CN113699098A - 三维血管化心肌组织及其制备方法和应用 - Google Patents

三维血管化心肌组织及其制备方法和应用 Download PDF

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CN113699098A
CN113699098A CN202111060823.XA CN202111060823A CN113699098A CN 113699098 A CN113699098 A CN 113699098A CN 202111060823 A CN202111060823 A CN 202111060823A CN 113699098 A CN113699098 A CN 113699098A
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cells
culture
myocardial
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刘中民
刘洋
乐文俊
胡益辉
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Shanghai East Hospital Tongji University Affiliated East Hospital
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Abstract

本发明公开了三维血管化心肌组织及其制备方法和应用，所述方法包括将人多能干细胞来源的早期血管细胞和心肌细胞进行三维共培养，培养中诱导分化及分化后的继续培养成熟；其中，三维共培养过程是人多能干细胞来源的早期血管细胞和心肌细胞在非粘附状态下聚集成细胞球进行共培养或以3D生物打印方法混合共培养。与现有技术相比，本发明具有以下优点：(1)所述方法结合细胞分化自组装功能和3D生物打印技术，无需在制备过程中手动调整位置，提高了试验效率和结果的稳定性；(2)所述方法制备的心肌组织不仅可以用于体外疾病研究模型，还可作为心肌梗死等疾病的损伤修复。

Description

三维血管化心肌组织及其制备方法和应用
技术领域
本发明属于生物技术领域，涉及一种人工结构体，具体为三维血管化心肌组织及其制备方法和应用。
背景技术
心肌梗死等缺血性心脏病是现代社会发病率和致死率最高的重大疾病之一，虽然目前的治疗方法可以延缓心力衰竭的进展，但不能再生心肌组织从而修复受损心脏。近年来，干细胞特别是人诱导多能干细胞技术的发展使我们看到了人心肌细胞移植对心肌损伤修复的广阔前景，但是由于心脏持续搏动的动态特殊性和移植心肌细胞的低粘附性，以单细胞形式注射的心肌细胞会面临随循环血流失的问题，近年来的移植策略更多倾向于构建相对成熟和稳定的心肌补片并移植到损伤部位，但目前构建的心肌组织仍面临血管化程度低，难以形成具有复杂三维血管网结构的问题，从而限制了心肌组织构建的厚度以及心肌细胞的存活率和驻留率。因此，体外构建具有复杂三维血管网结构的心肌组织以提高心肌组织的血管化程度，对于构建大尺寸心肌组织并提高心肌组织中细胞的存活率和驻留率都具有重要意义，是解决干细胞来源心肌细胞移植治疗瓶颈问题的关键。
中国专利CN112608878A公开了一种体外耳蜗微器官功能单元及其三维构建方法和应用，其解决的问题在于体外构建听觉发生模型，试验细胞为小鼠耳蜗前体细胞和小鼠神经细胞，其三维培养过程中需人为手动调整耳蜗类器官与螺旋神经元的位置。该现有技术方法需经手动调整，操作随机性较强且效率较低；另外，该方法构建的体外耳蜗微器官功能单元为小鼠来源，不能进一步用于临床移植和损伤修复。
中国专利CN111187749A公开了用于快速再生血管化组织的人工结构体及构建方法与应用，其解决的问题在于提供用于细胞三维培养的水凝胶微球，可用于多种细胞类型的包裹或附着生长，但该技术血管化结构的构建需要经过繁复的操作步骤且仅利用了永生化内皮细胞系HUVEC，无法通过祖细胞的三维分化方式实现血管网络在组织内部的自组装构建，同时局限于水凝胶材料的硬度和制作微球的方法，无法构建完整的大尺寸心肌组织片，与之不同的是，本发明应用3D生物打印技术结合可降解的纤维蛋白水凝胶材料可以构建大尺寸的心肌组织片，并且可以在心肌组织内部重建三维血管网络。
