是否无菌：是
本系列为LMH细胞通用型无血清全悬浮培养基，广泛应用于禽用疫苗的生产，包括以下两类：
[L1525352] LMH 生长培养基 ：用于LMH悬浮细胞的常规扩增培养，维持细胞高活力和快速增殖。
[L1525353] LMH 扩毒培养基 ：用于LMH细胞接种流感病毒后的病毒增殖阶段，优化病毒产量。
产品参数
参数 | 指标 | 外观（液体） | 澄清浅黄色液体 | pH | 6.8 ~ 7.2 | 渗透压 | 280 ~ 320 mOsm/kg | 内毒素 | < 2 EU/mL | 含谷氨酰胺 | 是 | 适用细胞 | LMH | 倍增时间（典型值） | 28 h | 保质期 | 2-8℃，12个月 |
|
使用范围
仅限科研及工业生产使用，不可用于人体。
培养基适应
直接适应：最初培养阶段，细胞按初始密度1.5×10⁶ cells/mL 接种，直接将培养基更换为LMH 生长培养基进行培养。待细胞培养2-3 代，且生长稳定后进行后续实验。LMH 细胞稳定生长时按1.5×10⁶ cells/mL 密度接种，培养72h 后，细胞密度≥9.0×10⁶cells/mL，细胞活率≥95%。
间接适应： LMH 生长培养基与细胞原培养基1：1 混合培养细胞，连续传2-3 代，细胞稳生长后可将培养基更换为LMH 生长培养基，再传2-3 代，细胞生长稳定后进行后续实验。LMH 细胞稳定生长时按照1.5×10⁶ cells/mL 密度接种，培养72h 后，细胞密度≥9.0×10⁶ cells/mL，细胞活率≥95%。
注意事项：
根据细胞具体情况选择进行培养基直接适应或者间接适应。
细胞驯化初期，若出现细胞生长密度较低、细胞结团等其他不稳定状态，根据当天细胞密度选择传代方式（稀释传代、离心传代）。
稀释传代：根据细胞密度计算传代时所需细胞悬液，去除多余细胞悬液并补加新鲜培养基至培养体积。
离心传代：根据细胞密度计算传代时所需细胞悬液，进行800rpm 离心5min，去除上清，用新鲜培养基重悬细胞。
细胞恢复正常状态传代：
| 细胞名称 | 培养基 | 接种密度
(cells/mL) | 第三天细胞密度
(cells/mL)
| 细胞活率
(%) | 平均倍增时间
(h) |
| LMH | LMH生长培养基
| 1.5×10⁶ | ≥9.0×10⁶ | ≥95% | 28 |
LMH 悬浮细胞冻存
1.待细胞恢复至正常状态后，扩大细胞培养体积准备冻存建库。尽量多建细胞库，保证备份充足。
2.选择处于对数生长期、活率＞95%的细胞进行冻存，冻存细胞密度:15-20×10⁶ cells/mL，推荐20×10⁶ cells/mL。
3.准备冻存盒：向程序降温盒注入适量异丙醇后，置于4℃冰箱预冷。
4.配制细胞冻存液：冻存液= 70%新鲜培养基+ 20% FBS(建议使用进口胎牛血清) + 10%DMSO，冻存液配好后置于4℃冰箱预冷（冻存液配制时，先加培养基再加入血清或DMSO，防止DMSO 浓度过高导致血清有效成分变性）。
5.取细胞悬液，800rpm 离心5min，弃去上清，用适量细胞冻存液重悬细胞，调整细胞密度至目标值。
6.快速分装细胞液至冻存管中。
7.将冻存管放入程序降温盒中，放于-80℃冰箱，24h 后转至液氮罐中储存。一段时间后取一只复苏，检测冻存是否成功。
LMH悬浮细胞复苏
1.预热培养基：取15mL 对应培养基置于摇瓶中，放入37℃二氧化碳培养箱中预热30min 以上（平衡培养基pH 值），保证预热充分。
2.将冻存管从液氮罐或-80℃冰箱中取出，迅速转移至37℃水浴中，快速摇晃，直至完全融化。
3.取预热好的培养基5mL 于无菌离心管中，并将解冻后的细胞悬液移入此离心管中。800rpm 离心5min（除去冻存液中DMSO）。
4.离心后去除上清，用10mL 预热好的培养基（保证较高细胞复苏密度）重悬细胞，移回相应摇瓶，放于培养箱中悬浮培养。
LMH悬浮细胞传代
1.预热培养基：取适量对应培养基置于37℃预热，保证预热充分。
2.细胞复苏初期传代：细胞复苏第一代，如果细胞密度大于2.5×10⁶ cells/mL，取部分细胞液离心传代，按初始密度2.0×10⁶ cells/mL接种；如果细胞密度小于2.5×10⁶ cells/mL，进行全部离心换液（细胞刚复苏会有细胞碎片，属正常现象），细胞复苏后，前4代为细胞恢复期，期间细胞密度和活率较低，出现细胞碎片，属正常现象）, 待细胞恢复稳定生长状态时，可进行正常稀释传代。
LMH悬浮细胞建库
1.待细胞恢复至正常状态后，扩大细胞培养体积准备冻存建库。尽量多建细胞库，保证备份充足。
2.选择处于对数生长期、活率大于90%的细胞进行冻存，冻存细胞密度:15-20×10⁶ cells/mL，推荐20×10⁶ cells/mL。
3.准备冻存盒：向程序降温盒注入适量异丙醇后，置于4℃冰箱预冷。
4.配制细胞冻存液：冻存液=70%对应培养基+20%FBS(建议使用进口胎牛血清)+10%DMSO，冻存液配好后置于4℃冰箱预冷（冻存液配制时，先加培养基再加入血清或DMSO，防止DMSO浓度过高导致某些有效成分变性）。
5.取细胞悬液，800rpm离心5min，弃去上清，用适量细胞冻存液重悬细胞，调整细胞密度至目标值。
6.快速分装细胞液至冻存管中。
7.将冻存管放入程序降温盒中，放于-80℃冰箱，24h后转至液氮罐中储存。一段时间后取一支复苏，检测冻存是否成功。
LMH悬浮细胞禽流感接毒方案：
1、摇瓶小试实验
由于毒株差异，需要通过摇瓶小试实验，确定自己毒株合适的接毒量及收获
细胞株 ① | 培养培养基 | 接毒量（体积比） ② | 接毒 方式 | 接毒培养基 | 接毒密度 （cells/mL） | 温度（℃） | 收获时间（h） ③ | TCID₅₀ /mL | LMH | [L1525352] LMH 生长培养基 | 2‰ | 稀释 | [L1525353] LMH 扩毒培养基 | 2.5×10⁶ | 35 | 72 | ≥8.3 |
|
注
① 健康LMH细胞培养条件为：温度为37℃、转速为140-160rpm、CO₂浓度5%。生长至第三天，细胞密度≥9.0×106 cells/mL，活率≥95%。
② 由于不同的毒株接毒量存在差异，可设计接毒量梯度，确定最优接毒量，工艺稳定性与种毒品质相关，建议选用高品质种毒。
③ 时间收获：由于不同毒株存在差异，需根据实际细胞病变情况确定最优的收获时间，建议收获时间为72h
This series is a universal serum‑free, fully‑suspended medium for LMH cells, specifically designed for influenza vaccine production and research applications. It includes the following two types:
