Diagnologix pioneers the development of microbubbles enabled separation method (BUBLES: buoyancy enabled separation) and system for clinical and research applications. This technology has several advantages over others in performing bulk cell isolation. First, its lipid shell microbubble is self-molding to external forces (e.g. ultrasound and bound cells), because it is the most compressible and flexible shell of microbubbles, for instance compared to albumin, polymer or glass. In conjunction with a gas core, it is a very gentle material for cell isolation. Second, the microbubble is a “self-driving vehicle”, as microbubble-bound cells automatically float to the top surface of a liquid. Third, the lipid-shelled microbubbles self-separate from water-based solutions and self-concentrate/aggregate to other microbubbles. Fourth, cell-bound microbubbles can be disrupted from their internal bonds, without causing cell damage, by
increasing ambient air pressure.
Given their cell-sized and flexible phospholipid surface, microbubbles are ideal as the long-sought-after scalable synthetic materials for artificial antigen-presenting cells (aAPC).
The following sections will describe how these unique advantages of microbubble can help resolve the cell therapy challenges. This technology has been applied to the following applications (with patents granted).
- Cell isolation and concentration (e.g. for isolating circulating tumor cellsa & hematopoietic stem cellsb)
- Serial multiple-marker cell sorting: iBUBLES (e.g. for naïve or regulatory T cell isolation)c
- Artificial antigen/ligand presenting cells (e.g. for T cell activation/expansion)d
- Artificial Virus presenting cells (e.g. for CAR-T cell production)c,e
Shi G, Cui W, Mukthavaram R, Liu YT, Simberg D. Binding and isolation of tumor cells in biological media with perfluorocarbon microbubbles. Methods 2013;64:102-7.
Wang G, Benasutti H, Jones JF, et al. Isolation of Breast cancer CTCs with multitargeted buoyant immunomicrobubbles. Colloids Surf B Biointerfaces 2018;161:200-9.
b Shi G, Wu CN, Darmadi S, Wang Z, Carson D, Liu Y-T. Buoyant Targeted Microbubble Technology Provides Efficient and Superior Quality of Hematopoietic Stem Cells Isolated from Umbilical Cord Blood. AACR 2020 Annual Meeting, #4397, 2020.
c Fu C, Shi G, Liu Y-T. Manufacturing Anti-CD19 CAR-Tscm Cells for Immunotherapy Using Innovative Microbubble-Based Technologies for Precision Cell Processing Blood. 2021;138 (Supplement 1):3889.
d Lustig A, Manor TK, Shi G, Li J, Wang Y-T, An Y, Liu Y-T, Weng N-p. Lipid Microbubble–Conjugated Anti-CD3 and Anti-CD28 Antibodies (Microbubble-Based Human T Cell Activator) Offer Superior Long-Term Expansion of Human Naive T Cells In Vitro. ImmunoHorizons. 2020;4(8):475-84.
Lu J, Chen G, Sorokina A, Nguyen T, Wallace T, Nguyen C, Dunn C, Wang S, Ellis S, Shi G, McKelvey J, Sharov A, Liu Y-T, Schneck J, Weng N-p. Cytomegalovirus infection reduced CD70 expression, signaling and expansion of viral specific memory CD8+ T cells in healthy human adults. Immunity & Ageing. 2022;19(1):54.
Choy C, Chen J, Li J, Gallagher DT, Lu J, Wu D, Zou A, Hemani H, Baptiste BA, Wichmann E, Yang Q, Ciffelo J, Yin R, McKelvy J, Melvin D, Wallace T, Dunn C, Nguyen C, Chia CW, Fan J, Ruffolo J, Zukley L, Shi G, Amano T, An Y, Meirelles O, Wu WW, Chou CK, Shen RF, Willis RA, Ko MSH, Liu YT, De S, Pierce BG, Ferrucci L, Egan J, Mariuzza R, Weng NP. SARS-CoV-2 infection establishes a stable and age-independent CD8(+) T cell response against a dominant nucleocapsid epitope using restricted T cell receptors. Nat Commun. 2023;14(1):6725.
Underwood S, Jin J, Shao L, Prochazkova M, Shi R, Song HW, Jin P, Shah NN, Somerville RP, Stroncek DF, Highfill SL. T Cell Activators Exhibit Distinct Downstream Effects on Chimeric Antigen Receptor T Cell Phenotype and Function. Immunohorizons. 2024 Jun 1;8(6):404-414. (External Validation)
e Liu Y, Yin M, Kunte N, Lopez-Ramirez M, Shi G. Targeted lentiviral transduction using microbubble-based artificial virus presenting cells. Cytotherapy. 2024;26(6, Supplement):S212-S3.
