CN102174501B - Preparation method of three-dimensional histioid cardiac muscular tissue for studying simulated microgravity effect - Google Patents
- ️Wed Dec 12 2012
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- CN102174501B CN102174501B CN 201110061542 CN201110061542A CN102174501B CN 102174501 B CN102174501 B CN 102174501B CN 201110061542 CN201110061542 CN 201110061542 CN 201110061542 A CN201110061542 A CN 201110061542A CN 102174501 B CN102174501 B CN 102174501B Authority
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Abstract
一种用于研究模拟微重力效应的三维类心肌组织的制备方法，涉及一种三维类心肌组织微球的制备方法。本发明是要解决目前用于研究模拟微重力效应的细胞三维培养方法中cytodex微载体材料具有毒性而对心肌细胞产生不利影响，培养时需要的细胞量大，收取细胞困难，细胞在回转器中培养时易受剪切力的干扰，心肌细胞的持续收缩时间短的问题。方法：制备混合溶液；制备心肌细胞；制成细胞悬液，加入二价阳性离子溶液中，得微球；将微球置于培养基中培养，即形成三维类心肌组织。使用本发明方法制备的三维类心肌组织可长期培养，无需传代，可长时间维持心肌细胞收缩功能。应用于空间生物学、航天医学和组织工程研究领域。A method for preparing a three-dimensional myocardium-like tissue for studying simulated microgravity effects, and relates to a preparation method for a three-dimensional myocardium-like tissue microsphere. The present invention aims to solve the problem that cytodex microcarrier materials are toxic and have adverse effects on cardiomyocytes in the three-dimensional cell culture method currently used to study simulated microgravity effects. The amount of cells required for culture is large, and it is difficult to collect cells. It is easily disturbed by shear force during culture, and the sustained contraction time of cardiomyocytes is short. Methods: prepare mixed solution; prepare cardiomyocytes; make cell suspension, add divalent positive ion solution to obtain microspheres; culture microspheres in culture medium to form three-dimensional myocardium-like tissue. The three-dimensional myocardium-like tissue prepared by the method of the present invention can be cultured for a long time without subculture, and can maintain the contraction function of cardiomyocytes for a long time. It is used in the fields of space biology, aerospace medicine and tissue engineering research.
Description
技术领域 technical field
本发明涉及一种三维类心肌组织的制备方法。The invention relates to a method for preparing a three-dimensional myocardium-like tissue.
背景技术 Background technique
外太空环境具有微重力、超低温、高真空和强辐射等特征，其中，微重力对人体生理功能有明显影响。国内外航天医学研究者进行了大量微重力对人体机能影响的医学观察和研究，发现长时间太空飞行可引起生理机能出现一定程度的病理学改变，主要包括心血管系统、肌肉系统、免疫系统和骨骼系统的病理性改变。由于心血管系统维持着人体的正常生命活动，因此研究微重力对人体心血管系统的影响，探讨其机理及制定有效防护措施，对于保证航天员在太空飞行时的健康和有效工作具有重要意义。The outer space environment has the characteristics of microgravity, ultra-low temperature, high vacuum and strong radiation. Among them, microgravity has a significant impact on human physiological functions. Aerospace medical researchers at home and abroad have conducted a large number of medical observations and studies on the effects of microgravity on human body functions, and found that long-term space flight can cause a certain degree of pathological changes in physiological functions, mainly including cardiovascular system, muscular system, immune system and Pathological changes of the skeletal system. Since the cardiovascular system maintains the normal life activities of the human body, it is of great significance to study the influence of microgravity on the human cardiovascular system, explore its mechanism and formulate effective protective measures to ensure the health and effective work of astronauts during spaceflight.
目前研究微重力效应的心肌细胞模型主要是通过在回转器中培养细胞，从而达到定性模拟微重力环境。心肌细胞的三维培养方法是目前用于研究模拟微重力效应最广泛的细胞培养方法，使心肌细胞生长在cytodex微载体的表面。这种方法存在许多不足：(1)cytodex微载体材料具有毒性，会对细胞产生不利影响；(2)培养时需要的细胞量大，收取细胞困难；(3)在回转器中培养时，易受气泡等因素产生的剪切力的干扰；(4)心肌细胞的持续收缩时间短。At present, the cardiomyocyte model for studying the effect of microgravity is mainly through culturing cells in a gyrator, so as to qualitatively simulate the microgravity environment. The three-dimensional culture method of cardiomyocytes is currently the most widely used cell culture method for studying the effect of simulating microgravity, allowing cardiomyocytes to grow on the surface of cytodex microcarriers. There are many deficiencies in this method: (1) the cytodex microcarrier material is toxic, which will adversely affect the cells; (2) the amount of cells required for cultivation is large, and it is difficult to collect cells; (3) when cultivating in a gyrator, it is easy to Interference by the shear force generated by air bubbles and other factors; (4) The sustained contraction time of cardiomyocytes is short.
发明内容 Contents of the invention
本发明是要解决目前用于研究模拟微重力效应的细胞三维培养方法中cytodex微载体材料具有毒性而对心肌细胞产生不利影响，培养时需要的细胞量大，收取细胞困难，细胞在回转器中培养时易受剪切力的干扰，心肌细胞的持续收缩时间短的问题，提供一种用于研究模拟微重力效应的三维类心肌组织微球的制备方法。The present invention aims to solve the problem that cytodex microcarrier materials are toxic and have adverse effects on cardiomyocytes in the three-dimensional cell culture method currently used to study simulated microgravity effects. The amount of cells required for culture is large, and it is difficult to collect cells. It is easy to be disturbed by shear force during culture, and the continuous contraction time of cardiomyocytes is short. A method for preparing three-dimensional myocardial tissue-like microspheres for studying the effect of simulating microgravity is provided.
