細(xì)胞培養(yǎng)：培養(yǎng)基特性及選擇
發(fā)布時(shí)間：
2022-12-24
作者：
預(yù)制備培養(yǎng)基生產(chǎn)廠家
細(xì)胞培養(yǎng)：培養(yǎng)基特性及選擇
常用培養(yǎng)基及基本特性
RPMI-1640 Medium：RPMI-1640 廣泛應(yīng)用于哺乳動物、特殊造血細(xì)胞、正常或惡性增生的白細(xì)胞，雜交瘤細(xì)胞的培養(yǎng)，是目前應(yīng)用十分廣泛的培養(yǎng)基。主要用于懸浮細(xì)胞培養(yǎng)。其它像 K-562、HL-60、Jurkat、Daudi、IM-9 等成淋巴細(xì)胞、T 細(xì)胞淋巴瘤細(xì)胞以及 HCT-15 上皮細(xì)胞等均可參考使用。
Minimum Essential Medium（MEM）：也稱最低必需培養(yǎng)基，它僅含有 12 種必需氨基酸、谷氨酰胺和 8 種維生素。成分簡單，可廣泛適應(yīng)各種已建成細(xì)胞系和不同地方的哺乳動物細(xì)胞類型的培養(yǎng)。MEM-Alpha 一般用于培養(yǎng)一些難培養(yǎng)細(xì)胞類型，而其它沒有特殊之處的細(xì)胞株則幾乎均可采用 MEM 來培養(yǎng)。
DMEM-高糖（標(biāo)準(zhǔn)型）是一種應(yīng)用十分廣泛的培養(yǎng)基，可用于許多哺乳動物細(xì)胞培養(yǎng), 更適合高密度懸浮細(xì)胞培養(yǎng)。適用于附著性較差，但又不希望它脫離原來生長點(diǎn)的克隆培養(yǎng)，也可用于雜交瘤中骨髓瘤細(xì)胞和 DNA 轉(zhuǎn)染的轉(zhuǎn)化細(xì)胞的培養(yǎng)。
DMEM-低糖（標(biāo)準(zhǔn)型）是一種應(yīng)用十分廣泛的培養(yǎng)基，可用于許多哺乳動物細(xì)胞培養(yǎng)。低糖適于依賴性貼壁細(xì)胞培養(yǎng)，特別適用于生長速度快、附著性較差的腫瘤細(xì)胞培養(yǎng)。
DMEM/F12：DMEM/F12 培養(yǎng)基適于克隆密度的培養(yǎng)。F12 培養(yǎng)基成分復(fù)雜，含有多種微量元素，和 DMEM 以 1：1 結(jié)合，稱為 DMEM/F12 培養(yǎng)基（DME/F12medium），作為開發(fā)無血清配方的基礎(chǔ)，以利用 F12 含有較豐富的成分和 DMEM 含有較高濃度的營養(yǎng)成分為優(yōu)點(diǎn)。該培養(yǎng)基適用于血清含量較低條件下哺乳動物細(xì)胞培養(yǎng)。為了增強(qiáng)該培養(yǎng)基的緩沖能力，改良之一是在 DMEM/F12（1：1）中加入 15 mMHEPES 緩沖液。
McCoy’s 5A：McCoy’s 5A Medium 主要為肉瘤細(xì)胞的培養(yǎng)所設(shè)計(jì)，可支持多種（如骨髓、皮膚、肺和脾臟等）的原代移植物的生長，除適于一般的原代細(xì)胞培養(yǎng)外，主要用于作組織活檢培養(yǎng)、一些淋巴細(xì)胞培養(yǎng)以及一些難培養(yǎng)細(xì)胞的生長支持。例如 Jensen 大鼠肉瘤成纖維細(xì)胞、人淋巴細(xì)胞、HT-29、BHL-100 等上皮細(xì)胞。
Iscove’s Modified Dulbecco Medium (IMDM)：Guilber 和 Iscove 將 Dulbecco’medium 改良為 Iscove’s Medium，用于培養(yǎng)紅細(xì)胞和巨噬細(xì)胞前體。此種培養(yǎng)液含有硒、額外的氨基酸和維生素、丙酮酸鈉和 HEPES。并用硝酸鉀取代了硝酸鐵。IMDM 還能夠促進(jìn)小鼠 B 淋巴細(xì)胞，LPS 刺激的 B 細(xì)胞，骨髓造血細(xì)胞，T 細(xì)胞和淋巴瘤細(xì)胞的生長。IMDM 為營養(yǎng)非常豐富的培養(yǎng)液，因此可以用于高密度細(xì)胞的快速增殖培養(yǎng)。
M-199 Medium：1950 年，Morgan 成功研制出具有確定化學(xué)成分的細(xì)胞培養(yǎng)液，即 M-199，主要用于雞胚成纖維細(xì)胞培養(yǎng)。此培養(yǎng)液必須輔以血清才能支持長期培養(yǎng)。M-199 可用于培養(yǎng)多種種屬來源的細(xì)胞，并能培養(yǎng)轉(zhuǎn)染的細(xì)胞。
