本PBMC 细胞冻存液是专为外周血单个核细胞 (Peripheral Blood Mononuclear Cell, PBMC)深低温冻存设计的即用型无血清冻存保护液。配方采用渗透性与非渗透性冷冻保护剂优化配比，兼顾降温过程中冰晶损伤与渗透压损伤的平衡，其特点有：
1、无需自行配制，即开即用；
2、 冻存及复苏过程对细胞损伤小，复苏后细胞存活率≥90% (以实际检定数据为准)；
3、 复苏后细胞表型 (CD3/CD4/CD8/CD19 等)与冻存前保持一致，增殖与功能活性不受显著影响；
4、适用于 PBMC 及多种悬浮免疫细胞的 -80℃ 短期保存与液氮（-196℃）长期保存。
主要成分：
| 成分类别 | 说明 |
| 冷冻保护剂 | DMSO (终浓度≤10%) |
| 血清替代成分 | 无动物源成分，蛋白水解物 / 白蛋白类替代物 |
| 缓冲体系 | 缓冲盐体系，维持生理 pH |
| 营养与稳定成分 | 氨基酸、糖类、抗氧化剂等 |
| 抗生素 | 不含（如需添加由使用者自行决定） |
使用方法：
1. 细胞冻存
1.1. 取新鲜分离或培养的 PBMC，计数并确认活率≥90% (台盼蓝或 AO/PI 法)；
1.2. 300 × g 离心 5–10 min，室温，弃上清；
1.3. 用预冷（2–8℃）的 PBMC 细胞冻存液重悬细胞，调整密度至 1×10⁷–1×10⁸ cells/mL，轻柔混匀；
1.4. 按每管 1 mL 分装至无菌冻存管，拧紧管盖并标记；
1.5. 程序降温：将冻存管置于程序降温盒（含异丙醇）于 -80℃ 放置≥4 h（或按 4℃ 10 min → -20℃ 30 min → -80℃ 过夜 的梯度降温）；
1.6. 长期保存：将冻存管转移至液氮（-196℃）中储存，并做好位置登记。
2. 细胞复苏
2.1. 将冻存管自液氮中取出，迅速放入 37℃ 水浴，轻摇使其在 1–2 min 内完全融化；
2.2. 用 75% 酒精擦拭管壁消毒，将细胞悬液缓慢转移至含预温完全培养基的离心管中 (滴加稀释，边加边轻摇)；
2.3. 300 × g 离心 5 min，弃上清，去除冻存保护液；
2.4. 用新鲜培养基重悬细胞，按实验需求接种培养或直接进行下游检测；
2.5. 复苏后 24 h 建议计数并检测存活率，确认复苏效果。
提示：本操作流程为推荐方案，客户可根据自身工艺 (如 CAR-T / NK 工艺、全血分离流程)进行优化。
注意事项：
1、本产品仅限科研使用（For Research Use Only），严禁用于临床诊断、治疗及人体回输；
2、操作全程需无菌环境（生物安全柜）进行，避免微生物污染；
3、配方含 DMSO，具有一定挥发性和皮肤刺激性，建议在通风条件下操作，避免直接接触皮肤与黏膜；
4、产品为即用型，请勿稀释、额外添加 DMSO 或反复冻融；
5、开启后建议一次用完，如需分装，请在无菌条件下操作并于 2–8℃ 短期保存；
6、若发现液体浑浊、沉淀、变色等异常，请勿使用并联系供应商；
7、废弃液体及接触耗材请按生物危害废弃物处理规范处置。
常见问题：
1、复苏后存活率偏低怎么办？
建议：检查降温程序是否规范 (避免直接 -80℃ 快速冻结)；确认水浴复苏在1–2 min 内完成；核实冻存前细胞状态。
2、冻存后细胞成团怎么办？
建议：复苏稀释时缓慢滴加、轻摇混匀；必要时使用 40 μm 滤网过滤。
3、能否用于 T 细胞 / NK 细胞？
建议：本产品针对 PBMC 优化，兼容 T、NK 等免疫细胞；大规模工艺建议选用对应专用冻存液。
4、可否用于治疗 / 临床？
建议：科研级不可用于临床，临床需求请选用治疗级产品。
This PBMC Cryopreservation Medium is a ready‑to‑use serum‑free cryoprotective solution specially designed for cryopreservation of Peripheral Blood Mononuclear Cells (PBMC). Formulated with an optimized ratio of permeable and non‑permeable cryoprotectants, it balances ice crystal damage and osmotic injury during cooling. Key features are as follows:
1. Ready‑to‑use, no on‑site preparation required;
2. Minimal cell damage during freezing and thawing; post‑thaw cell viability ≥90% (subject to actual test results);
3. Post‑thaw cell phenotypes (CD3/CD4/CD8/CD19, etc.) remain consistent with pre‑freezing status, with no significant impairment to proliferation and functional activity;
4. Suitable for short‑term storage at −80 °C and long‑term storage in liquid nitrogen (−196 °C) of PBMC and various suspended immune cells.
