细胞培养：你养的到底是谁
一个刚进组的人，第一次被交给一管细胞时，最该问的不是“怎么养”，而是“这是谁、从哪来、第几代”。本篇把两件常被分开讲的事放在一起：怎么从新生鼠脑里把原代小胶质分出来（精译 Bio-protocol 2025 的分离流程），以及细胞系的身份如何被污染、又如何在文献里继续流通（精译 PLoS ONE 2017 的引言与“Practical measures”）。正文中红线处可悬停或点击，看原文与译注；共 12 条。
细胞培养是生物医学里门槛最低的实验：买一管细胞，化开，放进培养箱，每隔几天传一次代。也正因为门槛低，它最容易藏住一个问题——你养的到底是谁。
这个问题有三层：类型（它是不是你以为的那种细胞）、身份（它是不是你以为的那一株）、状态（它现在是静息还是激活、第几代、有没有污染）。前两层是本页的主线：一篇讲怎么从新生鼠脑里把小胶质原代分出来，一篇讲细胞系的身份如何被污染、又如何在文献里阴魂不散。
本篇精译自 Bio-protocol 2025 的方法学文章《Revisiting Primary Microglia Isolation Protocol: An Improved Method for Microglia Extraction》，以及 PLoS ONE 2017 的《The ghosts of HeLa: How cell line misidentification contaminates the scientific literature》的引言与“Practical measures”两节；原文见文末附录。
一、原代与细胞系：两种“细胞”，两种诚实
原代细胞（primary cells）是直接取自动物或人组织、经机械或酶解分离、没有经过永生化处理的细胞。它们更接近生理状态：受体、离子通道、转录程序、代谢方式都还在原来的位置上。代价是批次差异：不同个体、不同日龄、不同消化时间、不同血清批号，做出来的细胞状态就不一样。原代细胞通常也只能传有限几代，越传越不像原来的组织。
细胞系（cell line）则是原代细胞经过永生化（病毒基因、端粒酶、自发转化等）后获得的、可以长期传代的群体。它们可重复、成本低、能做大通量筛选。代价是漂移：基因型会变，表型会变，培养条件会筛选出不同的亚群。更极端的情况是身份被替换——你以为在养 A，其实在养 B。
原文对小鼠 BV2 小胶质细胞系的评价很直接：the immortalization of BV2 cells may lead to aberrant gene expression, dysregulated signaling pathways, and altered metabolic states（BV2 的永生化可能导致基因表达异常、信号通路失调、代谢状态改变）；与原生小胶质相比，BV2 缺乏关键的稳态特征与免疫功能，用它模拟帕金森病里小胶质的生理反应并不合适。这不是说 BV2 没用，而是说：用它之前要知道它是什么、不是什么。
所以原代与细胞系的区别，不是“好”与“坏”，而是两种不同的诚实：原代诚实地承认“每次都不一样”，细胞系诚实地承认“我可能已经不是原来那个”。做实验的人要做的，是把这份诚实写进记录里。
| 维度 | 原代细胞 | 细胞系 |
|---|---|---|
| 来源 | 直接取自动物/人组织，酶解或机械分离 | 原代经永生化或转化，可长期传代 |
| 生理相关性 | 高：保留受体、通道与转录程序 | 低到中：随传代漂移，可能丢失关键表型 |
| 批次一致性 | 低：个体、日龄、消化、血清都影响 | 高（前提是身份正确、无污染） |
| 可传代性 | 有限（原代小胶质通常几代以内，经验值） | 近乎无限 |
| 主要风险 | 纯度不够、状态被激活、产量低 | 身份错误、支原体、基因型漂移 |
二、原代小胶质：从一只新生鼠到 1.6×106 个细胞
原代小胶质（microglia）是中枢神经系统的驻留免疫细胞，参与突触修剪、碎片清除与炎症反应。研究神经炎症、神经退行性疾病时，它是比细胞系更接近生理的模型。原文的定位很清楚：MACS 与 FACS 纯度高，但贵、回收率低；传统的混合胶质摇床法便宜，纯度却常常不够。这篇文章在摇床法上做了两处改动——解离与早期培养加 B27，后期加 M-CSF——把流程稳定在 9–10 天、每只新生鼠约 1.6×106 个细胞、纯度约 90%，并且全程不加抗生素。
先把材料摆齐。原文给的配方可以直接照抄：洗涤液是 49 mL DMEM/F12 加 1 mL B27（50×）；10% FBS 培养基是 45 mL DMEM/F12 加 5 mL 热灭活 FBS；20% FBS 培养基是 40 mL 加 10 mL。
| 试剂 / 材料 | 规格或浓度 | 在这套流程里的作用 |
|---|---|---|
| DMEM/F12（1:1） | 含 2 mM L-谷氨酰胺 | 基础培养基 |
| 胰酶-EDTA | 0.25% | 消化皮层与海马组织 |
| 胎牛血清（FBS） | 热灭活 | 终止消化；配 10% 与 20% 培养基 |
| B27 | 50×，无血清 | 解离与早期培养，抗氧化、维持静息形态 |
| M-CSF | 20 ng/mL（10–50 可调） | 促小胶质存活与增殖 |
| 多聚甲醛（PFA） | 4% | 固定细胞用于染色 |
| 细胞筛网 | 70 µm | 去除未消化组织块 |
| 动物 | C57BL/6J 或 SNCA A53T（#039167），P1–P2 | 取材 |
流程（原文的 Step-by-step，参数照抄）。
- 准备。器械高压灭菌；B27 避光、4°C 保存；培养箱设 37°C、5% CO2；备冰盒与冰冷 PBS。原代操作的时间大半花在准备上。
- 取材。取 P1–P2 新生鼠，颈椎脱臼，75% 乙醇表面消毒 5 min。在冰上无菌取全脑，放入冰冷 PBS 30 s 去血。显微镜下用无菌剪与眼科镊剥去脑膜（meninges）与表面血管，沿中线分左右两半球，取皮层（表面灰白薄层）与海马（内侧 C 形结构）。全程 ≤10 min。
- 消化。组织移入 1.5 mL 管，PBS 洗 1–2 次；加 1 mL 0.25% 胰酶（trypsin），37°C、3 min，轻吹打；加 1 mL 含 10% FBS 的 DMEM/F12 终止；过 70 µm 筛；300×g、5 min。
- 接种。3 mL 含 20% FBS 的 DMEM/F12 重悬，种入 T25 瓶，37°C、5% CO2 培养 24 h。
- 换液。次日吸去上清（碎片、死细胞、未贴壁细胞），PBS 洗 3 次，加 5 mL 含 20% FBS 的培养基，再养 5 天。
- 加 M-CSF。第 5 天，细胞已分层：星形胶质与成纤维细胞贴在底层，小胶质呈圆或椭圆、浮在上层。换液后加 M-CSF 20 ng/mL，维持到第 8 天；第 8 天换液。
- 摇床分离。第 9 天，封口，37°C、200 rpm、2 h。收集悬浮的小胶质，重铺到新的 T25 瓶。
- 收第二批。原瓶补 5 mL 含 20% FBS 的培养基，再养 5 天，同法摇床收第二批。
产量约 1.6×106 个细胞/只（皮层加海马），全程 9–10 天。如果第 9 天收不到足够的细胞，原文的处理办法是把 M-CSF 补到 10–50 ng/mL（troubleshooting 里建议 20 ng/mL），再养几天。
| 时间 | 做什么 | 看什么 |
|---|---|---|
| 第 0 天 | 取材、消化、接种 T25 | 组织是否消化完全、有无大块残留 |
| 第 1 天 | 去上清、洗 3 次、换 20% FBS 培养基 | 贴壁细胞密度、碎片多少 |
| 第 5 天 | 换液、加 M-CSF 20 ng/mL | 底层星形胶质是否铺满、上层小胶质是否圆亮 |
| 第 8 天 | 换液 | 小胶质数量是否够 |
| 第 9 天 | 200 rpm、2 h，收第一批 | 上清里悬浮细胞是否多、有无成团 |
| 第 14 天 | 原瓶补液后再摇，收第二批 | 产量是否值得再收 |
三、关键参数：为什么是 3 分钟、20% 血清与 200 rpm
原代流程最怕“照着做但不知道为什么”。下面每个参数都对应一个具体的失败模式；知道它在防什么，出问题时才知道先改哪里。
为什么用 P1–P2 新生鼠。出生后早期的小胶质仍处于增殖与成熟阶段，静息态比例高、贴壁行为稳定；成年脑组织髓鞘化、细胞外基质致密，消化更狠、产量更低，而且分离过程中容易被机械与缺血刺激激活。原文用 postnatal day 1–2，不是随手写的。