发明内容
发明目的：为了克服现有技术的不足，本发明的目的在于：
其一、提供一种人多能干细胞来源的早期血管细胞与心肌细胞三维共培养及诱导分化的方法；
其二、基于目的一的方法提供体外非治疗性的构建三维血管化心肌组织的方法，及通过该方法制备获得的三维血管化心肌组织；
其三、提供三维血管化心肌组织应用于制备治疗心脏病的药物或作为移植供体治疗心脏疾病。
技术方案：三维血管化心肌组织的制备方法，所述方法包括将人多能干细胞来源的早期血管细胞和心肌细胞进行三维共培养，培养中诱导分化及分化后的继续培养成熟；其中，三维共培养过程是人多能干细胞来源的早期血管细胞和心肌细胞在非粘附状态下聚集成细胞球进行共培养或以3D生物打印方法混合共培养。
优选的，三维共培养过程中的支持条件为水凝胶材料。
进一步的，所述水凝胶材料为Matrigel(基质胶)、Collagen I(I型胶原)、Fibrin(纤维蛋白)、Gelatin(明胶)、Hyaluronic acid(透明质酸)中的至少一种。
优选的，培养中诱导分化是将人多能干细胞来源的早期血管细胞诱导分化为血管内皮细胞和血管周细胞中的至少一种。
优选的，所述分化后的继续培养成熟采用以下方法之一：
其一、采用解剖方法将三维生长的血管化心肌组织从水凝胶中解离并继续在非粘附条件下培养成熟；
其二、采用3D生物打印技术构建的支架悬浮培养三维生长的血管化心肌组织并继续培养成熟。
优选的，所述方法的具体步骤包括：
(1)将扩增培养后的人多能干细胞经Accutase消化成单细胞并铺板到低粘附培养板中，此时培养液中添加Y-27632以抑制细胞凋亡；
(2)待干细胞聚集成球后更换细胞培养液为RPMI/B27 minus insulin并添加CHIR99021和BMP4以诱导中胚层特化；
(3)继续培养3天后更换细胞培养液为EGM2并添加VEGF-A以诱导血管谱系细胞分化；
(4)将步骤(3)诱导分化2天后的细胞球用Accutase消化成单细胞并与人多能干细胞来源的心肌细胞以3:7的比例混合，培养液为RPMI/B27 with insulin:EGM-2，随后用hanging drop方法将细胞聚集成球，并以悬浮状态继续培养2天；
(5)将步骤(4)获得的细胞球包埋到水凝胶材料中并以RPMI/B27 with insulin:EGM-2培养液培养，同时添加VEGF-A、FGF-2和SB43152以诱导血管谱系细胞分化和内皮细胞出芽；
(6)将水凝胶中跳动和出芽的细胞球以解剖方法解离并继续培养在低粘附培养板中以诱导血管网的自组装和血管化心脏类器官的成熟，每3天更换RPMI/B27 withinsulin:EGM-2培养液；
(7)实验过程中以免疫荧光染色方法检测步骤(5)获得的水凝胶中心肌细胞和血管内皮细胞的生长状态，以及步骤(6)获得的心脏类器官中心肌细胞和血管内皮细胞组成的三维血管化心肌组织结构；
(8)制备含有水凝胶材料的生物墨水，将步骤(3)诱导分化2天后的细胞球与新生大鼠心肌细胞混合到生物墨水中，并采用3D生物打印机进行混合打印以构建血管化的大尺寸功能心肌组织；
(9)以添加VEGF、bFGF、SB431542、FBS、L-glutamine、aprotinin和penicillin/streptomycin的Claycomb/EGM2混合培养液进行步骤(8)打印心肌组织的培养，3周后用免疫荧光染色检测心肌组织中的血管网结构、肌丝结构，并用钙成像方法检测血管化心肌组织的收缩频率和电生理功能。
由以上所述方法制备获得的三维血管化心肌组织。
以上所述三维血管化心肌组织在制备治疗心脏疾病的药物组合物中的应用。
以上所述三维血管化心肌组织在制备心脏移植供体中的应用。
有益效果：(1)所述方法结合细胞分化自组装功能和3D生物打印技术，无需在制备过程中手动调整位置，提高了试验效率和结果的稳定性；(2)所述方法制备的心肌组织不仅可以用于体外疾病研究模型，还可作为心肌梗死等疾病的损伤修复。
