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Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Published: October 12th, 2018

DOI:

10.3791/58332

1Biomedical Engineering, Translational Tissue Engineering Center, Institute for Nanobiotechnology, Johns Hopkins University School of Medicine, 2Biomedical Engineering, Translational Tissue Engineering Center, Institute for Nanobiotechnology, Ophthalmology, Oncology, Neurosurgery, Materials Science and Engineering, Chemical and Biomolecular Engineering, and the Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine
* These authors contributed equally

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.

To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for "off-the-shelf" immunotherapies.

Artificial antigen presenting cells (aAPC) have shown promise as immunomodulatory agents because they can generate a robust antigen-specific T cell response. Essential to these platforms are their ability to efficiently present crucial signals for T cell activation. Acellular aAPC are an attractive alternative to cell-based aAPC because they are easier and less costly to fabricate, face fewer challenges during scale-up and translation, and alleviate risks associated with cell-based therapies. Acellular aAPC also allow for a high degree of control over signal presentation parameters and physical properties of the surface that will interface with T cells.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

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All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Johns Hopkins University.

1. Fabrication of Spherical PLGA Particles of Tunable Size

  1. Preparation of materials for particle synthesis
    1. Prepare 5% w/w polyvinyl alcohol (PVA) solution.
      1. Add 500 mL of deionized (DI) water to an Erlenmeyer flask with a magnetic stir bar and place on hot plate stirrer at 500 rpm and monitor temperature with thermometer. Cov.......

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A schematic for the automated 2D thin film stretching device is given in Figure 1. A schematic and description for a 1D thin film stretching device is given in Ho et al.17 The stretcher is constructed from aluminum parts using standard milling and machining techniques. Similar to the 1D stretcher, the 2D stretcher consists of metallic grips and guide rails. Bidirectional lead screws are used to translate linear to rotational motion. Th.......

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This protocol details a versatile method for the precise generation of anisotropic polymeric particles. The thin film stretching technique described here is scalable, highly reproducible and inexpensive. Alternative techniques for generating anisotropic particles suffer from many limitations, including high cost, low throughput, and limited particle size. The thin film stretching approach is also advantageous because the particles are modified to be anisotropic after synthesis, and, as a result, is compatible with a wide.......

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EBA (DGE-1746891) and KRR (DGE-1232825) thank the NSF Graduate Research Fellowship program for support. RAM thanks the National Research Service Award NIH NCI F31 (F31CA214147) and the Achievement Rewards for College Scientists Fellowship for support. The authors thank the NIH (R01EB016721 and R01CA195503), the Research to Prevent Blindness James and Carole Free Catalyst Award, and the JHU Bloomberg-Kimmel Institute for Cancer Immunotherapy for support.

