本系列为MDCK细胞通用型无血清全悬浮培养基，专门用于流感疫苗的生产及科研应用。包括以下两类：
[M1525341] MDCK生长培养基 (不含酚红, 含谷氨酰胺) ：用于MDCK悬浮细胞的常规扩增培养，维持细胞高活力和快速增殖。
[M1525342] MDCK扩毒培养基 (不含酚红, 含谷氨酰胺)：用于MDCK细胞接种流感病毒后的病毒增殖阶段，优化病毒产量。
使用范围
仅限科研及工业生产使用，不可用于人体。
本产品含有两个组分：
| 组分 | 配方量 | | Part1 | 28.98g/L | | Part2 | 0.7g/L |
|
配制说明：
1.确定配制总体积，选择两个容器进行配制，其中容器 1 体积大于总体积，容器 2 体积大于总体积 10%；
2.按照配方量在容器 1 中加入 Part1 培养基粉末，再加入约配制总体积 80%的超纯水或注射用水，建议机械搅拌至少 30min；
3.按照配方量在容器 2 中加入 Part2 培养基粉末，再加入约配制总体积 10%的超纯水或注射用水，建议机械搅拌 30min；
4.将溶解后的容器 2 中的液体移入容器 1 中混合；
5.建议机械搅拌至少 60min 至粉末全部溶解；
6.充分溶解后测定培养基 pH，若测定值在正常范围 7.0-7.4 内则无需调整，若测定值超出正常范围，则用 1M 的 HCl 或 5M 的 NaOH 调节至正常范围；
7.使用注射用水或超纯水调整总体积至目标值；
8.无菌过滤，2-8℃避光保存
培养基适应
直接适应：最初培养阶段，细胞按初始密度1×10⁶ cells/mL 接种，直接将培养基更换为MDCK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。MDCK 细胞稳定生长时按1×10⁶ cells/mL 密度接种，培养72h 后，细胞密度≥6.0×10⁶cells/mL，细胞活率≥95%。
间接适应：MDCK S05 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MDCK生长培养基，再传2-3 代，细胞生长稳定后进行后续实验。MDCK 细胞稳定生长时按照1×10⁶ cells/mL 密度接种，培养72h 后，细胞密度≥6.0×10⁶ 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) |
| MDCK | MDCK生长培养基
| 1.0×10⁶ | ≥6.0×10⁶ | ≥95% | 28 |
MDCK 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＞90%的细胞进行冻存，冻存细胞密度:10-15×10⁶ cells/mL，推荐15×10⁶ 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= 80%新鲜培养基+ 10% 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。
MDCK 悬浮细胞复苏
1.预热培养基：取25mL 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20mL 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。
MDCK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2×10⁶ cells/mL，取部分细胞液离心传代，按初始密度1.5×10⁶ cells/mL接种；如果细胞密度小于2×10⁶ 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）。
MDCK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98%的细胞进行冻存，冻存细胞密度:5-15×10⁶ cells/mL，推荐15×10⁶ 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=80%对应培养基+10%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。
MDCK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③ | 毒株 | 接毒培养基 | 温度（℃） | 0h补加 普通胰酶 (mg/L) ④ | 收获时间(h)⑤ | HA （log2） | MDCK | [M1525341] MDCK生长培养基 | 0.2‰ | 按6×10⁶ cells/mL密度稀释 | H5、H7、H9 | [M1525342] MDCK扩毒培养基 | 33 | 70 | 72-96h | 10-11 |
|
注
①健康MDCK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≥10.0×10⁶ cells/mL，活率≥98%。
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（HA≥9(log2)、病毒含量≥10⁸‧⁵(EID₅₀/0.1mL)）。
③接毒方式：取生长至第三天的健康细胞悬液按照6×10⁶ cells/mL密度用病毒扩增培养基稀释后接毒，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稀释密度。
④普通胰酶建议配制浓度为10mg/mL，例：按70mg/L添加，30mL接毒体系加入210μL普通胰酶。
⑤收获时间：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-96h。
This series is a universal serum‑free, fully‑suspended medium for MDCK cells, specifically designed for influenza vaccine production and research applications. It includes the following two types:
[M1525341] MDCK Growth Medium (Phenol Red-Free, with L-Glutamine): used for routine expansion of MDCK suspension cells to maintain high viability and rapid proliferation.
[M1525342] MDCK Virus Propagation Medium (Phenol Red-Free, with L-Glutamine): used during the viral replication stage after MDCK cells are inoculated with influenza virus, to optimize virus yields.
Product Specifications
[M1525341] MDCK Growth Medium (for human influenza vaccine)
| Parameter | Specification |
|---|
| Appearance (liquid) | Clear, light‑yellow liquid | | pH | 7.0– 7.4 | | Osmolality | 280 – 320 mOsm/kg | | Endotoxin | < 2 EU/mL | | Contains glutamine | Yes | | Suitable cells | MDCK | | 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 stage, seed cells at a density of 1×10⁶ cells/mL and replace the medium directly with MDCK Growth Medium. After culturing for 2–3 passages and once cell growth is stable, proceed with subsequent experiments. When stably growing, MDCK cells are seeded at 1×10⁶ cells/mL and cultured for 72 h; the cell density should reach ≥6.0×10⁶ cells/mL with viability ≥95%.
