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The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.
The process of isolating T cells from peripheral blood mononuclear cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.
More than 500 chimeric antigen receptor (CAR)-T cell therapy clinical trials are currently being conducted across the world, and four CAR-T cell therapy products are available on the market1. However, numerous CAR-T cell research and manufacturing needs still exist that must be addressed to improve the efficacy, scalability, and long-term success of these potentially curative therapies2,3,4,5. Adoptive CAR-T cell clinical research and manufacturing begins with T cell isolation from a peripheral blood sample and the subsequent stimulation, transduction, and expansion of the isolated cells. Parameters such as T cell recovery, purity, and activation/exhaustion signals require careful consideration when choosing the cell isolation and stimulation techniques for CAR-T cell research and manufacturing3,4,6. Importantly, improvement in the therapeutic persistence of CAR-T cell therapies by minimizing the biological impediments that result from the current manufacturing processes, such as T cell exhaustion, is needed to enhance the therapeutic efficacy6,7.
As an alternative to traditional cell isolation methods such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), here, buoyancy-activated cell sorting (BACS) with microbubbles for T cell isolation is demonstrated. Microbubble separation uses buoyant, hollow microspheres (microbubbles) to bind the targets and float them to the surface of fluid samples8,9. By functionalizing microbubbles with antibodies (i.e., anti-CD3), the desired T cell populations can be positively selected from peripheral blood samples. Subsequently, the use of a different population of antibody-functionalized microbubbles (i.e., anti-CD28) to co-stimulate and activate positively selected T cells in suspension is demonstrated in this work. Microbubbles offer a simple and highly tunable isolation and activation workflow that generates T cells ready for suspended cell culture and downstream applications such as genetic modification and expansion. Critically, buoyant cell activation with microbubbles promotes restrained cell stimulation to prevent excessive T cell exhaustion7.
For this study, flow cytometry was the primary tool used to analyze the isolation, activation, and transduction success of the functionalized microbubbles, as well as to provide detailed information about the specific subpopulations present during the growth and expansion phases post-transduction. In addition to flow cytometry, brightfield and fluorescence microscopy were used to confirm the cell health, morphology, and transduction success. Based on these results, the microbubble technology and protocol provide a more tunable and gentler alternative to the traditional isolation and activation methods currently in use today; in particular, microbubble-activated cells show notably lower expression of T cell exhaustion markers than that typically observed with industry-standard tools and kits.
1. Isolation of T cells with microbubbles using positive selection
NOTE: This protocol details a small-scale CD3+ positive selection approach using SAMBs.
2. Co-stimulation (activation) of the positively selected T cells
3. Expansion of the co-stimulated cells in cell culture medium
4. Optional: Transduction of activated T cells with lentivirus
NOTE: The approach used here is adapted from Prommersberger et al.10.
5. Expansion of the T cells (with or without prior transduction)
6. Harvesting the T cells and flow cytometry
T cells were isolated from purchased PBMCs and plated for activation as described in the protocol. The negative control samples (purchased PBMCs) were not activated. These control samples were included to demonstrate the effect that the microbubble activation process had on the experimental samples as compared to the untouched and unstimulated T cell controls, ensuring that the activation markers observed were the result of the added activation factors and were not inherent to the T cells themselves. As per the experimen...
The described protocol allows for the isolation of T cells from PBMC samples and the activation of suspended T cells in culture media with microbubbles. This method relies on functionalized microbubbles whose inherent buoyancy offers a unique opportunity to introduce co-stimulatory signals to cells and activate them while they are suspended in a culture medium, thereby reducing the exposure of the expanding cells to prolonged stimulation; such overstimulation can result in the increased expression of T cell exhaustion ma...
All authors, at the time of submission, are employees of Akadeum Life Sciences, which manufactures and sells microbubble separation products.
None.
Name | Company | Catalog Number | Comments |
2-Mercaptoethanol | Gibco | 21985-023 | CAS: 60-24-2 |
Biologix Multi-Well Culture Plates 24-well plates | VWR | 76081-560 | |
Biotin anti-human CD28 (28.2) Antibody | Biolegend | 302904 | |
Biotin anti-human CD3 (OKT3) Antibody | Biolegend | 317320 | |
DPBS, no calcium, no magnesium | Gibco | 14190-136 | |
GlutaMAX Supplement | Thermofisher | 35050061 | |
Human Recombinant IL2 | BioVision (vwr) | 10006-122 | |
Lentiviral Particle rLV.EF1.zsGreen1-9 | Takara Bio | 0038VCT | |
Leukopak | BioIVT | HUMANLMX100-0001129 | |
Normal Human PBMCs | BioIVT | HUMANHLPB-0002562 | |
Penicillin/Streptomycin 100X for tissue culture | VWR | 97063-708 | CAS: 8025-06-7 |
Polybrene Infection/Transfection Reagent | Millipore Sigma | TR-1003-G | CAS:28728-55-4 |
Pooled Human AB Serum Plasma Derived Heat Inactivated | Innovative Research | ISERABHI100mL | |
RPMI 1640 Medium, GlutaMAX Supplement, HEPES | Gibco | 72400047 | |
Streptavidin Microbubble Kit (includes Akadeum's separation buffer) | Akadeum | 11110-000 |
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