开发一种细胞共培养模型以模拟心脏缺血/体外再灌注
Summary
空间距离是评估分离内皮和心肌细胞层共培养模型中缺氧/再氧合损伤的关键参数，首次表明优化共培养空间环境对于为测试内皮细胞在心肌细胞保护中的作用提供有利的 体外 模型是必要的。
Abstract
缺血性心脏病是全世界死亡和残疾的主要原因。再灌注可导致缺血以外的其他损伤。内皮细胞（EC）可以通过细胞间相互作用保护心肌细胞（CM）免受再灌注损伤。共培养可以帮助研究细胞 – 细胞相互作用的作用。混合共培养是最简单的方法，但由于分离处理和单细胞类型的下游分析不可行，因此受到限制。为了研究ECs是否可以以剂量依赖性方式衰减CM细胞损伤，以及是否可以通过改变两个细胞系之间的接触距离来进一步优化这种保护，我们使用小鼠原发冠状动脉内皮细胞和成年小鼠心肌细胞来测试三种类型的细胞培养插入物，其细胞间层距离在0.5， 分别为 1.0 毫米和 2.0 毫米。在仅CM中，通过乳酸脱氢酶（LDH）释放评估的细胞损伤在缺氧期间和再氧合后显着增加，当距离为2.0 mm时，与0.5和1.0 mm相比。当EC和CM几乎直接接触（0.5 mm）时，缺氧后CM的再氧合损伤只有轻度衰减。当空间距离为1.0 mm时，这种衰减显着增加，当距离为2.0 mm时，EC在缺氧和缺氧/再氧合期间都减轻了CM损伤，表明ECs需要足够的培养距离才能与CM串扰，以便分泌的信号分子可以循环并充分刺激保护途径。我们的研究结果首次表明，优化EC/CM共培养空间环境对于测试EC在模拟缺血/再灌注损伤CM保护中的作用提供了有利的 体外 模型。本报告的目标是为调查人员提供一种循序渐进的方法，以利用这一重要模型来发挥自己的优势。
Introduction
缺血性心脏病是全世界死亡和残疾的主要原因1，2。然而，再灌注的治疗过程本身可引起心肌细胞死亡，称为心肌缺血/再灌注（IR）损伤，对此仍然没有有效的补救措施3。内皮细胞（EC）已被建议通过分泌旁分泌以及细胞间相互作用来保护心肌细胞（CMs） 4。
细胞共培养模型已被广泛用于研究自分泌和/或旁分泌细胞 – 细胞相互作用对细胞功能和分化的作用。在共培养模型中，混合共培养是最简单的，其中两种不同类型的细胞以所需的细胞比5在单个培养室内直接接触。然而，鉴于混合群体，细胞类型之间的单独处理和单细胞类型的下游分析并不容易实现。
先前的研究表明，缺氧和缺血性损伤对细胞膜的完整性造成显着损害，通过乳酸脱氢酶（LDH）的释放来测量。这种损伤在复氧时加重，类似于再灌注损伤6，7，8。当前方案的目标是测试假设，即EC的存在可以剂量依赖性地衰减由缺氧和再氧合（HR）引起的CM的细胞膜泄漏，并且可以通过改变两个细胞系之间的接触距离来优化EC的保护作用。因此，我们采用了三种类型的细胞培养插入物和小鼠原发性冠状动脉内皮细胞和成年小鼠心肌细胞。这些由康宁、默克密理博和格瑞纳Bio-One品牌设计的插入物使我们能够创建三种不同的细胞培养串扰条件，细胞间距离分别为0.5、1.0和2.0 mm。在每种情况下，每个刀片镀100，000个EC。
此外，为了确定共培养中ECs的密度是否有助于该模型中的HR损伤衰减，我们研究了EC浓度与CM释放LDH之间的剂量 – 反应关系。
本报告为调查人员提供了一种循序渐进的方法，使他们能够利用这一重要模型。
Protocol
1. 实验准备/电镀 根据制造商的说明维护CM和EC。 当两个细胞系从供应商处到达时，解冻它们。用新鲜培养基洗涤后，将板放入T25烧瓶中。建议从购买细胞的同一供应商处购买每种细胞培养基。第二天，用培养基刷新细胞，并在汇合时使用。 将细胞培养箱保持在37°C，21%O2，5%CO2，74%N2 并保持加湿。注意：从C57BL / 6小鼠的冠状动脉中分离…
Representative Results
本实验中使用的所有三种类型的插入物（A，B，C）具有相同的0.4μm孔径。它们之间的唯一区别是插入物到碱基的高度，这使得两个共培养的细胞层之间的距离分别为0.5，1.0和2.0 mm（图3），并且它们来自不同的供应商（有关详细信息，请参阅 材料表）。 为了建立一种 体外 共培养模型，其中两个细胞系的单独层经历HR以模拟IR损伤，我?…
Discussion
协议中的关键步骤
细胞共培养模型已被用于研究心脏保护的细胞机制。因此，如何创建两个独立的层，它们之间有有意义的距离，对于开发合适的共培养模型至关重要。研究模拟IR（即HR损伤）的一个挑战是，不仅缺血（缺氧）本身，而且再灌注（再氧合）也会加重细胞功能障碍。因此，一个现实的模型需要反映这些特征，例如，证明缺氧后再氧合的损伤充分增加，而不是单独缺氧…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了美国退伍军人事务部生物医学实验室研发服务（I01 BX003482）和M.L.R.机构资金的支持。
Materials
| Adult Mouse Cardiomyocytes (CMs) | Celprogen Inc | 11041-14 | Isolated from adult C57BL/6J mouse cardiac tissue |
| Automated Cell Counter Countess II | Invitrogen | A27977 | Cell counting for calculating cell numbers |
| Bio-Safety Cabinet | Nuaire | NU425400 | Cell culture sterile hood |
| Cell Culture Freezing Medium | Cell Biologics Inc | 6916 | Used for cell freezing for long term cell line storage |
| Cell Culture Incubator | Nuaire | Nu-5500 | To provide normal cell living condition (21%O2, 5%CO2, 74%N2, 37°C, humidified) |
| Cell Culture Incubator Gas Tank | A-L Compressed Gases | UN1013 | Gas needed for cell culture incubator |
| Cell Culture Inserts A (0.5 mm) | Corning Inc | 353095 | Used for EC-CM co-culture |
| Cell Culture Inserts B (1.0 mm) | Millicell Millipore | PIHP01250 | Used for EC-CM co-culture |
| Cell Culture Inserts C (2.0 mm) | Greiner Bio-One | 662640 | Used for EC-CM co-culture |
| Centrifuge | Anstel Enterprises Inc | 4235 | For cell culture plating and passaging |
| CMs Cell Culture Flasks T25 | Celprogen Inc | E11041-14 | Used for CMs regular culture, coated by manufacturer |
