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

Wydajne wytwarzanie mysich chimerycznych receptorów antygenowych (CAR)-T

JoVE Journal
Bioengineering

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JoVE Journal Bioengineering

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

Wydajne wytwarzanie mysich chimerycznych receptorów antygenowych (CAR)-T

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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.

Ten protokół usprawnia produkcję wektorów retrowirusowych i transdukcję mysich limfocytów T, ułatwiając efektywne wytwarzanie mysich komórek CAR-T.

Nasze badania dotyczą skutecznych kombinacji zmodyfikowanych bakterii i komórek CAR-T w leczeniu guzów litych. Ważne jest dla nas, aby zbadać, jak te dwie terapie komórkowe działają w kontekście kompletnego układu odpornościowego przy użyciu syngenicznych modeli raka myszy. Badanie funkcji komórek CAR-T w syngenicznych modelach nowotworów było ograniczone przez brak znormalizowanych protokołów generowania mysich komórek CAR-T.

Mysie limfocyty T są szczególnie trudne do transdukcji i hodowli ex vivo przy użyciu tradycyjnych metod. Opracowaliśmy ten protokół, aby usprawnić produkcję mysich komórek CAR-T, aby umożliwić badanie w modelach syngenicznych, które zapewniają ważny kontekst do oceny aktywności nowych technologii CAR. Na początek należy odpipetować 3,75 mililitra zredukowanej pożywki surowicy do probówki oznaczonej A i rozcieńczyć pożywkę 105 mikrolitrami odczynnika do transfekcji.

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