污染细胞的支原体从哪里来

缔一生物导读： 在培养细胞时，人们常常关注细菌和真菌的污染，认为它们是细胞培养的主要干扰因素。但其实，更严重的是支原体 的感染，它的发生率非常高，而且不易被发现。不仅普通光镜下无法发现支原体，而且培养基也不容易变色。但支原体对细胞的各种干扰却是不容忽视的。它不仅导致细胞状态不佳，生长速度慢，而且会使细胞内DNA、RNA及蛋白表达发生改变，严重影响实验结果。因此，在预防和治疗支原体污染之前，有必要去了解细胞被支原体污染的可能途径。

　　支原体污染细胞的途径（点击这里，详细了解支原体污染防治类试剂）

　　支原体可以通过顶部的细胞器特异性和宿主细胞结合。这些顶部的细胞器含有高浓度的粘附蛋白，可粘附到真核细胞并穿入到细胞内部。支原体缺乏细胞壁，它们的胞膜可和宿主的细胞膜融合并交换其胞膜和胞浆成分。

　　图1：在细胞培养中出现的不同种类的支原体频率

　　McGarrity设计了一个模型，来发现支原体是如何在超净台里的细胞传代过程中发生传播的。他先故意用支原体感染细胞。在超净台中，用胰蛋白酶消化该污染的细胞后发现，活的支原体可从细胞培养瓶外的细胞计数板、移液器、废弃盘中被技术员分离出来。即使过了四至六天，活的支原体仍然可以存在于超净台表面，可被成功恢复。在超净台里，传代完被支原体污染的细胞后，再传代干净的不含支原体的细胞，结果仍然在6周后被检测出支原体阳性。这些结果表明，支原体的传播是多么的快速和容易！它也警告我们，要最大限度的避免支原体的污染，因为一瓶细胞发生污染，就会带来支原体传播的可能。

　　未真正消毒的耗材、培养基和溶液

　　不恰当的消毒导致污染的另一个原因。高压锅或干热烤箱中堆积太多的物品造成加热不均，使一小部分耗材未得到消毒。灭菌循环时间过短是另一个消毒上犯的错误，特别是对于超过500ml的液体，或者是包含固体成分的溶液，或胶体物质如琼脂、淀粉等。为了实现无菌，灭菌材料的尺寸、质量、性质和体积必须始终考虑。

　　在无菌和无昆虫区域存放消毒好的物品，使防止再次污染的前提。良好的无菌操作也是至关重要的。

　　实验室人员

　　在因人导致的细胞支原体污染中，口腔支原体发生率最高，它主要存在人的口咽部。发酵支原体和唾液支原体也在污染的细胞培养液中被检测出来，不过发生率更低。

　　被支原体污染的细胞

　　实验室内细胞支原体的相互传染，有必要对于从外源获得的新的细胞系进行支原体检测。一瓶细胞中的单个支原体的存在足以威胁到其他培养的细胞。支原体的污染能通过细胞操作产生的气溶胶或液滴传播。所以，应一次只操作一种细胞，为每种细胞系配置单独的培养基和试剂，这能避免支原体的污染。

　　细胞培养的正确操作和对新培养的细胞定期检查，可以减少支原体污染的机会。

　　英文原文：

　　Ways in which cells are contaminated by mycoplasma

　　Mycoplasmas can bind to their host cells using special tip organelles. These tip organelles have a high concentration of adhesins, to attach to eukaryotic cells and penetrate the host cell. The lack of a stiff wall in mycoplasma may help it to fuse with the membrane of the host cell and exchange its membrane and cytoplasmic components.

　　Frequency and sources of mycoplasma species

　　There are a number of different sources for mycoplasma contamination in cell cultures associated with human, bovine and swine species. Personnel in the laboratories are the main sources of M. orale, M. fermentans, and M. hominis. These species of mycoplasmas account for more than half of all mycoplasma infections in cell cultures and physiologically are found in the human oropharyngeal tract . M. arginini and A. laidlawii are two other mycoplasmas contaminating cell cultures and originate in fetal bovine serum （FBS） or newborn bovine serum （NBS）。 Trypsin solutions provided by swines are a major source of M. hyorhinis. Figure 1 is a diagram showing the normal host and frequency of different species of mycoplasma occurring in cell culture.

　　Different sources for the spreading of mycoplasma in the laboratory

　　McGarrity designed a model to find out how mycoplasmas spread in a laminar flow hood during a routine subculturing procedure. He intentionally infected a cell culture with mycoplasma. After trypsinization of the infected culture in a laminar flow hood, live mycoplasmas were isolated by the technician, outside of the flask, a hemocytometer, the pipettor, and outside of the pipette discard pan. Live mycoplasma could be successfully recovered from the surface of the laminar flow hood even four to six days later! A clean culture, that was subcultured once a week in the same hood following the work with the contaminated cells, tested positive for mycoplasma after only 6 weeks. These results show how quickly and easily mycoplasma can spread and also warn us against the possibility of contamination of most if not all of the other cultures after the entry of a single mycoplasma infected culture into the laboratory.

　　Nonsterile supplies, media and solutions

　　Improper sterilization is a major source of biological contaminants. Packing too much into an autoclave or dry heat oven will cause uneven heating, resulting in pockets of nonsterile supplies. Using too short a sterilization cycle, especially for autoclaving volumes of liquids greater than 500 ml per vessel or solutions containing solids or viscous materials such as agar or starches are other mistakes resulting in incorrect sterilization. To accomplish sterility, the size, mass, nature and volume of the materials for sterilization have to always be considered .

　　Storing sterilized supplies and solutions in a dust- and insect-free area is an obligation to prevent recontamination. Good aseptic technique is also crucial

　　Laboratory personnel

　　Laboratory personnel are considered a major source of mycoplasma contamination . Table 1 shows potential sources of cell culture contamination. M. orale, a species commonly found colonizing the human oral cavity and oropharynx, has been the leading contaminant in study after study. Two other human mycoplasma species, M. fermentans and M. salivarium, are also detected in contaminated cultures but at a much lower rate. Table 2 shows major mycoplasma species found in cell culture and also some of the research results reporting the percentage of contamination with different types of mycoplasma in previous years . Table 1: Potential sources of cell culture contamination.

　　Other mycoplasma contaminated cell cultures

　　A mycoplasma-infected cell culture is a major source of mycoplasma contamination of other cell cultures in the lab. To avoid mycoplasma contamination in cell cultures, it is recommended to test the new cell lines which are obtained from an outside source. A single mycoplasma contaminated cell culture is enough to endanger other cell cultures in the lab. The contamination can spread by means of aerosols and particulates generated during the handling of the mycoplasma infected cell culture. So, working with only one cell culture at a time and preparing separate media and reagents for each individual cell line can avert mycoplasma contamination .

　　A good cell culture practice and regular testing of all new cell cultures can decrease the risk of mycoplasma contamination .

　　文献出处：N Laleh，F Parvaneh. Prevention and Detection of Mycoplasma Contamination in Cell Culture. Cell Journal, 2012, 13（4）： 203