WO2022156456A1 - 用于肌肉干细胞培养的交联水凝胶及其制备方法和应用 - Google Patents

用于肌肉干细胞培养的交联水凝胶及其制备方法和应用 Download PDF

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WO2022156456A1
WO2022156456A1 PCT/CN2021/139539 CN2021139539W WO2022156456A1 WO 2022156456 A1 WO2022156456 A1 WO 2022156456A1 CN 2021139539 W CN2021139539 W CN 2021139539W WO 2022156456 A1 WO2022156456 A1 WO 2022156456A1
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cross
collagen
hydrogel
muscle stem
alginate
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胡静
尹健
汪翔
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江南大学
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Definitions

  • the invention relates to a cross-linked hydrogel for culturing muscle stem cells and a preparation method and application thereof, in particular to a novel enzymatic-physical method double-cross-linked network hydrogel preparation method and application thereof, belonging to the technical field of biological food materials .
  • Stem cell cultured meat is a new technology that uses muscle cells to culture meat in vitro.
  • the development of cultured meat has been accelerating due to its potential for sustainable production.
  • stem cell cultured meat can not only reduce the suffering of animals, but also reduce the pollution of animal husbandry to the environment (Food & Agriculture Organization of the United Nations, 2006).
  • cultured meat can solve this problem.
  • the lack of scalable cell culture substrates (scaffolds) has become one of the major challenges limiting stem cell culture meat.
  • hydrogels extracted from natural biomaterials such as polysaccharide-based materials (such as hyaluronic acid, chitosan, alginate, etc.) or protein-based materials (such as collagen, gelatin, etc.)
  • polysaccharide-based materials such as hyaluronic acid, chitosan, alginate, etc.
  • protein-based materials such as collagen, gelatin, etc.
  • Polylysine ( ⁇ -PL) is a typical natural polyamino acid biomaterial, containing about 25 repeating units of lysine, rich in positive charges, rich in biocompatibility, biodegradability and excellent water solubility.
  • Collagen is a natural protein-based material that retains abundant biologically active sequences (such as RGD) that promote cell adhesion and growth.
  • RGD biologically active sequences
  • natural hydrogels suffer from insufficient crosslinking and poor mechanical properties.
  • Most crosslinking agents, such as glutaraldehyde and epoxy resins have some toxicity that affects the biocompatibility of hydrogels. Therefore, nontoxic, mechanically stable, and cell-adhesive natural hydrogel scaffolds remain a challenge.
  • the present invention proposes a kind of natural hydrogel with strong adsorption force and not easy to collapse for culturing stem cells. Novel enzymatic-physical dual-network cross-linked hydrogels.
  • the present invention firstly prepares the enzymatic-physical method double network hydrogel of collagen/ ⁇ -PL/heparan sulfate proteoglycan/alginate by combining the enzymatic method and the physical method by using TG enzyme.
  • These modifying groups give the hydrogel the ability to adhere to cells and release growth factors in a controlled manner.
  • the cross-linked structure of the double network enables the synergistic effect of the two networks to enhance the mechanical properties and maintain the integrity of the hydrogel after a large amount of water swelling.
  • this hydrogel improves the cytotoxicity of the chemically crosslinked network due to toxic chemical crosslinking agents.
  • the present invention first provides a method for preparing a cross-linked hydrogel for muscle stem cell culture.
  • the method includes: first, dissolving collagen to make a solution, and then adding a certain amount of alginate and heparan sulfate protein Polysaccharide, mix it with the collagen solution; then add polylysine ⁇ -PL and TG enzyme to the solution, stir evenly, and place the slurry in a mold for cross-linking to obtain a hydrogel.
  • the method specifically includes the following steps:
  • Hydrogel put the slurry obtained in (5) into a mold, cross-link for 12-36 hours, and release the mold to obtain a hydrogel for muscle stem cell growth.
  • the water described in the method is preferably deionized water or ultrapure water.
  • the collagen includes one of collagen, gelatin, hydrolyzed collagen, collagen polypeptide, etc., extracted from the skin of cattle, sheep, pigs, donkeys, poultry, aquatic animals, etc., bovine Achilles tendon, bone tissue, etc. one or a mixture of two or more.
  • the concentration of the aqueous acetic acid solution described in step (1) is 0.02-0.05 mol/L.
  • the mass of collagen is 10-15% of the mass of water.
  • the quality of the alginate in step (2) is 15-25% of the water quality.
  • the viscosity of the alginate in step (2) is 4-12 cP.
  • the concentration of heparan sulfate proteoglycan in the collagen/alginate/heparan sulfate proteoglycan solution in step (3) is 200-500 ⁇ g/L.
  • the molar ratio of the carboxyl group of the alginate to the amino group of the polylysine ⁇ -PL is 1:1 to 1:2.
  • the amount of TG enzyme used in step (5) is 1-10% of the collagen mass.