本发明的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，按以下步骤进行：一、将质量浓度为0.05％～3％的海藻酸钠溶液与质量浓度为10％～50％的I型胶原蛋白溶液混合，得到混合溶液；二、将出生后1～3d的Wistar大鼠乳鼠心脏置于0～4℃的0.1mol/L的PBS溶液中，然后用0～4℃的0.1mol/L的PBS溶液清洗心脏3～5次，再将心脏组织剪成1～3mm3大小的组织块，再用0～4℃的0.1mol/L的PBS溶液清洗3～5次，然后将组织块用消化液消化10～30min，再加入组织块10倍体积的DMEM培养基，离心后弃上清，然后向沉淀中加入DMEM培养基制成细胞悬液A，将细胞悬液A接种到培养瓶中，差速贴壁90～120min，得到纯化的心肌细胞；三、将纯化的心肌细胞与步骤一制得的混合溶液混合，制成细胞密度为0.5×106～5×106个/mL的心肌细胞悬液，采用针头将心肌细胞悬液逐滴加入到25～100mmol/L的二价阳性离子溶液中，静置5～20min，得到包裹心肌细胞的微球；四、将包裹心肌细胞的微球加入质量浓度为0.1％～3％的海藻酸钠溶液中静置5～20min后，再放入质量浓度为0.5～2％的多聚赖氨酸溶液中静置5～20min，然后将经过静置的包裹心肌细胞的微球放于DMEM培养基进行三维培养1～2周，即形成具有持续搏动功能的三维类心肌组织；其中步骤一中I型胶原蛋白溶液的质量是海藻酸钠溶液质量的0.1％～50％；步骤二中消化液由胰酶和胶原酶II组成，消化液中胰酶的质量浓度为0.02～0.2％，胶原酶II的质量浓度为0.01～0.1％；步骤二中消化液的体积为组织块体积的9～12倍；步骤二和步骤四中的DMEM培养基中都含质量浓度为10％～15％的胎牛血清；步骤二中的细胞悬液A的细胞密度为0.5×106～5×106个/mL；步骤三中的二价阳离子溶液中二价阳离子为钙离子、钡离子或锶离子；步骤四中海藻酸钠溶液的体积为微球体积的9～12倍，多聚赖氨酸溶液的体积为微球体积的9～12倍。A method for preparing a three-dimensional myocardium-like tissue for studying the simulated microgravity effect of the present invention is carried out according to the following steps: 1. A sodium alginate solution with a mass concentration of 0.05% to 3% is mixed with a mass concentration of 10% to 50% % type I collagen solution was mixed to obtain a mixed solution; 2. Place the heart of Wistar rat suckling rat at 1 to 3 days after birth in a 0.1mol/L PBS solution at 0 to 4°C, and then use 0 to 4°C Wash the heart with 0.1mol/L PBS solution for 3-5 times, then cut the heart tissue into 1-3mm3 tissue pieces, and then wash with 0-4℃ 0.1mol/L PBS solution for 3-5 times. Then digest the tissue block with digestive solution for 10-30min, then add 10 times the volume of DMEM medium to the tissue block, discard the supernatant after centrifugation, and then add DMEM medium to the precipitate to make cell suspension A. Cell suspension A Inoculate into a culture bottle, adhere to the wall at a differential speed for 90-120 minutes, and obtain purified cardiomyocytes; 3. Mix the purified cardiomyocytes with the mixed solution prepared in step 1 to make a cell density of 0.5×10 6 to 5×10 6 cells/mL of cardiomyocyte suspension, use a needle to add the cardiomyocyte suspension dropwise into 25-100mmol/L divalent positive ion solution, and let it stand for 5-20min to obtain microspheres wrapped with cardiomyocytes; 4. Add the microspheres wrapped with cardiomyocytes into a sodium alginate solution with a mass concentration of 0.1% to 3% and let it stand for 5 to 20 minutes, then put it into a polylysine solution with a mass concentration of 0.5 to 2% and let it stand for 5 minutes. ~20min, and then put the static microspheres wrapped in cardiomyocytes in DMEM medium for three-dimensional culture for 1 to 2 weeks, and then form a three-dimensional myocardial tissue with continuous beating function; the type I collagen solution in step 1 The mass is 0.1% to 50% of the mass of the sodium alginate solution; the digestive juice in step 2 is composed of trypsin and collagenase II, the mass concentration of trypsin in the digestive juice is 0.02% to 0.2%, and the mass concentration of collagenase II is 0.01% ~0.1%; the volume of the digestive solution in step two is 9 to 12 times of the volume of the tissue block; the DMEM medium in step two and step four all contains fetal bovine serum with a mass concentration of 10% to 15%; The cell density of the cell suspension A is 0.5×10 6 to 5×10 6 cells/mL; the divalent cations in the divalent cation solution in step 3 are calcium ions, barium ions or strontium ions; the sodium alginate in step 4 The volume of the solution is 9-12 times of the volume of the microsphere, and the volume of the polylysine solution is 9-12 times of the volume of the microsphere.
本发明采用组织工程技术在体外构建具有心肌结构和功能的类心肌组织，该类心肌组织具有与真实的心脏组织十分相近的特点而且便于精确调控，利用这样的类心肌组织来代替心肌细胞用于研究模拟失重条件下的心肌结构和功能改变的分子生物学机制，搭建一个适用于空间生命科学研究和空间环境风险评价的三维心肌细胞培养平台。The present invention uses tissue engineering technology to construct myocardial-like tissue with myocardial structure and function in vitro. This type of myocardial tissue has characteristics very similar to real heart tissue and is convenient for precise regulation. Such myocardial tissue is used to replace cardiomyocytes for To study the molecular biological mechanism of myocardial structure and function changes under simulated weightlessness conditions, and build a three-dimensional cardiomyocyte culture platform suitable for space life science research and space environment risk assessment.