Main Components:
| Component Category | Description |
| Cryoprotectant | DMSO (final concentration ≤10%) |
| Serum replacement components | Animal-origin-free, protein hydrolysate / albumin substitutes |
| Buffer system | Buffered salt system to maintain physiological pH |
| Nutritional and stabilizing components | Amino acids, carbohydrates, antioxidants, etc. |
| Antibiotics | None (to be added by user if required) |
Instructions for Use:
1. Cell Cryopreservation
1.1 Harvest freshly isolated or cultured PBMC, count cells and confirm viability ≥90% (Trypan Blue or AO/PI method).
1.2 Centrifuge at 300 × g for 5–10 min at room temperature and discard the supernatant.
1.3 Resuspend the cell pellet with pre‑chilled (2–8 °C) PBMC Cryopreservation Medium, adjust cell density to 1×10⁷–1×10⁸ cells/mL, and mix gently.
1.4 Aliquot 1 mL into each sterile cryovial, cap tightly and label.
1.5 Controlled cooling: Place cryovials in a controlled‑rate cooling container (with isopropanol) at −80 °C for ≥4 h, or follow gradient cooling: 4 °C for 10 min → −20 °C for 30 min → overnight at −80 °C.
1.6 Long‑term storage: Transfer cryovials to liquid nitrogen (−196 °C) and record storage locations.
2. Cell Thawing
2.1 Remove the cryovial from liquid nitrogen and thaw rapidly in a 37 °C water bath with gentle agitation until completely melted within 1–2 min.
2.2 Disinfect the vial surface with 75% ethanol. Slowly transfer the cell suspension dropwise into a centrifuge tube containing pre‑warmed complete medium while swirling gently.
2.3 Centrifuge at 300 × g for 5 min and discard the supernatant to remove cryoprotectant.
2.4 Resuspend cells with fresh medium for culture or downstream assays as required.
2.5 It is recommended to count and test cell viability 24 h after thawing to verify recovery performance.
Tip: This protocol is for reference only. Users may optimize it according to their own workflows (e.g., CAR‑T / NK processes, whole‑blood isolation protocols).
Precautions:
1. For Research Use Only. Not for clinical diagnosis, therapy or human infusion.
2. All operations must be performed aseptically in a biosafety cabinet to prevent microbial contamination.
3. This formulation contains DMSO, which is volatile and irritating to skin. Operate under ventilation and avoid direct contact with skin and mucous membranes.
4. This product is ready‑to‑use. Do not dilute, add extra DMSO or subject it to repeated freeze‑thaw cycles.
5. It is recommended to use up the product once opened. If aliquoting is needed, perform aseptically and store the aliquots short‑term at 2–8 °C.
6. Do not use if the liquid appears turbid, precipitated, discolored or otherwise abnormal; contact your supplier.
7. Dispose of waste liquid and contaminated consumables in accordance with biohazard waste regulations.
FAQs:
1. What to do if post‑thaw viability is low?
Suggestion: Verify the cooling procedure (avoid direct rapid freezing at −80 °C); ensure thawing is completed within 1–2 min in a 37 °C water bath; check cell quality before freezing.
2. What to do if cells aggregate after thawing?
Suggestion: Add cell suspension slowly and mix gently during dilution; filtration through a 40 μm cell strainer may be applied if needed.
3. Can it be used for T cells / NK cells?
Suggestion: Optimized for PBMC and compatible with T, NK and other immune cells. Dedicated freezing medium is recommended for large‑scale T/NK manufacturing.
4. Can it be used for therapeutic / clinical purposes?
Suggestion: This research‑grade product is not for clinical use. Therapeutic‑grade products are required for clinical applications.
危险品化学品经营许可证（带存储）