CN108795921A - A kind of preparation method of calcium alginate multiphase microcarrier - Google Patents
A kind of preparation method of calcium alginate multiphase microcarrier Download PDFInfo
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- 235000010410 calcium alginate Nutrition 0.000 title claims abstract description 12
- 239000000648 calcium alginate Substances 0.000 title claims abstract description 12
- 229960002681 calcium alginate Drugs 0.000 title claims abstract description 12
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 26
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 26
- 239000000661 sodium alginate Substances 0.000 claims abstract description 26
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 26
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 19
- 239000007921 spray Substances 0.000 claims abstract description 7
- 238000007590 electrostatic spraying Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 abstract description 9
- 239000007788 liquid Substances 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 239000007864 aqueous solution Substances 0.000 abstract description 2
- 239000003102 growth factor Substances 0.000 abstract description 2
- 230000009881 electrostatic interaction Effects 0.000 abstract 1
- 230000005389 magnetism Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 26
- 239000004005 microsphere Substances 0.000 description 16
- 239000001110 calcium chloride Substances 0.000 description 12
- 229910001628 calcium chloride Inorganic materials 0.000 description 12
- 239000000203 mixture Substances 0.000 description 7
- 239000002994 raw material Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
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- 239000000049 pigment Substances 0.000 description 3
- 230000009471 action Effects 0.000 description 2
- 239000003181 biological factor Substances 0.000 description 2
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- 239000000017 hydrogel Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000004115 adherent culture Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
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Abstract
Description
技术领域technical field
本发明属于静电喷射技术领域，涉及一种海藻酸钙多相微载体的制备方法。The invention belongs to the technical field of electrostatic spraying, and relates to a preparation method of calcium alginate multiphase microcarriers.
背景技术Background technique
细胞培养技术作为研究细胞结构和功能的基础与批量化生产商品型细胞及其产物的重要方法，作用十分关键。微载体培养兼具贴壁培养和悬浮培养的优势，具有很高的比表面积，扩散路径短，易于操作和监控等特点。国内目前常见的微载体类型有实心微载体和多孔微载体。已有的单相微载体培养极大地限制了不同细胞或生物因子等相互作用下的功能表达(周燕等.微载体培养技术的研究与进展[J].中国组织工程研究与临床康复,2010,(16):2945-2948.)。将微载体培养的细胞注入生物体内时，由于体内成型困难和微球游走，其定位性与固定性不强。同时由于生物体内免疫系统的排斥，使其细胞的功能表达明显减少。另外，在选择制造微载体的生物材料时，必须考虑其是否具有良好的生物安全性、生物相容性、生物降解性与可加工性。As an important method for studying the basis of cell structure and function and mass production of commercial cells and their products, cell culture technology plays a key role. Microcarrier culture has the advantages of both adherent culture and suspension culture, and has the characteristics of high specific surface area, short diffusion path, and easy operation and monitoring. At present, the common types of microcarriers in China include solid microcarriers and porous microcarriers. The existing single-phase microcarrier culture greatly limits the functional expression under the interaction of different cells or biological factors (Zhou Yan et al. Research and progress of microcarrier culture technology[J]. Chinese Tissue Engineering Research and Clinical Rehabilitation, 2010 , (16):2945-2948.). When cells cultured on microcarriers are injected into living organisms, their positioning and immobilization are not strong due to the difficulty in forming in vivo and the migration of microspheres. At the same time, due to the rejection of the immune system in the organism, the functional expression of the cells is significantly reduced. In addition, when choosing biomaterials for manufacturing microcarriers, it must be considered whether they have good biosafety, biocompatibility, biodegradability and processability.
海藻酸钠，一种天然多糖，具有药物制剂辅料所需的稳定性、溶解性、粘性和安全性。目前海藻酸钠主要用于食品工业、制药业等，而未用于细胞培养方面，特别是多相细胞微载体的构建。Sodium alginate, a natural polysaccharide, has the stability, solubility, viscosity and safety required for excipients of pharmaceutical formulations. At present, sodium alginate is mainly used in the food industry, pharmaceutical industry, etc., but not in cell culture, especially in the construction of multiphase cell microcarriers.