为什么全程 ≤10 min、全程冰上。缺血、缺氧、机械剪切、内源蛋白酶释放，都会让小胶质从分枝状（ramified）变成阿米巴状（amoeboid），也就是“被激活”。你在取材时多花五分钟，后面所有炎症指标的基线就抬高了五分钟的量。剥脑膜与血管要在显微镜下做，宁可慢一点、剥干净，也不要为了赶时间把成纤维细胞带进来。
为什么胰酶只 3 min。胰酶切的是膜蛋白。消化不足，组织块残留、产量低；消化过度，膜上的受体与表位被切掉——CX3CR1、P2RY12、CD11b 这些你后面要染的指标，可能还没上机就已经丢了。终止必须用含血清的培养基：血清里的蛋白酶抑制剂能真正把酶停住，单用 PBS 稀释只是把酶冲淡。原文还有一句提醒：不要延长消化时间。
为什么初期用 20% FBS。高血清促贴壁、促存活，帮助混合胶质铺成一层。但血清是这套流程里最大的变量：不同批次的生长因子、激素、内毒素水平不同，细胞长得就不一样。经验做法是：同一批实验锁定同一批血清，记录批号；换批号时先做一次小规模对比。
为什么加 B27。B27 是神经元培养的添加剂，含多种抗氧化剂与激素样成分（如维生素 E、皮质酮）。原文的解释是：它可以降低培养体系里的氧化应激与炎症信号，帮助小胶质维持静息的、分枝状的形态，避免过度激活。换句话说，B27 不是“营养”，是“压住激活”。
为什么第 5 天才加 M-CSF。M-CSF 是巨噬细胞集落刺激因子，受体是 CSF1R，小胶质的存活与增殖依赖这条信号。前两天是混合胶质贴壁、星形胶质铺层的阶段，过早加 M-CSF 会打乱这个分层；第 5 天加，正好在小胶质开始需要增殖支持的时候。
为什么是 200 rpm、2 h。混合胶质里，星形胶质贴得牢，小胶质只是半贴附在上层。摇床的剪切力把上层的小胶质“晃”下来，这叫差速贴壁（differential adherence）的一种变体。转速过高，星形胶质也被晃下来，纯度下降；转速过低或时间太短，收不到细胞，产量下降。200 rpm、2 h 是原文的参数；不同摇床的实际振幅不同，1–3 h 是常见的优化区间（经验值）。
为什么全程不加抗生素。原文明确不加青霉素/链霉素与抗真菌剂。抗生素会掩盖低水平的污染，让细胞带着支原体或细菌继续“看起来正常”；它也会改变细胞生理，干扰炎症与代谢读数；对支原体，多数抗生素根本无效，只会让检测更难。不加抗生素的代价是操作必须更干净——这也是对技术的训练。
| 这一步错了 | 你会看到什么 | 后果 |
|---|---|---|
| 脑膜/血管没剥净 | 瓶底出现旋涡状、长条形成纤维细胞 | 纯度下降，小胶质比例被稀释 |
| 消化过度或时间过长 | 碎片多、活率低、贴壁差 | 产量低，膜受体/表位丢失 |
| 血清换了批号 | 生长变慢、形态变扁、批次间差异大 | 结果不可比 |
| 摇床转速过高 | 收下的细胞里混着星形胶质 | 纯度低，GFAP 阳性 |
| 摇床时间不足 | 上清里细胞很少 | 产量低 |
| 加了抗生素 | 培养基不浑浊，但细胞状态越来越差 | 污染被掩盖，支原体阴性假象 |
四、纯度与功能验证：90% 是怎么算出来的
“分出来了”和“分对了”是两件事。原文用三步验证：形态、免疫荧光、流式，再加一步功能。这三步缺一不可，因为形态可以骗人（圆的小胶质和圆的死细胞在明场里很像），标记可以非特异（死细胞粘抗体），只有功能能说明这批细胞还活着、通路还通。
形态。静息的小胶质呈分枝状（ramified），有细长突起；激活后变成阿米巴状（amoeboid），圆而亮，突起回缩。原代培养里大多数细胞应该是分枝状或至少带短突起；如果一摇下来全是圆球、贴壁后也不伸突起，先怀疑消化过度或取材时被激活。
免疫荧光。原文的做法是：收细胞，用含 20% FBS 的 DMEM/F12 重悬，按 2×105 个/mL 种到 6-cm 皿或含玻片的 12 孔板，过夜贴壁；4% PFA、4°C 过夜固定；然后做 Iba1（小胶质标志物）与 GFAP（星形胶质标志物）双染。Iba1 阳性、GFAP 阴性的比例，就是纯度。计数时至少随机取 3–5 个视野、累计 200 个以上细胞，报告“阳性细胞数/总细胞数”和视野数——只写“纯度约 90%”而不给计数方法，别人无法判断这个 90% 是怎么来的。原文报的纯度就是约 90%。
流式。原文用 CD11b 做流式验证。CD11b 是髓系标志物，小胶质与巨噬细胞都表达；如果要区分脑内小胶质与浸润的单核/巨噬细胞，还要看 CD45 的亮度——小胶质通常是 CD45 低表达（CD45low）、CD11b 阳性，而外周巨噬细胞 CD45 高表达（经验补充，常见 panel 写法）。流式的好处是样本量大、能同时看活率；坏处是消化本身会切表位，所以流式结果要和免疫荧光对得上。
功能。原文用 LPS 1 µg/mL 刺激 24 h，然后看两件事：流式测 CD40 的表面上调，qPCR 测 IL-6、IL-1β、TNF-α 与 TGF-β 的转录变化。能对 LPS 起反应，说明细胞是活的、TLR4 通路是通的、培养过程没有把它们“养木”。这一步比纯度更重要：一批 95% 纯但完全不响应 LPS 的细胞，做炎症实验没有意义。
关于那 10% 的杂质，要说清楚：混合胶质培养里总会有星形胶质、成纤维细胞、少突胶质前体细胞残留。90% 的纯度对 bulk 实验（Western blot、qPCR、ELISA）通常够用；对单细胞测序、极微量蛋白组，10% 的“别人”可能主导信号，需要再纯化（MACS 分选 CD11b，或 FACS）。如果有人告诉你“原代小胶质 99% 纯”，先问他要流式图和门控策略。
| 验证手段 | 看什么 | 合格的样子 | 局限 |
|---|---|---|---|
| 明场形态 | 分枝状/阿米巴状比例 | 多数为分枝状或带短突起 | 死细胞、碎片也会是圆的 |
| 免疫荧光 | Iba1+/GFAP− 比例 | ≥90%（原文值），计数方法与视野数要报告 | 抗体特异性、固定/通透影响表位 |
| 流式 | CD11b+、CD45low | 主群 CD11b 阳性、CD45 低 | 消化切表位；死细胞非特异结合 |
| 功能 | LPS 后 CD40、IL-6、IL-1β、TNF-α、TGF-β | 刺激组显著上调（n=3，统计学检验） | 只证明“有反应”，不证明“是原代” |
五、细胞系的身份危机：HeLa 的幽灵
1951 年，Henrietta Lacks 的宫颈癌细胞在实验室里长成了第一株人类永生化细胞系。七十多年后，HeLa 仍然是细胞培养史上最著名的名字，但它的名声有一部分来自另一件事：它污染了无数别的细胞系，而且这些被污染的细胞系至今还在文献里流通。原文的标题叫“HeLa 的幽灵”（The ghosts of HeLa），这个比喻不是修辞——一株死了五十年的细胞，通过冻存管、细胞库和引用链，还在今天的论文里“活动”。
先说规模。原文估计，所有细胞系里被误认的比例在五分之一到三分之一之间；国际细胞系认证委员会（International Cell Line Authentication Committee, ICLAC）的误认清单里，仅“没有已知原种”的第一类就有 451 株；用 Web of Science 检索，基于误认细胞系发表的原始文献有 32,755 篇，这些文章保守估计被引用超过 50 万次。更刺眼的是时间分布：其中 57% 发表于 2000 年之后；2016 年一年，引用这些污染文献的论文超过 4 万篇；到 2017 年 2 月，仍有 58 篇新的污染文献出现。问题没有随时间消失。
历史上出现过三次“本该让人醒悟”的节点。第一次是 Gartler 在 1967 年用遗传标记证明多种细胞系其实是同一株，随后 1968 年《Nature》报道了多起 HeLa 污染；第二次是 Nelson-Rees 等人在 1970 年代把污染问题推上全球议程，1981 年在《Science》公布了大规模被 HeLa 污染的细胞培养清单；第三次是 2001 年 STR（short tandem repeat，短串联重复）分型被引入细胞系鉴定。但原文指出：即便在 STR 之后，每年基于误认细胞系的论文数量也没有下降。
为什么一株细胞能污染那么多？机制有四层，一层比一层难防。
- 源头污染。建系时，培养皿里可能已经有一株生长更快的细胞（最常见的就是 HeLa）。原代细胞长得慢，HeLa 长得快，几代之后原代细胞被“挤出去”，而实验者以为自己在传自己的细胞。
