从鱼类垂体制备优质原代细胞培养
Summary
在这里, 我们描述了一个协议, 以准备和维护主要的垂体细胞培养从鳉 (Oryzias latipes)。该协议中的优化条件是通过模拟鱼类的生理条件来考虑温度、渗透和 pH 等重要参数, 从而使生理上有更有意义的结果。
Abstract
主细胞培养是科学家常用的一种强有力的工具, 用于研究受控环境中孤立细胞的细胞性质和机制。尽管哺乳动物和鱼类之间的生理学存在巨大差异, 但鱼类的初级细胞培养协议往往是以哺乳动物的文化条件为基础的, 通常只进行少量的修饰。环境的差异不仅影响体温, 而且对血清中渗透、ph 值和 ph 值的缓冲能力等指标也有很显著的作用。由于细胞培养培养基和类似的工作解决方案旨在模仿细胞外液和/或血清的特性, 对其进行适应, 关键是这些参数是专门针对所讨论的动物进行调整的。
当前协议描述优化的鳉 (Oryzias latipes) 的主要区域性条件。该议定书提供了有关如何隔离和维持健康的分离垂体细胞超过一周的详细步骤, 包括以下步骤: 1. 渗透压对鳉血浆中发现的值的调节, 2. 调整孵化温度到正常的鳉温度 (这里在水族馆设施), 和3。将 pH 值和碳酸氢盐缓冲量调整到与生活在类似温度下的其他鱼类相比的价值。使用描述的协议的结果促进了生理上有意义的结果为鳉并且可以作为参考指南由科学家做主要细胞文化从其他非哺乳动物种类。
Introduction
细胞培养是分子生物学研究的主要工具之一, 它为回答从正常细胞生理学到药物筛选和致癌1等不同生物学问题提供了一个优秀的模型系统。从动物组织直接分离的主要细胞使用酶法和/或机械方法, 通常被认为比细胞系更具生物学意义, 因为生物反应可能更接近体内的情况。为了模拟细胞适应的特性, 获得生理上有意义的结果, 应针对每个物种和细胞类型对准备初级细胞培养的协议进行优化。
许多协议描述哺乳动物细胞系统的文化条件, 而类似的协议描述鱼类细胞的主要培养条件是相当稀少的比较。细胞容易受到温度、pH 值和渗透压的快速变化, 在离解过程中尤其脆弱。用于哺乳动物细胞培养的商业盐溶液和培养基不适合硬骨鱼类鱼, 特别是在 pH 缓冲系统和渗透压方面。因此, 重要的是要测量和调整这些参数的生理相关水平的解决方案的兴趣品种。
主要的垂体培养由几种硬骨鱼类鱼类组成, 包括鲤鱼 (鲤鱼)2,3, 草鱼 (草鱼艾达拉)4, 金鱼 (鲫鱼)5, 虹鳟 (虹鳟鱼虹鳟鱼)6, 欧洲鳗鲡 (安圭拉)7, 罗非鱼 (罗非鱼)8, 斑马鱼 (斑马斑马)9, 和大西洋鳕鱼 (Gadus morhua)10。除了将孵化温度调整到感兴趣的物种之外, 这些协议中的一些还在哺乳动物样的条件下孵化出细胞, 这种情况对感兴趣的物种可能是不理想的, pH 值从7.2 到7.5 在湿润的大气层中。包含 3-5% CO2。此外, 目前尚不清楚, 在不同的溶液中, 用于制备几种主要细胞培养液的渗透压是否调整和稳定。
目前的议定书是根据以前与大西洋鳕鱼10的初级文化有关的工作, 包括孵化温度、渗透力、ph 值和 ph 值缓冲系统的调整, 包括二氧化碳的分压 (2), 以鳉的生理学 (latipes)。鳉是一种小 (3–4厘米) 淡水鱼, 原产于东亚。这些天, 它被作为一个模型物种在世界各地的许多研究实验室, 因为它是相对容易繁殖和高度抵抗许多常见的鱼类疾病11。使用鳉作为模型有几个优点, 包括温度耐受度从4–40°c11, 短世代时间, 透明胚胎, 性别确定基因12, 和序列化基因组13, 以及许多其他可利用的遗传资源。
本协议中的主要养殖条件被优化, 以符合 medakas 在鱼设施中保存的26摄氏度的温度。此外, 渗透压从生活在盐水中的大西洋鳕鱼的 320 mOsm/千克减少到 290 mOsm/千克, 鳉生活在淡水中, 并符合鳉等离子14的正常渗透压。相比之下, 哺乳动物血浆的典型渗透压在 275–295 mOsm15的范围内。鱼类生活在各种温度下, 鳃与水直接接触, 使鱼的血液和胞外液的 pH 值和缓冲能力与哺乳动物不同。哺乳动物的文化媒介通常包括缓冲系统, 导致 pH 值约 7.4, 当媒体被平衡到标准大气 5% CO2在湿润空气在37摄氏度。ph 值是温度依赖性的, 中性酸碱度 (水中) 的价值随温度的降低而增加16。典型的硬骨鱼类鱼血浆 pH 值从7.7 到 7.917不等。该协议的优化包括减少从 ph 值为7.85 的 cod 保持在12°c, ph 为7.75 的鳉保持在26°c 通过增加 CO2从0.5% 到1%。
此外, 在鱼类和哺乳动物中, 碳酸氢盐的缓冲能力是相当不同的。CO2容易地被交换在鳃在鱼和2在水中是仅一小部分的2在肺18。改变温度或2会改变介质的 pH 值和缓冲度。因此, 对于孵化哺乳动物细胞的 pH 值和2的不均, 都不是最佳的鱼细胞, 因此, 培养基应优化与含有生理相关的鱼类价值的缓冲系统和特殊种类的兴趣。该协议描述了如何从鳉垂体中制备主细胞培养, 包括孵化温度、渗透、ph 和 ph 缓冲系统的调整, 以及在准备主细胞时需要考虑的其他重要参数。非哺乳动物物种的文化。
Protocol
Representative Results
Discussion
体外细胞培养系统为研究人员提供了强有力的工具, 如果使用正确的方式1, 就可以回答过多的不同的生物学问题。重要的是要记住, 失去了与邻近细胞的连接的分离细胞可能已经获得了不同的功能性质比他们原来在体内。为了避免有误解从体外实验获得的结果的风险, 重要的是考虑调整原细胞培养过程的物种和细胞类型的兴趣。即使是对标准的文化程序和解决?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这一项目由挪威生命科学大学和挪威研究理事会资助, 拨款243811和 244461 (水产养殖项目)。我们感谢挪威生命科学大学的卢尔德卡伦谭维持鱼类设施。
Materials
| Dulbecco’s Phosphate Buffered Saline (dPBS), without calcium chloride and magnesium chloride | Sigma (Merck, Damstadt, Germany) | D8537 | Adjust solution to pH 7.75 with 1M NaOH and 290 mOsm with mannitol. |
