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Tips for Use (1) | Amplification Tips for the NK Expansion Kit (Cytokine-Based Method)
Background Introduction
During ex vivo expansion and culture of NK cells, challenges often arise, including low expansion rates, prolonged culture durations, and the initial overgrowth of conventional T cells, which can lead to phenotypic exhaustion of the NK cell population. The Heyousheng NK Cell Expansion Kit (Cytokine-Based Method) enables rapid expansion and culture of NK cells from diverse sources under feeder-free conditions, making it the preferred choice for clinical trials and research in cell therapy. It is suitable for the expansion and culture of NK cells derived from human peripheral blood and umbilical cord blood as starting samples.
| Number | Name | Specifications |
| NK882-1L | HiXpan™ NK Cell Expansion Kit (Cytokine) | 1 kit |
| NK882-1L | HiXpan™ NK Cell Expansion Kit (Cytokine) | 1 kit |
The Heyousheng NK Cell Expansion Kit (Cytokine-Based Method) has earned widespread acclaim from users for its robust expansion capacity, high purity of expanded NK cells, and potent cytotoxic activity. We extend our heartfelt gratitude to all our partners for their continued support and recognition. In response, the Heyousheng technical team has carefully collected and systematically organized the most frequently raised and critical questions regarding the use of this kit, helping more collaborators swiftly achieve their desired experimental outcomes.
NK Cell Expansion Kit (Cytokine-Based) Expansion Tips
Q: Can the kit be aliquoted for use? If so, how should it be aliquoted?
A: Splitting into aliquots is permissible; however, due to minor volume variations between batches, it is recommended to centrifuge each component prior to aliquoting and to use a pipette to measure the total volume of the components before dividing them proportionally.
For example, the kit recommends starting with 30–60 mL of peripheral blood or umbilical cord blood; in this experiment, we initiated expansion using 5 million cells. First, centrifuge the sample to pellet the cells, then use a pipette to measure the supernatant volume. Divide the supernatant into four equal aliquots based on volume, and add one-quarter of each factor (A, B, C, and D) to the corresponding aliquot. After adding one-quarter of factor A to 250 mL of basal culture medium, take 2.5 mL of this mixture and supplement it with 10% autologous plasma (blood substitute/FBS). Subsequently, add one-quarter of factors B, C, and D at the time points recommended in the product manual to initiate expansion.
Q: How long can factor A be stored after being added to the entire bottle of culture medium?
A: It is recommended to use the factor within one month, as its potency decreases when diluted to lower concentrations. If you cannot use the entire amount at once, it is advisable to aliquot the factor rather than preparing the full volume and using it in one go.
Q: Is it permissible to gently pipette or blow on the cells during the early stages of expansion?
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, it is recommended to replenish the medium based on the cell count and then perform flow cytometry analysis.
Q: How should the culture medium be brought to room temperature?
A: The prepared culture medium can be aliquoted into the amount needed for this experiment and allowed to equilibrate at 37°C; however, it is not recommended to place the entire bottle in a 37°C incubator for rapid warming, as repeated exposure to high temperatures may compromise the potency of the factors. If the entire bottle needs to be warmed, it is advisable to remove it from the freezer and allow it to equilibrate at room temperature for 1 hour beforehand.
Q: When should fluid replacement be administered?
A: For the first rehydration, it is recommended to perform rehydration on Day 3 or Day 4. The specific timing of rehydration should be determined based on the degree of cell aggregation; under the microscope, rehydration can be initiated when more than approximately 30% of the cells are visibly clumped.
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.
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.
Q: What is the usage of plasma, blood substitutes, and culture media?
A: Plasma, blood substitute, or FBS can all be used. In the early stage, 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 yield better expansion results.
Q: 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 specially formulated immune cell cryopreservation medium that provides excellent cryopreservation performance for fragile cell types such as NK cells, monocytes, and GDT cells.
Q: DAY7 NK What is the typical ratio?
A ： NK Cell expansion and Donor Highly correlated, in part. Donor Day 7 Can achieve 60%-70% Purity, partial Donor Perhaps only 30%-40% 。
Q: What is the ultimate purity achievable upon 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.
Q: What is the maximum amplification fold?
A: On Day 14, the donor-derived NK cells exhibit robust expansion, achieving up to a 100-fold increase compared with the initial PBMCs.
Q: Which basal medium should be used?
A : It is recommended to use hycells Serum-free basal medium; other serum-free media available on the market include Corning 581/TARAKA H3/X-VIVO Better results can also be achieved.
NK Cell Expansion Kit (Cytokine-Based) – Expansion Tips (PDF Format):
NK Cell Expansion Kit (Cytokine-Based) Expansion Tips.pdf
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