Targeted Microbubbles (A) The basic components of a targeted microbubble. (B) The image shows characteristic rosettes when microbubbles (MB) bind to target cells, enabling separation by flotation. (C) With anti-CD3/CD28 conjugated MBs (indicated by pink arrows) as artificial APC (aAPC) for T cell activation (T1-T3), immunological synapse formation is evident by the polarized elongated cell shape (T1 and T2, left panels) and F-actin staining by fluorescein labeled phalloidin (middle panels) at the contact zone. The T cell that is not activated by MB (T3) has typical cortical F-actin staining. The cell nucleus is stained with DAPI (right panels).
Cell Isolation and Concentration
A patented cone-shaped syringe-like tube is used to isolate microbubbles/cells in a small-volume sample collecting tube.
Patents granted: US10302536, CN106255537, JP6557681, EP3142764
Serial Multiple-Marker Cell Sorting: iBUBLES
The Principle and Method of BUBLES-Based Serial Multi-Marker Cell Isolation: Iterative BUBLES or iBUBLES (US Patent: 10479976). The Conceptual Scheme (top panel) illustrates that targeted balloons (microbubbles) can find the objects of interest (cells) (A), and separate the targeted objects (red marker) by floatation from the background (B). After the background objects are removed, the balloons are burst, which result in the targeted objects falling (C). Therefore, the first set of the objects of interest can be targeted by the 2nd type of balloons (targeting green marker) for the 2nd iteration (D). The practical Method is shown in the bottom panel (animation explaining the method). Microbubbles (MB) targeting a first cell surface marker of the targeted cells are mixed with PBMC and incubate for 30 minutes with rotation (A). The container is then inverted and non-targeted cells in waste are discarded by pushing down the plunger (B). Optional cell washing steps may be applied. The container is turned around again and the plunger is pulled out, increasing the airspace volume within the container (C). Subsequently, the cap is installed again, sealing the container, so that the air pressure is increased by pushing up the plunger (D), which results in disruption of MB, and returns cells back to solution (E). After optionally adding again a diluent or media, the steps A – E can be repeated for a second set of microbubbles targeting a second cell surface marker of the targeted cells. FACS analysis shows an example of serial CD8+/CD45RA+/CD62L+ naïve T cell enrichment (indicated by red arrows) using this technology. The efficiency for each cycle was > 90%.
Patents granted: US10479976, EP3555267, JP7061125, CN110072993, AU2017378413B2
The dissolution of cell-bound lipid-shelled microbubbles occurs due to air compression at 2 atm, as depicted in a series of time-lapse images captured over 2 seconds.
Microbubbles (MB) based T cell activations. MB_1X: PBMCs were activated once (at day 0) by MB human T-activator and then incubated for 30 days. MB_2X: PBMCs were sequentially activated by MB human T-activator at day 0 and day 14, respectively. The leading commercial magnetic microbeads (Bead) based T cell activation was used as control. Notably, T cell proliferation persisted after 2nd stimulation by MBs (MB_2X), but not by microbeads (Bead_2X).
Patents granted: US10479976, EP3555267, JP7061125, CN110072993, AU2017378413B2
Artificial Virus Presenting Cell
Microbubble-based artificial virus presenting cell (aVPC) (WO/2022/103756). Forming a “virological synapse” is an efficient mechanism for direct cell-to-cell transmission by viruses, such as HIV-1. Microbubble (MB)-based artificial virus presenting cells (aVPCs) mimic this interaction to facilitate efficient and convenient viral transduction (A). A MB conjugated with excess ligands that bind both viral particles and target cells bridges their interactions. The virus and cell binding domains can be in the same molecule, such as RetroNectin (RN), or different ones. Unlike free viral particles in solution (B), the interaction of the bound viral vectors on an aVPC and the viral receptors on the target cell are in 2D space. In contrast to the solid surface of a microbead (C), the viral particles on the fluid lipid shell of a microbubble are mobile. An example of applying MB-based aVPCs is presented (D). Recombinant retroviruses expressing GFP were incubated with MB-anti-CD3/CD28 treated PBMCs in the absence (D1) or presence (D2) of RN. The transduction rates were poor for both conditions. Conveniently, incubating MB-RN with viruses and PBMCs (D4) resulted in equivalent (or better) transduction efficiency, compared to the conventional method of using an equal amount of RN coated plates in conjunction with 2-hour spinoculation (D3).
Patents: pending