Description

Natural Killer Cells Induction Culture Kit 3.0 is a GMP-grade in vitro induced expansion culture kit, which is suitable for natural killer cell expansion without feeder cells. The kit includes NK Cell Coating Solution, NK Cell Induction Solution, NK Cell Activator, NK Cell Culture Supplement and N300 Serum-Free Medium for NK Cells. This product has stable inter-batch quality and is used to induce and expand NK cells from umbilical cord blood in vitro.

Specification

Product Name	Cat. No.	Size	Component Name	Component Size	Quantity
Natural Killer Cells Induction Culture Kit 3.0	6813551	3 L/kit	NK Cell Coating Solution	300 μL	1 pcs
			NK Cell Induction Solution	600 μL	1 pcs
			NK Cell Activator	500 μL	3 pcs
			NK Cell Culture Supplement	42 mL	1 bottle
			N300 Serum-Free Medium for NK Cells	1 L	3 bottle
	6813552	2 L/kit	NK Cell Coating Solution	200 μL	1 pcs
			NK Cell Induction Solution	400 μL	1 pcs
			NK Cell Activator	500 μL	2 pcs
			NK Cell Culture Supplement	28 mL	1 bottle
			N300 Serum-Free Medium for NK Cells	1 L	2 bottle
	6813553	1 L/kit	NK Cell Coating Solution	100 μL	1 pcs
			NK Cell Induction Solution	200 μL	1 pcs
			NK Cell Activator	500 μL	1 pcs
			NK Cell Culture Supplement	14 mL	1 bottle
			N300 Serum-Free Medium for NK Cells	1 L	1 bottle

Storage Conditions and Validity Period

NK Cell Coating Solution, NK Cell Induction Solution, NK Cell Activator, NK Cell Culture
Supplement: Store at -15℃ to -25℃, valid for one year.

N300 Serum-Free Medium for NK Cells: Keep away from light at 2 to 8℃, valid for one year.

Directions for Use

Take 1 L system (Cat.No.6813553) as an example:

Sample Requirements

Fresh or cryopreserved human umbilical cord blood mononuclear cells (UBMCs) .

Preparation

Flask Coating

On Day -1, mix 100 μL NK Cell Coating Solution with 3 mL PBS thoroughly and add the mixture into a T25 flask. Shake the T25 flask horizontally to cover the mixture over the bottom of the flask and coat it overnight at 4℃ (coat for 2 h at 37℃ in case of an emergency condition). After the coating, remove excess coating solution and wash the bottom of the flask gently with 10 mL PBS. Be careful not to scour the bottom of the culture flask directly.
Prepare Amplification Medium for NK Cells

On Day 0, add 1 vial of 500 μL NK Cell Activator to 1 L of N300 Serum-Free Medium for NK Cells to prepare the Amplification Medium for NK Cells. Keep the medium away from light at 2 to 8℃, valid for 3 weeks.
Prepare Induction Medium for NK Cells

On Day 0, add 200 μL NK Cell Induction Solution to 25 mL of Amplification Medium for NK Cells to prepare the Induction Medium for NK Cells which is used on D0 and D3 of NK cell culture. Keep the medium away from light at 2 to 8℃.

NK Cell Culture

On Day 0, inoculate UBMCs with 10 mL Induction Medium for NK Cells (containing 10% heat-inactivated cord blood plasma and 10% NK Cell Culture Supplement) at a density of 2.5–3.0×10^6 cells/mL into the T25 coated flask. After shaking, incubate the cells in a 37℃ incubator containing 5% CO2.
On Day 3, observe the cell status, then supplement 15 mL of fresh Induction Medium for NK Cells (containing 10% heat-inactivated cord blood plasma and 10% NK Cell Culture Supplement) to the original T25 flask gently. Do not disturb the cells at the bottom.
Note: T25 flask should be placed slightly tilted to avoid liquid overflow.
On Day 5, observe the cell status and count the number. If the cell density is less than 1.4×10^6 cells/mL, add 25 mL of Amplification Medium for NK Cells (containing 5% heat-inactivated cord blood plasma and 5% NK Cell Culture Supplement) to the culture. If the cell density is greater than 1.4×10^6 cells/mL, add 55 mL of Amplification Medium for NK Cells (containing 5% heat-inactivated cord blood plasma and 5% NK Cell Culture Supplement).
On Day 7, observe the cell status and count the number. Add Amplification Medium for NK Cells (containing 2.5% heat-inactivated cord blood plasma and 2.5% NK Cell Culture Supplement) to the culture according to medium supplementation reference procedure in Table 1.
On Day 9, observe the cell status and count the number. Add Amplification Medium for NK Cells (containing 1% NK Cell Culture Supplement) to the culture according to medium supplementation reference procedure in Table 1.
On Day 11, observe the cell status and count the number. Add Amplification Medium for NK Cells (containing 0.5% NK Cell Culture Supplement) to the culture according to medium supplementation reference procedure in Table 1.
On Day 14, observe the cell status and count the number. Add the rest of Amplification Medium for NK Cells (containing 0.5% NK Cell Culture Supplement) to the culture according to medium supplementation reference procedure to achieve a final culture volume of 1000 mL.
On Day 16–18, observe the cell status, take photos, count cell number and harvest cells for subsequent use.

Note:

(1) Detect NK proportion on D0, D7 and D17 by flow cytometry, and the harvest time could be advanced or delayed appropriately according to the growth status of cells.