- 分发网络。细胞系通过细胞库、个人赠予、同一实验室的共用试剂与操作台扩散。一次气溶胶、一支没换枪头的移液器、一瓶共用的培养基，就够完成一次交叉污染。进入细胞库之后，它会被分发到全世界。
- 标签与记录错误。冻存管写错、传代记录断链、复苏时拿错盒子——这些“低级错误”在细胞系历史上一点都不罕见。
- 文献自我复制。一旦污染细胞系的“表型”被发表，后续研究就建立在这个结论上；引用链越长，错误越像常识。原文把这叫 secondary literature 的污染：2016 年就有超过 4 万篇论文引用了污染文献。
为什么作者发现不了？因为污染后的细胞往往“看起来很正常”。HeLa 是上皮样、贴壁、生长快，和很多癌细胞形态接近；实验者看到细胞长得好，会把它当成“状态好”，而不是“换了个物种”。更根本的是，过去几十年里大多数实验室没有做身份鉴定——同工酶、核型、HLA 分型、DNA 指纹，直到 STR 才成为标准。在那之前，大家依赖的其实是“这管细胞来自可靠的细胞库”这个假设。而细胞库本身也可能出错。
原文给了三个案例，最能说明“知道了也没用”。ALVA-31 于 1993 年被建立为前列腺癌细胞系，2001 年被发现其实就是 PC-3；原文检索到 56 篇使用 ALVA-31 的论文，其中 22 篇发表于误认被揭穿之后，最后一篇到 2016 年还在发——距离揭穿已经 15 年。更讽刺的是，有作者用 ALVA-31 与 PC-3 互相比较，声称“排除了细胞类型特异效应”，实际上是在比较两管同一株细胞。另外两个案例（胸腺细胞系家族、JCA-1）的情节类似：揭穿之后，仍有大量论文继续以错误身份使用它们。
原文还引了一组关于中国实验室的数据：有调查估计细胞系污染率在 25%–46% 之间，中国开发的“新”细胞系里有 85% 实际是 HeLa。同时原文也指出，多数污染文献来自科研传统深厚的国家，美国占 36%——这不是某个国家或某个实验室的问题，而是整个细胞培养生态的问题。把这两组数字放在一起看，比只看其中一组更接近事实。
那么，已经发表的东西怎么办？原文的“Practical measures”给了几条具体建议，语气很克制：
- 加“关注声明”（expression of concern），而不是动辄撤稿。原文引用的定义是：Neither retractions nor corrections, they alert readers that there may be an issue with a paper, when the full story is not yet clear.（既不是撤稿也不是更正，而是在事情还没完全清楚时提醒读者“这篇可能有问题”。）模板句也给了：“Cell line X in this study is known to be misidentified and is actually Y. See Z for more information.”（本研究中的细胞系 X 已知被误认，实际是 Y，详见 Z。）只有结论被严重动摇时才撤稿；对“诚实错误”，还可以用 Fanelli 提出的 self-retraction（作者主动撤稿）。
- 让文章可被检索。把细胞系名写进关键词或摘要；使用 RRID（Research Resource Identifier）；把 Cellosaurus、ICLAC、NCBI BioSamples 与论文数据库交叉链接。
- 留下“细胞护照”。原文叫 paper trail：来源、做过的验证试验、参与过的实验与结果。这样一旦某株细胞被揭穿，可以快速定位哪些研究受影响；它也能顺带暴露知识空白，并为阴性结果提供发表的位置。
原文的结尾说，距离第一次对细胞系误认的担忧已经近半个世纪，认证技术已经普及，但对“已经污染的文献”的处理仍然缺位，而且任务规模大、紧迫。这句话值得每个准备用细胞系做实验的人读一遍：你论文里的“HeLa”，确定是 HeLa 吗？
六、STR、支原体与 RRID：三张身份证
细胞系的问题，说到底是没有“身份证”。一管细胞传到你手里，标签上写着名字，但名字不等于身份。现代实验室有三样工具来补这个缺口：STR 分型回答“这株细胞是不是它自己”，支原体检测回答“它有没有被污染”，RRID 回答“论文里写的这株细胞，别人能不能找到同一株”。
STR 分型（short tandem repeat profiling）。STR 是染色体上 2–7 个碱基为一个重复单位的短序列，比如“GATA”重复 9 次或 12 次；不同个体、不同细胞系在十几个位点上的重复次数组合不同，扩增后跑毛细管电泳，就得到一张“长度图谱”。人源细胞常用 21 个位点（含 Amelogenin 用于判断性别），小鼠常用 18 个位点（常用 panel，具体位点数因试剂盒而异，经验值）。把图谱与 ATCC、DSMZ、Cellosaurus 的参考图谱比对，按 ANSI/ATCC ASN-0002 标准，匹配度达到 80% 以上可判为同一株。什么时候做：收到新细胞时、建立种子库时、每年一次、论文投稿前；换过培养箱、换过操作人、细胞状态突变时也要做。
STR 也有盲区：它检测的是“主群”的图谱，如果一株细胞里混入了 10%–20% 以下的另一株细胞，图谱可能仍显示为原来的细胞（经验值，检测下限因方法和混合比例而异）；降解样本、PCR 抑制物也会让结果不可靠；参考数据库本身也可能有错。所以 STR 是必要条件，不是充分条件。
支原体（mycoplasma）检测。支原体是没有细胞壁的最小原核生物，污染细胞后培养基不浑浊、细胞不立刻死，但会悄悄改变代谢、基因表达、生长速度、转染效率与病毒产量——你的实验“做不出来”，可能不是你的问题。细胞培养的支原体污染率，文献里常见的估计是 15%–35%，不同调查差异很大（估计值，随实验室与检测方法变化）。检测方法有四类：PCR（最常用，1–2 天出结果，灵敏度高）、Hoechst 33258 荧光染色（看细胞表面/周围的小点状 DNA 荧光，需要经验）、培养法（2–3 周，金标准但太慢）、商品化生化/荧光素酶试剂盒（快，但可能漏检低浓度）。频率：新细胞入场必检、每次冻存前、常规每月一次、细胞状态异常时立即检。注意：抗生素不能清除支原体，只会让培养“看起来干净”，检测更难。
RRID（Research Resource Identifier）。RRID 是一个全局唯一的标识符，指向数据库里的资源记录。细胞系用 Cellosaurus 的编号，例如 HeLa 是 RRID:CVCL_0030；抗体用 Antibody Registry 的编号，例如 RRID:AB_xxxxxx。它的价值在于消歧：全世界有太多“HEK293”“HeLa”“PC-3”的变体、拼写和亚克隆，光写名字无法定位到具体来源。写论文时给出 RRID，读者可以顺着指针查到这株细胞的来源、STR 图谱、同义词与相关文献；审稿人也能据此判断你用的到底是不是你以为的那一株。
三者的关系可以这样记：STR 是“体检报告”，支原体检测是“传染病筛查”，RRID 是“身份证号”。体检报告证明身份，筛查证明干净，身份证号让别人找得到你——缺一样，档案就不完整。
| 工具 | 回答什么问题 | 什么时候做 | 主要局限 |
|---|---|---|---|
| STR 分型 | 这株细胞是不是它声称的那一株 | 收到新细胞、建库、每年、投稿前 | 检测不到低比例混合；依赖参考数据库 |
| 支原体检测 | 培养物有没有被支原体污染 | 新细胞入场、冻存前、每月、状态异常时 | 不同方法灵敏度差异大；抗生素会掩盖 |
| RRID | 论文里的这株细胞能否被唯一检索到 | 写论文、建库存档时 | 只保证“指向的记录”，不保证细胞本身正确 |
七、日常维护：培养基、传代、冻存与抗生素
原代与细胞系的日常维护，大部分工夫在“不变”两个字上：让每次传代的条件尽量一致，差异才能归因于实验处理。下面这些参数，每一条都值得写进实验室的 SOP。
培养基。DMEM 高糖与 DMEM/F12 是两种常见基础培养基：前者葡萄糖高、适合高代谢细胞；后者营养更全面，常用于神经细胞与低血清培养。L-谷氨酰胺在溶液中不稳定，会自行降解为氨，几周后浓度就不够了——可以用 GlutaMAX 替代，或每周新鲜补加。HEPES 提供额外缓冲，但过量对某些细胞有毒；碳酸氢钠依赖 CO2 培养箱维持 pH，酚红只是指示剂，颜色变黄说明酸了，变紫说明碱了。培养基开封后 4°C 避光保存，不要反复温浴。