2.Indirect adaptation
Mix MDCK S05 cells with the original medium and MDCK Growth Medium at a 1:1 ratio, and culture for 2–3 consecutive passages. Once cells grow stably, replace the medium completely with MDCK Growth Medium and passage for another 2–3 generations. After stable growth is achieved, seed at 1×10⁶ cells/mL and culture for 72 h; the cell density should reach ≥6.0×10⁶ cells/mL with 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) |
|---|
| MDCK | MDCK Growth Medium | 1.0×10⁶ | ≥6.0×10⁶ | ≥95% | 28 |
|
Cryopreservation of MDCK Suspension Cells
1.After cells return to normal growth, expand the culture volume to prepare for cryopreservation and banking. Prepare as many vials as possible to ensure sufficient backups.
2.Select cells in the logarithmic growth phase with viability >90%. Recommended cryopreservation density: 10‑15×10⁶ cells/mL, with 15×10⁶ cells/mL 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 the freezing medium: 80% fresh medium + 10% FBS (preferably imported fetal bovine serum) + 10% DMSO. After preparation, keep at 4°C. (When preparing, add serum and DMSO after the medium to prevent denaturation of serum components due to high DMSO concentration.)
5.Harvest the cell suspension, centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend the cells in an appropriate volume of freezing medium to reach the target cell density.
6.Quickly aliquot the cell suspension into cryovials.
7.Place the cryovials in the controlled‑rate freezing container, store at ‑80°C, and transfer to liquid nitrogen after 24 h. After some time, thaw one vial to verify successful cryopreservation.
Thawing of MDCK Suspension Cells
1.Pre‑warm the medium: place 25 mL of the corresponding medium in a shake flask and incubate in a 37°C CO₂ incubator for at least 30 min to equilibrate the pH.
2.Remove the cryovial from liquid nitrogen or ‑80°C, and immediately transfer to a 37°C water bath. Shake rapidly until completely thawed.
3.Transfer 5 mL of pre‑warmed medium into a sterile centrifuge tube, then add the thawed cell suspension. Centrifuge at 800 rpm for 5 min (to remove DMSO).
4.After centrifugation, discard the supernatant and resuspend the cells in 20 mL of pre‑warmed medium (to ensure a high recovery density). Transfer back to the shake flask and culture in suspension.
Subculturing of MDCK Suspension Cells
1.Pre‑warm the medium: place an appropriate volume of the corresponding medium at 37°C until fully pre‑warmed.
2.Initial passage after thawing: if the cell density is >2×10⁶ cells/mL, take an aliquot for centrifugation and passage at an initial density of 1.5×10⁶ cells/mL. If the density is <2×10⁶ cells/mL, perform complete medium exchange by centrifugation (some cell debris is normal after thawing).
Cell Banking for MDCK Suspension Cells
1.After cells recover to normal growth, expand the culture volume for banking. Prepare as many vials as possible.
2.Select cells in logarithmic growth phase with viability >98%. Recommended cryopreservation density: 5‑15×10⁶ cells/mL, with 15×10⁶ cells/mL recommended.
3.Prepare the freezing container: add isopropanol and pre‑cool at 4°C.
4.Prepare the freezing medium: 80% corresponding medium + 10% FBS + 10% DMSO. Pre‑cool at 4°C.
5.Harvest the cells, centrifuge at 800 rpm for 5 min, discard supernatant, resuspend in freezing medium to target density.
6.Quickly aliquot into cryovials.
7.Place vials in the freezing container, store at ‑80 °C for 24 h, then transfer to liquid nitrogen. Thaw one vial after a period to confirm successful freezing.
Avian Influenza Virus Infection Protocol for MDCK Suspension Cells
Small‑scale shake‑flask trial
Due to strain variations, a small‑scale trial is necessary to determine the optimal virus inoculation volume and harvest time for your specific virus strain.
| Cell line① | Growth medium | Virus inoculum (v/v) ② | Infection method③ | Virus strain | Infection medium | Temperature (°C) | 0 h supplement | Trypsin(mg/L) ④ | Harvest time (h) ⑤ | HA titer (log₂) |
|---|
| MDCK | [M1525341] MDCK Growth Medium | 0.2‰ | Dilute to 6×10⁶ cells/mL | H5, H7, H9 | [M1525342] MDCK Propagation Medium | 33 | – | 70 | 72‑96 | 10‑11 |
|
Notes:
① Healthy MDCK cells are cultured at 37 °C, 140‑160 rpm, 5% CO₂. By day 3, cell density should reach ≥10.0×10⁶ cells/mL with viability ≥98%.
② Because different virus strains require different inoculum amounts, design a gradient of inoculum concentrations to determine the optimal amount. Process stability is related to seed virus quality; use high‑quality seed virus (HA ≥9 log₂, virus titre ≥10⁸·⁵ EID₅₀/0.1 mL).
③ Infection method: take healthy cell suspension on day 3, dilute to 6×10⁶ cells/mL using virus propagation medium, then inoculate with virus. Because strains differ, optimize the dilution density based on cytopathic effect (CPE) observations.
④ Prepare trypsin stock solution at 10 mg/mL. For example, for 70 mg/L addition, add 210 μL of stock trypsin to a 30 mL infection system.
⑤ Harvest time: because strains differ, determine the optimal harvest time based on CPE. The recommended harvest window is 72‑96 h.
危险品化学品经营许可证（带存储）