附图说明
图1是分化和鉴定人多能干细胞来源的早期血管细胞结果图。其中，图(A)显示了由H9人多能干细胞通过拟胚体(embryoid body,EB)诱导方法分化获得早期血管细胞的过程，比例尺为200μm。(B)显示了EB通过Collagen I-Matrigel水凝胶包埋后构建获得的血管网和血管类器官，比例尺为100μm。(C)免疫荧光染色检测血管网和血管类器官显示其中具有典型的以CD31为标志的血管内皮细胞网络结构，比例尺分别为100μm和200μm。(D)对EB分化各阶段的细胞进行基因表达检测显示从EB分化的第5天开始表达早期血管细胞特异的CD34和KDR基因，并且可以进一步分化获得以CD31和CD144为标志的血管内皮细胞。
图2是三维共培养及分化后构建血管化心肌组织结果图。其中，(A)免疫荧光检测结果显示H9分化的心肌细胞具有高比例表达的心肌细胞特异心肌肌钙蛋白T(cTnT)，比例尺为50μm。(B)免疫荧光检测心肌细胞肌丝结构蛋白α辅肌动蛋白(α-actinin)显示分化的心肌细胞具有比较完善的肌丝结构，比例尺为20μm。(C)单细胞的人EVC与心肌细胞在非粘附状态下聚集成细胞球进行共培养并经Collagen I-Matrigel水凝胶包埋后显示典型的血管出芽现象，比例尺为200μm。(D)通过对共培养细胞球中心肌细胞cTnT和血管内皮细胞CD31的免疫荧光染色检测显示，与未经Collagen I-Matrigel水凝胶包埋的共培养细胞球相比，包埋后的细胞球表现典型的以CD31为标志的血管内皮细胞出芽结构，比例尺分别为100μm和200μm。(E)细胞球血管内皮细胞出芽结构的定量统计与比较，分别显示了血管交叉点数量、血管密度和总出芽长度在经Collagen I-Matrigel水凝胶包埋后均有显著提升。(F)构建获得的血管化心脏类器官的典型形态及其中心肌细胞和血管内皮细胞的免疫荧光检测结果，比例尺为200μm。
图3是利用人早期血管细胞球的原位分化和3D生物打印自组装构建具有血管网的功能心肌组织。其中，(A)显示了3D生物打印机及其温控打印头和对应生物墨水材料。(B)显示了利用3D生物打印方法将新生大鼠心肌细胞和EVC细胞球混合进行共培养后构建血管化心肌组织的流程图。(C)免疫荧光染色显示3D打印的心肌组织中，EVC细胞球分化并出芽形成三维血管化结构，比例尺为200μm。(D)免疫荧光染色显示培养3周的3D打印心肌组织中，EVC细胞球分化并自组装形成三维血管网结构，比例尺分别为100μm和200μm。(E)对心肌组织中α-actinin的免疫荧光染色显示培养3周的3D打印心肌组织中心肌细胞具有整齐的肌丝结构，比例尺分别为100μm和50μm。(F)对培养3周的3D打印心肌组织进行钙成像检测显示心肌细胞与EVC细胞球混合共培养后构建的血管化心肌组织表现正常的搏动频率。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改和替换，均属于本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1
三维血管化心肌组织的制备方法，所述方法包括以下步骤：
(1)将扩增培养后的人多能干细胞经Accutase消化成单细胞并以40万/孔的密度铺板到低粘附6孔板中，此时培养液中添加10μM Y-27632以抑制细胞凋亡；
(2)待干细胞聚集成球后更换细胞培养液为RPMI/B27 minus insulin并添加12μMCHIR99021和30ng/mL BMP4以诱导中胚层特化；
(3)继续培养3天后更换细胞培养液为EGM2并添加100ng/mL VEGF-A以诱导血管谱系细胞分化；
(4)将步骤(3)诱导分化2天后的细胞球即早期血管细胞用Accutase消化成单细胞并与准备好的心肌细胞以3:7的比例混合，培养液为RPMI/B27 with insulin:EGM-2(1:1)，随后用hanging drop方法将细胞聚集成球(每个细胞球含1万细胞)，并以悬浮状态继续培养2天；