血清。FBS 是最大的变量之一：批次之间的生长因子、激素、内毒素、血红蛋白水平不同，细胞形态和生长速度就会变。做法是：一次买够同一批号，做小规模测试后再用；记录批号；换批号时对比生长曲线。热灭活（56°C、30 min）灭活补体，免疫实验或某些细胞需要；但不是所有细胞都需要，过度加热会破坏生长因子，反而让细胞长得慢。
传代。贴壁细胞在汇合度 70%–80% 时传代最稳：太稀，细胞间缺少接触信号、长得慢；太密，接触抑制、表型漂移、营养耗尽。0.25% 胰酶消化 1–3 min，在显微镜下看到细胞回缩、变圆、开始脱落就终止，不要等全部漂起来；消化过度会切掉表面受体，消化不足则吹打造成机械损伤。终止用含血清培养基，300×g、5 min 离心，弃上清，按比例重悬。每次记录代次——细胞系的代次是有限资源，超过一定代次（常见 20–30 代，因细胞而异，经验值）就该从种子库重新起用。
冻存与复苏。冻存液常用 10% DMSO + 90% FBS（或商品化无血清冻存液），细胞密度约 1×106 个/mL；用程序降温盒以约 −1°C/min 降到 −80°C，过夜后转入液氮。DMSO 有毒，冻存前要预冷、动作要快。复苏时 37°C 水浴快速融化（1–2 min 内），加预温培养基稀释，离心去掉 DMSO，次日换液去除死细胞；复苏活率低于 70% 时，先养一两代再用于实验（经验值）。
抗生素该不该常规加。原文的答案是明确的：不加。原因在上面已经说过——掩盖污染、改变细胞生理、让支原体检测出现假阴性。很多实验室只在原代初期、建系、复苏后的一两代短期使用抗生素，之后撤掉，靠无菌操作与定期检测维持。如果你发现“撤了抗生素细胞就死”，那不是抗生素的问题，是培养体系里一直有污染。
记录。每一批细胞都该有一份“细胞护照”：来源（细胞库/赠予者/建系文章）、RRID、代次、日期、操作人、培养基与血清批号、STR 结果、支原体检测结果、冻存位置与管数。听起来繁琐，但当你三个月后想找回“那批长得特别好的细胞”，或者审稿人问“你们验证过细胞系吗”，这份记录就是答案。细胞培养里最贵的一句话是“我记得当时是……”——记忆不可靠，记录才可靠。
八、本科生守则：先记录，再动手
这一页讲了很多“怎么防止出错”，最后把能直接执行的部分收成三条守则。它们不高级，但每一条都对应前面某一节的事故。
第一条：先记录，再动手。进细胞房之前，先想清楚今天要做什么、用哪一管、第几代、培养基和血清是哪一批。操作时把日期、操作人、细胞系名与代次、处理、换液/传代/冻存写在记录本上。不要相信“我等会儿补”——补出来的记录一定会漏掉当时觉得“显然”的信息。三个月后你会感谢现在的自己。
第二条：污染要第一时间坦白。培养基突然浑浊、pH 快速变黄、镜下出现大量颗粒或丝状物、细胞成片脱落——先隔离，再告诉带教老师或实验室负责人，按规程处理（高压灭菌、丢弃、清洁培养箱与台面）。不要偷偷加抗生素“压一压”，也不要假装没看见继续用。污染在早期处理是小事，拖到整批细胞、整个培养箱、整个实验室都是事。瞒报比污染本身更严重。
第三条：别用“看起来还行”的细胞做关键实验。形态变扁、突起消失、生长变慢、贴壁变差、传代后恢复不了——这些都是“细胞状态不对”的信号。关键实验（要写进论文、要用于组会汇报、要决定下一步方向）之前，至少确认：代次在合理范围、STR 结果与来源一致、支原体阴性、形态与文献一致。任何一条不确定，就先别做，或者把不确定性写进结果里。
| 频率 | 检查什么 | 合格标准 |
|---|---|---|
| 每次进细胞房 | 紫外/酒精擦拭、手套、枪头、瓶口过火、开盖角度 | 无菌操作习惯稳定 |
| 每次传代 | 形态、汇合度、代次、培养基颜色 | 形态与文献一致、代次可追溯 |
| 每周 | 培养箱温度、CO2、水盘、液氮罐液位 | 记录在案、无报警 |
| 每月 | 支原体检测、细胞系 STR（按需） | 阴性、图谱与参考一致 |
| 每次冻存 | 代次、密度、冻存液、位置 | “细胞护照”完整 |
最后回到标题：你养的到底是谁？每次传代前问三个问题——这是第几代？来源是哪、上次验证是什么时候？形态和行为对不对？三个问题都能答上来，你养的才真的是“你的”细胞；答不上来，你养的只是培养箱里一管会分裂的东西。
▲ 本页翻译难点（对照学习用）
附录 · 英文原文
以下为两篇原文的英文转录：其一为 Bio-protocol 2025 年小胶质分离方案全文；其二为 PLoS ONE 2017 年 HeLa 论文的 Introduction（含 “The process of distributing cell lines” 小节）与 “Practical measures” 一节。原文中的图片、参考文献编号与部分项目符号在文本抽取时未保留；中文译文按原文顺序与内容逐节译出，英文版权归原作者与期刊所有，此处仅作学习对照。
Revisiting Primary Microglia Isolation Protocol: An Improved Method for Microglia Extraction
Abstract
Microglia, the resident immune cells of the central nervous system, play a crucial role in maintaining neural homeostasis and in regulating neurodevelopment, neuroinflammation, tissue repair, and neurotoxicity. They are also key contributors to the pathogenesis of various neurodegenerative disorders, underscoring the need for in vitro models that accurately recapitulate disease-relevant conditions. Among the available isolation methods, the classical mixed glial culture shaking technique remains the most commonly employed, while alternatives such as magnetic bead separation and fluorescence-activated cell sorting (FACS) offer higher purity but are often constrained by technical complexity and cost. In this study, we refined the traditional shaking method by supplementing specific cytokines during culture to enhance microglial viability and proliferation. Our optimized protocol produced primary microglia with higher purity, greater yield, and improved viability compared with the conventional approach, thereby increasing experimental efficiency while substantially reducing time, animal usage, and overall cost.
Key features
• The microglial cells obtained using this protocol achieve a purity of approximately 90%.
• This protocol maximizes the viability of primary microglial cells.
• The entire procedure requires a minimum of 9 days to complete.
• Antibiotic or antifungal solutions are not used in this protocol.