(2) The result of in vitro expansion of NK cells derived from umbilical cord blood varies from donor to donor, and the above culture protocol is a relatively stable culture procedure after optimization. Due to differences in NK donors, NK cells may expand faster or slower than expected. It is recommended to carefully observe cell growth during the culture cycle and flexibly adjust the medium supplementation and harvest steps to ensure that the in vitro expansion performance of the kit can be achieved.

Protocol for the Separation of UBMCs

Pour the anticoagulant blood into 50 mL centrifuge tubes, centrifuge at 700 g for 15 min (the acceleration is set to eighth gear and the deceleration is set to fourth gear). Then transfer the plasma layer to a new centrifuge tube. Inactivate the plasma at 56℃ for 30 min, then centrifuge at 900 g for 10 min. Place supernatant at -20℃ for 20 min to cool down and centrifuge again at 900 g for 10 min. Afterwards, keep supernatant (i.e., heat-inactivated cord blood plasma) at 4℃ for future use.
Add PBS in the same volume as inactivated plasma to the previous tube after the plasma was removed. Mix the tube thoroughly and add the Erythrocyte Sedimentation Solution in the ratio of 1:3 to 1:4 according to the volume of mixture in the tube. Mix the tube thoroughly and allow the tube to stand for about 30 min, until the interface between supernatant and RBC accounted for about 50% of the total volume, and collect the sedimentation supernatant into a 50 mL centrifuge tube and mix inversely.
Add a certain volume of the Human Lymphocyte Separation Medium (the volume ratio of the separation medium to the sedimentation supernatant is 1:1) into the centrifuge tube, carefully layer the sedimentation supernatant over the separation medium, centrifuge at 800 g for 20 minutes at 20℃ (the acceleration and the deceleration are set to third gear) to obtain a high-purity mononuclear cell layer.
Collect the UBMCs layer, wash with RPMI 1640 twice (250 g, 10 min, 20℃), and re-suspend UBMCs with medium. Count after dilution, then obtain the UBMCs.

Table 1: Medium Supplementation Reference Procedure

Days /d	Culture Flask or Culture Bag	Supplementation				Total Volume /mL	Notes
Days /d	Culture Flask or Culture Bag	Medium	Medium Volume /mL	Plasma Volume /mL	NK Cell Culture Supplement /mL	Total Volume /mL	Notes
-1	T25	/	/	/	/	/	Coat the T25 flask overnight at 4℃.
0	T25	Induction Medium for NK Cells	8	1 (10%)	1 (10%)	10	Inoculation density is 2.5–3.0 × 10⁶ cells/mL.
3	T25	Induction Medium for NK Cells	12	1.5 (10%)	1.5 (10%)	25	Do not pipette cells to avoid interfering with regular cell activation.
5	T175	Amplification Medium for NK Cells	22.5/49.5	1.25/2.75 (5%)	1.25/2.75 (5%)	50/80	Maintain the cell density at 0.5–1.0 × 10⁶ cells/mL after supplementation.
7	T175/Cell Culture Bag	Amplification Medium for NK Cells	95/152	2.5/4 (2.5%)	2.5/4 (2.5%)	150/240	Maintain the cell density at 0.5–1.0 × 10⁶ cells/mL after supplementation.
9	Cell Culture Bag	Amplification Medium for NK Cells	150/160	0	1.5/1.6 (1%)	300/400	Maintain the cell density at about 1.0 × 10⁶ cells/mL after supplementation.
11	Cell Culture Bag	Amplification Medium for NK Cells	250	0	1.25 (0.5%)	550/650
14	Cell Culture Bag	Amplification Medium for NK Cells	450/350	0	2.25/1.75 (0.5%)	1000	Add the rest of medium.
16-18	Cell Culture Bag	/	/	/	/	1000	Harvest cells.

In Table 1, the percentage in parentheses refers to the percentage of plasma or NK Cell Culture

Notes

Avoid repeated freezing and thawing of this product, and pay attention to operate aseptically during use.
NK Cell Culture Supplement can support the efficient expansion of NK cells by adding to the medium in a certain proportion. Defrost it at 37℃ and prepare aliquots. Do not freeze and thaw the supplement repeatedly.
The recommended initial inoculation cell density is 2.5–3.0 × 10^6 cells/mL. But the initial inoculation cell density can be increased to 3.0–3.5 × 10^6 cells/mL when dealing with cryopreserved UBMCs . Too high or too low inoculation density would affect the effect of NK cell expansion.
Dilute the NK Cell Coating Solution with PBS and coat the T25 flask overnight at 4℃ (coat for 2 h at 37℃ in case of an emergency condition).
Before use, the medium should be equilibrated at room temperature. Otherwise, please transfer the estimated amount for use to a separate container and prewarm to 37℃. Do not place the whole bottle of medium at 37℃ for repeated prewarming.
Cell passage shall be carried out gently to avoid mechanical damages to cells.
3 L system (Cat.No.6813551) is applicable to expansion of 7.5–9.0 × 10^7 UBMCs; 2 L system (Cat.No.6813552) is applicable to expansion of 5.0–6.0 × 10^7 UBMCs; 1 L system (Cat.No.6813553) is applicable to expansion of 2.5–3.0 × 10^7 UBMCs.
NK cells tend to form clumps during the early stage of culture and expansion. Gently add supplemental medium as much as possible and do not shake the cells to avoid interfering with the regular activation of NK cells. At the same time, note that Induction Medium for NK Cells is used for D0 and D3, followed by Amplification Medium for NK Cells.
When NK cells are cultured in the culture bag, it is recommended to fold the bag in half according to the culture volume.

Documents

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