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* 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
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
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.......
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.......
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.
....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 | Â 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+Â 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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