  • the cross-linking temperature in step (6) is 37-50°C.
  • the present invention provides the cross-linked hydrogel for muscle stem cell culture prepared by the above preparation method.
  • the present invention provides a medium comprising the above-described cross-linked hydrogel for muscle stem cell culture.
  • the present invention provides a method for culturing muscle stem cells.
  • the method uses the above-mentioned cross-linked hydrogel for culturing muscle stem cells as a culture medium.
  • the muscle stem cells include, but are not limited to, porcine muscle stem cells, bovine muscle stem cells, and the like.
  • the present invention provides the above preparation method or the application of the above cross-linked hydrogel for muscle stem cell culture in the field of cultured meat.
  • the slurry used for preparing hydrogel of the present invention is based on collagen and alginate, and polylysine and heparan sulfate proteoglycan are added. Heparan proteoglycans are linked to form covalent cross-links, and through the physical electrostatic interaction between polylysine and alginate, a dense three-dimensional "egg box" network structure is formed. Through enzymatic and physical cross-linking, a dense double-cross-linked structure hydrogel is formed inside and outside.
  • polylysine and TG enzyme into the hydrogel system in the present invention facilitates the double cross-linking of the enzymatic method and the physical method, and can obtain a hydrogel with higher mechanical strength.
  • collagen is introduced into the hydrogel system, which is beneficial to the adhesion of stem cells and improves the biocompatibility of the hydrogel.
  • heparan sulfate proteoglycan is introduced into the hydrogel system, which is beneficial to the immobilization of stem cell growth factors and the long-term release of growth factors.
  • Figure 1 is a flow chart of the preparation of the enzymatic-physical method double network hydrogel of collagen/ ⁇ -PL/heparan sulfate proteoglycan/alginate.
  • Figure 2 is a graph showing the in vitro binding efficiency of three heparins and their derivatives to bFGF.
  • Example 4 is a microscopic view of culturing primary porcine muscle stem cells using the hydrogel prepared in Example 1. a) Microscopic image of primary porcine muscle stem cells before undifferentiated (4X); b) Microscopic image of primary porcine muscle stem cells after 72h differentiation (4X).
  • FIG. 5 is a SEM image of the hydrogel prepared in Example 1.
  • FIG. 6 is a microscopic view of culturing primary muscle stem cells using the hydrogel prepared in Comparative Example 1.
  • FIG. 7 is a microscopic view of culturing primary muscle stem cells using the hydrogel prepared in Comparative Example 2.
  • FIG. 8 is a microscopic view of culturing primary muscle stem cells using the hydrogel prepared in Comparative Example 3.
  • FIG. 9 is a microscopic view of culturing primary muscle stem cells using the hydrogel prepared in Comparative Example 4.
  • FIG. 10 is a microscopic view of culturing primary muscle stem cells using the hydrogel prepared in Comparative Example 5.
  • TG enzyme was purchased from Anhui Datang Bioengineering Co., Ltd., and the enzyme activity was 109U/g.
  • Alginate was purchased from Sigma-Aldrich with a viscosity of 8 cP.
  • Example 1 Preparation of collagen/ ⁇ -PL/heparan sulfate proteoglycan/alginate double network hydrogel by enzymatic-physical method
  • the hydrogel was prepared according to the flow chart in Figure 1: 10 g of collagen was added to 100 mL of 0.02 mol/L acetic acid solution, and the collagen solution was obtained by stirring and dissolving. Add 15 g of alginate to the collagen solution, stir until the alginate solution, to obtain a collagen/alginate solution; then add 20 ⁇ g of heparan sulfate proteoglycan to the prepared solution to obtain collagen/alginate / Heparan sulfate proteoglycan solution; add ⁇ -PL with a molar ratio of amino groups to carboxyl groups in alginate of 1:1 into the solution, stir evenly, and obtain the first slurry prepared by physical cross-linking; 0.1 g of TG enzyme was added to the slurry and stirred evenly to obtain the second slurry prepared by enzyme cross-linking.
  • the slurry was put into a mold, cross-linked at 37 °C for 12 h, and then demolded to obtain an enzymatic-physical double network hydrogel of collagen/ ⁇ -PL/heparan sulfate proteoglycan/alginate.
  • a large number of porcine muscle stem cells were observed after culturing porcine muscle stem cells on the hydrogel for 7 days.
  • the hydrogels were lyophilized in a vacuum freeze dryer (-80°C) and observed by scanning electron microscopy that the hydrogels were porous structures with various pore sizes.
  • the hydrogel was prepared according to the flow chart in Figure 1: 15 g of collagen was added to 100 mL of 0.05 mol/L acetic acid solution, and the collagen solution was obtained by stirring and dissolving. Add 25 g of alginate to the collagen solution, stir until the alginate solution, to obtain a collagen/alginate solution; then add 50 ⁇ g of heparan sulfate proteoglycan to the prepared solution to obtain collagen/alginate / Heparan sulfate proteoglycan solution; add ⁇ -PL with a molar ratio of amino groups to carboxyl groups in alginate of 2:1 into the solution, stir evenly, and obtain the first slurry prepared by physical cross-linking; 1.5 g of TG enzyme was added to the slurry and stirred evenly to obtain the second slurry prepared by enzyme cross-linking.
