PSC Cryopreservation Kit - FAQs

您可按照冻存任意多能干细胞的实验方案来冻存iPSC。推荐使用含10% DMSO的生长培养基来冻存细胞。或使用我们的PSC冻存试剂盒（货号 A2644601）。

我们尚未尝试此种操作。不过，这款培养基可在4度条件下稳定保存6个月。我们的研发部门认为本品中的各种成份均不会受冻融过程的很大影响；不过，这一操作确实未经正式测试。

PSC冻存试剂盒包含了无异源成份的PSC冻存培养基——该产品是用于冻存早期传代的多能干细胞（PSC）的即用型溶液，以及Gibco RevitaCell添加剂（100X）——应用于解冻后培养基的化学成份明确的复苏添加剂。这些试剂组合使用时，有助于将细胞活力的丧失降至最小，使细胞复苏后的恢复最大化，并尽可能减少PSC不期望的分化情况发生。本试剂盒也可用于冻存和复苏外周血单核细胞（PBMCs ），增强其复苏后的细胞活力及恢复情况。

不可以，不能使用冻存的组织。

下列步骤以单管冻存细胞进行培养的实验方案为例。

1.准备一烧杯37°C的水。
2. 从液氮储存中取出一管细胞，注意保护手和眼睛。
3. 拧松管盖1/4圈，等待10秒钟以释放螺纹中可能残留的液氮，再重新拧紧管盖。
4. 将冻存管的下半部分置于37°C水浴中进行解冻，直至冻存管内仅剩余一小块冰芯，使细胞迅速解冻。
5. 用消毒液擦拭管体外表面，再将其移至II级A型层流细胞培养通风橱中。
6. 打开管盖，使用1 mL移液器上下吹打细胞悬液以分散细胞。
7. 从管中吸取20 uL细胞悬液，并将其稀释于20 uL台盼蓝溶液中（如：Gibco台盼蓝，货号 15250-061）。
8. 使用血细胞计数板来确定每毫升悬液中的活细胞数。
9. 将管中悬液（1 mL）稀释至产品说明中的推荐浓度（例如1.25 x 10E4活细胞/毫升，Gibco 新生人表皮角质形成细胞）。
10. 将5 mL细胞悬液加入25 cm²培养瓶，或将15 mL细胞悬浮液加入75cm²培养瓶中。
11. 摇匀培养瓶中的培养基以彻底分散细胞。许多类型的细胞都会迅速贴壁，如果没有在传代后即刻摇匀细胞，细胞可能生长不均匀。
12. 在37°C、5% CO2/95%空气、湿度细胞培养箱中培养细胞。为了获得最佳结果，培养开始后至少在24小时之内不要扰动细胞。

请点击此处(https://www.thermofisher.com/cn/zh/home/references/gibco-cell-culture-basics/cell-culture-protocols/thawing-cells.html)查看我们的细胞复苏方案。

请点击此处（https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/freezing-cells.html）查看我们的细胞冻存方案。

No. Combination of RevitaCell Supplement with a traditional ROCK inhibitor will result in deleterious effects on PSCs.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Yes, it is normal that the RevitaCell Supplement has a strong smell.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

The CTS PSC Cryopreservation Kit (Cat. No. A4239301) provides the same efficient recovery, consistency, and reduced variability as the PSC Cryopreservation Kit (Cat. No. A2644601). However, these two kits have different intended use statements, i.e., “For Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. CAUTION: Not intended for direct administration into humans or animals” and “For Research Use Only”, respectively. Furthermore, the CTS PSC Cryopreservation Kit has full USP sterility testing info and a Drug Master file (DMF), whereas the sterility testing of the PSC Cryopreservation is via a modified USP method, and it does not have a DMF.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

You can cryopreserve iPSCs just as you would cryopreserve any pluripotent stem cells. Growth medium with 10% DMSO is recommended for freezing, or our PSC cryopreservation kit (Cat. No. A2644601) can be used

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

We have not tested this. However, the medium is stable when stored at 4 degrees C for up to 6 months. There are no components that R&D would be concerned about during a freeze thaw; however, this was not formally tested.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

The PSC cryopreservation kit contains xeno-free PSC Cryopreservation Medium, which is a ready-to-use solution for the cryopreservation of early passage pluripotent stem cells (PSCs), and Gibco Revitacell Supplement (100X), a chemically defined recovery supplement for use in the post-thaw culture medium. When used in combination, these reagents help minimize loss of cell viability, maximize post-thaw recovery, and minimize unwanted differentiation of PSCs. This kit can also be used to cryopreserve and recover peripheral blood mononuclear cells (PBMCs) to improve post-thaw cell viability and recovery.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

No, the tissue was not cryopreserved.

The procedure given below is a sample protocol for establishing cultures from the contents of one vial.

1. Prepare a beaker of water at 37 degrees C.
2. Remove a vial of cells from liquid nitrogen storage, taking care to protect hands and eyes.
3. Loosen the cap on the vial 1/4 turn for 10 seconds to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
4. Dip the lower half of the vial into the 37 degrees C water to thaw.
5. When the contents of the vial have thawed, wipe the outside of the vial with disinfecting solution and move to a Class II, type A laminar flow culture hood.
6. Open the vial and pipette the suspension up and down with a 1 mL pipette to disperse the cells.
7. Remove 20 µL from the vial and dilute the cell suspension in 20 µL of trypan blue solution (for example: Gibco Trypan Blue, Cat. No. 15250-061).
8. Use a hemacytometer to determine the number of viable cells per mL.
9. Dilute the contents of the vial (1 mL) to the concentration recommended by the product instructions (for example 1.25 X 10E4 viable cells/mL for Gibco neonatal human epidermal keratinocytes ).
10. Add 5 mL of cell suspension to each 25 cm² culture flask or 15 mL of cell suspension to each 75 cm² culture flask.
11. Following inoculation, swirl the medium in the flasks to distribute the cells. Many cell types attach to culture surfaces quickly, and if the medium is not distributed immediately following inoculation, the cells may grow in uneven patterns.
12. Incubate the cultures in a 37 degrees C, 5% CO²/95% air, humidified cell culture incubator. For best results, do not disturb the culture for at least 24 hours after the culture has been initiated.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Please see our protocol here for thawing frozen cells (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/thawing-cells.html).

Please see our protocol here for freezing cells (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/freezing-cells.html).