[L1525352] LMH Growth Medium: used for routine expansion of LMH suspension cells to maintain high viability and rapid proliferation.
[L1525353] LMH Virus Propagation Medium: used during the viral replication stage after LMH cells are inoculated with influenza virus, to optimize virus yields.
Product Specifications
[M1525341] LMH Growth Medium (for human influenza vaccine)
| Parameter | Specification |
|---|
| Appearance (liquid) | Clear, light‑yellow liquid | | pH | 6.8 – 7.2 | | Osmolality | 280 – 320 mOsm/kg | | Endotoxin | < 2 EU/mL | | Contains glutamine | Yes | | Suitable cells | LMH | | Doubling time (typical) | 28 h | | Shelf life | 2‑8 °C, 12 months |
|
Intended use
For research and industrial production only. Not for human use.
Medium Adaptation
1.Direct adaptation
In the initial culture phase, cells are seeded at an initial density of 1.5×10⁶ cells/mL, and the medium is directly replaced with LMH growth medium. After 2–3 passages, when cell growth is stable, subsequent experiments can be performed. For stable LMH cell growth, cells are seeded at 1.5×10⁶ cells/mL; after 72 h of culture, the cell density should be ≥9.0×10⁶ cells/mL with a viability ≥95%.
2.Indirect adaptation
LMH growth medium and the cell's original medium are mixed at a 1:1 ratio for culture. Cells are passaged continuously for 2–3 generations. After stable growth is achieved, the medium is replaced with LMH growth medium, and cells are passaged for another 2–3 generations. Once growth is stable, subsequent experiments can be performed. For stable LMH cell growth, cells are seeded at 1.5×10⁶ cells/mL; after 72 h of culture, the cell density should be ≥9.0×10⁶ cells/mL with a viability ≥95%.
3.Precautions
①Choose either direct or indirect adaptation based on the specific condition of the cells.
②During the initial adaptation phase, if cells show low density, aggregation, or other unstable states, select the appropriate subculturing method (dilution passaging or centrifugation passaging) based on the cell density on that day.
③Dilution passaging: Calculate the required cell suspension volume based on cell density, remove the excess cell suspension, and add fresh medium to reach the culture volume.
④Centrifugation passaging: Calculate the required cell suspension volume, centrifuge at 800 rpm for 5 min, remove the supernatant, and resuspend the cell pellet in fresh medium.
4. Normal cell passage after recovery:
| Cell line | Medium | Seeding density (cells/mL) | Day‑3 cell density (cells/mL) | Viability (%) | Average doubling time (h) |
|---|
| LMH | LMH Growth Medium | 1.5×10⁶ | ≥9.0×10⁶ | ≥95% | 28 |
|
Cryopreservation of LMH Suspension Cells
1.After cells return to normal status, expand the culture volume for cryopreservation and cell banking. Prepare as many cell banks as possible to ensure sufficient backups.
