Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

Summary

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Abstract

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.

Introduction

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 therapies^2,3,4,5. Adoptive CAR-T cell clinical research and manufacturing begins with T cell isolation from a peripheral blood sample and the su....

Protocol

1. Isolation of T cells with microbubbles using positive selection

NOTE: This protocol details a small-scale CD3⁺ positive selection approach using SAMBs.

1. Incubate 3 x 10⁸ commercially obtained PBMCs in 2.5 mL of separation buffer with biotinylated anti-CD3 (OKT3) antibody at a concentration of 25 ng of antibody per 1 million cells (25 ng/M). Gently mix by pipetting up and down, and incubate at room temperature for 10 min.
2. Add streptavidin microbubbles (SAMBs) at a ratio of 0.5 (SAMB quantity):1 (cell quantity) according to the manufacturer's reported SAMB concentration.
....

Results

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.......

Discussion

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.......

Materials

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