  • the slurry was put into a mold, cross-linked at 37 °C for 36 h, and then demolded to obtain an enzymatic-physical double network hydrogel of collagen/ ⁇ -PL/heparan sulfate proteoglycan/alginate.
  • a large number of porcine muscle stem cells were observed after culturing porcine muscle stem cells on the hydrogel for 7 days.
  • the hydrogels were lyophilized in a vacuum freeze dryer (-80°C) and observed by scanning electron microscopy that the hydrogels were porous structures with various pore sizes.
  • Example 1 and Example 2 can adsorb all growth factors, indicating that the hydrogels prepared by the method of the present invention are helpful for adsorbing growth factors.
  • Growth factor release test put the hydrogel adsorbed on growth factor in Example 3 into 1 mL of sterile PBS solution, use a pipette to collect the PBS liquid in the experiment every 24 hours and replace it with a new equal volume of sterile PBS solution , the collected liquid in the well plate is stored in an EP tube and placed in a -20°C refrigerator, waiting for detection.
  • concentration of bFGF (450nm) and the content of vitamin C (536nm) in the collected solution were detected by enzyme-linked immunosorbent assay (ELISA).
  • Example 1 Through the growth factor release test, it can be seen that the bFGF and vitamin C adsorbed by the hydrogel in Example 1 were still detected on the 12th day, and the bFGF and vitamin C adsorbed by the hydrogel in Example 2 were still detected on the 12th day. was detected, and the results of Example 1 and Example 2 were not significantly different.
  • the results show that the hydrogel prepared by the present invention is beneficial to immobilize stem cell growth factors and can release growth factors for a long time.
  • Example 4 Using the hydrogel of Example 1 to carry out the hydrogel containing growth factors obtained after the experiment in Example 3, cells were seeded on the prepared enzymatic-physical double network hydrogel at a density of 1500 cells/mm 2 , and Cultured in growth medium (79% DMEM, 10% FBS, 1% dual antibody, 79% DMEM) for 24 h. The medium was then changed to differentiation medium (97% DMEM, 2% horse serum, 1% double antibody) and the cells were cultured for an additional 7 days. Significantly proliferating large numbers of cells were observed after 7 days of culture. The results are shown in Figure 4.
  • the hydrogels were tested in uniaxial compression using an Instron mechanical test frame (Model 5565A). Stress is based on the force curve Calculated, with F and A 0 being the force used to compress the sample and the initial area of the sample. The modulus of the gel passes through calculated. Samples were repeated at least three times. Before testing, carefully inspect the hydrogel for cracks or deformation. Align the gel in the center of the stainless steel compression plate. The gel is slippery, allowing the gel to expand freely when compressed. The stress relaxation of the samples was investigated under compression of 5%, 10% and 20% strain using an initial crosshead speed of 4% strain/sec.
  • the stress response of the hydrogel prepared by the present invention is as long as 290 s when it is relaxed, and the stress response of the hydrogel of Example 2 when it is relaxed is as long as 300 s.
  • the morphology of the lyophilized hydrogel was imaged using a Hitachi S-4800 SEM (Hitachi, Japan) with an accelerating voltage of 5 kV. Before testing, the cross-section of the hydrogel was fixed on a metal substrate with conductive tape and sputter-coated with gold. Studies have found that the hydrogel prepared by the present invention is a porous structure with various pore sizes (as shown in Figure 5), and these structures are conducive to the swelling of growth factors and promote the diffusion of growth factors into the hydrogel. Moreover, these pores provide a large specific surface area, which is conducive to the adhesion of muscle stem cells.
  • the growth factor adsorption experiment was carried out according to the method of Example 3, and it was found that after 24 hours of growth factor adsorption, the hydrogel collapsed, and the collapse ratio accounted for 8%.
  • the as-prepared hydrogel was subjected to stress testing, and its stress response was only 150 s.
  • a small number of porcine muscle stem cells were observed after culturing porcine muscle stem cells on the hydrogel for 7 days.
  • Example 3 The growth factor adsorption experiment of Example 3 was carried out on the prepared hydrogel, and it was found that after adsorbing the growth factor for 24 hours, the hydrogel collapsed, and the collapse ratio accounted for 5%.
  • the as-prepared hydrogel was subjected to stress testing, and its stress response was only 180 s. A small number of porcine muscle stem cells were observed after culturing porcine muscle stem cells on the hydrogel for 7 days.
  • Example 2 When there is no heparan sulfate proteoglycan, other steps are the same as in Example 1, and a hydrogel is prepared.