| CMs Cell Culture Medium Complete | Celprogen Inc | M11041-14S | CMs culture complete medium |
| CMs Cell Culture Medium Complete Phenol free | Celprogen Inc | M11041-14PN | CMs culture medium without phenol red used during LDH measurement |
| CMs Cell Culture Plates 96 well | Celprogen Inc | E11041-14-96well | Used for experiments of LDH measurement, coated by manufacturer |
| CMs Hypoxia Cell Culture Medium | Celprogen Inc | M11041-14GFPN | CMs cell culture under hypoxic condition (glucose- and serum-free) |
| Countess cell counting chamber slides | Invitrogen | C10283 | Counting slides used for cell counter |
| Cyquant LDH Cytotoxicity Kit | Thermo Scientific | C20301 | LDH measurement kit |
| ECs Cell Culture Flasks T25 | Fisher Scientific | FB012935 | Used for ECs regular culture |
| ECs Cell Culture Medium Complete | Cell Biologics Inc | M1168 | ECs culture complete medium |
| ECs Cell Culture Medium Complete Phenol free | Cell Biologics Inc | M1168PF | ECs culture medium without phenol red used during LDH measurement |
| ECs Cell Culture Plates 96 well | Fisher Scientific (Costar) | 3370 | Used for experiments of LDH measurement |
| ECs Culture Gelatin-Based Coating Solution | Cell Biologics Inc | 6950 | Used for coating flasks and plates for ECs |
| ECs Hypoxia Cell Culture Medium | Cell Biologics Inc | GPF1168 | ECs cell culture under hypoxic condition (glucose- and serum-free) |
| Fetal Bovine Serum (FBS) | Fisher Scientific | MT35011CV | FBS-HI USDA-approved for cell culture and maintenance |
| Hypoxia Chamber | StemCell Technologies | 27310 | To create a hypoxic condition with 0.01%O2 environment |
| Hypoxia Chamber Flow Meter | StemCell Technologies | 27311 | To connect with hypoxic gas tank for a consistent gas flow speed |
| Hypoxic Gas Tank (0.01%O2 Cylinder) | A-L Compressed Gases | UN1956 | Used to flush hypoxic medium and chamber (0.01%O2/5%CO2/94.99N2) |
| Microscope | Nikon | TMS | To observe cell condition |
| Mouse Primary Coronary Artery Endothelial Cells (ECs) | Cell Biologics Inc | C57-6093 | Isolated from coronary artery of C57BL/6 mice |
| NUNC 15ML CONICL Tubes | Fisher Scientific | 12565269 | For cell culture process, experiments, solution preparation etc. |
| NUNC 50ML CONICL Tubes | Fisher Scientific | 12565271 | For cell culture process, experiments, solution preparation etc. |
| Phosphate Buffered Saline (PBS) | Sigma-Aldrich | D8662 | Used for cell washing during culture or experiments |
| Plate Reader | BioTek Instrument | 11120533 | Colorimetric or fluorometric plate reading |
| Reaction 96 Well Palte (clear no lid) | Fisher Scientific | 12565226 | Used for LDH measurement plate reading |
| Trypsin/EDTA for CMs | Celprogen Inc | T1509-014 | 1 x sterile filtered and tissue culture tested |
| Trypsin/EDTA for ECs | Cell Biologics Inc | 6914/0619 | 0.25%, cell cuture-tested |
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Cite This Article
Li, Z., Hampton, M. J. W., Barajas, M. B., Riess, M. L. Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro. J. Vis. Exp. (176), e62913, doi:10.3791/62913 (2021).