发明内容Contents of the invention
本发明的目的是提供一种海藻酸钙多相微载体的制备方法。该方法采用电喷技术，将海藻酸钠水溶液在静电作用下双针头并喷形成均匀液滴，调控承接液氯化钙溶液的浓度，海藻酸钠液滴经螯合作用形成球状微球。本发明方法制得的微载体由两相组成，可同时担载多种细胞和多种生长因子，为细胞的共培养创造良好的条件，为组织工程、细胞治疗等奠定良好的基础。The purpose of the present invention is to provide a preparation method of calcium alginate heterogeneous microcarrier. The method adopts electrospray technology, and the sodium alginate aqueous solution is sprayed with double needles under electrostatic action to form uniform droplets, and the concentration of the receiving liquid calcium chloride solution is regulated, and the sodium alginate droplets are chelated to form spherical microspheres. The microcarrier prepared by the method of the invention is composed of two phases, can simultaneously carry various cells and various growth factors, creates good conditions for co-cultivation of cells, and lays a good foundation for tissue engineering, cell therapy and the like.
实现本发明目的的技术解决方案是：The technical solution that realizes the object of the present invention is:
一种海藻酸钙多相微载体的制备方法，包括以下步骤：A preparation method of calcium alginate heterogeneous microcarrier, comprising the following steps:
采用双针头并喷的方式，设置电压为8～12kv，接收距离为10～15cm，推进速度为0.8～1.0mL/h，将质量分数为0.2～0.75％的海藻酸钠溶液进行静电喷射，喷出的液滴进入到质量分数为1.0～2.0％的氯化钙溶液中，制得海藻酸钙多相微载体。Double-needle parallel spraying method is adopted, the voltage is set at 8-12kv, the receiving distance is 10-15cm, the propulsion speed is 0.8-1.0mL/h, and the sodium alginate solution with a mass fraction of 0.2-0.75% is electrostatically sprayed. The liquid droplet is put into the calcium chloride solution with the mass fraction of 1.0-2.0%, and the calcium alginate multiphase microcarrier is prepared.
优选地，所述的海藻酸钠溶液的质量分数为0.5％。Preferably, the mass fraction of the sodium alginate solution is 0.5%.
优选地，所述的氯化钙溶液的质量分数为1.5％。Preferably, the mass fraction of the calcium chloride solution is 1.5%.
优选地，针头直径为0.60mm。Preferably, the needle diameter is 0.60mm.
本发明利用海藻酸钠具有形成凝胶的稳定性，采用双针头静电喷射的方法，再加以利用适宜浓度的承接液氯化钙溶液的螯合作用，形成球形水凝胶。本发明方法简单，成本低廉，反应条件温和，能够有效调控水凝胶形状、大小及其分相区。本发明的海藻酸钙微载体具有多相区域，有利于不同细胞或生物因子等相互作用下的功能表达。The present invention utilizes the gel-forming stability of sodium alginate, adopts the method of double-needle electrostatic spraying, and further utilizes the chelating action of the accepting liquid calcium chloride solution of suitable concentration to form spherical hydrogel. The method of the invention is simple, low in cost, mild in reaction conditions and capable of effectively regulating the shape, size and phase separation region of the hydrogel. The calcium alginate microcarrier of the present invention has a heterogeneous region, which is beneficial to the functional expression under the interaction of different cells or biological factors.
附图说明Description of drawings
图1为海藻酸钙两相微球的示意图。Figure 1 is a schematic diagram of calcium alginate two-phase microspheres.
图2为海藻酸钙两相微球的光学显微镜图。Figure 2 is an optical microscope image of calcium alginate two-phase microspheres.
具体实施方式Detailed ways
下面结合实施例和附图对本发明作进一步详述。The present invention will be described in further detail below in conjunction with the embodiments and accompanying drawings.