本发明的方法具有以下优点：The method of the present invention has the following advantages:
1、心肌细胞在微球内较少受到剪切力干扰；1. Cardiomyocytes are less disturbed by shear force in the microsphere;
2、心肌细胞在微球内形成球状细胞团，在生物学性能上与在体组织更为接近；2. Cardiomyocytes form spherical cell clusters in the microspheres, which are closer to in vivo tissue in terms of biological performance;
3、微球具有一定的孔隙率，心肌细胞在微球内既能接受营养物质又能将代谢废物排出；3. Microspheres have a certain porosity, and cardiomyocytes can receive nutrients and discharge metabolic waste in the microspheres;
4、心肌细胞在微球内可长期培养，无需传代，更适于空间搭载；4. Cardiomyocytes can be cultured in microspheres for a long time without passage, and are more suitable for space loading;
5、使用本发明方法制备的三维类心肌组织可以长时间维持心肌细胞收缩功能，持续时间为6个月；而目前的二维培养方法心肌细胞持续收缩时间是130天，目前的三维培养方法心肌细胞持续收缩时间是2个月。5. The three-dimensional myocardium-like tissue prepared by the method of the present invention can maintain the contractile function of myocardial cells for a long time, and the duration is 6 months; while the continuous contraction time of myocardial cells in the current two-dimensional culture method is 130 days, the current three-dimensional culture method myocardial The duration of cell shrinkage was 2 months.
附图说明 Description of drawings
图1为具体实施方式二十四中步骤三得到的包裹心肌细胞的微球的照片；图2为具体实施方式二十四中步骤三得到的包裹心肌细胞的微球在显微镜下放大40倍的照片；图3为具体实施方式二十四中经DAPI染色后的微球在显微镜下放大40倍的照片；图4为具体实施方式二十四中培养2个月的包裹三维类心肌组织的微球的照片；图5为具体实施方式二十四中培养2个月的包裹三维类心肌组织的微球在显微镜下放大400倍的照片；图6为具体实施方式二十四中培养2个月的三维类心肌组织放大500倍的扫描电镜照片；图7为具体实施方式二十四中培养2个月的三维类心肌组织放大4000倍的扫描电镜照片；图8为具体实施方式二十四中三维类心肌组织在不同的培养时间同步搏动频率的检测图。Fig. 1 is a photo of the microspheres wrapped cardiomyocytes obtained in step 3 of the specific embodiment 24; Fig. 2 is a 40-fold magnification of the microspheres wrapped cardiomyocytes obtained in step 3 of the specific embodiment 24 Photo; Fig. 3 is a 40-fold magnified photo of the microspheres stained with DAPI in Embodiment 24; Fig. 4 is a microsphere of wrapped three-dimensional myocardium-like tissue cultured in Embodiment 24 for 2 months The photo of the ball; Fig. 5 is a 400 times magnified photo of the microspheres wrapped with three-dimensional myocardium-like tissue cultured for 2 months in the specific embodiment 24; Fig. 6 is the 2 month culture in the specific embodiment 24 500 times magnification of the three-dimensional myocardium-like tissue; FIG. 7 is a 4000-time magnification scanning electron micrograph of the three-dimensional myocardium-like tissue cultured in the twenty-fourth specific embodiment; FIG. 8 is the twenty-fourth specific embodiment Three-dimensional myocardium-like tissue for detection of synchronized beating frequency at different culture times.
具体实施方式 Detailed ways
本发明技术方案不局限于以下所列举具体实施方式，还包括各具体实施方式间的任意组合。The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.
具体实施方式一：本实施方式一种用于研究模拟微重力效应的三维类心肌组织的制备方法，按以下步骤进行：一、将质量浓度为0.05％～3％的海藻酸钠溶液与质量浓度为10％～50％的I型胶原蛋白溶液混合，得到混合溶液；二、将出生后1～3d的Wistar大鼠乳鼠心脏置于0～4℃的0.1mol/L的PBS溶液中，然后用0～4℃的0.1mol/L的PBS溶液清洗心脏3～5次，再将心脏组织剪成1～3mm3大小的组织块，再用0～4℃的0.1mol/L的PBS溶液清洗3～5次，然后将组织块用消化液消化10～30min，再加入组织块10倍体积的DMEM培养基，离心后弃上清，然后向沉淀中加入DMEM培养基制成细胞悬液A，将细胞悬液A接种到培养瓶中，差速贴壁90～120min，得到纯化的心肌细胞；三、将纯化的心肌细胞与步骤一制得的混合溶液混合，制成细胞密度为0.5×106～5×106个/mL的心肌细胞悬液，采用针头将心肌细胞悬液逐滴加入到25～100mmol/L的二价阳性离子溶液中，静置5～20min，得到包裹心肌细胞的微球；四、将包裹心肌细胞的微球加入质量浓度为0.1％～3％的海藻酸钠溶液中静置5～20min后，再放入质量浓度为0.5～2％的多聚赖氨酸溶液中静置5～20min，然后将经过静置的包裹心肌细胞的微球放于DMEM培养基进行三维培养1～2周，即形成具有持续搏动功能的三维类心肌组织；其中步骤一中I型胶原蛋白溶液的质量是海藻酸钠溶液质量的0.1％～50％；步骤二中消化液由胰酶和胶原酶II组成，消化液中胰酶的质量浓度为0.02～0.2％，胶原酶II的质量浓度为0.01～0.1％；步骤二中消化液的体积为组织块体积的9～12倍；步骤二和步骤四中的DMEM培养基中都含质量浓度为10％～15％的胎牛血清；步骤二中的细胞悬液A的细胞密度为0.5×106～5×106个/mL；步骤三中的二价阳离子溶液中二价阳离子为钙离子、钡离子或锶离子；步骤四中海藻酸钠溶液的体积为微球体积的9～12倍，多聚赖氨酸溶液的体积为微球体积的9～12倍。Specific Embodiment 1: In this embodiment, a method for preparing a three-dimensional myocardium-like tissue for simulating microgravity effects is carried out according to the following steps: 1. A sodium alginate solution with a mass concentration of 0.05% to 3% is mixed with a mass concentration of Mixing 10% to 50% type I collagen solution to obtain a mixed solution; 2. Place the heart of a Wistar rat suckling mouse 1 to 3 days after birth in a 0.1mol/L PBS solution at 0 to 4°C, and then Wash the heart with 0.1mol/L PBS solution at 0-4°C for 3-5 times, then cut the heart tissue into 1-3mm3 tissue pieces, and wash with 0.1mol/L PBS solution at 0-4°C 3 to 5 times, then digest the tissue block with digestive solution for 10 to 30 min, then add DMEM medium 10 