Zhu Xuejing, Liu Jian Corning Incorporated

Corning Life Sciences Asia Technology Center Shanghai 201206, China

本文介绍了Corning NK 细胞活化扩增套装快速扩增NK细胞的使用方法，以及验证套装的培养性能。验证结果表明Corning NK 细胞活化扩增套装可以用于NK细胞的大量扩增，并且具有高活力和高杀伤能力。Corning NK 细胞活化扩增套装包含预包被T-75培养瓶，KBM NK活化培养基，KBM NK活化添加剂和KBM NK扩增培养基。前三个特别设计用于NK活化，zui后一个KBM NK扩增培养基于NK细胞扩增。NK活化培养基和扩增培养基生产都是使用高质量试剂和cGMP级的原料。培养基唯二所含蛋白是注射级的血清白蛋白和重组人胰岛素。

介绍

自然杀伤细胞（NK）是重要的免疫细胞，能识别并清除肿瘤和病毒感染。体外大量培养NK细胞，满足过继性免疫治疗所需一向比较困难。据资料，现有的培养方法都是采用扩增培养基搭配细胞因子（IL-2，IL-7，IL-15）的方案，需要研究者自行优化后使用。

Corning NK 细胞活化扩增套装提供了NK 细胞活化扩增的全套材料和标准操作方法，所得NK细胞扩增能力强，活性和杀伤能力高。研究者无需优化，而且使用步骤十分精简。本文还和竞争产品比较了NK细胞产量，活力和NK 细胞体外 阳性表达比率和杀伤能力。

材料和方法

健康供体血液使用淋巴细胞分离液分离PBMCs（外周血单个核细胞）。冲洗缓冲液是PBS，培养容器是透气细胞培养袋。NK 细胞活化扩增的细胞因子是白介素 2（IL-2）。

NK 细胞活化和扩增

1. 热灭活自体血浆，56°C，30min。离心， 800 xg ， 20 min，去除沉淀。4°C 保存上清血浆，培养备用。

2.至少5倍体积的PBS（不含钙镁）清洗PBMCs。室温离心500xg，10min，收集PBMCs。 PBMCs稀释到 1 x 106 cells/mL，使用NK活化培养基配比1.8mL NK活化添加剂和10%自体血浆。

3.预包被T-75培养瓶使用前，用PBS（不含钙镁）清洗两次。添加30 mL PBMC悬液到预包被T-75培养瓶，37°C，5% CO2培养。

注意：第六天之前，如果培养基变黄，细胞密度超过2.0 x 10⁶ cells/mL，需要添加新鲜的NK活化培养基配比10%自体血浆，保持细胞密度在在5 x 10⁵到 2.0 x 10⁶ cells/mL之间。

4.培养第六天，培养液离心，适量NK扩增培养基（1000 IU/mL IL-2 和10%自体血浆）重悬细胞，再转移到T-225培养瓶或透气细胞培养袋。

5.每隔两三天，根据细胞扩增状态，培养体系中添加新鲜的NK扩增培养基（1000 IU/mL IL-2 和10%自体血浆）保持细胞密度在5 x 10⁵到 2.0 x 10⁶ cells/mL之间。

6.细胞总量到达2 x 10⁹ 后收获细胞用于CCK8 杀伤力和免疫表型检测。

注意：整个实验过程中，细胞悬液吹打和培养容器拍打都要轻柔，以避免细胞损伤，保持细胞活性。

免疫表型

收获细胞表面标记表达评估方法：CD3-CD56+阳性率。

100 μL PBS 加入 1 x 10⁶ 个细胞，CD3 FITC +CD56 PE抗体染色 20min，染色过程 室温避光。孵育后，PBS 缓冲液清洗细胞，200 μL PBS 重悬，用于流式细胞分析。每个样本收集1万个events，再用histogram overlay subtraction method using BD Accuri C6 软件。

CCK-8 细胞毒性检测

 K-562 细胞作为靶细胞。1 x 10⁵ cells/mL的K-562 细胞和 5 x 10⁵ cells/mL的效应细胞共培养（96孔培养板三份，终体积200 μL/孔），37°C 过夜。分析前，每孔添加20 μL细胞计数试剂（ Cell Counting Kit-8 solution ，CCK8, Sigma Cat. No. 96992）， 37°C ，5% CO2孵育1 h。数据读取：SpectraMax® M4 (Molecular Devices) multifunction microplate reader。

结论
Corning® NK细胞活化扩增培养套装支持NK细胞的快速扩增，NK细胞产量高（约8.6 万亿，扩增290倍），细胞杀伤力强（效应细胞：靶细胞=5:1 下杀伤力77.9%），与竞争产品相比很有优势。

Corning NK 细胞活化扩增套装提供了NK 细胞活化扩增的全套材料和标准操作方法。无需优化，而且使用步骤十分精简。

重庆市华雅干细胞技术有限公司 ，  ：

Zhu Xuejing, Liu Jian Corning Incorporated

Corning Life Sciences Asia Technology Center Shanghai 201206, China

This application note describes the protocol for rapid expansion of NK cells using the Corning NK Expansion kit, as well as tests that validate the use of the kit. The results support the use of the Corning NK Expansion kit by researchers who need high expan- sion capacity, high viability, and high cytotoxicity of NK cells. The Corning NK Expansion kit contains a pre-coated T-75 flask, KBM NK Primary medium, KBM NK Primary supplement, and KBM NK Expansion medium. The first three components are specifically designed for NK cell activation, while the KBM NK Expansion

medium is utilized for NK cell expansion. Both the KBM NK Primary medium and KBM NK Expansion medium are manufactured using high quality reagents and GMP-grade raw materials. The only pro- teins present in the media are injectable levels of serum albumin and recombinant human insulin.

Introduction

Natural killer (NK) cells are crucial immune cells that can recognize and kill tumors and virus infection. It is difficult to obtain the large numbers of NK cells ex vivo that are necessary for adoptive immu- notherapy. Several methods of NK activation/expansion have been reported that use expansion medium plus various cytokines (e.g., IL-2, IL-7, and IL-15). However, these require further optimization by researchers.

The Corning NK Expansion kit provides a complete set of materials and a standard operating procedure to expand NK cells with high expansion capacity, high viability, and high cytotoxicity. Researchers have no need to further optimize it, and the procedure has mini- mal consumables cost. In this application note, we provide com- petitive data, including cell yields, cell viability, and positive NK ratio and cytotoxicity capability in vitro, that compares the Corning NK expansion kit with other commercially available expansion media.

Materials and Methods

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor whole blood using lymphocyte separation medium

(Corning Cat. No. 25-072-CI) according to the manufacturer’s instructions. The wash buffer was PBS (Corning Cat. No. 21-040-CV), and the culture vessel was a gas-permeable cell culture media bag (Corning Cat. No. 88-610-20). Interleukin 2 (IL-2, Corning Cat. No. 354043) was used as an in vitro cytokine for NK cell activation and expansion.