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Tips for Use (3) | Amplification Tips for the NK Amplification Kit (Trophoblast Cell Method)
Immunotherapy is currently one of the most promising approaches for cancer treatment, with non-specific immune cells—particularly natural killer (NK) cells—emerging as a highly promising cell type. NK cells can exert their cytotoxic activity without prior stimulation, enabling faster responses; they typically do not mediate graft-versus-host disease (GVHD), thus offering a higher safety profile. NK cells can be derived from a wide range of sources, including peripheral blood, umbilical cord blood, human embryonic stem cells, induced pluripotent stem cells, and even the NK-92 cell line, making them among the preferred options for off-the-shelf immune cell therapies. The trophoblast-based expansion method allows for rapid NK cell proliferation, yielding high-purity, high-quality NK cells with excellent stability, high purity, and fast expansion rates.
The IL-15/IL-21 NK Cell Expansion Reagent (Trophoblast Cell-Based Method) was developed by the HeyouSheng R&D team for the expansion and culture of human natural killer (NK) cells. It enables highly efficient expansion of NK cells derived from human peripheral blood and umbilical cord blood—up to 300–400-fold, based on the final harvested NK cell yield relative to the initial PBMC input—and yields NK cells with high purity, with a CD3−CD56+ expression rate exceeding 90%.
| Number | Name | Specifications |
| NK881-F | HiXpan™ NK Cell Expansion Reagent (Feeder Cell, IL-15) | 1mL |
| NK881-21-F | HiXpan™ NK Cell Expansion Reagent (Feeder Cell, IL-21) | 1mL |
| NK881-2L | HiXpan™ NK Cell Expansion Kit (Feeder Cells, IL-15) | 1 kit |
| NK881-21-2L | HiXpan™ NK Cell Expansion Kit (Feeder Cells, IL-21) | 1 kit |
NK Cell Expansion Kit (Trophoblast cell method) Product advantages:
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An independently developed, domestically produced original research system;
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Suitable for the culture of NK cells from multiple sources;
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After 13 days of culture, NK cell expansion reaches 300–400-fold.
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The purity of expanded NK cells can reach as high as 90%.
Through continuous optimization, validation, and summarization by the Heyousheng team and our client partners, the Heyousheng technical team has compiled and organized the key issues that users are most focused on and concerned about during the use of the NK amplification kit (trophoblast cell method).
NK Cell Expansion Kit (Trophoblast Cell Method) – Expansion Tips
Q1, Can the kit be aliquoted for use?
A: We do not recommend aliquoting the expansion reagent. The expansion reagent is prepared using irradiated trophoblast cells, and one vial of the reagent is sufficient for 8–10 × 10^7 PBMCs at the start. If you wish to reduce the initial PBMC count, simply decrease the volume of expansion reagent proportionally. For example, if starting with 2 × 10^7 PBMCs, use only one-quarter of the standard reagent volume and set up the expansion culture at a concentration of 2 × 10^6 PBMC/mL in a total culture volume of 10 mL.
Q2, Is it permissible to gently pipette or blow on the cells during the pre-amplification stage?
A: It is not recommended to gently pipette or disperse the cells during the first 7 days after expansion; simply replenish the culture medium according to the appropriate ratio. This is because, in the early stages, the cells are aggregating and being activated; vigorous pipetting to break up these aggregates may reduce the activation efficiency. Sampling for flow cytometry analysis is also not advised during the first 7 days. After Day 7, replenish the medium based on the cell count and then proceed with flow cytometry analysis.
Q3, What is the added concentration of IL-2?
A: Add IL-2 at 100 IU/mL (final concentration) for the first 7 days, then increase the concentration to 1,000 IU/mL for the subsequent 7 days. Alternatively, the culture medium can be prepared in advance; however, it is not recommended to store the prepared medium at 4°C for an extended period (use within one month) to prevent a decline in cytokine potency.
Q4, When to administer fluid replacement?
A: For the first media change, it is recommended to perform it on Day 3 or Day 4. The specific timing should be determined based on cell aggregation; under the microscope, a clear aggregate of more than approximately 30% of the cells indicates that a media change is warranted. If no obvious cell aggregates are observed on Day 3, the first media change can be performed on Day 4.
Note: If no obvious clumping is observed by Day 4, further waiting is not recommended; fluid replacement may be administered directly, with subsequent monitoring of activation status.
On Day 3, the cluster formation is not yet obvious; you can wait another day for fluid replacement.
Clumping is evident, and fluid replacement can be administered.
Q5, What is the usage of plasma, blood substitutes, and culture media?
A: Plasma, blood substitute, or FBS can all be used. In the early stages, it is recommended to add 10%; during the mid-expansion phase, 5% can be added; subsequently, the remaining autologous plasma can be added, or the amount of blood substitute/FBS can be reduced. After Day 9, autologous plasma (or blood substitute) may no longer be added, but doing so will further enhance the expansion outcome.
Q6, Sample requirements?
A: Fresh samples yield the best expansion results, and the initial NK cell proportion significantly influences the final expansion outcome. We recommend that the initial NK cell proportion be greater than 15% and the T cell proportion less than 60% to achieve optimal expansion. For cryopreserved samples, the cryopreservation medium plays a critical role in NK cell expansion; Hycells offers a superior cryopreservation medium for immune cells (catalog no.: HYS01013) that provides excellent cryopreservation performance for fragile cell types such as NK cells, monocytes, and γδ T cells.
Q7, What is the typical NK ratio on Day 7?
A: NK cell expansion is highly dependent on the donor; some donors achieve a purity of 60%–70% by day 7, while others may only reach 30%–40%.
Q8, What is the ultimate purity that can be achieved through amplification?
A: NK cell expansion is highly dependent on the donor; donors with superior expansion can achieve a purity of over 90% by Days 14–18.
Q9, How high can the amplification factor be?
A: On Day 14 post-donation, the expansion efficiency is excellent, with NK cells expanded from initial PBMCs achieving a fold expansion of 300–400.
Q10, Which basal medium should be used?
A: We recommend using Hycells serum-free basal medium; other serum-free media available on the market, such as Corning 581 and TARAKA H3/X-VIVO, can also deliver good results.
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