times the volume of the tissue block, centrifuge and discard the supernatant, then add DMEM medium to the pellet to make cell suspension A, Inoculate the cell suspension A into the culture flask, adhere to the wall at a differential speed for 90-120 minutes, and obtain purified cardiomyocytes; 3. Mix the purified cardiomyocytes with the mixed solution prepared in step 1 to make a cell density of 0.5×10 6-5 × 106 /mL cardiomyocyte suspension, add the cardiomyocyte suspension drop by drop into 25-100mmol/L divalent positive ion solution with a needle, let it stand for 5-20min, and obtain the cardiomyocyte-encapsulated Microspheres; 4. Add the microspheres wrapped in cardiomyocytes into a sodium alginate solution with a mass concentration of 0.1% to 3% and let it stand for 5 to 20 minutes, then add polylysine with a mass concentration of 0.5 to 2%. Stand still in the solution for 5-20 minutes, and then put the static microspheres wrapped in cardiomyocytes in DMEM medium for three-dimensional culture for 1-2 weeks, and then form a three-dimensional myocardial tissue with continuous beating function; wherein step I The quality of type collagen solution is 0.1%～50% of the sodium alginate solution quality; In step 2, digestive juice is made up of trypsin and collagenase II, and the mass concentration of trypsin in the digestive juice is 0.02～0.2%, collagenase II The mass concentration is 0.01-0.1%; the volume of the digestive solution in step 2 is 9-12 times the volume of the tissue block; the DMEM medium in step 2 and step 4 contains fetal bovine with a mass concentration of 10%-15%. Serum; the cell density of cell suspension A in step 2 is 0.5×10 6 to 5×10 6 cells/mL; the divalent cation in the divalent cation solution in step 3 is calcium ion, barium ion or strontium ion; step The volume of sodium alginate solution in No. 4 is 9 to 12 times the volume of the microspheres, and the volume of the polylysine solution is 9 to 12 times the volume of the microspheres.
本实施方式步骤一中的I型胶原蛋白为大鼠I型胶原蛋白，为购买得到。The type I collagen in step 1 of this embodiment is rat type I collagen, which is purchased.
本实施方式的方法具有以下优点：心肌细胞在微球内较少受到剪切力干扰；心肌细胞在微球内形成球状细胞团，在生物学性能上与在体组织更为接近；微球具有一定的孔隙率，心肌细胞在微球内既能接受营养物质又能将代谢废物排出；心肌细胞在微球内可长期培养，无需传代，更适于空间搭载；使用本发明方法制备的三维类心肌组织可以长时间维持心肌细胞收缩功能，持续时间为6个月；而目前的二维培养方法心肌细胞持续收缩时间是130天，目前的三维培养方法心肌细胞持续收缩时间是2个月。The method of this embodiment has the following advantages: cardiomyocytes are less disturbed by shear force in microspheres; cardiomyocytes form spherical cell clusters in microspheres, which are closer to in vivo tissue in biological properties; microspheres have With a certain porosity, cardiomyocytes can receive nutrients and discharge metabolic waste in the microspheres; cardiomyocytes can be cultured in the microspheres for a long time without passage, and are more suitable for space loading; the three-dimensional analogues prepared by the method of the present invention Myocardial tissue can maintain the contractile function of cardiomyocytes for a long time, lasting for 6 months; while the current two-dimensional culture method has a continuous contraction time of cardiomyocytes for 130 days, and the current three-dimensional culture method has a continuous contraction time of cardiomyocytes for 2 months.
具体实施方式二：本实施方式与具体实施方式一不同的是：步骤一中海藻酸钠溶液的质量浓度为0.5％～2％。其它与具体实施方式一相同。Embodiment 2: This embodiment is different from Embodiment 1 in that: the mass concentration of the sodium alginate solution in step 1 is 0.5%-2%. Others are the same as in the first embodiment.
具体实施方式三：本实施方式与具体实施方式一或二不同的是：步骤一中海藻酸钠溶液的质量浓度为1％。其它与具体实施方式一或二相同。Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that the mass concentration of the sodium alginate solution in step 1 is 1%. Others are the same as in the first or second embodiment.
具体实施方式四：本实施方式与具体实施方式一至三之一不同的是：步骤一中I型胶原蛋白溶液的质量浓度为20％～40％。其它与具体实施方式一至三之一相同。Embodiment 4: This embodiment differs from Embodiment 1 to Embodiment 3 in that: the mass concentration of the type I collagen solution in step 1 is 20%-40%. Others are the same as those in the first to third specific embodiments.
具体实施方式五：本实施方式与具体实施方式一至三之一不同的是：步骤一中I型胶原蛋白溶液的质量浓度为30％。其它与具体实施方式一至三之一相同。Embodiment 5: This embodiment differs from Embodiment 1 to Embodiment 3 in that the mass concentration of the type I collagen solution in step 1 is 30%. Others are the same as those in the first to third specific embodiments.
