• Developing an improved method for generation of human T cell clones
  • Mahdie Jafari,1 Mohammad Ali Shokrgozar,2 Shahriyar Abdoli,3 Masoud Moghaddam Pour,4 Zahra Sharifzadeh,5,*
    1. Immunology Department, Pasteur Institute of Iran, Tehran, Iran
    2. National Cell bank of Iran, Pasteur Institute of Iran, Tehran, Iran
    3. School of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran
    4. Scientific board of Zist Fanavaran Pajoh Alborz Company, R&D Departmen, Karaj, Iran
    5. Immunology Department, Pasteur Institute of Iran, Tehran, Iran


  • Introduction: T cell-based immunotherapy is a promising treatment for cancer, human immunodeficiency virus infection/acquired immunodeficiency syndrome, and other diseases. Current immunotherapies, such as the generation of chimeric antigen receptor (CAR)-T cells or gene editing using clustered regularly interspaced short palindromic repeat (CRISPR) technology, often require the genetic manipulation of T cells. Peripheral blood T lymphocytes are a pool of cells with extremely different characteristics and, therefore, it may be difficult to obtain clear results and to attribute a certain function to a defined T cell population in several experimental settings. The availability of a population of human T lymphocytes deriving from the same progenitor with a unique phenotype and function (clone) may therefore be of help. At present, the isolation and growing of single cells is still a technically challenging task; especially in suspended cells used in a wide range of CAR T cell studies. Therefore, researchers are still trying to establish an easy and effective method for isolation and expansion of single T clones. In this regard, we aimed to develop a simple and cost-effective method to enable isolation of human T cells at high yields and viability.
  • Methods: In this study, we explored the factors affecting T cell growing and assessed their ability to develop homogeneous clone from a single cell. At first, single T cells were isolated by serial dilution and seeded in 96 cell plates. Different factors affecting cell growth and survival and factors increasing the interaction between cells to facilitate clone formation including FBS, Matrigel and feeder layer cells were selected for this project. These factors were used for T cell expansion, separately or simultaneously, in ten different groups. The microplate wells were coated with the defined factors and then single T cells were added. Every two to three days, about 20 microliters of medium containing 20% FBS was added to cells. All wells were examined for two weeks and the single clones were transferred to a new well and propagated.
  • Results: By examining the microscopic images of T cells in terms of morphology and comparing the survival of clones formed within the experimental groups, three groups of pre-coated wells with 300 μl FBS, 100 μl Matrigel and FBS-Matrigel showed the best results. Among them, wells coated with FBS and Matrigel for 48 and 3 hours, respectively, showed the best results after cell seeding and clone formation. The T cell clones developed in these wells were successfully transferred to the new wells and expanded.
  • Conclusion: Due to the cost-effectiveness of FBS compared to Matrigel and the simplicity of this method compared to feeder layer cells, as well as its easy and short-term preparation that can reduce the risk of contamination, this method can be considered as a simple and cost-effective method to isolate and expand T cell clones. This protocol can replace time-consuming and expensive methods for T cell expansion to address the challenges of T cell proliferation and cell line development.
  • Keywords: Chimeric Antigen Receptor, T cell clone expansion, single cell isolation, cancer immunotherapy, Limi