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Name Company Catalog Number Comments
Poly(vinyl alcohol), MW 25000, 88% hydrolyzed Polysciences, Inc. 02975-500
Glycerol Sigma-Aldrich G9012
Digital Thermometer Fluke N/A Model name: Fluke 52 II
Immersion Temperature Probe Fluke N/A Model name: Fluke 80PK 22
Digital Hotplate & Stirrer Benchmark Scientific H3760-HS
Multipoint stirrer Thermo Fisher Scientific 50093538
Resomer RG 504 H, Poly(D,L-lactide-co-glycolide) Sigma-Aldrich 719900
Dichloromethane Sigma-Aldrich D65100
Homogenizer IKA  0003725001
Sonicator Sonics & Materials, Inc. N/A Model number: VC 505
Sonicator sound abating enclosure Sonics & Materials, Inc. N/A Part number: 630-0427
Sonicator probe Sonics & Materials, Inc. N/A Part number: 630-0220
Sonicator microtip Sonics & Materials, Inc. N/A Part number: 630-0423
High speed centrifuge Beckman Coulter N/A Model number: J-20XP (discontinued), alternative model: J-26XP
High speed centrifuge rotor Beckman Coulter 369691 Model number: JA-17
High speed polycarbonate centrifuge tubes Thermo Fisher Scientific 3118-0050 50 mL, screw cap
Rectangular disposable petri dish VWR International 25384-322 75 x 50 x 10 mm
Square disposable petri dish VWR International 10799-140 100 mm x 100 mm
LEAF Purified anti-mouse CD3ε Antibody Biolegend 100314
InVivoMab anti-mouse CD28, clone 37.51 Bio X Cell BE0015-1
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride Sigma-Aldrich E6383
N-Hydroxysulfosuccinimide sodium salt Sigma-Aldrich 56485
MES Sigma-Aldrich M3671
Alexa Fluor 488 anti-mouse CD3 Antibody Biolegend 100212
APC anti-mouse CD28 Antibody Biolegend 102109
Corning 96 Well Solid Polystyrene Microplate Sigma-Aldrich CLS3915 flat bottom, black polystyrene
Protein LoBind Tubes, 1.5 mL Eppendorf 22431081
RPMI 1640 Medium (+ L-Glutamine) ThermoFisher Scientific 11875093
Fetal Bovine Serum Sigma-Aldrich F4135 Heat Inactivated, sterile-filtered
Ciprofloxacin Sigma-Aldrich 17850
2-Mercaptoethanol Sigma-Aldrich M6250
Recombinant Human IL-2 (carrier-free) Biolegend 589102
Sodium Pyruvate (100 mM) ThermoFisher Scientific 11360070
MEM Non-Essential Amino Acids Solution (100X) ThermoFisher Scientific 11140050
MEM Vitamin Solution (100X) ThermoFisher Scientific 11120052
CD8a+ T Cell Isolation Kit, mouse Miltenyi Biotech 130-104-075
CellTrace CFSE Cell Proliferation Kit ThermoFisher Scientific C34554
LS Columns Miltenyi Biotech 130-042-401
MidiMACS Separator Miltenyi Biotech 130-042-302
MACS Multistand Miltenyi Biotech 130-042-303
Flow Cytometer Accuri C6
Synergy 2 Multi-Detection Microplate Reader BioTek
autoMACS Running Buffer Miltenyi BIotech 130-091-221
Cell Strainer ThermoFisher Scientific 22363548 Sterile, 70 µm nylon mesh
ACK Lysing Buffer ThermoFisher Scientific A1049201
C57BL/6J (Black 6) Mouse The Jackson Laboratory 000664 Male, at least 7 weeks old
U-Bottom Tissue Culture Plates VWR 353227 Sterile, 96-well tissue culture treated polystyrene plates
40 V DC Power Supply Probotix LPSK-4010
PTFE Coated Wire Mouser 602-5858-100-01 This is for a 100 ft. spool but an equivalent wire will work
Stepper Motor Driver Probotix MondoStep5.6
IDC Connector Kit Probotix IDCM-10-12
Microcontroller Probotix PBX-RF
4A Fuses Radio Shack 2701026 Equivalent fuses will work as well
DB25 Male to Male Cable Probotix DB25-6
USB-A to USB-B Cable Staples 2094915 Equivalent cable will work as well
8-Pin Amphenol Connectors Male and Female Mouser 654-97-3100A-20-7P and 654-97-3106A20-7S
Stepper Motor Probotix HT23-420-8
Right Hand Lead Screw Roton 60722
Left Hand Lead Screw Roton 60723
Screws McMaster Carr 92196A151
Neoprene Rubber McMaster Carr 8698K51
Right Handed Flanged Lead Nut Roton 91962
Left Handed Flanged Lead Nut Roton 91963
Linux Control Computer Probotix LCNC-PC Any computer with matching specification and Linux operating system will work
Corning bottle-top vacuum filter system Sigma-Aldrich CLS431097
Trypan Blue Solution, 0.4 % ThermoFisher Scientific 15250061

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