具体实施方式六：本实施方式与具体实施方式一至五之一不同的是：步骤一中I型胶原蛋白溶液的质量是海藻酸钠溶液质量的5％～40％。其它与具体实施方式一至五之一相同。Embodiment 6: This embodiment differs from Embodiment 1 to Embodiment 5 in that: the quality of the type I collagen solution in step 1 is 5% to 40% of that of the sodium alginate solution. Others are the same as one of the specific embodiments 1 to 5.
具体实施方式七：本实施方式与具体实施方式一至五之一不同的是：步骤一中I型胶原蛋白溶液的质量是海藻酸钠溶液质量的10％～30％。其它与具体实施方式一至五之一相同。Embodiment 7: The difference between this embodiment and one of Embodiments 1 to 5 is that the quality of the type I collagen solution in step 1 is 10% to 30% of that of the sodium alginate solution. Others are the same as one of the specific embodiments 1 to 5.
具体实施方式八：本实施方式与具体实施方式一至五之一不同的是：步骤一中I型胶原蛋白溶液的质量是海藻酸钠溶液质量的20％。其它与具体实施方式一至五之一相同。Embodiment 8: The difference between this embodiment and one of Embodiments 1 to 5 is that the quality of the type I collagen solution in step 1 is 20% of that of the sodium alginate solution. Others are the same as one of the specific embodiments 1 to 5.
具体实施方式九：本实施方式与具体实施方式一至八之一不同的是：步骤二的消化液中胰酶的质量浓度为0.05～0.15％。其它与具体实施方式一至八之一相同。Embodiment 9: This embodiment is different from Embodiment 1 to Embodiment 8 in that: the mass concentration of trypsin in the digestive juice in step 2 is 0.05-0.15%. Others are the same as one of the specific embodiments 1 to 8.
具体实施方式十：本实施方式与具体实施方式一至八之一不同的是：步骤二的消化液中胰酶的质量浓度为0.1％。其它与具体实施方式一至八之一相同。Embodiment 10: This embodiment is different from Embodiment 1 to Embodiment 8 in that: the mass concentration of trypsin in the digestive juice in step 2 is 0.1%. Others are the same as one of the specific embodiments 1 to 8.
具体实施方式十一：本实施方式与具体实施方式一至十之一不同的是：步骤二的消化液中胶原酶II的质量浓度为0.05％。其它与具体实施方式一至十之一相同。Embodiment 11: This embodiment differs from Embodiments 1 to 11 in that: the mass concentration of collagenase II in the digestive juice in step 2 is 0.05%. Others are the same as those in Embodiments 1 to 11.
具体实施方式十二：本实施方式与具体实施方式一至十一之一不同的是：步骤二中消化液的体积为组织块体积的10倍。其它与具体实施方式一至十一之一相同。Embodiment 12: This embodiment differs from Embodiments 1 to 11 in that the volume of the digestive fluid in step 2 is 10 times the volume of the tissue block. Others are the same as those of the specific embodiments 1 to 11.
具体实施方式十三：本实施方式与具体实施方式一至十二之一不同的是：步骤二中将组织块用消化液消化20min。其它与具体实施方式一至十二之一相同。Embodiment 13: This embodiment differs from Embodiments 1 to 12 in that: in step 2, the tissue block is digested with digestive juice for 20 minutes. Others are the same as one of the specific embodiments 1 to 12.
具体实施方式十四：本实施方式与具体实施方式一至十三之一不同的是：步骤二中差速贴壁100min。其它与具体实施方式一至十三之一相同。Embodiment 14: This embodiment is different from Embodiment 1 to Embodiment 13 in that: in step 2, the differential speed adheres to the wall for 100 minutes. Others are the same as those of the first to thirteenth specific embodiments.
具体实施方式十五：本实施方式与具体实施方式一至十四之一不同的是：步骤二中的细胞悬液A的细胞密度为1×106～3×106个/mL。其它与具体实施方式一至十四之一相同。Embodiment 15: This embodiment differs from Embodiments 1 to 14 in that: the cell density of the cell suspension A in step 2 is 1×10 6 -3×10 6 cells/mL. Others are the same as one of the specific embodiments 1 to 14.
具体实施方式十六：本实施方式与具体实施方式一至十四之一不同的是：步骤二中的细胞悬液A的细胞密度为2×106个/mL。其它与具体实施方式一至十四之一相同。Embodiment 16: This embodiment differs from Embodiment 1 to Embodiment 14 in that: the cell density of the cell suspension A in step 2 is 2×10 6 cells/mL. Others are the same as one of the specific embodiments 1 to 14.
具体实施方式十七：本实施方式与具体实施方式一至十六之一不同的是：步骤三中制成细胞密度为1×106～3×106个/mL的心肌细胞悬液。其它与具体实施方式一至十六之一相同。Embodiment 17: This embodiment differs from Embodiments 1 to 16 in that: in step 3, a cardiomyocyte suspension with a cell density of 1×10 6 -3×10 6 cells/mL is prepared. Others are the same as one of the specific embodiments 1 to 16.
具体实施方式十八：本实施方式与具体实施方式一至十六之一不同的是：步骤三中制成细胞密度为2×106个/mL的心肌细胞悬液。其它与具体实施方式一至十六之一相同。Embodiment 18: This embodiment differs from Embodiments 1 to 16 in that: in Step 3, a cardiomyocyte suspension with a cell density of 2×10 6 cells/mL is prepared. Others are the same as one of the specific embodiments 1 to 16.
具体实施方式十九：本实施方式与具体实施方式一至十八之一不同的是：步骤三中将心肌细胞悬液逐滴加入到40～80mmol/L的二价阳性离子溶液中。其它与具体实施方式一至十八之一相同。Embodiment 19: This embodiment differs from Embodiment 1 to Embodiment 18 in that: in step 3, the cardiomyocyte suspension is added dropwise to the 40-80 mmol/L divalent positive ion solution. Others are the same as those of the first to eighteenth specific embodiments.