实施例1Example 1
以氯化钙及浓度为0.2％的海藻酸钠为原料制备多相微载体：Preparation of heterogeneous microcarriers with calcium chloride and 0.2% sodium alginate as raw materials:
(1)配制质量分数为0.2％的海藻酸钠溶液，并在磁力搅拌下至溶液澄清均一，然后静置一夜；(1) Prepare a sodium alginate solution with a mass fraction of 0.2%, and stir it under magnetic force until the solution is clear and uniform, and then let it stand overnight;
(2)将1.5克氯化钙溶于100mL水中，溶解混匀用作承接液；(2) Dissolve 1.5 grams of calcium chloride in 100mL of water, dissolve and mix well and use it as a receiving solution;
(3)在电喷装置中进行电喷，采用两针头并喷，用两支1.0mL针管吸取海藻酸钠溶液，针头直径为0.60mm，电喷参数：在8kv的电压下，接收距离为15cm，以0.8mL/h的推进速度进行电喷，将微球承接于氯化钙承接液中即得到两相微球。(3) Perform electrospray in the electrospray device, use two needles to spray together, use two 1.0mL needle tubes to absorb sodium alginate solution, the diameter of the needles is 0.60mm, and the electrospray parameters: under the voltage of 8kv, the receiving distance is 15cm , carry out EFI at a propulsion speed of 0.8mL/h, and accept the microspheres in the calcium chloride receiving solution to obtain two-phase microspheres.
(4)取微量朱红色和酞箐蓝丙烯颜料分别加入海藻酸钠溶液中混匀(加入量为5-10mg/mL)，用染色后的海藻酸钠溶液再重复步骤(2)和步骤(3)，即可在显微镜下观察到微球的红、蓝色的两相区域分布。(4) Take a small amount of vermilion and phthalocyanine blue acrylic pigments and add them to the sodium alginate solution and mix them evenly (the addition amount is 5-10mg/mL), and repeat steps (2) and steps ( 3), the distribution of the red and blue two-phase regions of the microspheres can be observed under the microscope.
实施例2Example 2
以氯化钙及浓度为0.5％的海藻酸钠为原料制备多相微载体：Prepare multi-phase microcarriers with calcium chloride and 0.5% sodium alginate as raw materials:
(1)配制质量分数为0.5％的海藻酸钠溶液，并在磁力搅拌下至溶液澄清均一，然后静置一夜；(1) Prepare a sodium alginate solution with a mass fraction of 0.5%, and stir it under magnetic force until the solution is clear and uniform, and then let it stand overnight;
(2)将1.5克氯化钙溶于100mL水中，溶解混匀用作承接液；(2) Dissolve 1.5 grams of calcium chloride in 100mL of water, dissolve and mix well and use it as a receiving solution;
(3)在电喷装置中进行电喷，采用两针头并喷，用两支1.0mL针管吸取已配制好的海藻酸钠溶液，针头直径为0.60mm，电喷参数：在8kv的电压下，接收距离为15cm，以1.0mL/h的推进速度进行电喷，将微球承接于氯化钙承接液中即得到两相微球。(3) Perform electrospray in the electrospray device, use two needles to spray together, use two 1.0mL needles to absorb the prepared sodium alginate solution, the diameter of the needles is 0.60mm, the parameters of electrospray: under the voltage of 8kv, The receiving distance is 15cm, electrospray is carried out at a propulsion speed of 1.0mL/h, and the microspheres are accepted in the calcium chloride receiving solution to obtain two-phase microspheres.
(4)取微量朱红色和酞箐蓝丙烯颜料分别加入海藻酸钠溶液中混匀(加入量为5-10mg/mL)，用染色后的海藻酸钠溶液再重复步骤(2)和步骤(3)，即可在显微镜下观察到微球的红、蓝色的两相区域分布。(4) Take a small amount of vermilion and phthalocyanine blue acrylic pigments and add them to the sodium alginate solution and mix them evenly (the addition amount is 5-10mg/mL), and repeat steps (2) and steps ( 3), the distribution of the red and blue two-phase regions of the microspheres can be observed under the microscope.