2.Select cells in the logarithmic growth phase with viability >95% for cryopreservation. Cryopreservation density: 15–20×10⁶ cells/mL; 20×10⁶ cells/mL is recommended.
3.Prepare the freezing container: Add an appropriate amount of isopropanol to the controlled-rate freezing container and pre‑cool at 4°C.
4.Prepare cell cryopreservation medium: 70% fresh medium + 20% FBS (preferably imported fetal bovine serum) + 10% DMSO. After preparation, pre‑cool at 4°C. (When preparing the freezing medium, add medium first, then serum and DMSO, to prevent protein denaturation caused by excessively high DMSO concentration.)
5.Collect cell suspension, centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend cells with an appropriate volume of cryopreservation medium to adjust to the target cell density.
6.Quickly aliquot the cell suspension into cryovials.
7.Place the cryovials into the controlled-rate freezing container and store at –80°C. After 24 h, transfer to liquid nitrogen for long‑term storage. After a period of time, thaw one vial to verify successful cryopreservation.
Thawing of LMH Suspension Cells
1.Pre‑warm medium: Place 15 mL of corresponding medium in a shake flask and pre‑warm in a 37°C CO₂ incubator for at least 30 min (to equilibrate the pH) to ensure thorough pre‑warming.
2.Remove the cryovial from liquid nitrogen or –80°C storage, and quickly transfer to a 37°C water bath with rapid shaking until completely thawed.
3.Transfer 5 mL of pre‑warmed medium into a sterile centrifuge tube, then transfer the thawed cell suspension into this tube. Centrifuge at 800 rpm for 5 min (to remove DMSO).
4,After centrifugation, discard the supernatant, resuspend the cells in 10 mL of pre‑warmed medium (ensuring a relatively high seeding density for recovery), transfer back to the corresponding shake flask, and culture in the incubator under suspension conditions.
Subculture of LMH Suspension Cells
1.Pre‑warm medium: Place an appropriate volume of the corresponding medium at 37°C to ensure thorough warming.
2.Subculture in early recovery: For the first passage after thawing, if cell density is >2.5×10⁶ cells/mL, take a portion of the cell suspension for centrifugation and subculture at an initial density of 2.0×10⁶ cells/mL. If cell density is <2.5×10⁶ cells/mL, perform complete centrifugation and medium exchange (cell debris is normal right after thawing). The first four passages constitute the recovery period; during this time, cell density and viability may be low, and debris may appear – this is normal. Once cells recover to stable growth, normal dilution subculture can be performed.
Cell Banking of LMH Suspension Cells
1.After cells return to normal status, expand the culture volume for cryopreservation and banking. Prepare as many cell banks as possible to ensure sufficient backups.
2.Select cells in logarithmic growth phase with viability >90% for cryopreservation. Cryopreservation density: 15–20×10⁶ cells/mL; 20×10⁶ cells/mL is recommended.
3.Prepare the freezing container: Add an appropriate amount of isopropanol to the controlled‑rate freezing container and pre‑cool at 4°C.
4.Prepare cell cryopreservation medium: 70% corresponding medium + 20% FBS (preferably imported fetal bovine serum) + 10% DMSO. Pre‑cool at 4°C after preparation. (Add medium first, then serum and DMSO, to prevent denaturation of certain active components due to high DMSO concentration.)
5.Collect cell suspension, centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend cells with an appropriate volume of cryopreservation medium to the target density.
6.Quickly aliquot into cryovials.
7.Place cryovials in the controlled‑rate freezing container, store at –80°C for 24 h, then transfer to liquid nitrogen. After a period, thaw one vial to verify successful banking.
Avian Influenza Virus Infection Protocol for LMH Suspension Cells
Shake flask small‑scale experiment
Because viral strains differ, it is necessary to conduct shake flask experiments to determine the optimal virus inoculation volume and harvest time for your specific strain.
| Cell Line ① | Culture Medium | Inoculum Volume (v/v) ② | Inoculation Method | Infection Medium | Seeding Density (cells/mL) | Temperature (°C) | Harvest Time (h) ③ | TCID₅₀ /mL |
|---|
| LMH | [L1525352] LMH growth medium | 2‰ | Dilution | [L1525353] LMH virus propagation medium | 2.5×10⁶ | 35 | 72 | ≥8.3 |
|
Notes:
① Healthy LMH cell culture conditions: temperature 37°C, agitation speed 140–160 rpm, CO₂ concentration 5%. On day 3 of growth, cell density ≥9.0×10⁶ cells/mL, viability ≥95%.
② Because different virus strains require different inoculum volumes, design a gradient of inoculation amounts to determine the optimal volume. Process stability is related to seed virus quality; it is recommended to use high‑quality seed virus.
③ Harvest time: Since different strains vary, determine the optimal harvest time based on actual cytopathic effects. The recommended harvest time is 72 h.
ZJ26F0838680
危险品化学品经营许可证（带存储）