计算溶液所需的质量、体积或浓度。
CHO无血清细胞培养基（含长效谷氨酰胺）
有货
为什么选择此级别
BioReagent,无菌,用于细胞培养 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,避光。低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
CHO无血清细胞培养基是化学成份明确、无血清及无动物源成分的基础高密度CHO细胞培养液，含葡萄糖和丙氨酰谷氨酰胺（L-Alanyl-L-Glutamine，长效谷氨酰胺），适用于CHO-K1细胞及其它CHO细胞的高密度悬浮培养，以及基于此培养基的配方优化和工艺开发。
产品特点
- 成分明确，不含动物源成分，无动物源性重组蛋白及蛋白水解物；
- 支持细胞高密度悬浮培养，适用于重组抗体、重组蛋白因子等蛋白表达技术研究；
- 性能稳定，品质可控，批间差异小。
仅限于科研使用，不适于临床诊断和治疗。
使用方法
1. 细胞的换液与传代
1.1 新收到的常温细胞应立即转移至合适的摇瓶中（培养液体积应控制在摇瓶容量规格的五分之一以内），置于摇床中震荡培养。待细胞密度恢复至3-6×10⁶ cells/ml时，可进行传代培养或细胞冻存；
1.2 从液氮罐中取出的细胞或用干冰运输的细胞请依照下述（2. 细胞的冻存与复苏）方法进行细胞复苏和培养；
1.3 传代时，需先做细胞计数，确认密度后无需离心细胞，可直接将细胞悬液依照所需比例兑入培养液中。传代后的细胞密度应控制在在0.3×10⁶ cells/ml，一般每4天需传代1次；或传代密度为0.6×10⁶ cells/ml，每隔3天传代1次。本培养液可支撑的最高细胞密度约为1×10⁷ cells/ml，细胞在达到此密度时存活率一般仍可保持在95%以上；
1.4 若在震荡培养过程中出现死细胞数过多的状况，可接种低细胞密度进行静置培养，待细胞恢复到正常活率后再进行震荡培养。
2. 细胞的冻存与复苏
2.1 冻存
(1) 用细胞培养液制备7.5% 二甲基亚砜（DMSO）细胞冻存液；
(2) 将细胞培养至对数生长期（密度约为6×10⁶ cells/ml），计数并离心收集细胞；
(3) 用细胞冻存液将离心的细胞以5-20×10⁶ cells/ml的浓度重悬；
(4) 将细胞悬液分装到标记好的冻存管内，确保拧紧管盖使其完全密封；
(5) 将冻存管放置于-80℃冰箱中缓慢降温；
(6) 次日将冻存管转移至液氮内长期保存。此过程需尽可能快速完成（建议在2 min内），如果在此过程中冻存管温度升至-50℃以上，细胞则可能会迅速受损。
2.2 复苏
(1) 从液氮罐、干冰或超低温冰箱中取出细胞冻存管，立刻放置于37℃的温水中，直至管内冰晶完全融化；
(2) 解冻后用75%乙醇彻底擦拭冻存管，用移液枪将管内细胞悬液全部转移至一个50 mL的无菌离心管中，缓慢加入细胞培养液，至其终体积为20 mL；
(3) 1000 rpm离心5 min，弃去含有冻存液的上清液；
(4) 用新鲜的CHO无血清细胞培养基重悬细胞，使其密度为0.6-1.0×10⁶ cells/ml，接种于摇瓶中震荡培养。培养3-6天后对细胞进行计数，观察细胞密度和存活率是否上升。若细胞密度达到3-6×10⁶ cells/ml，可对细胞进行传代，一般细胞传代1-2次后即可恢复正常的生长状态；
(5) 使用无血清细胞冻存液冻存的细胞，可直接复苏融化后转移至已添加CHO无血清细胞培养基的摇瓶中重悬即可，培养3-6天后对细胞进行计数，观察细胞密度和存活率是否上升。
(6) 若化冻后细胞密度过低，可先接种于培养瓶中静置培养，待其密度恢复至0.6-1.0×10⁶ cells/ml时，再置于培养箱摇床中震荡培养。
操作要点及注意事项
1. 摇床参数设置建议
温度36-37℃，CO₂浓度为5%，转速≤120rpm（振幅26）。
不同振幅摇床计算公式如下：
如振幅50按照公式计算：m×ω1²×26²=m×ω2²×50² ，所以ω1和ω2的关系是：ω1≈1.4ω2，就是86rpm左右。
2. 如细胞此前在其它品牌培养基培养，建议逐步过渡到CHO无血清细胞培养基，让细胞持续传代数周时间，等细胞恢复正常生长状态再做下一步实验。
3. 细胞密度及活率降低
(1) 当细胞存活率为80%-95%时
将细胞离心去上清，用新鲜培养液重悬，使其密度为3×10⁶ cells/ml，接种于摇瓶中振荡培养。每天观察细胞是否增殖，若有增殖，继续培养，直至细胞密度为9×10⁶ cells/ml时方可传代。传代密度继续使用3×10⁶ cells/ml，直至细胞存活率恢复至95%以上时，可将传代密度恢复至0.3-0.6×10⁶ cells/ml。
(2) 当细胞存活率为60%-80%时
将细胞离心去上清，用新鲜CHO无血清细胞培养基重悬，使其密度为0.5×10⁶ cells/ml，接种于培养瓶中静置培养。每2天观察一次细胞是否生长。若细胞有生长，继续培养至细胞密度为2.0×10⁶ cells/ml后，传代至0.5×10⁶ cells/ml于培养瓶中静置培养。重复上述过程，直至细胞活率大于90%时，可转移至摇瓶中培养，此时接种密度应不少于2.0×10⁶ cells/ml。在静置培养过程中若发现有细胞贴壁，可继续培养。当细胞密度达到一定程度时，部分细胞会自然脱落，继续贴壁的细胞则可以借助移液管的轻微吹打脱离培养瓶。
(3) 细胞存活率小于60%时
丢弃细胞，重新获取细胞。
4. 细胞结团
检查是否往培养液中添加了其它物质，若使用可重复使用的玻璃摇瓶，需检查瓶子是否清洗干净。如果结团细胞成球状，可能是因为添加了其它外源物质或洗涤剂；如果结团细胞成片状或不规则的松散结构，可能是因为摇瓶有上一次培养时没洗干净的残留物粘附于瓶身或细胞成活率较低。
CHO Serum-Free Cell Culture Medium is a chemically defined, serum-free, and animal-derived component-free basal high-density CHO cell culture medium. It contains glucose and L-Alanyl-L-Glutamine (a stable dipeptide that serves as a long-acting glutamine source). This medium is suitable for high-density suspension culture of CHO-K1 cells and other CHO cell lines, as well as for formulation optimization and process development based on this medium.
