6.6.2　Production of CAR-T Cells

Several steps are required for the production of CAR-T cells.It is also very important to conduct quality control testing throughout the entire protocol [149].

First, leukocytes are taken from the patient’s or donor’s body using leukapheresis.Second, The T cells are enriched and washed to separate them from the leukocytes.Third, the T cell subsets at the level of CD4/CD8 composition are separated using specific antibody bead conjugates or markers.Culture is then needed to activate the T cells.This process requires purifying autologous antigen-presenting cells (APCs) from the patients or donors, or beads coated with anti-CD3/anti-CD28 monoclonal antibodies, or anti-CD3 antibodies alone or in combination with feeder cells and growth factors, such as IL-2.IL-2 is the most commonly used agent because it induces rapid T cell growth.In order to polarize T cells to a specific phenotype, the culture conditions are further refined [161,162].

CARs are encoded with viral vectors, which guide the RNA to reverse-transcribe into DNA and permanently integrate into the genome of the patient cells.The viral vector is washed out of the culture via medium exchange and/or dilution during the activation process.Lentiviral vectors are more commonly used than gammaretroviral vectors in clinical trials because of their safer integration site profile.The other methods include Sleeping Beauty transposon system and mRNA transfection.However, many concerns remain, such as the requirement for several rounds of infusion using transient mRNA transfection and the unknown potential of insertional mutagenesis and remobilization of transposons when using the Sleeping Beauty transposon system [163,164].(https://www.daowen.com)

Three bioreactor culture systems are used to culture CAR-T cells: WAVE Bioreactor, G-Rex and CliniMACS Prodigy.The major drawback of the former two systems is that the flask must be opened during cell inoculation [164].

However, the CliniMACS Prodicy system is a single device that can effectively enrich, activate,transducer and expand the cells.After the cells reach the numbers required for clinical uses, they are collected and transfused into the patients [164].