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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Poly(vinyl alcohol), MW 25000, 88% hydrolyzedPolysciences, Inc.02975-500
GlycerolSigma-AldrichG9012
Digital ThermometerFlukeN/AModel name: Fluke 52 II
Immersion Temperature ProbeFlukeN/AModel name: Fluke 80PK 22
Digital Hotplate & StirrerBenchmark ScientificH3760-HS
Multipoint stirrerThermo Fisher Scientific50093538
Resomer RG 504 H, Poly(D,L-lactide-co-glycolide)Sigma-Aldrich719900
DichloromethaneSigma-AldrichD65100
HomogenizerIKA 0003725001
SonicatorSonics & Materials, Inc.N/AModel number: VC 505
Sonicator sound abating enclosureSonics & Materials, Inc.N/APart number: 630-0427
Sonicator probeSonics & Materials, Inc.N/APart number: 630-0220
Sonicator microtipSonics & Materials, Inc.N/APart number: 630-0423
High speed centrifugeBeckman CoulterN/AModel number: J-20XP (discontinued), alternative model: J-26XP
High speed centrifuge rotorBeckman Coulter369691Model number: JA-17
High speed polycarbonate centrifuge tubesThermo Fisher Scientific3118-005050 mL, screw cap
Rectangular disposable petri dishVWR International25384-32275 x 50 x 10 mm
Square disposable petri dishVWR International10799-140100 mm x 100 mm
LEAF Purified anti-mouse CD3ε AntibodyBiolegend100314
InVivoMab anti-mouse CD28, clone 37.51Bio X CellBE0015-1
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorideSigma-AldrichE6383
N-Hydroxysulfosuccinimide sodium saltSigma-Aldrich56485
MESSigma-AldrichM3671
Alexa Fluor 488 anti-mouse CD3 AntibodyBiolegend100212
APC anti-mouse CD28 AntibodyBiolegend102109
Corning 96 Well Solid Polystyrene MicroplateSigma-AldrichCLS3915flat bottom, black polystyrene
Protein LoBind Tubes, 1.5 mLEppendorf22431081
RPMI 1640 Medium (+ L-Glutamine)ThermoFisher Scientific11875093
Fetal Bovine SerumSigma-AldrichF4135Heat Inactivated, sterile-filtered
CiprofloxacinSigma-Aldrich17850
2-MercaptoethanolSigma-AldrichM6250
Recombinant Human IL-2 (carrier-free)Biolegend589102
Sodium Pyruvate (100 mM)ThermoFisher Scientific11360070
MEM Non-Essential Amino Acids Solution (100X)ThermoFisher Scientific11140050
MEM Vitamin Solution (100X)ThermoFisher Scientific11120052
CD8a+ T Cell Isolation Kit, mouseMiltenyi Biotech130-104-075
CellTrace CFSE Cell Proliferation KitThermoFisher ScientificC34554
LS ColumnsMiltenyi Biotech130-042-401
MidiMACS SeparatorMiltenyi Biotech130-042-302
MACS MultistandMiltenyi Biotech130-042-303
Flow CytometerAccuri C6
Synergy 2 Multi-Detection Microplate ReaderBioTek
autoMACS Running BufferMiltenyi BIotech130-091-221
Cell StrainerThermoFisher Scientific22363548Sterile, 70 µm nylon mesh
ACK Lysing BufferThermoFisher ScientificA1049201
C57BL/6J (Black 6) MouseThe Jackson Laboratory000664Male, at least 7 weeks old
U-Bottom Tissue Culture PlatesVWR353227Sterile, 96-well tissue culture treated polystyrene plates
40 V DC Power SupplyProbotixLPSK-4010
PTFE Coated WireMouser602-5858-100-01This is for a 100 ft. spool but an equivalent wire will work
Stepper Motor DriverProbotixMondoStep5.6
IDC Connector KitProbotixIDCM-10-12
MicrocontrollerProbotixPBX-RF
4A FusesRadio Shack2701026Equivalent fuses will work as well
DB25 Male to Male CableProbotixDB25-6
USB-A to USB-B CableStaples2094915Equivalent cable will work as well
8-Pin Amphenol Connectors Male and FemaleMouser654-97-3100A-20-7P and 654-97-3106A20-7S
Stepper MotorProbotixHT23-420-8
Right Hand Lead ScrewRoton60722
Left Hand Lead ScrewRoton60723
ScrewsMcMaster Carr92196A151
Neoprene RubberMcMaster Carr8698K51
Right Handed Flanged Lead NutRoton91962
Left Handed Flanged Lead NutRoton91963
Linux Control ComputerProbotixLCNC-PCAny computer with matching specification and Linux operating system will work
Corning bottle-top vacuum filter systemSigma-AldrichCLS431097
Trypan Blue Solution, 0.4 %ThermoFisher Scientific15250061

References

  1. Eggermont, L. J., Paulis, L. E., Tel, J., Figdor, C. G. Towards efficient cancer immunotherapy: Advances in developing artificial antigen-presenting cells. Trends in Biotechnology. 32 (9), 456-465 (2014).
  2. Maus, M. V., Riley, J. L., Kwok, W. W., Nepom, G. T., June, C. H.

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