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Overview Of Cell Separation And Isolation

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Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

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Leucine Zipper-based Cell Sorting for Purification of Dual-vector-transduced Cells.

Katerina Su¹, Bintao Wang¹, Adhithi Rajagopalan¹

¹City of Hope Los Angeles and National Medical Center.

Journal of Visualized Experiments : Jove

|November 10, 2025

Summary

Chimeric antigen receptor (CAR) T cell therapy shows promise but faces resistance. This study introduces a novel dual-vector transduction and sorting method to enhance CAR T cell therapy for cancer treatment.

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Area of Science:

Immunology
Cancer Biology
Biotechnology

Background:

Adoptive T cell therapies, including CAR T cells, are effective against hematologic cancers but face resistance in solid tumors.
Mechanisms of resistance include antigen loss, T cell exhaustion, and immunosuppressive tumor microenvironments.
Overcoming resistance often requires combining multiple engineered functions in T cells.

Purpose of the Study:

To develop a method for co-delivering multiple genetic constructs into T cells for enhanced anti-tumor activity.
To address vector packaging limitations that hinder the co-expression of multiple engineered functions.
To improve the efficiency and specificity of engineering T cells for combination immunotherapies.

Main Methods:

Co-transduction of T cells using two distinct viral vectors to deliver multiple transgenes.
Implementation of a leucine zipper-based cell sorting methodology (Zip-sort).
Selective immunomagnetic purification of dual-transduced T cells harboring two vectors.

Main Results:

Successfully co-transduced T cells with two vectors, enabling the expression of multiple engineered functions.
Demonstrated the efficacy of Zip-sort for purifying dual-transduced cells.
Increased the number of engineered functions that can be simultaneously delivered to T cells.

Conclusions:

The described protocol overcomes vector packaging constraints for T cell engineering.
This dual-vector transduction and Zip-sort method facilitates the development of combination T cell immunotherapies.
This approach holds potential for improving therapeutic success against challenging malignancies.