  • the growth factor adsorption experiment was carried out on the prepared hydrogel. After 24 h of growth factor adsorption, it was found that the hydrogel only adsorbed a small amount of growth factor. Then, the growth factor release test was performed, and no growth factor was detected in the hydrogel after 2 days. The prepared hydrogel was subjected to stress test, and its stress response was 285 s. A small number of porcine muscle stem cells were observed after culturing porcine muscle stem cells on the hydrogel for 7 days.
  • the first slurry dissolve 10 g of collagen with 50 mL of 0.04mol/L acetic acid solution to obtain a collagen solution, add 20 ⁇ g of heparan sulfate proteoglycan to the collagen solution, and stir to obtain a solution; pour the solution into the first slurry In the first slurry, mix with the first slurry, stir evenly, add 0.1 g of TG enzyme to the slurry, and stir evenly to obtain the second slurry prepared by enzyme cross-linking. The slurry was put into a mold, cross-linked at 37 °C for 12 h, and demolded to obtain a hydrogel. A small number of porcine muscle stem cells were observed after culturing porcine muscle stem cells on the hydrogel for 7 days.

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Abstract

本发明公开了用于肌肉干细胞培养的交联水凝胶及其制备方法和应用，属于生物食品材料技术领域。本发明将胶原蛋白溶解制成溶液，然后加入一定量的海藻酸盐和硫酸乙酰肝素蛋白多糖，将其与胶原蛋白溶液混匀；然后向溶液中加入ε-PL和TG酶，搅拌均匀，将浆料放置在模具中进行交联，得到水凝胶。本发明的水凝胶通过TG酶将胶原蛋白和聚赖氨酸以及硫酸乙酰肝素蛋白聚糖连接，形成共价交联，在通过聚赖氨酸与海藻酸盐之间的物理静电作用，形成致密的三维"鸡蛋盒"网状结构。利用这种水凝胶，可以加强肌肉干细胞对营养物质的吸收并有利于其生长。双交联网络水凝胶有潜力作为干细胞培养肉的肌肉干细胞生长的支架。

Description

用于肌肉干细胞培养的交联水凝胶及其制备方法和应用 技术领域
本发明涉及用于肌肉干细胞培养的交联水凝胶及其制备方法和应用，特别涉及一种新型酶法-物理法双交联网络水凝胶制备方法及其应用，属于生物食品材料技术领域。
背景技术
肉类的大量消费是一个重大的环境负担，世界上超过90％的人口吃肉。干细胞培养肉是一种利用肌肉细胞离体培养肉的新技术。目前，由于培养肉的可持续生产的潜力，其开发进程一直在不断加速。干细胞培养肉与传统肉类生产相比，不仅可以减轻动物的痛苦，而且能够减少畜牧业生产对环境的污染(Food&Agriculture Organization of the United Nations,2006)。同时，畜牧行业中一直存在抗生素滥用的情况。显然，培养肉可以解决这一问题。然而，缺乏可扩展的细胞培养底物(支架)成为限制干细胞培养肉的主要挑战之一。
