Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Rosa L. Vincent; Fangda Li; Edward R. Ballister; Nicholas Arpaia; Tal Danino

doi:10.3791/65887

JoVE Journal
Bioengineering

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JoVE Journal Bioengineering

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

マウスキメラ抗原受容体(CAR)-T細胞の効率的な作製

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06:22 min

February 2, 2024

DOI: 10.3791/65887-v

Rosa L. Vincent¹, Fangda Li², Edward R. Ballister¹, Nicholas Arpaia^2,3, Tal Danino^1,3,4

¹Department of Biomedical Engineering,Columbia University, ²Department of Microbiology & Immunology, Vagelos College of Physicians and Surgeons,Columbia University, ³Herbert Irving Comprehensive Cancer Center,Columbia University, ⁴Data Science Institute,Columbia University

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Overview

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells. The study focuses on the integration of engineered bacteria and CAR-T cells for solid tumor treatment in syngeneic models.

Key Study Components

Area of Science

Neuroscience
Immunology
Oncology

Background

CAR-T cell therapy is a promising approach for cancer treatment.
Murine models are essential for studying CAR-T cell efficacy.
Standardized protocols for generating murine CAR-T cells are lacking.
Transduction of murine T-cells is challenging with traditional methods.

Purpose of Study

To develop a streamlined protocol for murine CAR-T cell production.
To enable effective study of CAR-T cells in syngeneic tumor models.
To evaluate the activity of new CAR technologies in a complete immune system context.

Methods Used

Preparation of reduced serum medium for transduction.
Dilution of media with transfection reagent.
Application of the protocol to murine T cells.
Evaluation of CAR-T cell functionality in syngeneic models.

Main Results

The protocol successfully enhances murine T cell transduction efficiency.
Generated CAR-T cells exhibit improved functionality in tumor models.
Standardization allows for reproducibility in research.
Facilitates further exploration of CAR technologies.

Conclusions

This protocol provides a reliable method for generating murine CAR-T cells.
It supports the study of CAR-T cell interactions within the immune system.
Future research can build on this foundation to enhance cancer therapies.

Frequently Asked Questions

What are CAR-T cells?

CAR-T cells are genetically engineered T cells designed to target and kill cancer cells.

Why are murine models used in this research?

Murine models provide a relevant biological context to study immune responses and cancer therapies.

What challenges exist in murine T cell transduction?

Murine T cells are often difficult to transduce and culture using traditional methods.

How does this protocol improve CAR-T cell production?

The protocol streamlines the production process, enhancing transduction efficiency and reproducibility.

What implications does this research have for cancer treatment?

It paves the way for improved CAR-T cell therapies and better understanding of their mechanisms in solid tumors.

Can this protocol be adapted for other types of cells?

While designed for murine T cells, the principles may be applicable to other cell types with modifications.

このプロトコルは、レトロウイルスベクターの生産とマウスT細胞の形質導入を合理化し、マウスCAR-T細胞の効率的な生成を促進します。

私たちの研究は、固形腫瘍治療のための遺伝子組換え細菌とCAR-T細胞の効果的な組み合わせを探求しています。卵巣がんの同系モデルを用いて、これら2つの細胞療法が完全な免疫系の中でどのように機能するかを研究することは重要です。同系腫瘍モデルにおけるCAR-T細胞の機能の研究は、マウスCAR-T細胞を作製するための標準化されたプロトコルの欠如によって制限されてきました。

マウスT細胞は、従来の方法を用いたex vivoでの培養に形質導入することが特に困難です。このプロトコルは、マウスCAR-T細胞の生産を合理化し、新しいCAR技術の活性を評価するための重要なコンテキストを提供する同系モデルでの研究を可能にするために開発されました。

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