Product Highlight

• The high-performance version can obtain more than 180 times of cell expansion and more than 85% of positive rate after 14~16 days of culturing. It can be specifically used for the activation and culture of NK cells in unconventional cord blood or peripheral blood samples, such as blood samples with a long-distance transportation time of more than 8 hours or unknown cryopreservation quality and supports the culture protocol without the addition of autologous plasma.
• Induction with pure factors, with no trophoblastic cells, simple and feasible culture method, basically uniform cell state, fed batch of solutions according to the recommended process, without the need for cell counting.
• When combined with the newly upgraded NK5.0 basic culture medium, it significantly improves the continuous expansion ability of cells after 7 consecutive days of cultivation, and the continuous support ability of cells that expand from 2 L system to 3 L or even 4 L system after 14 days.
• It can be paired with the research-grade basal medium (NC0102) or the chemically defined basal medium (NC0102.H). The basal medium has been filed with the US FDA DMF with the filing No. of 37874.
• Stable culture results: This product has been on the market for 7 years, with highly reliable stability.
• Lower requirement for initial sample positive rate: the product has a strong induction capability for special NK blood samples (initial positive rate below 10%)
• It is used for the in vitro induction and amplification of NK cells from peripheral blood samples or umbilical cord blood samples.
• Standard operation for different samples: Provided that the initial PBMC inoculation conditions are controlled, the subsequent cultures can be carried out according to the recommended fluid replacement process without monitoring density after culture refilled.

Product Background

The NK Serum-free Kit is a type of chemically defined, animal component-free medium designed for NK (natural killer) cell expansion optimized to deliver cell growth and maintaining NK cell functionality and potency.

Natural killer cells (NK cells or large granular lymphocytes) are a kind of cytotoxic lymphocyte critical to the innate immune system that belong to the rapidly expanding family of known innate lymphoid cells and represent 5–20% of all circulating lymphocytes in humans.

The NK Cells Serum-free Medium is the best solution for NK cell culture and expansion from peripheral blood mononuclear cells (PBMCs) or cord blood (CB）. It's chemically defined, xeno-free component ensures reproducible results. With the minimum to expansion the cells from PBMCs and CB. Expanded NK cells are fully functional and can be used in any downstream research.

Killing K562

Incubate NK cells cultured for 14 days with CFSE stained K562 for 4 hours and then measure the apoptosis rate of K562.
Killing rate of K562=(number of K562 cells inoculated - remaining K562 cells after 4 hours of incubation)/number of K562 cells inoculated.

Group	Gate	Residual cells	K562 (%)	K562 residual cells	K562 inoculum size	K562 killing cells	Killing rate
0:1	84.85%	1.16E+05	99.83%	1.16E+05	1.00E+05	/	/
0.5:1	28.50%	3.94E+04	62.59%	2.47E+04	5.00E+04	2.53E+04	51%
1 : 1	25.26%	7.22E+04	27.85%	2.01 E+04	5.00E+04	2.99E+04	60%
2:1	27.56%	9.74E+04	11.22%	1.09E+04	5.00E+04	3.91 E+04	78%

Reference Culture Procedure

When using Yocon FEP culture bags, the final volume is 2.2 liters.

Day	Culture Consumables	Culture Medium	Fluid refill volume (mL)	Total Vol (mL)	Plasma proportion (%)	Plasma volume (mL)
0	1x T75 flask	Activation medium	30	30	10	3
3	2x T25 flask	Activation medium	30	60	5	1.5
5	2x T175 flask	Activation medium	140	200	5	7
7	1x culture bag	Expansion medium	400	600	1	4
9	1x culture bag	Expansion medium	600	1200	0	0
11	1x culture bag	Expansion medium	1000	2200	0	0
14-16	1x culture bag	Harvest cells

On the 7th day, if the density is < 1.5×10⁶/mL, the final volume is 2.2L.

Day	Culture Consumables	Culture Medium	Fluid refill volume (mL)	Total Vol (mL)	Plasma proportion (%)	Plasma volume (mL)
0	1x T75 flask	Activation medium	30	30	10	3
3	2x T25 flask	Activation medium	30	60	5	1.5
5	2x T175 flask	Activation medium	140	200	5	7
7	1x culture bag	Expansion medium	200	400	1	2
9	2x culture bag	Expansion medium	400	800	0	0
11	2x culture bag	Expansion medium	800	1600	0	0
13	2x culture bag	Expansion medium	600	2200	0	0
16-18	2x culture bag	Harvest cells

Special Instructions

1. Isolation of PBMC
   Two points should be particularly noted when isolating mononuclear cells. First, before isolating mononuclear cells, the blood and all reagents should be pre-warmed to 20°C and centrifuged at room temperature. Second, PBMC should be isolated within 8 hours after blood collection.
2. Inoculation density
   The recommended inoculation density for peripheral blood mononuclear cells is 2×10⁶/mL. For fresh umbilical cord blood mononuclear cells, the recommended inoculation density is 3×10⁶/mL, and for cryopreserved umbilical cord blood mononuclear cells, the recommended inoculation density is 3.5×10⁶/mL. An inoculation density that is too low or too high will have an impact on the final number of harvested cells and the purity of NK cells.
3. Reagent preservation
   The factors in the NK cell kit are prohibited from being repeatedly frozen and thawed, otherwise their activity will be reduced, resulting in a lower positive rate. If they are not to be used temporarily, please store them at -20°C. For the factors that have already been thawed, if they will be used within one week, they can be stored
   at 4°C.
4. Preservation of mononuclear cells
   When PBMCs are not to be used temporarily, such as during counting, pre-warming the culture medium, and waiting for the factors to thaw, PBMC should be stored in a 37°C incubator to avoid being chilled.
5. Washing after coating with YC00A
   For the culture flasks coated with YC00A, PBMC, YC00B, plasma, and activation medium should be prepared in advance before washing, and inoculation should be carried out immediately after washing. Do not add the medium and leave it for a long time after washing or leave it to dry after washing.