Product Features
Chemically defined, free from animal-derived components, and contains no animal-derived recombinant proteins or protein hydrolysates.
Supports high-density suspension culture of cells, suitable for research in protein expression technologies such as recombinant antibodies and recombinant protein factors.
Stable performance, quality-controlled, with low batch-to-batch variability.
For research use only. Not for use in diagnostic or therapeutic procedures.
Instructions for Use
1. Cell Medium Exchange and Subculture
1.1 Newly received cells shipped at ambient temperature should be immediately transferred to an appropriate shake flask (culture volume should be controlled within one-fifth of the flask's nominal capacity) and cultured with agitation on a shaker. When the cell density recovers to 3-6×10⁶ cells/ml, subculture or cryopreservation can be performed.
1.2 For cells taken from a liquid nitrogen tank or shipped on dry ice, please follow the method described in section (2. Cell Cryopreservation and Recovery) for cell recovery and culture.
1.3 For subculture, perform a cell count first. After confirming the density, centrifugation is not required. The cell suspension can be directly diluted into fresh culture medium at the required ratio. The cell density after subculture should be controlled at 0.3×10⁶ cells/ml, typically requiring subculture every 4 days; alternatively, seed at 0.6×10⁶ cells/ml and subculture every 3 days. This medium can support a maximum cell density of approximately 1×10⁷ cells/ml, with viability generally remaining above 95% upon reaching this density.
1.4 If excessive cell death occurs during shake culture, seed at a low cell density for static culture. Once the cells recover normal viability, resume shake culture.
2. Cell Cryopreservation and Recovery
2.1 Cryopreservation
(1) Prepare cell freezing medium containing 7.5% (v/v) dimethyl sulfoxide (DMSO) using the cell culture medium.
(2) Culture cells to the logarithmic growth phase (density approximately 6×10⁶ cells/ml), count, and collect the cells by centrifugation.
(3) Resuspend the pelleted cells at a concentration of 5-20×10⁶ cells/ml in the prepared freezing medium.
(4) Dispense the cell suspension into labeled cryovials, ensuring the caps are tightly sealed.
(5) Place the cryovials in a -80°C freezer for slow cooling.
(6) The next day, transfer the cryovials to liquid nitrogen for long-term storage. This transfer must be done as quickly as possible (recommended within 2 minutes). If the temperature of the cryovials rises above -50°C during this process, the cells may be rapidly damaged.