具体实施方式二十：本实施方式与具体实施方式一至十八之一不同的是：步骤三中将心肌细胞悬液逐滴加入到60mmol/L的二价阳性离子溶液中。其它与具体实施方式一至十八之一相同。Embodiment 20: This embodiment differs from Embodiments 1 to 18 in that: in step 3, the cardiomyocyte suspension is added dropwise to the 60 mmol/L divalent positive ion solution. Others are the same as those of the first to eighteenth specific embodiments.
具体实施方式二十一：本实施方式与具体实施方式一至二十之一不同的是：步骤四中海藻酸钠溶液的质量浓度为0.5％～2％。其它与具体实施方式一至二十之一相同。Embodiment 21: This embodiment differs from Embodiments 1 to 21 in that the mass concentration of sodium alginate solution in step 4 is 0.5%-2%. Others are the same as the first to twenty-first specific embodiments.
具体实施方式二十二：本实施方式与具体实施方式一至二十之一不同的是：步骤四中海藻酸钠溶液的质量浓度为1％。其它与具体实施方式一至二十之一相同。Embodiment 22: This embodiment differs from Embodiments 1 to 21 in that the mass concentration of the sodium alginate solution in Step 4 is 1%. Others are the same as the first to twenty-first specific embodiments.
具体实施方式二十三：本实施方式与具体实施方式一至二十二之一不同的是：步骤四中多聚赖氨酸溶液的质量浓度为1％。其它与具体实施方式一至二十二之一相同。Embodiment 23: This embodiment is different from Embodiment 1 to Embodiment 22 in that the mass concentration of the poly-lysine solution in step 4 is 1%. Others are the same as one of the specific embodiments 1 to 22.
具体实施方式二十四：本实施方式一种用于研究模拟微重力效应的三维类心肌组织的制备方法，按以下步骤进行：一、将质量浓度为2％的海藻酸钠溶液与质量浓度为30％的I型胶原蛋白溶液混合，得到混合溶液；二、将出生后3d的Wistar大鼠乳鼠心脏置于4℃的0.1mol/L的PBS溶液中，然后用4℃的0.1mol/L的PBS溶液清洗心脏5次，再将心脏组织剪成1～3mm3大小的组织块，再用4℃的0.1mol/L的PBS溶液清洗3次，然后将组织块用消化液消化30min，再加入组织块10倍体积的DMEM培养基，离心后弃上清，然后向沉淀中加入DMEM培养基制成细胞悬液A，将细胞悬液A接种到培养瓶中，差速贴壁120min，得到纯化的心肌细胞；三、将纯化的心肌细胞与步骤一制得的混合溶液混合，制成细胞密度为2×106个/mL的心肌细胞悬液，采用针头将心肌细胞悬液逐滴加入到100mmol/L的二价阳性离子溶液中，静置5min，得到包裹心肌细胞的微球；四、将包裹心肌细胞的微球加入质量浓度为1％的海藻酸钠溶液中静置10min后，再放入质量浓度0.5％的多聚赖氨酸溶液中静置10min，将经过静置的包裹心肌细胞的微球放于DMEM培养基进行三维培养2周，即形成具有持续搏动功能的三维类心肌组织；其中步骤一中I型胶原蛋白溶液的质量是海藻酸钠溶液质量的20％；步骤二中消化液由胰酶和胶原酶II组成，消化液中胰酶的质量浓度为0.02％，胶原酶II的质量浓度为0.01％；步骤二中消化液的体积为组织块体积的10倍；步骤二和步骤四中的DMEM培养基中都含质量浓度为15％的胎牛血清；步骤二中的细胞悬液A的细胞密度为2×106个/mL；步骤三中的二价阳离子溶液中二价阳离子为钙离子、钡离子或锶离子；步骤四中海藻酸钠溶液的体积为微球体积10倍，多聚赖氨酸溶液的体积为微球体积的10倍。Specific Embodiment 24: In this embodiment, a method for preparing a three-dimensional myocardium-like tissue for simulating microgravity effects is carried out according to the following steps: 1. Mix a sodium alginate solution with a mass concentration of 2% and a mass concentration of 30% type I collagen solution was mixed to obtain a mixed solution; 2. Place the Wistar rat suckling mouse heart at 4°C in 0.1mol/L PBS solution at 4°C, and then use 0.1mol/L at 4°C to obtain a mixed solution; Wash the heart with PBS solution for 5 times, then cut the heart tissue into 1-3mm3 tissue pieces, wash with 0.1mol/L PBS solution at 4℃ for 3 times, then digest the tissue pieces with digestive juice for 30min, and then Add 10 times the volume of DMEM medium to the tissue block, centrifuge and discard the supernatant, then add DMEM medium to the pellet to make cell suspension A, inoculate cell suspension A into a culture bottle, and adhere to the wall at a differential speed for 120 minutes to obtain Purified cardiomyocytes; 3. Mix the purified cardiomyocytes with the mixed solution prepared in step 1 to make a cardiomyocyte suspension with a cell density of 2× 106 cells/mL, and add the cardiomyocyte suspension dropwise with a needle In the divalent positive ion solution of 100mmol/L, stand still 5min, obtain the microsphere that wraps cardiomyocyte; 4, add the microsphere that wraps cardiomyocyte into the sodium alginate solution that mass concentration is 1% after standing for 10min, Then put it in a polylysine solution with a mass concentration of 0.5% and let it stand for 10 minutes, put the microspheres wrapped with cardiomyocytes in DMEM medium for three-dimensional culture for two weeks, and then form a three-dimensional class with continuous beating function. Myocardial tissue; wherein the quality of type I collagen solution in step 1 is 20% of the quality of sodium alginate solution; In step 2, the digestive juice is made up of trypsin and collagenase II, and the mass concentration of trypsin in the digestive juice is 0.02%, The mass concentration of collagenase II is 0.01%; the volume of the digestive solution in step 2 is 10 times of the volume of the tissue block; the DMEM medium in step 2 and step 4 contains fetal bovine serum with a mass concentration of 15%; step 2 The cell density of the cell suspension A in the method is 2×10 6 cells/mL; the divalent cation in the divalent cation solution in the step 3 is calcium ion, barium ion or strontium ion; the volume of the sodium alginate solution in the step 4 is The volume of the microsphere is 10 times, and the volume of the polylysine solution is 10 times of the volume of the microsphere.
