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

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. Cover flask with tinfoil to prevent evaporation.
      2. When water temperature reaches approximately 70 °C, add 25 g total of PVA in small batc....

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.......

Materials

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