2.2 Recovery
(1) Remove the cell cryovial from liquid nitrogen, dry ice, or an ultra-low temperature freezer and immediately place it in a 37°C water bath until the ice crystals are completely thawed.
(2) After thawing, thoroughly wipe the cryovial with 75% ethanol. Using a pipette, transfer the entire cell suspension to a sterile 50 mL centrifuge tube. Slowly add fresh cell culture medium to a final volume of 20 mL.
(3) Centrifuge at 1000 rpm for 5 minutes and discard the supernatant containing the freezing medium.
(4) Resuspend the cell pellet in fresh CHO Serum-Free Cell Culture Medium to a density of 0.6-1.0×10⁶ cells/ml and inoculate into a shake flask for culture with agitation. Count the cells after 3-6 days of culture to observe whether cell density and viability have increased. If the cell density reaches 3-6×10⁶ cells/ml, the cells can be subcultured. Generally, cells return to normal growth status after 1-2 passages.
(5) For cells frozen using a serum-free cell freezing medium, they can be directly thawed, transferred to a shake flask containing pre-warmed CHO Serum-Free Cell Culture Medium, and resuspended. Culture for 3-6 days, then count the cells to observe whether cell density and viability have increased.
(6) If the cell density is too low after thawing, they can be initially seeded in a culture flask for static culture. Once the density recovers to 0.6-1.0×10⁶ cells/ml, transfer them back to shake flasks for culture with agitation.
Key Operating Points and Precautions
1. Recommended Shaker Parameters
Temperature: 36-37°C, CO₂ Concentration: 5%, Rotation Speed: ≤120 rpm (for 26 mm orbit).
For shakers with different orbit diameters, the calculation formula is as follows:
For a 50 mm orbit, calculate using the formula: m × ω₁² × 26² = m × ω₂² × 50², thus the relationship between ω₁ and ω₂ is: ω₁ ≈ 1.4 ω₂. This corresponds to approximately 86 rpm.
2. Adaptation Protocol
If cells were previously cultured in another brand's medium, it is recommended to gradually adapt them to the CHO Serum-Free Cell Culture Medium. Continue subculturing for several weeks until the cells return to normal growth status before proceeding with further experiments.
3. Troubleshooting Reduced Cell Density and Viability
(1) When cell viability is 80%-95%:
Centrifuge the cells, remove the supernatant, and resuspend the pellet in fresh culture medium at a density of 3×10⁶ cells/ml. Inoculate into a shake flask and culture with agitation. Observe daily for cell proliferation. If proliferation occurs, continue culturing until the cell density reaches 9×10⁶ cells/ml before subculturing. Continue subculturing at 3×10⁶ cells/ml until cell viability recovers above 95%. Then, the seeding density can be returned to 0.3-0.6×10⁶ cells/ml.
(2) When cell viability is 60%-80%:
Centrifuge the cells, remove the supernatant, and resuspend the pellet in fresh CHO Serum-Free Cell Culture Medium at a density of 0.5×10⁶ cells/ml. Inoculate into a culture flask for static culture. Observe for cell growth every 2 days. If growth is observed, continue static culture until the cell density reaches 2.0×10⁶ cells/ml. Then, subculture by seeding at 0.5×10⁶ cells/ml into a new culture flask for static culture. Repeat this process until cell viability exceeds 90%. At this point, cells can be transferred to shake flask culture with an initial seeding density of no less than 2.0×10⁶ cells/ml. During static culture, if cells are observed attaching, continue culturing. When cell density reaches a certain level, some cells will naturally detach. Cells remaining attached can be dislodged by gentle pipetting.
(3) When cell viability is less than 60%:
Discard the cells and obtain a fresh batch.
4. Cell Clumping
Check if any additional substances have been added to the culture medium. If using reusable glass shake flasks, verify that the flasks are cleaned properly. If clumped cells form spherical aggregates, it may be due to the addition of foreign substances or detergent residue. If clumped cells form sheet-like or irregular loose structures, it may be due to residual material from previous cultures adhering to the flask wall or low cell viability.
规格
别名
ProCHO 无血清培养基 | CHO无血清专用培养基
英文别名
CD CHO Medium | ProCHO | CHO Serum-Free Suspension Specific Medium
规格或纯度
BioReagent,无菌,用于细胞培养
稳定性与储存
Store at 2-8℃ long term (9 months). Store in the dark.
英文名称
CHO Serum-Free Medium (with L-Alanyl-L-Glutamine)
储存条件
2-8°C储存,避光
运输条件
低温运输
产品线
biological_products
名称和识别符
分子类型
培养基
技术文档
高级数据
化学和物理性质
敏感性
Light-sensitive
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