Graphical overview
Flowchart of the isolation procedure for primary microglial cells from mice
Background
Microglia are the resident immune effector cells of the central nervous system (CNS), endowed with essential functions such as phagocytosis of pathogens and clearance of apoptotic cells. They are primarily responsible for maintaining neural homeostasis, mediating inflammatory responses, and contributing to tissue repair. Increasing evidence has highlighted their pivotal roles in pathological conditions, including neurodegenerative diseases, brain injury, and psychiatric disorders, which have become major focuses of contemporary neuroscience research [1–4]. Although several established in vitro models, such as the murine BV2 microglial cell line, are widely used, they exhibit intrinsic limitations as experimental systems [5]. In detail, the immortalization of BV2 cells may lead to aberrant gene expression, dysregulated signaling pathways, and altered metabolic states. Compared with primary microglia, BV2 cells lack key homeostatic features and immune functionalities, making them less suitable for accurately modeling the physiological responses of microglia in Parkinson’s disease (PD) [6]. Consequently, obtaining primary microglia with both high yield and high purity is of great importance for mechanistic studies in molecular neuroscience. Furthermore, in vitro experiments employing purified primary microglia provide a controllable and physiologically relevant model for dissecting activation pathways and intercellular interactions, thereby facilitating a deeper understanding of their functions under diverse pathological states.
Several approaches have been developed for the isolation of primary microglia from mice, including differential adherence, magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS), and the mixed glial culture shaking method [7–9]. Among them, MACS and FACS offer high specificity and yield microglia of greater purity; however, these methods are limited by their high cost and relatively low overall recovery. In contrast, differential adherence and shaking methods are technically straightforward and cost-effective for routine use, yet they typically produce cells of lower purity. Therefore, developing an isolation protocol that allows rapid, efficient, and cost-effective acquisition of highly purified primary microglia would be highly valuable for experimental model establishment and subsequent mechanistic studies.
In this protocol, we optimized the traditional mixed glial culture shaking method for microglial isolation. During the initial brain tissue dissociation and early mixed glial culture stage, the neuronal supplement B27 was added to enhance cell survival and preserve microglial function [10,11]. In the subsequent culture phase, macrophage colony-stimulating factor (M-CSF) was supplemented to promote microglial proliferation and expansion [12,13]. This modified protocol not only produced microglia with higher purity but also markedly shortened the isolation timeline, enabling the acquisition of usable primary microglia within as few as 10 days. Moreover, no antibiotic or antifungal agents were used in this protocol. Importantly, the improved method yielded sufficient numbers of more viable cells to support common biochemical analyses—including western blot, polymerase chain reaction (PCR), and proteomic studies—without requiring large numbers of neonatal mice. In addition, the functional competence of the isolated primary microglia was validated through co-culture with lipopolysaccharide (LPS) stimulation (1 μg/mL) [14].