如今，从天然生物材料中提取的水凝胶，例如多糖基材料(如透明质酸、壳聚糖、海藻酸盐等)或蛋白质基材料(如胶原蛋白、明胶等)，由于其良好的生物相容性和生物降解性，在干细胞支架中得到了广泛的应用(Small,2020,16,1-17)。海藻酸盐来源广泛且廉价，具有生物相容性等多种优良特性，并且海藻酸盐可以和正离子相互作用形成一个密集的三维“鸡蛋盒”结构，以改善机械性能。聚赖氨酸(ε-PL)是一种典型的天然聚氨基酸生物材料，含有约25个重复单位的赖氨酸，具有丰富的正电荷，具有丰富的生物相容性，生物降解性和优异的水溶性。胶原蛋白的一种天然蛋白质基材料，保留了丰富的生物活性序列(如RGD)，可以促进细胞粘附和生长。然而，天然水凝胶存在交联不足和机械性能差问题。大多数交联剂，如戊二醛和环氧树脂，都有一定的毒性，影响水凝胶的生物相容性。因此，无毒，机械稳定性好，细胞粘附性强的天然水凝胶支架仍是挑战。
此外，用于培养肉的肌肉干细胞的干性维持和增加粘附性成为水凝胶材料的新挑战，使得其对水凝胶的性能提出了新的要求。通过添加生长因子bFGF，可以使得肌肉干细胞激活和增殖。然而，生长因子在培养基中存留时间短。因此，无毒，机械稳定性好，肌肉干细胞粘附性强，可以促进肌肉干细胞维持干性的天然水凝胶支架仍未有很好的解决办法。
发明内容
技术问题：
本发明针对上述如何制备无毒，机械稳定性高，细胞粘附性强且维持肌肉干细胞干性的天然水凝胶的难题，提出了一种具有强吸附力且不易崩塌的用于培养干细胞的新型酶法-物理 法双网络交联水凝胶。
技术方案：
本发明首先通过将酶法和物理法相结合，利用TG酶制备了胶原蛋白/ε-PL/硫酸乙酰肝素蛋白聚糖/藻酸盐的酶法-物理法双网络水凝胶。这些修饰基团使得水凝胶具有粘附细胞和控释生长因子的能力。并且双网络交联的结构使得水凝胶在大量水溶胀后，两个网络的协同作用也可以增强水凝胶的机械性能并保持完整性。同时，这种水凝胶改善了化学交联网络所需有毒化学交联剂而产生细胞毒性的特点。
本发明首先提供了一种用于肌肉干细胞培养的交联水凝胶的制备方法，所述方法包括：首先，将胶原蛋白溶解制成溶液，然后加入一定量的海藻酸盐和硫酸乙酰肝素蛋白多糖，将其与胶原蛋白溶液混匀；然后向溶液中加入聚赖氨酸ε-PL和TG酶，搅拌均匀，将浆料放置在模具中进行交联，得到水凝胶。
优选的，所述方法具体包括以下步骤：
(1)制备胶原蛋白溶液：将胶原蛋白和醋酸水溶液混匀溶解得到胶原蛋白溶液；
(2)制备胶原蛋白/海藻酸盐溶液：向步骤(1)中制备的胶原蛋白溶液中加入海藻酸盐，搅拌至海藻酸盐溶解，得到胶原蛋白/海藻酸盐溶液；
(3)制备胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液：向步骤(2)中制备的胶原蛋白/海藻酸盐溶液加入硫酸乙酰肝素蛋白聚糖多糖，搅拌至溶解，得到胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液；
(4)制备物理交联的第一浆料：向(3)中制备的胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液加入聚赖氨酸ε-PL，搅拌均匀，得到物理交联制备的第一浆料；
(5)制备酶交联的第二浆料：向(4)中制备的第一浆料加入TG酶，搅拌均匀，得到酶交联制备的第二浆料；
(6)水凝胶：将(5)中得到的浆料放入模具中，交联12～36h，脱模，得到用于肌肉干细胞生长的水凝胶。
优选的，方法中所述的水优选为去离子水或超纯水。
优选的，所述的胶原蛋白包括牛、羊、猪、驴、禽类、水产动物等的皮肤、牛跟腱、骨组织等部位提取的胶原蛋白、明胶、水解胶原蛋白、胶原多肽等中的一种或两种以上的混合物。
优选的，步骤(1)中所述的醋酸水溶液的浓度为0.02～0.05mol/L。
优选的，步骤(1)中，胶原蛋白的质量是水质量的10～15％。
优选的，步骤(2)中海藻酸盐的质量是水质量的15～25％。
优选的，步骤(2)中所述海藻酸盐的粘度为4～12cP。
优选的，步骤(3)中胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液中硫酸乙酰肝素蛋白聚糖的浓度为200～500μg/L。
优选的，步骤(4)中海藻酸盐的羧基和聚赖氨酸ε-PL的氨基的摩尔比为1:1～1:2。
优选的，步骤(5)中TG酶的用量为胶原蛋白质量的1～10％。
优选的，步骤(6)中所述交联温度为37～50℃。
本发明提供了上述制备方法制备得到的用于肌肉干细胞培养的交联水凝胶。
本发明提供了包含上述用于肌肉干细胞培养的交联水凝胶的培养基。
本发明提供了一种肌肉干细胞培养的方法，所述方法以上述用于肌肉干细胞培养的交联水凝胶作为培养基。
优选的，所述肌肉干细胞包括但不限于猪肌肉干细胞、牛肌肉干细胞等。
本发明提供了上述制备方法或上述用于肌肉干细胞培养的交联水凝胶在培养肉领域的应用。
本发明取得的有益效果：
1.本发明用于制备水凝胶的浆料以胶原蛋白和海藻酸盐为基础，增加了聚赖氨酸和硫酸乙酰肝素蛋白聚糖，通过TG酶将胶原蛋白和聚赖氨酸以及硫酸乙酰肝素蛋白聚糖连接，形成共价交联，在通过聚赖氨酸与海藻酸盐之间的物理静电作用，形成致密的三维“鸡蛋盒”网状结构。通过酶法和物理法的交联，形成了内外致密的双交联结构水凝胶。