本实施方式步骤三制得的包裹心肌细胞的微球如图1和2所示，图1为包裹心肌细胞的微球的照片，图2为微球在显微镜下放大40倍的照片，可看出微球的大小均匀，直径约为2.3mm。将微球进行DAPI染色，如图3所示，图3为经DAPI染色后的微球在显微镜下放大40倍的照片，可以看到心肌细胞在微球内分布均匀。The microspheres wrapped cardiomyocytes prepared in Step 3 of this embodiment are shown in Figures 1 and 2. Figure 1 is a photo of the microspheres wrapped cardiomyocytes, and Fig. 2 is a photo of the microspheres magnified 40 times under a microscope. The microspheres were uniform in size and about 2.3 mm in diameter. The microspheres were stained with DAPI, as shown in Figure 3, which is a 40-fold magnified photo of the DAPI-stained microspheres under a microscope, and it can be seen that cardiomyocytes are evenly distributed in the microspheres.
将本实施制备的三维类心肌组织继续培养至2个月，形成肉眼可见的三维类心肌组织，如图4和图5所示，图4为培养2个月的包裹三维类心肌组织的微球的照片，图5为培养2个月的包裹三维类心肌组织的微球在显微镜下放大400倍的照片。Continue to culture the three-dimensional myocardium-like tissue prepared in this practice until 2 months to form a three-dimensional myocardium-like tissue visible to the naked eye, as shown in Figure 4 and Figure 5, Figure 4 shows the microspheres wrapped with three-dimensional myocardium-like tissue cultured for 2 months Figure 5 is a 400-fold magnified photo of microspheres wrapped with three-dimensional myocardium-like tissue cultured for 2 months under a microscope.
将培养2个月的三维类心肌组织从微球中释放出来，进行扫描电镜观察，图6为培养2个月的三维类心肌组织放大500倍的扫描电镜照片，图7为培养2个月的三维类心肌组织放大4000倍的扫描电镜照片，可以看到类心肌组织具有多层致密的细胞结构。The three-dimensional myocardoid tissue cultured for 2 months was released from the microspheres and observed with a scanning electron microscope. Figure 6 is a 500-fold scanning electron micrograph of the three-dimensional myocardoid tissue cultured for 2 months. The three-dimensional myocardium-like tissue is magnified 4000 times in the scanning electron microscope photo, and it can be seen that the myocardium-like tissue has a multi-layer dense cell structure.
将本实施制备的三维类心肌组织继续培养，观察其搏动情况，图8为三维类心肌组织在不同的培养时间同步搏动频率的检测图，图中表明类心肌细胞在三维状态下形成了细胞间通讯连接，其搏动时间可持续2个月，搏动频率维持在20～30次左右。Continue to culture the three-dimensional myocardium-like tissue prepared in this implementation, and observe its pulsation. Figure 8 is a detection diagram of the synchronous beating frequency of the three-dimensional myocardium-like tissue at different culture times. Communication connection, the pulsation time can last for 2 months, and the pulsation frequency is maintained at about 20 to 30 times.
Claims (10)
1.一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于用于研究模拟微重力效应的三维类心肌组织的制备方法，按以下步骤进行：一、将质量浓度为0.05％～3％的海藻酸钠溶液与质量浓度为10％～50％的I型胶原蛋白溶液混合，得到混合溶液；二、将出生后1～3d的Wistar大鼠乳鼠心脏置于0～4℃的0.1mol/L的PBS溶液中，然后用0～4℃的0.1mol/L的PBS溶液清洗心脏3～5次，再将心脏组织剪成1～3mm3大小的组织块，再用0～4℃的0.1mol/L的PBS溶液清洗3～5次，然后将组织块用消化液消化10～30min，再加入组织块10倍体积的DMEM培养基，离心后弃上清，然后向沉淀中加入DMEM培养基制成细胞悬液A，将细胞悬液A接种到培养瓶中，差速贴壁90～120min，得到纯化的心肌细胞；三、将纯化的心肌细胞与步骤一制得的混合溶液混合，制成细胞密度为0.5×106～5×106个/mL的心肌细胞悬液，采用针头将心肌细胞悬液逐滴加入到25～100mmol/L的二价阳性离子溶液中，静置5～20min，得到包裹心肌细胞的微球；四、将包裹心肌细胞的微球加入质量浓度为0.1％～3％的海藻酸钠溶液中静置5～20min后，再放入质量浓度为0.5～2％的多聚赖氨酸溶液中静置5～20min，然后将经过静置的包裹心肌细胞的微球放于DMEM培养基进行三维培养1～2周，即形成具有持续搏动功能的三维类心肌组织；其中步骤一中I型胶原蛋白溶液的质量是海藻酸钠溶液质量的0.1％～50％；步骤二中消化液由胰酶和胶原酶II组成，消化液中胰酶的质量浓度为0.02～0.2％，胶原酶II的质量浓度为0.01～0.1％；步骤二中消化液的体积为组织块体积的9～12倍；步骤二和步骤四中的DMEM培养基中都含质量浓度为10％～15％的胎牛血清；步骤二中的细胞悬液A的细胞密度为0.5×106～5×106个/mL；步骤三中的二价阳离子溶液中二价阳离子为钙离子、钡离子或锶离子；步骤四中海藻酸钠溶液的体积为微球体积的9～12倍，多聚赖氨酸溶液的体积为微球体积的9～12倍。1. A method for preparing a three-dimensional myocardium-like tissue for studying the simulated microgravity effect, characterized in that the method for preparing the three-dimensional myocardium-like tissue for studying the simulated microgravity effect is carried out in the following steps: one, the mass concentration is 0.05% to 3% sodium alginate solution was mixed with type I collagen solution with a mass concentration of 10% to 50% to obtain a mixed solution; 2. Place the hearts of Wistar rat suckling mice 1 to 3 days after birth at 0 to 0.1mol/L PBS solution at 4°C, then wash the heart with 0.1mol/L PBS solution at 0-4°C for 3-5 times, then cut the heart tissue into 1-3mm3 tissue pieces, and then use Wash with 0.1 mol/L PBS solution at 0-4°C for 3-5 times, then digest the tissue block with digestive solution for 10-30 min, then add DMEM medium 10 times the volume of the tissue block, centrifuge, discard the supernatant, and pour Add DMEM medium to the precipitation to make cell suspension A, inoculate cell suspension A into a culture bottle, adhere to the wall at a differential speed for 90-120 minutes, and obtain purified cardiomyocytes; 3. Prepare purified cardiomyocytes with step 1 Mix the mixed solution to make a suspension of cardiomyocytes with a cell density of 0.5×10 6 to 5×10 6 cells/mL, and