Zhu Xuejing, Liu Jian Corning Incorporated

Corning Life Sciences Asia Technology Center Shanghai 201206, China

本文介紹了Corning NK 細(xì)胞活化擴(kuò)增套裝快速擴(kuò)增NK細(xì)胞的使用方法，以及驗證套裝的培養(yǎng)性能。驗證結(jié)果表明Corning NK 細(xì)胞活化擴(kuò)增套裝可以用于NK細(xì)胞的大量擴(kuò)增，并且具有高活力和高殺傷能力。Corning NK 細(xì)胞活化擴(kuò)增套裝包含預(yù)包被T-75培養(yǎng)瓶，KBM NK活化培養(yǎng)基，KBM NK活化添加劑和KBM NK擴(kuò)增培養(yǎng)基。前三個特別設(shè)計用于NK活化，zui后一個KBM NK擴(kuò)增培養(yǎng)基于NK細(xì)胞擴(kuò)增。NK活化培養(yǎng)基和擴(kuò)增培養(yǎng)基生產(chǎn)都是使用高質(zhì)量試劑和cGMP級的原料。培養(yǎng)基唯二所含蛋白是注射級的血清白蛋白和重組人胰島素。

介紹

自然殺傷細(xì)胞（NK）是重要的免疫細(xì)胞，能識別并清除腫瘤和病毒感染。體外大量培養(yǎng)NK細(xì)胞，滿足過繼性免疫治療所需一向比較困難。據(jù)資料，現(xiàn)有的培養(yǎng)方法都是采用擴(kuò)增培養(yǎng)基搭配細(xì)胞因子（IL-2，IL-7，IL-15）的方案，需要研究者自行優(yōu)化后使用。

Corning NK 細(xì)胞活化擴(kuò)增套裝提供了NK 細(xì)胞活化擴(kuò)增的全套材料和標(biāo)準(zhǔn)操作方法，所得NK細(xì)胞擴(kuò)增能力強(qiáng)，活性和殺傷能力高。研究者無需優(yōu)化，而且使用步驟十分精簡。本文還和競爭產(chǎn)品比較了NK細(xì)胞產(chǎn)量，活力和NK 細(xì)胞體外 陽性表達(dá)比率和殺傷能力。

材料和方法

健康供體血液使用淋巴細(xì)胞分離液分離PBMCs（外周血單個核細(xì)胞）。沖洗緩沖液是PBS，培養(yǎng)容器是透氣細(xì)胞培養(yǎng)袋。NK 細(xì)胞活化擴(kuò)增的細(xì)胞因子是白介素 2（IL-2）。

NK 細(xì)胞活化和擴(kuò)增

1. 熱滅活自體血漿，56°C，30min。離心， 800 xg ， 20 min，去除沉淀。4°C 保存上清血漿，培養(yǎng)備用。

2.至少5倍體積的PBS（不含鈣鎂）清洗PBMCs。室溫離心500xg，10min，收集PBMCs。 PBMCs稀釋到 1 x 106 cells/mL，使用NK活化培養(yǎng)基配比1.8mL NK活化添加劑和10%自體血漿。

3.預(yù)包被T-75培養(yǎng)瓶使用前，用PBS（不含鈣鎂）清洗兩次。添加30 mL PBMC懸液到預(yù)包被T-75培養(yǎng)瓶，37°C，5% CO2培養(yǎng)。

注意：第六天之前，如果培養(yǎng)基變黃，細(xì)胞密度超過2.0 x 10⁶ cells/mL，需要添加新鮮的NK活化培養(yǎng)基配比10%自體血漿，保持細(xì)胞密度在在5 x 10⁵到 2.0 x 10⁶ cells/mL之間。

4.培養(yǎng)第六天，培養(yǎng)液離心，適量NK擴(kuò)增培養(yǎng)基（1000 IU/mL IL-2 和10%自體血漿）重懸細(xì)胞，再轉(zhuǎn)移到T-225培養(yǎng)瓶或透氣細(xì)胞培養(yǎng)袋。

5.每隔兩三天，根據(jù)細(xì)胞擴(kuò)增狀態(tài)，培養(yǎng)體系中添加新鮮的NK擴(kuò)增培養(yǎng)基（1000 IU/mL IL-2 和10%自體血漿）保持細(xì)胞密度在5 x 10⁵到 2.0 x 10⁶ cells/mL之間。

6.細(xì)胞總量到達(dá)2 x 10⁹ 后收獲細(xì)胞用于CCK8 殺傷力和免疫表型檢測。

注意：整個實(shí)驗過程中，細(xì)胞懸液吹打和培養(yǎng)容器拍打都要輕柔，以避免細(xì)胞損傷，保持細(xì)胞活性。

免疫表型

收獲細(xì)胞表面標(biāo)記表達(dá)評估方法：CD3-CD56+陽性率。

100 μL PBS 加入 1 x 10⁶ 個細(xì)胞，CD3 FITC +CD56 PE抗體染色 20min，染色過程 室溫避光。孵育后，PBS 緩沖液清洗細(xì)胞，200 μL PBS 重懸，用于流式細(xì)胞分析。每個樣本收集1萬個events，再用histogram overlay subtraction method using BD Accuri C6 軟件。

CCK-8 細(xì)胞毒性檢測

 K-562 細(xì)胞作為靶細(xì)胞。1 x 10⁵ cells/mL的K-562 細(xì)胞和 5 x 10⁵ cells/mL的效應(yīng)細(xì)胞共培養(yǎng)（96孔培養(yǎng)板三份，終體積200 μL/孔），37°C 過夜。分析前，每孔添加20 μL細(xì)胞計數(shù)試劑（ Cell Counting Kit-8 solution ，CCK8, Sigma Cat. No. 96992）， 37°C ，5% CO2孵育1 h。數(shù)據(jù)讀?。篠pectraMax® M4 (Molecular Devices) multifunction microplate reader。

結(jié)論
Corning® NK細(xì)胞活化擴(kuò)增培養(yǎng)套裝支持NK細(xì)胞的快速擴(kuò)增，NK細(xì)胞產(chǎn)量高（約8.6 萬億，擴(kuò)增290倍），細(xì)胞殺傷力強(qiáng)（效應(yīng)細(xì)胞：靶細(xì)胞=5:1 下殺傷力77.9%），與競爭產(chǎn)品相比很有優(yōu)勢。

Corning NK 細(xì)胞活化擴(kuò)增套裝提供了NK 細(xì)胞活化擴(kuò)增的全套材料和標(biāo)準(zhǔn)操作方法。無需優(yōu)化，而且使用步驟十分精簡。

重慶市華雅干細(xì)胞技術(shù)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.