2.本发明向水凝胶体系中引入聚赖氨酸和TG酶有利于实施酶法和物理法双交联，可以得到机械强度更高的水凝胶。
3.本发明向水凝胶体系中引入胶原蛋白，有利于粘附干细胞，提高水凝胶的生物相容性。
4.本发明向水凝胶体系中引入硫酸乙酰肝素蛋白聚糖，有利于固存干细胞生长因子，并可以长期释放生长因子。
附图说明
图1为胶原蛋白/ε-PL/硫酸乙酰肝素蛋白聚糖/藻酸盐的酶法-物理法双网络水凝胶制备流程图。
图2为三种肝素及其衍生物与bFGF体外结合效率图。
图3位三种肝素及其衍生物与bFGF体外释放曲线。
图4为利用实施例1制备得到的水凝胶培养原代猪肌肉干细胞的显微镜图。a)原代猪肌肉干细胞未分化前的显微镜图(4X)；b)原代猪肌肉干细胞分化72h后的显微镜图(4X)。
图5为实施例1制备得到的水凝胶的SEM图。a)500μm下的SEM图；b)200μm下的SEM图。
图6为利用对比例1制备得到的水凝胶培养原代肌肉干细胞的显微镜图。
图7为利用对比例2制备得到的水凝胶培养原代肌肉干细胞的显微镜图。
图8为利用对比例3制备得到的水凝胶培养原代肌肉干细胞的显微镜图。
图9为利用对比例4制备得到的水凝胶培养原代肌肉干细胞的显微镜图。
图10为利用对比例5制备得到的水凝胶培养原代肌肉干细胞的显微镜图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
TG酶购自安徽大唐生物工程有限公司，酶活为109U/g。
海藻酸盐购自Sigma-Aldrich，粘度为8cP。
实施例1：胶原蛋白/ε-PL/硫酸乙酰肝素蛋白聚糖/藻酸盐的酶法-物理法双网络水凝胶的制备
按照图1的流程图制备水凝胶：向100mL的0.02mol/L醋酸溶液中加入10g胶原蛋白，搅拌溶解得到胶原蛋白溶液。向胶原蛋白溶液中加入15g海藻酸盐，搅拌至海藻酸盐溶液，得到胶原蛋白/海藻酸盐溶液；接着向制备好的溶液中加入20μg硫酸乙酰肝素蛋白聚糖，得到胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液；向溶液中加入氨基与海藻酸盐中的羧基的摩尔比为1:1的ε-PL，搅拌均匀，得到物理交联制备的第一浆料；向第一浆料中加入0.1gTG酶，搅拌均匀，得到酶交联制备的第二浆料。将浆料放入模具中，在37℃下交联12h，脱模，得到胶原蛋白/ε-PL/硫酸乙酰肝素蛋白聚糖/藻酸盐的酶法-物理法双网络水凝胶。在水凝胶上培养猪肌肉干细胞7天后观察到大量猪肌肉干细胞。将水凝胶放在真空冷冻干燥机中冻干(-80℃)后利用扫描电镜观察到水凝胶是具有多种孔径的多孔结构。
实施例2：胶原蛋白/ε-PL/硫酸乙酰肝素蛋白聚糖/藻酸盐的酶法-物理法双网络水凝胶的制备
按照图1的流程图制备水凝胶：向100mL的0.05mol/L醋酸溶液中加入15g胶原蛋白，搅拌溶解得到胶原蛋白溶液。向胶原蛋白溶液中加入25g海藻酸盐，搅拌至海藻酸盐溶液，得到胶原蛋白/海藻酸盐溶液；接着向制备好的溶液中加入50μg硫酸乙酰肝素蛋白聚糖，得 到胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液；向溶液中加入氨基与海藻酸盐中的羧基的摩尔比为2:1的ε-PL，搅拌均匀，得到物理交联制备的第一浆料；向第一浆料中加入1.5gTG酶，搅拌均匀，得到酶交联制备的第二浆料。将浆料放入模具中，在37℃下交联36h，脱模，得到胶原蛋白/ε-PL/硫酸乙酰肝素蛋白聚糖/藻酸盐的酶法-物理法双网络水凝胶。在水凝胶上培养猪肌肉干细胞7天后观察到大量猪肌肉干细胞。将水凝胶放在真空冷冻干燥机中冻干(-80℃)后利用扫描电镜观察到水凝胶是具有多种孔径的多孔结构。
实施例3：水凝胶生长因子的吸附
生长因子的吸附实验：用PBS对酶法-物理交联双网络水凝胶(实施例1和实施例2的水凝胶)进行清洗，将得到的水凝胶浸渍于75％的乙醇中20分钟，随后反复浸渍在无菌的去离子水中5分钟，无菌水洗涤乙醇的操作进行三次，以除去所有残余的乙醇(Food Hydrocolloids,2017,72,210-218)，然后将水凝胶转移到含生长因子维生素C(0.05μg/mL)和bFGF(10ng/mL)的溶液中，溶胀24h后既得。最后通过酶联免疫方法(ELISA)检测剩余溶液中bFGF(450nm)和维生素C(536nm)的含量，并根据溶液中bFGF和维生素C的初始浓度和剩余溶液浓度的差值计算出水凝胶对生长因子的吸附。
结果显示实施例1和实施例2的水凝胶能够将生长因子全部吸附，说明本发明方法制备得到的水凝胶有助于吸附生长因子。
实施例4：水凝胶生长因子的释放