(5)将步骤(4)获得的细胞球包埋到Collagen I-Matrigel水凝胶中并以RPMI/B27with insulin:EGM-2(1:1)培养液培养，同时添加100ng/mL VEGF-A，100ng/mL FGF-2和10μM SB43152以诱导血管谱系细胞分化和内皮细胞出芽；
(6)将水凝胶中跳动和出芽的细胞球以解剖方法解离并继续培养在低粘附6孔板中以诱导血管网的自组装和血管化心脏类器官的成熟，每3天更换RPMI/B27 withinsulin:EGM-2(1:1)培养液；
(7)实验过程中以免疫荧光染色方法检测步骤(5)获得的水凝胶中心肌细胞和血管内皮细胞的生长状态和步骤(6)获得的心脏类器官中心肌细胞和血管内皮细胞组成的三维血管化心肌组织结构。
本实施例成功将人多能干细胞三维分化获得了血管网和血管类器官构建的种子细胞，即人早期血管细胞，并证明含有细胞外基质成分的水凝胶对早期血管细胞分化和自组装形成三维血管网具有重要作用(图1)。另外，本实施例利用早期血管细胞和心肌细胞在水凝胶中共培养同时诱导早期血管细胞分化自组装的策略构建具有微血管网络的心肌组织，已成功构建了具有微血管网结构的微小心肌组织并参考血管类器官构建方法成功获得了具有三维微血管网结构的心脏类器官(图2)。
实施例2
三维血管化大尺寸功能心肌组织构建方法，所述方法利用实施例1中步骤(1)-(3)构建的细胞球即早期血管细胞球进行以下步骤实验：
(1)制备含有Fibrin(纤维蛋白)、Gelatin(明胶)和Hyaluronic acid(透明质酸)的水凝胶生物墨水，将实施例1步骤(3)诱导分化2天后的细胞球即早期血管细胞球与准备好的新生大鼠心肌细胞以500细胞球/107心肌细胞/mL的比例混合到生物墨水中；
(2)利用BIO-X 3D生物打印机进行有序混合打印以构建包含早期血管细胞球的大尺寸心肌组织；
(3)以添加100ng/mL VEGF，50ng/mL bFGF，10μM SB431542，10％FBS，1％L-glutamine，10mg/mL aprotinin，和1％penicillin/streptomycin的Claycomb/EGM2(1:1)混合培养液进行打印心肌组织的培养，以诱导早期血管细胞球的原位分化并自组装形成三维血管网络；
(4)3周后用免疫荧光染色检测心肌组织中的血管网结构、肌丝结构，并用钙成像方法检测血管化心肌组织的收缩频率和电生理功能。
为了实现在大尺寸功能心肌组织中建立血管网结构的目的，本实施例应用3D生物打印策略将新生大鼠心肌细胞和人多能干细胞来源的早期血管细胞混合共培养，验证早期血管细胞在功能心肌组织中自组装形成血管网的能力。目前，我们已经成功应用早期血管细胞球的分化自组装功能在大尺寸心肌组织中构建了血管网结构，并且证明与早期血管细胞球的共培养不会影响成熟心肌组织的功能(图3)。
实施例3
三维共培养用水凝胶材料的制备，所述水凝胶材料包括实施例1中Collagen I-Matrigel水凝胶以及实施例2中含有Fibrin(纤维蛋白)、Gelatin(明胶)和Hyaluronicacid(透明质酸)的水凝胶生物墨水。
具体的，在实施例1中，所用水凝胶材料及制备步骤包括：
(1)将Collagen I溶解于含有NaOH和PBS的水溶液中；
(2)加入1/3体积Matrigel制备Collagen I-Matrigel水凝胶；
(3)将早期血管细胞球或者通过hanging drop方法制备的心肌细胞与早期血管细胞混合细胞球包埋到Collagen I-Matrigel水凝胶中，铺板后培养在37℃细胞培养箱中使水凝胶凝固并进行后续细胞培养操作。
在实施例2中，3D生物打印所用水凝胶生物墨水及制备步骤包括：
(1)通过将20mg/mL纤维蛋白原，30mg/mL明胶，20mg/mL抑肽酶和3mg/mL透明质酸溶解在含有1％青霉素/链霉素的DMEM培养基中来制备基于纤维蛋白的水凝胶；
(2)通过将10％明胶溶解在含有1％青霉素/链霉素的DMEM培养基中来制备牺牲水凝胶；(3)将所有物质在37℃的温控振荡器中轻轻溶解，并通过0.2μm过滤器过滤灭菌；