Materials and reagents
Biological materials
1. C57BL/6-Sncatm2.1(SNCA*A53T)Mdk/J (The Jackson Laboratory, strain: #039167)
2. C57BL/6J (The Jackson Laboratory, strain: #000664)
Reagents
1. Phosphate-buffered saline (PBS) 1×, pH 7.2–7.4 (Solarbio, catalog number: P1020)
2. Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12), 1:1, 1×, supplemented with 2 mM L-glutamine (Gibco, catalog number: 11320033)
3. Trypsin-EDTA solution, 0.25%, 1× (NCM Biotech, catalog number: C100C1)
4. 4% paraformaldehyde fixative solution (Solarbio, catalog number: P1110)
5. B-27TM supplement (50×), serum-free (Thermo Fisher, catalog number: 17504044)
6. Macrophage colony-stimulating factor (M-CSF) (Sigma-Aldrich, catalog number: SRP3221)
7. Fetal bovine serum (FBS), qualified, heat-inactivated (Thermo Fisher, catalog number: 10091148)
Solutions
1. Wash buffer (see Recipes)
2. DMEM/F12 + 10% FBS (see Recipes)
3. DMEM/F12 + 20% FBS (see Recipes)
Recipes
1. Wash buffer
ReagentFinal concentrationQuantity or volumeDMED/F12n/a49 mLB27 (50×)1×1 mLTotaln/a50 mL
2. DMEM/F12 + 10% FBS
ReagentFinal concentrationQuantity or volumeDMED/F12n/a45 mLFBS2.5 mL/L5 mLTotaln/a50 mL
3. DMEM/F12 + 20% FBS
ReagentFinal concentrationQuantity or volumeDMED/F12n/a40 mLFBS5 mL/L10 mLTotaln/a50 mL
Laboratory supplies
1. 6-cm cell culture dish (JETBIOFIL, catalog number: TCD010060)
2. 12-well cell culture plate (JETBIOFIL, catalog number: TCP001012)
3. 15-mL centrifuge tube (JETBIOFIL, catalog number: CFT411150)
4. T25 cell culture flask (JETBIOFIL, catalog number: TCF002025)
5. 70-μm cell strainer (JETBIOFIL, catalog number: CSS013070)
6. Glass coverslips (Biosharp, catalog number: BS-18-RC)
Equipment
1. BB15 CO2 incubator (Thermo Scientific, catalog number: 51023126)
2. Multifuge X1 Pro centrifuge (Thermo Scientific, catalog number: 75009750)
3. CS-200 orbital shaker (Yooning, catalog number: CS-200)
4. Dissecting scissors (Fine Science Tools, catalog number: 14160-10)
5. Spring scissors (Fine Science Tools, catalog number: 15018-10)
6. Ophthalmic forceps (Fine Science Tools, catalog number: 11053-10)
7. Dissecting forceps (Fine Science Tools, catalog number: 11000-12)
8. Tissue forceps (Fine Science Tools, catalog number: 11021-12)
9. Scalpel handle and blades (Integra Miltex, catalog number: 4-7)
Procedure
A. Isolation of primary mouse glial cells
1. Mixed glial cell preparation: One neonatal (postnatal day 1–2) SNCA A53T transgenic mouse was euthanized by cervical dislocation and surface-sterilized in 75% ethanol for 5 min.
2. Place the sterilized mouse on a sterile surgical platform and perform all procedures on ice under aseptic conditions. Make a midline incision along the scalp and skull to expose the brain tissue. Using sterile forceps, gently separate and carefully remove the whole brain intact. Immediately transfer the excised brain into pre-chilled PBS and incubate for 30 s to remove residual blood and debris. Throughout the procedure, strict aseptic technique should be maintained.
3. Transfer the brain tissue into a 6-cm culture dish containing 5 mL of DMEM/F12 medium supplemented with 2.5 mM L-glutamine and 1× B-27 supplement.
Note: The B-27 supplement is widely used in culture systems for the differentiation of induced pluripotent stem cells (iPSCs) into neurons and their supporting cells, including microglia. In three-dimensional or multicellular co-culture models, B-27 primarily serves as a culture additive that supports neuronal survival and maturation, while providing a stable, serum-free environment conducive to neuronal maintenance [15]. Because the formulation of B-27 contains multiple antioxidants and hormone-like components (such as vitamin E and corticosterone), it can reduce oxidative stress and inflammatory signaling within the culture system. Consequently, B-27 helps microglia maintain a resting, ramified morphology and prevents excessive activation [7,16]. Therefore, in culture systems containing B-27, microglia typically exhibit low-inflammatory, homeostatic characteristics, which contribute to the establishment of a neuroprotective microenvironment.
4. Under a dissection microscope, carefully remove the meninges and surface blood vessels using sterile dissecting scissors and ophthalmic forceps. Place the brain with the dorsal side facing upward and divide it along the midline into two hemispheres. Under the microscope, identify the cerebral cortex (a thin, grayish-white layer on the surface) and the underlying hippocampus (located on the medial side of each hemisphere, with a curved “C” shape). Gently separate along the interface between the cortex and the white matter to remove the cortex and expose the hippocampus. Carefully dissect out the hippocampus along its outer boundary. Transfer the isolated cortical and hippocampal tissues into pre-labeled 1.5 mL microcentrifuge tubes containing PBS and keep them on ice. The entire dissection process should be completed within 10 min to ensure tissue viability.
5. Transfer the 1.5 mL microcentrifuge tubes from the sterile surgical bench to a biosafety cabinet. After washing the tissues with PBS 1–2 times, digest them with 1 mL of 0.25% trypsin at 37 °C for 3 min with gentle pipetting, followed by neutralization with 1 mL of DMEM/F12 medium containing 10% FBS.
6. In a biosafety cabinet, pass the homogenate through a 70-μm cell strainer, collect the filtrate into a clean 15 mL tube, and centrifuge at 300× g for 5 min. Discard the supernatant and resuspend the pellet in 3 mL of DMEM/F12 supplemented with 20% FBS. Then, seed into a T25 culture flask and maintain at 37 °C in a humidified 5% CO2 incubator for 24 h ().
7. After 24 h, numerous floating cell aggregates and debris are observed. Remove the supernatant and wash the culture three times with PBS (), followed by the addition of 5 mL of DMEM/F12 + 20% FBS and further incubation for 5 days.
[Figure 1.] Morphological characteristics of brain tissue–derived cells from nude mice observed under a microscope before and after medium change on day 2 of culture.(A) Day 1: Schematic workflow for isolating primary cells from mice; (B) Day 2: Cell morphology before and after medium change under light microscopy.
8. By day 5, cells exhibit stratified growth: astrocytes and fibroblasts predominate in the adherent layer, while microglia appear as round or oval cells in the upper layer. Following medium replacement, add M-CSF (20 ng/mL) to promote microglial proliferation and maintain the cultures until day 8.
Notes:
1. Since antibiotics and antifungal agents are not used during the culture process, strict aseptic techniques must be maintained throughout dissection. All surgical instruments should be thoroughly sterilized before use.
2. The entire dissection should be performed at low temperature to preserve cell viability.
3. The duration of trypsin digestion should not be prolonged to avoid cell damage.
B. Isolation and purification of primary microglia
1. By day 8 of culture, cellular debris was markedly reduced, and clear stratification was observed. The adherent layer consisted predominantly of astrocytes and fibroblasts, whereas the upper layer was composed of morphologically uniform round or oval-shaped microglia. The culture medium was replaced on day 8.
2. On day 9, seal the T25 flasks and place them on an orbital shaker at 200 rpm for 2 h at 37 °C. Collect the resulting suspended cells and reseed them into fresh T25 flasks to obtain isolated microglia. Replenish the original culture flasks with 5 mL of DMEM/F12 supplemented with 20% FBS and maintain for an additional 5 days; then, harvest a second batch of microglia using the same procedure (, B).
