預防支原體污染的細胞培養方法有哪些？

支原體污染幾乎可影響所有細胞之生長參數，代謝及研究之任一數據。本文威正翔禹/締一生物為您分析預防支原體污染的細胞培養方法有哪些？

提高無菌操作技術

在實驗室中，熟練的技術人員應細心監督新來人員是否遵循無菌操作的規範，這可以幫助減少支原體污染的風險。強烈建議對所有細胞污染事故要進行記錄和存檔，以解決污染問題。

檢查培養基是否污染

由於支原體污染的主要來源之一是從實驗室外帶進來的細胞，建議使用可靠的細胞庫提供的細胞。

如果存在應當隔離的支原體污染的細胞，而沒有單獨的細胞培養箱的情況下，在隔離期內，應使用有蓋的塑料細胞培養瓶。不要使用培養板和未密封的培養皿。對懷疑有污染的培養的細胞，應在每天所有其他細胞培養工作結束后再對它進行處理。應使用單獨分裝的培養基和試劑。強烈建議完工後要對生物安全櫃進行消毒。

合理使用抗生素

用於細胞培養的理想的抗生素應能消除所有的污染物，對宿主細胞無毒，而不干擾實驗，但沒有一種抗生素能滿足以上所有的標準。因此，用於細胞培養的抗生素應有限制。相反的，一個良好的無菌操作對於預防污染起著更重要的作用。

細胞培養中的微生物污染是可以通過細胞培養基的混濁或顏色變化檢測到。在細胞培養中使用抗生素的情況下，有四種可能：

1.對抗生素敏感。

2.對抗生素耐葯。

3.對抗生素部分耐葯。

4.只有支原體對抗生素耐葯。

最後一種情況是最差的，因為支原體可以通過氣溶膠傳播。在抗生素敏感的情況下，抗生素能防止細菌和真菌繁殖，但從一開始就沒有能力預防支原體。因此，在細胞培養中長時間連續使用抗生素，不僅是沒有幫助的，而且也會引起很多的問題。然而，在原代培養中，短期（前兩周）使用抗生素（青霉素/鏈黴素），是至關重要的。因為抗生素在培養基中不穩定，強烈建議每隔兩三天進行一次換液。

丟棄或治療支原體污染的細胞

如果被支原體污染的細胞較為珍貴或稀少，建議進行清除支原體的治療。否則，建議丟棄支原體污染的細胞，因為它們被認為是實驗室污染的來源。

隔離任何來源的新細胞

正如前面章節中描述，從其他實驗室帶來的新細胞是污染的主要來源。因此，在新細胞作任何用途之前，有必要隔離細胞，並檢查是否有支原體污染。

減少氣溶膠的產生

在生物安全櫃或細胞培養箱中細胞操作產生的氣溶膠，以及人員自身產生的氣溶膠，在實驗室中都會傳播支原體污染。因此，避免產生氣溶膠的活動有助於預防細胞培養中的支原體污染。

支原體檢查的重要性

根據一些報告，在美國和歐洲，正在培養的細胞或細胞庫凍存的細胞中，支原體污染的幾率大約是15%至35%。因此，經常進行支原體檢查是非常必要的。

英文原文：

Methods for prevention of mycoplasma contamination in cell culture

Improve aseptic techniques and practices

A precise supervision of new workers in the lab by a skilled operator to follow good aseptic techniques can help to reduce the risk of mycoplasma contamination. It is highly recommended to prepare the reports of all cell contamination incidents to solve the problem of contamination .

Test cultures for contamination

Since one of the main sources of mycoplasma is the cell cultures brought from outside, it is suggested to supply cells from reliable cell banks.

In the case of the existence of mycoplasma contaminated cell culture in quarantine and the absence of separate incubators, only flasks in a plastic box with lid should be used. Never use plates and unsealed dishes in quarantine. In the case of suspected cultures, handling them at the end of the workday after all other cell culture work is completed, using separated media and reagents, and finally disinfecting the laminar flow hood after working is strongly suggested .

Only use antibiotics responsibly

Although ideally antibiotics used for cell culture should eradicate all contaminants, be nontoxic for the host cells and not interfere with experiments, none of the available antibiotics meet the mentioned criteria. Therefore, application of antibiotics in cell culture should be limited. Instead, a good aseptic practice plays an important role in prevention of contamination.

Microbial contamination in cell culture which is antibiotic free is detectable by turbidity or color changes in cell culture medium. In the case of using antibiotics in a cell culture, there are four possibilities: 1. susceptibility to antibiotics, 2. resistance to antibiotics, 3. partial resistance to antibiotics, 4. resistance to antibiotics only by mycoplasma. The last one is the worst contamination since mycoplasmas can be spread by aerosols. In the case of antibiotic susceptibility, antibiotics prevent the cultivation of bacteria and fungi, but are incapable of precluding mycoplasma from the beginning. Therefore, continuous use of antibiotics for a long time in cell cultures not only is not helpful, but also can cause more problems. However, the use of antibiotics (Penicillin/ Streptomycin) for a short term (the first two weeks) in primary culture is vital. Since antibiotics are unstable in the medium, it is highly suggested to replace antibiotic containing medium with fresh medium every two or three days.

Discard or treat mycoplasma contaminated cells

In the case of mycoplasma contamination of cells which are really valuable and rare, it is suggested to treat them to eliminate mycoplasma infection. Otherwise, it is recommended to discard mycoplasma contaminated cells since they are considered as a source of contamination in the lab .

Quarantine new cells of any origin

As described in previous sections, new cells which are brought from other laboratories are characterized as a main source of contamination. So, it is necessary to quarantine cells and check for mycoplasma contamination before using them for any purposes .

Reduce aerosol generation

Aerosols during manipulation of cells in the laminar hood or in the incubators and also aerosols made by personnel can transfer mycoplasma contamination in the lab. Therefore, avoiding activities which result in making aerosols can help to prevent mycoplasma contamination in cell cultures .

Importance of mycoplasma tests

Mycoplasma contamination rate in cells which are in use or are banked in cell banks in USA as well as in Europe is about 15 to 35%, according to reports .

文章引自：NCBI;版權聲明：版權歸原作者所有，如有版權問題，請與我們聯繫。