Leibovitz Medium（L-15）：L-15 培養(yǎng)液適用于快速增殖瘤細(xì)胞的培養(yǎng)，用于在 CO2 缺乏的情況下培養(yǎng)腫瘤細(xì)胞株。此培養(yǎng)液采用磷酸鹽緩沖體系，氨基酸組成進(jìn)一步改良，并由半乳糖替代了葡萄糖。
如何配制 DMEM
在李玲、李雪峰編著的《細(xì)胞生物學(xué)實(shí)驗(yàn)》中配制方法如下：
1. 制備新鮮三蒸水或 Millipore 超純水。
2. 稱取所需量的干粉培養(yǎng)基，加入約終體積一半的三蒸水中；若配制一個(gè)包裝的培養(yǎng)液，在將整個(gè)包裝的干粉倒入三蒸水后，需用水洗包裝袋內(nèi)面 2 次，倒入培養(yǎng)液中，以保證所有干粉都溶解成培養(yǎng)液。磁力攪拌或人工攪拌使之完全溶解。
3. 根據(jù)包裝袋上的要求，補(bǔ)加所需量的碳酸氫鈉。根據(jù)實(shí)驗(yàn)需要，添 HEPES（5-20 mmol/L）、谷氨酰胺和其他特殊物質(zhì)。
4. 加水定容到終體積。
5. 必要時(shí)用 1 mol/L 鹽酸和 1 mol/L 氫氧化鈉調(diào)節(jié) pH。
6. 用無菌 0.22 um 濾膜過濾除菌，分裝于無菌血清瓶中，4℃ 冰箱保存。
配制好的培養(yǎng)液用前加入 100 U/mL 青霉素和 100 U/mL 鏈霉素，并根據(jù)需要加入血清（5% - 20%）。
如何選擇培養(yǎng)基
指導(dǎo)原則：尚未明確各種特定細(xì)胞培養(yǎng)的最適培養(yǎng)基，可參考各實(shí)驗(yàn)室已證明的或有文獻(xiàn)報(bào)道的方法來確定（經(jīng)驗(yàn)法），或采用選擇性實(shí)驗(yàn)來證明其是否適用（實(shí)驗(yàn)法）。
Cell culture: characteristics and selection of culture medium
Common medium and basic characteristics
RPMI-1640 Medium:RPMI-1640 is widely used in mammals, special hematopoietic cells, normal or malignant proliferation of white cells. The culture of hybridoma cells is a widely used medium at present. It is mainly used for suspension cell culture. Other cells such as K-562, HL-60, Jurkat, Daudi, IM-9 and other lymphoblastic cells, T cell lymphoma cells and HCT-15 epithelial cells can be used for reference.
Minimum Essential Medium (MEM): also known as the minimum essential medium, it contains only 12 essential amino acids, glutamine and 8 vitamins. The ingredients are simple, and can be widely adapted to the cultivation of mammalian cell types in various established cell lines and different places. MEM-Alpha is commonly used to cultivate some difficult to cultivate cell types, while other cells with no special characteristics can be almost MEM cultured.