(4)通过将基于纤维蛋白的水凝胶与新生大鼠心肌细胞以107个细胞/mL的密度混合来制备包含心肌细胞的水凝胶；
(5)为了制造血管化的大尺寸心肌组织，将早期血管细胞以单细胞或者细胞球的形式分别以2×107细胞/mL或500细胞球/mL的比例添加到包含心肌细胞的水凝胶中，将生物墨水加入3D打印喷头并进行后续3D生物打印和细胞培养操作。

Claims (9)

1.三维血管化心肌组织的制备方法，其特征在于，所述方法包括将人多能干细胞来源的早期血管细胞和心肌细胞进行三维共培养，培养中诱导分化及分化后的继续培养成熟；其中，三维共培养过程是人多能干细胞来源的早期血管细胞和心肌细胞在非粘附状态下聚集成细胞球进行共培养或以3D生物打印方法混合共培养。
2.根据权利要求1所述的三维血管化心肌组织的制备方法，其特征在于，三维共培养过程中的支持条件为水凝胶材料。
3.根据权利要求2所述的三维血管化心肌组织的制备方法，其特征在于，所述水凝胶材料为Matrigel、Collagen I、Fibrin、Gelatin、Hyaluronic acid中的至少一种。
4.根据权利要求1所述的三维血管化心肌组织的制备方法，其特征在于，培养中诱导分化是将人多能干细胞来源的早期血管细胞诱导分化为血管内皮细胞和血管周细胞中的至少一种。
5.根据权利要求1所述的三维血管化心肌组织的制备方法，其特征在于，所述分化后的继续培养成熟采用以下方法之一：
其一、采用解剖方法将三维生长的血管化心肌组织从水凝胶中解离并继续在非粘附条件下培养成熟；
其二、采用3D生物打印技术构建的支架悬浮培养三维生长的血管化心肌组织并继续培养成熟。
6.根据权利要求1所述的三维血管化心肌组织的制备方法，其特征在于，所述方法的具体步骤包括：
(1)将扩增培养后的人多能干细胞经Accutase消化成单细胞并铺板到低粘附培养板中，此时培养液中添加Y-27632以抑制细胞凋亡；
(2)待干细胞聚集成球后更换细胞培养液为RPMI/B27 minus insulin并添加CHIR99021和BMP4以诱导中胚层特化；
(3)继续培养3天后更换细胞培养液为EGM2并添加VEGF-A以诱导血管谱系细胞分化；
(4)将步骤(3)诱导分化2天后的细胞球用Accutase消化成单细胞并与人多能干细胞来源的心肌细胞以3:7的比例混合，培养液为RPMI/B27 with insulin:EGM-2，随后用hangingdrop方法将细胞聚集成球，并以悬浮状态继续培养2天；
(5)将步骤(4)获得的细胞球包埋到水凝胶材料中并以RPMI/B27 with insulin:EGM-2培养液培养，同时添加VEGF-A、FGF-2和SB43152以诱导血管谱系细胞分化和内皮细胞出芽；
(6)将水凝胶中跳动和出芽的细胞球以解剖方法解离并继续培养在低粘附培养板中以诱导血管网的自组装和血管化心脏类器官的成熟，每3天更换RPMI/B27 with insulin:EGM-2培养液；
(7)实验过程中以免疫荧光染色方法检测步骤(5)获得的水凝胶中心肌细胞和血管内皮细胞的生长状态，以及步骤(6)获得的心脏类器官中心肌细胞和血管内皮细胞组成的三维血管化心肌组织结构；
(8)制备含有水凝胶材料的生物墨水，将步骤(3)诱导分化2天后的细胞球与新生大鼠心肌细胞混合到生物墨水中，并采用3D生物打印机进行混合打印以构建血管化的大尺寸功能心肌组织；
(9)以添加VEGF、bFGF、SB431542、FBS、L-glutamine、aprotinin和penicillin/streptomycin的Claycomb/EGM2混合培养液进行步骤(8)打印心肌组织的培养，3周后用免疫荧光染色检测心肌组织中的血管网结构、肌丝结构，并用钙成像方法检测血管化心肌组织的收缩频率和电生理功能。
7.由权利要求1-6任一所述方法制备获得的三维血管化心肌组织。
8.权利要求7所述三维血管化心肌组织在制备治疗心脏疾病的药物组合物中的应用。
9.权利要求7所述三维血管化心肌组织在制备心脏移植供体中的应用。
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