black marker and a 1.5 ml EP tube as a selection tool.

[0058] (3) After the nude mouse skin is prepared, place a copper sheet (0.5 inch diameter, 0.219 inch thickness, 3.5 grams weight) in crushed dry ice (-80°C) and allow to cool for 15 minutes. Use your fingers to lift the mouse's back skin into a skin fold, then place two frozen copper sheets on the opposite sides of the skin fold so that they coincide with the center of the apex marked on the fold. Each group of two cooled copper sheets is placed for 1 minute, then removed and waited for 3 minutes. The above cycle is repeated 3 times in total.

[0059] (4) After three freeze-thaw cycles, mice were given a subcutaneous injection of buprenorphine (0.05 mg / kg) for analgesia. No dressing was used on the wound.

[0060] (5) On day 0, mice were killed by CO2, and the skin of the mice at the frostbite site was taken for pathological staining. The epidermis was necrotic, with a large number of inflammatory cells infiltrating (monocytes and macrophages), ruptured capillaries in the dermis, and a large number of red blood cells overflowed. The dermis collagen was partially necrotic, and the hair follicles were destroyed. The fat cells atrophied. The above showed that the full-thickness skin frostbite model was successfully established.

[0061] 2. Transplantation of skin cell components loaded with gelatin hydrogel into frostbitten skin:

[0062] (1) Mice with full-thickness skin frostbite were randomly divided into a frostbite group and a treatment group. The frostbite group did not receive any treatment to provide baseline data for comparison between the treatment group and the natural recovery group. A normal control group (no frostbite) was also set up. The affected skin of the mice in the treatment group was disinfected three times and local anesthesia was performed.

[0063] (2) A 0.4 cm opening was made in the skin below the frostbite (to the depth of the dermis) and the skin cell component seeds loaded with gelatin hydrogel were transplanted into the skin. The incision was closed and sutured, disinfected again, and the wound was covered with 3M dressing and changed daily.

[0064] (3) From day 1 to day 7, the wounds of mice in the treatment group and the frostbite group were observed every day, and the average frostbite area was calculated. The results are shown in Figure 4 The mice were killed on the 1st, 3rd and 7th day after treatment, and the skin tissues of the frostbitten areas were collected for single-cell sequencing, immunofluorescence staining and Masson staining.

[0065] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a gelatin hydrogel containing skin cell component seeds, characterized in that: The preparation method comprises the following steps: immersing skin cell component seeds obtained by differentiation of iPSCs cells into a gelatin hydrogel with a mass volume fraction of 40-50% and a temperature of 30° C.-40° C. to prepare a gelatin hydrogel containing skin cell component seeds.

2. The preparation method according to claim 1, characterized in that: The mass volume fraction of the gelatin hydrogel is 45%.

3. The preparation method according to claim 2, characterized in that: The temperature of the gelatin hydrogel is 35°C.

4. The preparation method according to any one of claims 1 to 3, characterized in that The process of differentiating iPSCs into skin cell components includes the following steps: (1) On day -2, iPSCs were dissociated into single cells and collected by centrifugation to obtain embryoid bodies; (2) On day 0 of differentiation, differentiation medium was added to induce ectoderm formation; the differentiation medium was Essential 6 medium + 1.5-2.5% Matrigel + 7.5-12.5M SB-431542 + 3-5ng / mL bFGF + 2-3ng / mL BMP4; (3) On the third day of differentiation, E6LF medium was added to induce the formation of non-epithelial mesenchymal cells; the E6LF medium was Essential 6 medium + 200-300 ng / mL bFGF + 0.75-1.25 μM LDN; (4) On day 6 of differentiation, add Essential 6 medium; on days 8 and 10, replace half of the medium with fresh Essential 6 medium; (5) On the 12th day of differentiation, the culture obtained in step (4) was collected, and OMM medium containing 1.5-2.5% Matrigel was added for shaking culture; the OMM medium was prepared by mixing Advanced DMEM-F12 medium, Neurobasal medium, B27, Glutamax, N2, 2-mercaptoethanol, and Normocin, wherein the volume ratio of Advanced DMEM-F12 medium to Neurobasal medium was 1:1, so that the final concentrations of B27, Glutamax, N2, 2-mercaptoethanol, and Normocin were (0.4-0.6)×, 1×, (0.4-0.6)×, 0.09-0.11 mM, and 90-110 μg / mL, respectively; (6) On the 15th day of differentiation, replace half of the medium with OMM medium containing 0.8-1.2% Matrigel; starting from the 18th day, replace half of the medium with fresh OMM medium every 2-3 days until the skin cell component seeds mature.

5. The preparation method according to claim 4, characterized in that: The differentiation medium in step (2) is Essential 6 medium + 2% Matrigel + 10M SB-431542 + 4ng / mL bFGF + 2.5ng / mL BMP4.

6. The preparation method according to claim 5, characterized in that: The E6LF medium in step (3) is Essential 6 medium + 250 ng / mL bFGF + 1 μM LDN.

7. The preparation method according to claim 6, characterized in that: The amount of Matrigel added in step (5) is 2%; the final concentrations of B27, Glutamax, N2, 2-mercaptoethanol, and Normocin in the OMM culture medium are 0.5×, 1×, 0.5×, 0.1 mM, and 100 μg / mL, respectively.

8. The preparation method according to claim 7, characterized in that: The concentration of Matrigel in the OMM culture medium in step (6) is 1%.

9. A gelatin hydrogel containing skin cell component seeds prepared by the preparation method described in any one of claims 1-3 and 5-8.

10. Use of the gelatin hydrogel according to claim 9 in preparing a product for treating frostbite.

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