add the suspension of cardiomyocytes dropwise to a divalent positive ion solution of 25 to 100 mmol/L using a needle. , standing still for 5-20 minutes to obtain microspheres wrapped with cardiomyocytes; 4. Add the microspheres wrapped with cardiomyocytes to a sodium alginate solution with a mass concentration of 0.1% to 3% and let stand for 5-20 minutes before putting them into Let stand in poly-lysine solution with mass concentration of 0.5-2% for 5-20 minutes, then place the static microspheres wrapped with cardiomyocytes in DMEM medium for three-dimensional culture for 1-2 weeks, and then form a persistent Three-dimensional myocardial tissue with beating function; wherein the quality of type I collagen solution in step 1 is 0.1% to 50% of that of sodium alginate solution; in step 2, the digestive juice is composed of trypsin and collagenase II, and the pancreatic The mass concentration of the enzyme is 0.02 to 0.2%, and the mass concentration of collagenase II is 0.01 to 0.1%; the volume of the digestive solution in step 2 is 9 to 12 times the volume of the tissue block; in the DMEM medium in steps 2 and 4, Both contain fetal bovine serum with a mass concentration of 10% to 15%; the cell density of cell suspension A in step 2 is 0.5×10 6 to 5×10 6 cells/mL; the divalent cation solution in step 3 contains two The valent cations are calcium ions, barium ions or strontium ions; in step 4, the volume of the sodium alginate solution is 9 to 12 times the volume of the microspheres, and the volume of the polylysine solution is 9 to 12 times the volume of the microspheres. 2.根据权利要求1所述的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于步骤一中海藻酸钠溶液的质量浓度为0.5％～2％。2. A method for preparing a three-dimensional myocardium-like tissue for studying simulated microgravity effects according to claim 1, wherein the mass concentration of the sodium alginate solution in step 1 is 0.5% to 2%. 3.根据权利要求1或2所述的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于I型胶原蛋白溶液的质量浓度为20％～40％。3. A method for preparing a three-dimensional myocardium-like tissue for studying simulated microgravity effects according to claim 1 or 2, characterized in that the mass concentration of the type I collagen solution is 20% to 40%. 4.根据权利要求3所述的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于步骤一中I型胶原蛋白溶液的质量是海藻酸钠溶液质量的5％～40％。4. A kind of preparation method for studying the three-dimensional myocardial tissue of simulating microgravity effect according to claim 3, it is characterized in that the quality of type I collagen solution in step 1 is 5%～of the quality of sodium alginate solution 40%. 5.根据权利要求4所述的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于步骤二的消化液中胰酶的质量浓度为0.05～0.15％。5. A method for preparing a three-dimensional myocardium-like tissue for studying simulated microgravity effects according to claim 4, characterized in that the mass concentration of trypsin in the digestive juice in step 2 is 0.05-0.15%. 6.根据权利要求5所述的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于步骤二的消化液中胶原酶II的质量浓度为0.05％。6. A method for preparing a three-dimensional myocardium-like tissue for studying simulated microgravity effects according to claim 5, characterized in that the mass concentration of collagenase II in the digestive juice in step 2 is 0.05%. 7.根据权利要求6所述的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于步骤三中制成细胞密度为1×106～3×106个/mL的心肌细胞悬液。7. A method for preparing a three-dimensional myocardium-like tissue for studying simulated microgravity effects according to claim 6, characterized in that in step 3, the cell density is 1×10 6 to 3×10 6 cells/mL cardiomyocyte suspension. 8.根据权利要求7所述的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于步骤三中将心肌细胞悬液逐滴加入到40～80mmol/L的二价阳性离子溶液中。8. A method for preparing a three-dimensional myocardium-like tissue for studying simulated microgravity effects according to claim 7, characterized in that in step 3, the cardiomyocyte suspension is added dropwise to 40～80mmol/L divalent positive ion solution. 9.根据权利要求8所述的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于步骤四中海藻酸钠溶液的质量浓度为0.5％～2％。9. A method for preparing a three-dimensional myocardium-like tissue for studying simulated microgravity effects according to claim 8, characterized in that the mass concentration of the sodium alginate solution in step 4 is 0.5% to 2%. 10.根据权利要求9所述的一种用于研究模拟微重力效应的三维类心肌组织的制备方法，其特征在于步骤四中多聚赖氨酸溶液的质量浓度为1％。10. A method for preparing a three-dimensional myocardium-like tissue for studying simulated microgravity effects according to claim 9, wherein the mass concentration of the polylysine solution in step 4 is 1%.
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