Notes:
1. According to quantitative analysis, approximately 1.6 × 106 primary microglial cells can be isolated from the cerebral cortex and hippocampal regions of a single mouse.
2. If the yield of suspended microglia on day 9 is insufficient, supplementation with M-CSF (10–50 ng/mL) may be applied to promote microglial expansion.
[Figure 2.] Morphological characteristics observed under a microscope on day 5 and day 8 of maintenance culture after medium change, and on day 9 after harvesting primary microglia.(A) Days 5–9: Schematic workflow of primary cell culture; (B) Days 5, 8, and 9: Cell morphological changes under light microscopy.
C. Identification of primary microglia
1. Cell preparation: Harvest cells by trypsin digestion, resuspend in complete DMEM/F12 supplemented with 20% FBS, and seed at a density of 2 × 105 cells/mL into 6-cm dishes and 12-well plates containing sterile glass coverslips. Incubate overnight at 37 °C in a humidified atmosphere with 5% CO2.
2. After adherence, remove the medium, rinse cells once with PBS, and subsequently fix with 4% paraformaldehyde at 4 °C overnight.
3. Subject cells grown on coverslips to immunofluorescence staining using the microglial marker Iba1 and the astrocytic marker GFAP ().
4. In parallel, the purity of primary microglia is further verified by flow cytometry using the microglia-specific surface marker CD11b ().
5. To evaluate functional activity, treat primary microglia with PBS or lipopolysaccharide (LPS) (1 μg/mL) for 24 h. Subsequently, analyze CD40 surface expression by flow cytometry to assess microglial activation markers (). Then, extract total RNA for qPCR analysis to determine the expression levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and the anti-inflammatory factor TGF-β ().
[Figure 3.] Identification of primary microglia.(A) Immunofluorescence staining of isolated cells with the microglial marker Iba1 and the astrocytic marker GFAP. (B) Flow cytometric analysis of primary microglia proportion using direct CD11b-PE labeling. (C) Flow cytometry analysis showing changes in CD40 surface expression in primary microglial cells from the two groups. (D) Functional validation of the model by stimulating isolated primary microglia with LPS (1 μg/mL). Data are presented as mean ± SD; n = 3 independent biological replicates per group. One-way ANOVA followed by Tukey’s post hoc test was used for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). Analyses were performed in GraphPad Prism 9.
Data analysis
Flow cytometry data were analyzed using FlowJo software, and statistical analyses were performed with GraphPad Prism. Quantitative data are presented as mean ± standard deviation (mean ± SD). Comparisons between two groups were conducted using Student’s t-test, while differences among multiple groups were assessed by one-way analysis of variance (one-way ANOVA), according to the experimental design. A P-value < 0.05 was considered statistically significant.
Validation of protocol
1. The experimental protocol was reproducibly validated, and all data were analyzed using FlowJo for flow cytometry and GraphPad Prism for statistical evaluation.
2. This protocol has been used and validated in the following research article:
• Liu et al. [17]. LRRK2 Mediates α-Synuclein-Induced Neuroinflammation and Ferroptosis through the p62-Keap1-Nrf2 Pathway in Parkinson’s Disease. Inflammation.: e1007/s10753–025–02291–8. https://doi.org/10.1007/s10753-025-02291-8 (Figure 13).
General notes and troubleshooting
General notes
1. This experimental protocol is applicable to C57BL/6-Sncatm2.1(SNCA*A53T)Mdk/J transgenic mice, standard C57BL/6 mice, and transgenic mice on a C57BL/6 background.
2. According to quantitative analysis, approximately 1.6 × 106 primary microglial cells can be isolated from the cerebral cortex and hippocampal regions of a single mouse.
Troubleshooting
Problem 1: Insufficient yield of primary microglia.
Possible cause: Reduced viability of primary microglia.
Solution: If the number of microglia obtained after initial extraction and transfer into T25 culture flasks (day 9) is insufficient, supplementation with 20 ng/mL M-CSF during medium replacement is recommended. This treatment helps maintain normal dendritic or pseudopod-like morphology and promotes the proliferation of primary microglia.
Acknowledgements
Jianwei Li, Menglin Zhang, Xinran Zhang, Xiao Wu: Conceptualization, Methodology, Writing—Original Draft, Supervision, Formal Analysis. Zijian Zheng, Cheng Xue, Lan Shen: Investigation, Data Curation, Writing—Review & Editing. Guohui Lu: Review and proofreading. Jianwei Li, Zijian Zheng, and Menglin Zhang made the primary contributions to this work and are designated as first authors. All authors contributed to the literature search, read, and approved the final manuscript. All authors confirm their accountability for the research presented in this manuscript, and no further changes to the authorship will be made.Funding: This study was supported by the National Natural Science Foundation of China (82460265), The Health and Wellness Innovation Talent Project of Jiangxi Province's "Gan po Talent Program" (gpyc20240210), Key Project of Scientific and Technological Innovation of Jiangxi Provincial Health Commission (2025ZD005), and the Yang Fan Project of the First Affiliated Hospital of Nanchang University (RSC-0036).This protocol was used in [17].
Second source: the ghosts of HeLa (selected sections)
Introduction
The misidentification of cell lines is a stubborn problem in the biomedical sciences, contributing to the growing concerns about errors, false conclusions and irreproducible experiments [[1], [2]]. As a result of mislabelled samples, cross-contaminations, or inadequate protocols, some research papers report results for lung cancer cells that turn out to be liver carcinoma, or human cell lines that turn out to be rat [[3], [4]]. In some cases, these errors may only marginally affect results; in others they render results meaningless [[4]].
The problems with cell line misidentification [[5]] have been known for decades, commencing with the controversies around HeLa cells in the 1960s [[6]–[10]]. In spite of several alarm calls and initiatives to remedy the problem, misidentification continues to haunt biomedical research, with new announcements of large-scale cross-contaminations and widespread use of misidentified cell lines appearing even recently [[11]–[13]]. Although no exact numbers are known, the extent of cell line misidentification is estimated between one fifth and one third of all cell lines [[4], [14]]. (Although currently only 488 or 0.6% of over 80,000 known cell lines have been reported as misidentified, most cell lines are used infrequently [[15]].) In addition, misidentified cell lines keep being used under their false identities long after they have been unmasked [[16]], while other researchers continue to build on their results. Considering the biomedical nature of research conducted on these cell lines, consequences of false findings are potentially severe and costly [[17]], with grants, patents and even drug trials based on misidentified cells [[18]]. Several case studies performed by the International Cell Line Authentication Committee (ICLAC) highlight some of the potential consequences of using misidentified cell lines [[19], [20]]. Especially in the last decade, the gravity of the problem has been widely acknowledged, with several calls for immediate action in journal articles [[3], [12], [21]–[23]], requirements for grant applications (e.g. [[24], [25]]) and even an open letter to the US secretary of health [[26]].