实施例3Example 3
以氯化钙及浓度为0.75％的海藻酸钠为原料制备多相微载体：Multiphase microcarriers were prepared from calcium chloride and 0.75% sodium alginate as raw materials:
(1)配制质量分数为0.75％的海藻酸钠溶液，并在磁力搅拌下至溶液澄清均一，然后静置一夜；(1) Prepare a sodium alginate solution with a mass fraction of 0.75%, and stir it under magnetic force until the solution is clear and uniform, and then let it stand overnight;
(2)将1.5克氯化钙溶于100mL水中，溶解混匀用作承接液。(2) Dissolve 1.5 grams of calcium chloride in 100 mL of water, dissolve and mix well and use it as a receiving solution.
(3)在电喷装置中进行电喷，采用两针头并喷，用两支1.0mL针管吸取已配制好的海藻酸钠溶液，针头直径为0.60mm，电喷参数：在12kv的电压下，接收距离为10cm，以1.0mL/h的推进速度进行电喷，将微球承接于氯化钙承接液中即得到两相微球。(3) Perform electrospray in the electrospray device, use two needles to spray together, use two 1.0mL needle tubes to absorb the prepared sodium alginate solution, the needle diameter is 0.60mm, electrospray parameters: under the voltage of 12kv, The receiving distance is 10cm, electrospray is carried out at a propulsion speed of 1.0mL/h, and the microspheres are accepted in the calcium chloride receiving solution to obtain two-phase microspheres.
(4)取微量朱红色和酞箐蓝丙烯颜料分别加入海藻酸钠溶液中混匀(加入量为5-10mg/mL)，用染色后的海藻酸钠溶液再重复步骤(2)和步骤(3)，即可在显微镜下观察到微球的红、蓝色的两相区域分布。(4) Take a small amount of vermilion and phthalocyanine blue acrylic pigments and add them to the sodium alginate solution and mix them evenly (the addition amount is 5-10mg/mL), and repeat steps (2) and steps ( 3), the distribution of the red and blue two-phase regions of the microspheres can be observed under the microscope.
对比例1Comparative example 1
以氯化钙及浓度为1.5％的海藻酸钠为原料制备多相微载体：Multiphase microcarriers were prepared from calcium chloride and 1.5% sodium alginate as raw materials:
(1)配制质量分数为1.5％的海藻酸钠溶液，并在磁力搅拌下至溶液澄清均一，然后静置一夜；(1) Prepare a sodium alginate solution with a mass fraction of 1.5%, and stir it under magnetic force until the solution is clear and uniform, and then let it stand overnight;
(2)将1.5克氯化钙溶于100mL水中，溶解混匀用作承接液。(2) Dissolve 1.5 grams of calcium chloride in 100 mL of water, dissolve and mix well and use it as a receiving solution.
(3)在电喷装置中进行电喷，采用两针头并喷，用两支1.0mL针管吸取已配制好的海藻酸钠溶液，针头直径为0.60mm，电喷参数：在8kv的电压下，接收距离为15cm，以1.0mL/h的推进速度进行电喷，将微球承接于氯化钙承接液中即得到两相微球。在喷射过程中，可以观察到针头喷射液滴断断续续，且微球尺寸较大不易控制。(3) Perform electrospray in the electrospray device, use two needles to spray together, use two 1.0mL needles to absorb the prepared sodium alginate solution, the diameter of the needles is 0.60mm, the parameters of electrospray: under the voltage of 8kv, The receiving distance is 15cm, electrospray is carried out at a propulsion speed of 1.0mL/h, and the microspheres are accepted in the calcium chloride receiving solution to obtain two-phase microspheres. During the spraying process, it can be observed that the needle sprays droplets intermittently, and the size of the microspheres is large and difficult to control.
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