生长因子释放试验：将实施例3中吸附过生长因子的水凝胶至于1mL的无菌PBS溶液中，每24h使用移液器采集实验中的PBS液体并更换新的等体积的无菌PBS溶液，收集的孔板中的液体使用EP管保存并放置于-20℃冰箱中，等待检测。采用酶联免疫法(ELISA)检测采集液中bFGF浓度(450nm)和维生素C(536nm)的含量。
通过生长因子释放试验可知，实施例1中的水凝胶吸附的bFGF和维生素C在第12天仍有检出，实施例2中的水凝胶吸附的bFGF和维生素C在第12天仍有检出，并且实施例1和实施例2的结果无显著性差异。结果表明本发明制备得到的水凝胶有利于固存干细胞生长因子，并可以长期释放生长因子。
实施例5：在双网络水凝胶上培养猪肌肉干细胞
利用实施例1的水凝胶进行实施例3中实验后得到的含生长因子的水凝胶，细胞以1500个/mm 2的密度接种到制备的酶法-物理双网络水凝胶上，并在生长培养基(79％DMEM，10％FBS，1％双抗，79％DMEM)中培养24h。随后将培养基换成分化培养基(97％DMEM，2％马血清，1％双抗)，并将细胞再培养7天。培养7天后可观察到明显增殖的大量细胞。结 果见图4。
实施例6：水凝胶的机械测试
使用Instron机械测试框架(型号5565A)对水凝胶进行单轴压缩测试。应力是根据力曲线
Figure PCTCN2021139539-appb-000001
计算得出的，与F和A 0是用来压缩样品的力和样品的初始面积。凝胶的模量通过
Figure PCTCN2021139539-appb-000002
计算得出的。样品至少重复三遍。在测试之前，仔细检查水凝胶是否有裂纹或变形。将凝胶在不锈钢压缩板的中心对齐。凝胶很滑，压缩时允许凝胶自由膨胀。使用4％应变/秒的初始十字头速度，在5％，10％和20％应变的压缩下研究了样品的应力松弛。
研究发现本发明制备得到的实施例1水凝胶松弛时的应力响应长达290s，实施例2水凝胶松弛时的应力响应长达300s。
实施例7：水凝胶的扫描电镜样品制备
使用Hitachi S-4800 SEM(日本日立)对冻干水凝胶进行5kV的加速电压，对水凝胶的形态进行成像。在测试前，将水凝胶的横截面用导电带固定在金属基底上，并用金进行溅射镀膜。研究发现本发明制备的水凝胶是具有多种孔径的多孔结构(如图5)，这些结构有利于生长因子的溶胀，促进生长因子扩散到水凝胶内部。并且，这些孔提供较大的比表面积，有利于肌肉干细胞的粘附。
对比例1
当仅不加海藻酸盐，其他步骤与实施例1相同，得到酶法交联的水凝胶。
按照实施例3的方式进行生长因子的吸附实验，发现吸附生长因子24h后，水凝胶出现塌陷，塌陷比例占8％。对制备的水凝胶进行应力测试，其应力响应仅为150s。在水凝胶上培养猪肌肉干细胞7天后观察到少量猪肌肉干细胞。
结果表明，当无海藻酸盐的存在时，制备得到的水凝胶对生长因子的吸附量明显变差，且溶胀和机械性能均明显变差，不利于肌肉干细胞的培养。
对比例2
当仅不加胶原蛋白和TG酶时，其他步骤与实施例1相同，制备得到水凝胶。
对制备的水凝胶进行实施例3的生长因子吸附实验，发现吸附生长因子24h，水凝胶出现塌陷，塌陷比例占5％。对制备的水凝胶进行应力测试，其应力响应仅为180s。在水凝胶上培养猪肌肉干细胞7天后观察到少量猪肌肉干细胞。
结果表明，当无胶原蛋白和TG酶的存在时，制备得到的水凝胶对生长因子的吸附量明显变差，且溶胀和机械性能均明显变差，不利于肌肉干细胞的培养。
对比例3
当无硫酸乙酰肝素蛋白聚糖时，其他步骤与实施例1相同，制备得到水凝胶。
对制备的水凝胶进行生长因子吸附实验，吸附生长因子24h后，发现水凝胶仅对生长因子进行了少量吸附。并之后进行生长因子释放试验，水凝胶在2天后就检测不到生长因子。对制备的水凝胶进行应力测试，其应力响应为285s。在水凝胶上培养猪肌肉干细胞7天后观察到少量猪肌肉干细胞。
对比例4
向50mL去离子水中加入15g海藻酸盐，搅拌得到海藻酸盐溶液，向溶液中加入氨基与海藻酸盐中的羧基的摩尔比为1:1的ε-PL，搅拌均匀，得到物理交联制备的第一浆料；接着用50mL的0.04mol/L醋酸溶液溶解10g胶原蛋白，得到胶原蛋白溶液，向胶原蛋白溶液中加入20μg硫酸乙酰肝素蛋白聚糖，搅拌均匀得到溶液；将溶液倒入第一浆料中，与第一浆料混合，搅拌均匀，向其中加入0.1gTG酶，搅拌均匀，得到酶交联制备的第二浆料。将浆料放入模具中，在37℃下交联12h，脱模，得到水凝胶。在水凝胶上培养猪肌肉干细胞7天后观察到少量猪肌肉干细胞。
对比例5
向50mL的0.04mol/L醋酸溶液中加入10g胶原蛋白，搅拌溶解得到胶原蛋白溶液。向胶原蛋白溶液中加入0.1gTG酶，搅拌均匀，得到凝胶状物体；向50mL的去离子水中加入15g海藻酸盐，搅拌得到海藻酸盐溶液，向溶液中加入20μg硫酸乙酰肝素蛋白聚糖，搅拌均匀，接着加入氨基与海藻酸盐中的羧基的摩尔比为1:1的ε-PL，搅拌均匀，得到物理交联的浆料，将浆料与凝胶状物体混合，将胶料放入模具中，在37℃下交联12h，脱模，得到水凝胶。在水凝胶上培养猪肌肉干细胞7天后观察到少量猪肌肉干细胞。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (13)