The current calls for action and remediation activities are almost exclusively concerned with avoiding future contaminations, such as through systems for easier verification of cell line identities. Various solutions have been proposed [[27]–[29]], among others employing genotypic identification through short tandem repeats (STR) [[30]]. In addition, authors are expected to check overviews of misidentified cells (such as [[12], [15], [27], [31]]) before conducting their experiments. However, little attention is currently paid to the damage that has already been done through the past distribution of research articles based on misidentified cells. Although systems such as retractions and corrections are available to alert other researchers of potential problems in publications, these systems are rarely used to flag problems with cell lines [[20], [32]]. Even if future misidentifications could be avoided completely–which is not likely given the track record of earlier attempts–these ‘contaminated’ articles will therefore continue to affect research.
Before any action can be taken, it is essential that we get a sense of the size and nature of the problem of contaminated literature. This raises several questions. First, how many research articles have been based on misidentified or contaminated cell lines? How wide is their influence on the scientific literature? Second, what can we say about origins and trends in the contaminated literature? Is the problem getting better, or restricted to peripheral regions of the world’s research, where perhaps protocols are less strict? Third, what could be appropriate ways to deal with the contaminated literature? To answer these questions, we searched the literature for research papers using cell lines that are known to have been misidentified. In order to put the results of this search in perspective, we analysed the precise complications of misidentification for three particular cell lines.
The process of distributing cell lines
To study the scale of literature contamination, we need to understand the process of setting up, distributing, and publishing about cell lines. This process is illustrated in.
[Fig 1] The creation, distribution and literature of a cell line: A cultured sample of cells (blue cells) may produce an immortal cell line (red cells), sometimes announced in ‘an establishing paper’ (in white).Cells may then be distributed to other researchers and reported in research papers, the ‘primary literature’. If misidentification of cells is reported in ‘a notifying paper’ (in red, bottom left), this may raise questions about the entire cell line (question marks) and the papers based on it, since misidentification commonly occurs at the source. Notifying papers should be reported to ICLAC, which will decide whether cell lines should be added to the ICLAC misidentified cell line register. Meanwhile, the contaminated primary literature is cited (dotted lines) by ‘secondary literature’, spreading the contamination further.
Briefly, setting up a novel cell line commences with a tissue sample from an organism, human or other. If this culture grows successfully, the establishment of a new cell line is sometimes reported in what we will call an ‘establishing paper’. Subsequently, scientists may share or obtain this cell line, either via their personal network or via cell banks. These scientists may perform research on this cell line and publish their findings in scientific journals, thereby setting up what we call the primary literature based on a cell line.
At some point, a cell line may be found to be misidentified. This observation may be published in a ‘notifying paper’, leading to a registration in the International Cell Line Authentication Committee’s (ICLAC) database of cross-contaminated or misidentified cell lines [[12]]. Based on available data, cell lines can be added to one of two tables. The first is reserved for cell lines with no known authentic stock, and the second for cell lines where authentic stock is known to exist.
In this paper, we focus on the first category only: cell lines without any reported original stock. In this case, it must be assumed that all primary literature could be based on false grounds and should at least be treated with caution. In addition, we tried to estimate the size of the secondary literature: those articles referring to the primary literature, hence potentially building on questionable materials.
Practical measures
Over the past decades thorough attention has been paid to the improvement of authentication testing for cell lines. Authentication of cell lines, and hence the ability to demonstrate cross contamination, became possible by the introduction of genetic markers by Gartler in 1967 [[9]]. Subsequently, many techniques for cell line authentication were introduced, starting off with inspection of banded marker chromosomes [[8]], and the visualization of chromosomal pattern and architecture in general [[50]], subsequently followed by the methods of Human Leukocyte Antigen (HLA) typing [[51]], enzyme polymorphisms [[52]] and DNA polymorphisms [[53]]. More recently, the techniques of ‘DNA fingerprinting’ [[54]] and the usage of locus-specific probes were introduced. Finally, this led to the now accepted standard method of short tandem repeat profiling [[45]]. As has been pointed out recently, the techniques for proper cell line authentication are now widely available [[55]]. However, implementation of these techniques is still falling short for multiple reasons, including time and financial constraints, lack of training and lack of (international) standards [[55]].
Despite measures to authenticate new and existing cell lines [[27]], research based on the wrong cells is still present in the literature and in fact continues to be published. Some form of precautionary labelling of contaminated articles seems unavoidable. However, this remedial action should be proportionate and not cause unnecessary damage. For some individual scientists, research departments, or scientific journals, rash measures could turn out to be painful. Indeed, some researchers have authored over a hundred articles in our set of contaminated primary literature. Even though the problem with these articles almost exclusively [[56]] falls under the heading of ‘honest error’, with no intention to deceive, notifying all those articles as potentially erroneous, or worse: retracting them, would have a disproportionate impact on several scientists’ careers. This would undermine, rather than support, an effective clean-up operation. However, in addition to catching cell line contamination at the source, initiatives to label contaminations ‘downstream’ in the published literature are direly needed. We can make several suggestions.
First, notifications should be posted alongside previously published articles using misidentified cell lines. This could be done in the form of ‘expressions of concern’, which are described as “Neither retractions nor corrections, they alert readers that there may be an issue with a paper, when the full story is not yet clear.” [[57]] If clear and uncontended, the consequences of the misidentification for the article’s conclusions could be reported, but otherwise the expression of concern could merely state: “Cell line X in this study is known to be misidentified and is actually Y. See Z for more information.” The interpretation of this warning is then entirely up to the expert reader. Such notifications would also serve to preserve as much valuable data as possible: data reported on a misidentified cell line might still be entirely valid, provided the real origin of the cell line is clear. Hence it might be a waste of funds and efforts to automatically dismiss these data. In cases where the use of these cell lines leads to (severely) false conclusions that could have a major impact on future research, articles could be retracted. For recent cases, a system of self-retractions, as proposed by Fanelli [[58]], could be employed.
Second, to allow for simple future identification of articles using misidentified cell lines, we recommend that authors mention the employed cell lines in easily searchable parts of their article, such as the keywords or abstract. Some journals have already suggested measures in this direction, but implementation seems to be slow [[30]]. However, some journals have currently installed a system of Research Resource Identifiers (RRIDs), which might assist in tackling the cell line misidentification issues [[59]]. Alternatively, a system of cross-reference between databases of cell lines and scientific journal publications could be set up. Linking the NCBI databases of ‘BioSamples’ and ‘research articles’ would be a natural candidate for such a system. In similar ways, the Cellosaurus database, the ICLAC database and Research Resource Initiative are already cross-linked.
In addition, better use could be made of paper trails for cell line provenance [[60]]. A clear and complete overview of the origin of a cell line, as well as the various verification tests, the experiments that it has been part of, and the results that these yielded, would be of great benefit in examining the status and quality of a cell line. In addition, this would allow for easy identification of potentially erroneous research when a cell line is found to be misidentified.
Besides being of use in terms of recognition of erroneous research, the paper trail might also serve other purposes, such as mapping the existing knowledge on a certain cell line (thereby also allowing for simple identification of knowledge gaps) and providing a stage for the publication of negative results of experiments on cell lines. The publication of such results has long been proposed as a way of fostering integrity in research [[61]].
Nearly half a century after the first concerns about misidentified cell lines, the initiatives to improve authentication need to be complemented by attention to the already contaminated literature. Our analysis shows that the task is sizeable and urgent.