DMEM- high glucose (standard form) is a widely used medium, which can be used in many mammalian cell cultures and is more suitable for high-density suspension cell culture. It is suitable for the poor adhesion, but does not want it to be isolated from the original growth point. It can also be used in the culture of myeloma cells in hybridoma and transformed cells transfected by DNA.
DMEM- low sugar (standard form) is a widely used culture medium, which can be used in many mammalian cell cultures. Low sugar is suitable for adherent cell culture, especially for tumor cell culture with high growth rate and poor adhesion.
DMEM/F12:DMEM/F12 medium is suitable for culture of clonal density. The F12 medium is complex, contains a variety of trace elements, and DMEM is combined with 1:1, known as the DMEM/F12 medium (DME/F12medium), as the basis for developing a serum-free formula, with the advantage of F12 containing a richer component and DMEM containing a higher concentration of nutrients. The medium is suitable for mammalian cell culture under low serum concentration. In order to enhance the buffering capacity of the medium, one of the improvements is to add 15 mMHEPES buffer into DMEM/F12 (1:1).
McCoy 's 5A:McCoy' s 5A Medium, designed mainly for the culture of sarcomas cells, can support the growth of a variety of primary grafts (such as bone marrow, skin, lung, and spleen). Apart from the general primary cell culture, it is mainly used for tissue biopsy, some lymphatic cell culture, and some difficult cells. Long support. For example, Jensen rat sarcoma fibroblasts, human lymphocytes, HT-29, BHL-100 and other epithelial cells.
Iscove 's Modified Dulbecco Medium (IMDM):Guilber and Iscove have Dulbecco' medium modified to Iscove 's, for the cultivation of red blood cells and macrophage precursors. The culture medium contains selenium, extra amino acids and vitamins, sodium pyruvate and HEPES. The iron nitrate was replaced with potassium nitrate. IMDM can also promote the growth of B lymphocytes, LPS stimulated B cells, bone marrow hematopoietic cells, T cells and lymphoma cells. IMDM is a nutrient rich culture medium, so it can be used for rapid proliferation of high-density cells.
In M-199 Medium:1950, Morgan successfully developed a cell culture medium with definite chemical composition, that is, M-199, which is mainly used for chicken embryo fibroblast culture. The culture medium must be supplemented with serum to support long-term culture. M-199 can be used to culture various kinds of cells and can transfect cells.
Leibovitz Medium (L-15): L-15 culture medium is suitable for rapid proliferation of tumor cells, and is used for culturing tumor cell lines in the absence of CO2. The culture medium was phosphate buffered, and amino acid composition was further improved, and galactose was replaced by galactose.
How to make up DMEM
In Li Ling and Li Xuefeng's experiment of cell biology, the following methods are formulated.
1. fresh three fresh water or Millipore ultra pure water was prepared.
2. is called the dry powder medium for the required amount, adding three steam water with a final volume of about half of the final volume; if a packaged culture liquid is prepared, the whole packing powder is poured into three water, and the inner surface of the bag is used for 2 times and poured into the culture liquid to ensure that all dry powder is dissolved into cultured liquid. Magnetic stirring or artificial agitation makes it completely dissolving.
3. according to the requirements of the bags, add the amount of sodium bicarbonate required. According to the experimental requirements, add HEPES (5-20 mmol/L), glutamine and other special substances.
4. water is fixed to the final volume.
5. it is necessary to adjust pH with 1 mol/L hydrochloric acid and 1 mol/L sodium hydroxide.
6. sterilize 0.22 um filter membrane, filtrate and remove bacteria, separate it in sterile serum bottle, and store it in refrigerator at 4 degrees.
The prepared culture medium was added 100 U/mL penicillin and 100 U/mL streptomycin before adding the serum (5% - 20%) as required.
How to select the culture medium
Guiding principles: the optimum medium for various specific cell cultures has not been identified, which can be identified by the laboratory, or by literature, to determine (empirical), or to use selective experiments to prove its application (experimental method).