  1. 一种用于肌肉干细胞培养的交联水凝胶的制备方法，其特征在于，所述方法包括：首先，将胶原蛋白溶解制成溶液，然后加入一定量的海藻酸盐和硫酸乙酰肝素蛋白多糖，将其与胶原蛋白溶液混匀；然后向溶液中加入ε-PL和TG酶，搅拌均匀，将浆料放置在模具中进行交联，得到水凝胶。
  2. 根据权利要求1所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法，其特征在于，所述方法具体包括以下步骤：
    (1)制备胶原蛋白溶液：将胶原蛋白和醋酸水溶液混匀溶解得到胶原蛋白溶液；
    (2)制备胶原蛋白/海藻酸盐溶液：向步骤(1)中制备的胶原蛋白溶液中加入海藻酸盐，搅拌至海藻酸盐溶解，得到胶原蛋白/海藻酸盐溶液；
    (3)制备胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液：向步骤(2)中制备的胶原蛋白/海藻酸盐溶液加入硫酸乙酰肝素蛋白聚糖多糖，搅拌至溶解，得到胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液；
    (4)制备物理交联的第一浆料：向(3)中制备的胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液加入ε-PL，搅拌均匀，得到物理交联制备的第一浆料；
    (5)制备酶交联的第二浆料：向(4)中制备的第一浆料加入TG酶，搅拌均匀，得到酶交联制备的第二浆料；
    (6)水凝胶：将(5)中得到的浆料放入模具中，交联12～36h，脱模，得到用于肌肉干细胞生长的水凝胶。
  3. 根据权利要求2所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法，其特征在于，步骤(1)中所述的醋酸水溶液的浓度为0.02～0.05mol/L，胶原蛋白的质量是水质量的10～15％。
  4. 根据权利要求2或3所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法，其特征在于，步骤(2)中海藻酸盐的质量是水质量的15～25％。
  5. 根据权利要求2或3所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法，其特征在于，步骤(3)中胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液中硫酸乙酰肝素蛋白聚糖的浓度为200～500μg/L。
  6. 根据权利要求4所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法，其特征在于，步骤(3)中胶原蛋白/海藻酸盐/硫酸乙酰肝素蛋白聚糖溶液中硫酸乙酰肝素蛋白聚糖的浓度为200～500μg/L。
  7. 根据权利要求2或3所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法，其特征在于，步骤(4)中海藻酸盐的羧基和ε-PL的氨基的摩尔比为1:1～1:2；步骤(5)中TG酶 的用量为胶原蛋白质量的1～10％。
  8. 根据权利要求4所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法，其特征在于，步骤(4)中海藻酸盐的羧基和ε-PL的氨基的摩尔比为1:1～1:2；步骤(5)中TG酶的用量为胶原蛋白质量的1～10％。
  9. 根据权利要求4所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法，其特征在于，步骤(6)中所述交联温度为37～50℃。
  10. 根据权利要求1～9任一项所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法制备得到的用于肌肉干细胞培养的交联水凝胶。
  11. 包含权利要求10所述的用于肌肉干细胞培养的交联水凝胶的培养基。
  12. 一种肌肉干细胞培养的方法，其特征在于，所述方法以权利要求10所述的用于肌肉干细胞培养的交联水凝胶作为培养基。
  13. 权利要求1～9任一项所述的一种用于肌肉干细胞培养的交联水凝胶的制备方法或权利要求10所述的用于肌肉干细胞培养的交联水凝胶在培养肉领域的应用。
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