| L-15 medium (Leibovitz), witout L-glutamine | Sigma (Merck, Damstadt, Germany) | L5520 | Supplement 500 ml culture medium with 10mM NaHCO3, 4.5 mM glucose, 2 mM Glutamax. Adjust solution to 290 mOsm with mannitol and filter solution through a 0.2 µm PES sterile filter, before adding 2.5 ml Penicillin-Streptomycin solution (see below for details). |
| NaOH | Sigma (Merck, Damstadt, Germany) | S5881 | Add drops of 1M solution to increase pH of dPBS and culture medium to 7.75. |
| NaHCO3 | Sigma (Merck, Damstadt, Germany) | S5761 | 10 mM NaHCO3 equals 420 mg per 500 ml culture medium. |
| D-mannitol | Sigma (Merck, Damstadt, Germany) | 63565 | Use to increase osmolality of dPBS and culture medium. Calculate correct amount needed to reach an osmolality of 290 mOsm. |
| D-glucose | Sigma (Merck, Damstadt, Germany) | G5400 | 4.5 mM D-glucose equals 405 mg per 500 ml culture medium. |
| GlutaMAX Supplement | Gibco (Life Technologies, Paisley, UK) | 35050-061 | Alternative to L-glutamine, with increased stability. 2 mM Glutamax equals 5 ml of 100X stock in 500 ml culture medium. |
| Penicillin-Streptomycin | Sigma (Merck, Damstadt, Germany) | P0781 | Stock solution 10,000 units penicillin and 10 mg streptomycin per mL. Use 2.5 ml of stock solution in 500 ml L-15 medium (equivalent of 50 U/ml Penicillin and 50 µg/ml Streptomycin). |
| Trypsin type II-S | Sigma (Merck, Damstadt, Germany) | T7409 | Prepare 1 mg/ml in dPBS solution. |
| Trypsin inhibitor type I-S | Sigma (Merck, Damstadt, Germany) | T6522 | Prepare 1 mg/ml in dPBS solution, supplement with 2 µg/ml Dnase I (see details below). |
| Dnase I | Sigma (Merck, Damstadt, Germany) | D5025 | Use in trypsin inhibitor solution (see above). |
| 0.2 µm Polyethersulfone (PES) sterile filter system | Corning Inc. (Corning, NY) | 431097 | Use for sterile filtration of dPBS and L-15 medium after adjustments. |
| 35 mm cell culture dish with glass bottom, poly d-lysine coated | MatTek Corporation (Ashland, MA) | P35GC-1.5-10-C | Can also be replaced by plastic dish, depending on downstream application. |
| Dumont #5 fine forceps | Fine Science Tools (CA) | 11254-20 | Straigt tip |
| Dumont #5/45 fine forceps | Fine Science Tools (CA) | 11253-25 | Angled 45° tip |
| Stereo microscope SZ61 | Olympus Corp. (Tokyo, Japan) | Use for dissection of pituitaries. | |
| Alegra X-22R Centrufuge | Beckman Coulter Inc. (Brea, CA) | With cooling option (similar to current model XR-30). | |
| Water bath | Techne (Staffordshire, UK) | Any water bath with the possibility of adjusting temperature will do. | |
| Pasteur glass pipettes | VWR (NY) | 612-1701 | Outer diameter 1.6 mm, fire polish and autoclave before use. |
| Galaxy MiniStar table centrifuge | VWR (NY) | 521-2844 | Any small table centrigue will do. |
| Fine needles / insect pins | Fine Science Tools (CA) | 26001-40 | Diameter 0.03 mm. Other fine needles can be used instead. |
| Wax plate | Custom made by adding melted paraffin wax in large petri dish. |
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