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Immunology and Infection

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Published: June 27th, 2016

DOI:

10.3791/54062

1Institute of Cellular Therapeutics, Allegheny Health Network, 2Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University, 3Department of Biological Sciences, Carnegie Mellon University

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

The thymus is the primary lymphoid organ responsible for the generation of a diverse population of pathogen-reactive, self-tolerant T cells that is essential to the function of the acquired immune system. It is a dynamic organ where developing thymocytes, immigrated from the bone marrow as lymphocyte progenitor cells, migrate through the sponge-like three-dimensional (3-D) matrix of the thymic stroma, undergo lineage-specification and differentiation, and eventually emigrate as mature T-cells. The success of this well programmed process depends largely on the cross-talk between the migrating thymocytes and the residential thymic epithelial cells (TECs), the predominan....

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All the animals used in the experiments were housed in the animal facility at Allegheny-Singer Research Institute under the protocol reviewed and approved by the Institutional Animal Care and Use Committee of the Allegheny Health Network/Allegheny Singer Research Institute.

1. Digesting the Thymus with Collagenase

  1. Harvest thymus glands.
    1. Euthanize 3-5 week-old C57BL6/J mice in a carbon dioxide (CO2) chamber to harvest thymus glands. In detail, place the mice i.......

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To examine the effectiveness of using the density gradient separation protocol to enrich the CD45- stromal cells, cells harvested from both the interface and the precipitated lymphocyte pellets were stained with anti-CD45 and anti-EpCAM antibodies. Both anti-Ulex Europaeus Agglutinin 1 (UEA1) and anti-MHC Class II antibodies were also included in the staining cocktail to further identify the cTEC and mTEC subsets. As shown in Figure 1, we were able to routinely achieve cl.......

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While TECs are the predominant population of the thymic stroma and play essential roles for the structure and function of the thymus glands, they represent only about 0.1-0.5% of the total thymic cellularity. They are also fragile cells as high percentages of cell death are occasionally observed following collagenase digestion, i.e., the treatment to dissociate TECs from the extracellular matrix (ECM). Their rarity (~200,000 per mouse thymus) and fragility made it a challenging task to isolate TECs. The recent u.......

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This work was supported in part by the National Institutes of Health grants R21 AI113000 (W.S.M) and R01 AI123392 (Y.F.).

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Name Company Catalog Number Comments
1. TEC Isolation
70% Ethanol Decon Laboratories 2701(1 Gallon) Ethanol 200 Proof, deionized water
Dissecting scissors, straight Fine Science Tools, Inc. 91460-11
Graefe forceps, straight Fine Science Tools, Inc. 11053-10
Graefe forceps, curved Fine Science Tools, Inc. 11052-10
Washing solution 1X PBS, 0.1% BSA, 2mM EDTA
1x PBS (Phosphate buffered saline) Gibco 10010-023
BSA (Bovine serum albumin) Sigma A1470-100G
EDTA (ethylenediaminetetraacetic acid) Invitrogen 15575-038
Digestion solution 9 mL RPMI-1640, 0.025 mg/mL Liberase TM Research grade, 10 mM HEPES, 0.2 mg/mL DNaseI
RPMI-1640 Gibco 11879-020
Liberase TM Research Grade Roche 05 401 127 001 referred as "purified collagenase"
1M HEPES Lonza 17-737E
Dnase I Roche 10 104 159 001
50mL Centrifuge tube Corning 430290
60mm tissue culture dish Falcon 353002
1/2cc U-100 Insulin syringe 28G1/2 Becton Dickinson 329461
5mL Polystyrene round-bottom tube Falcon 352058
5ml glass pipet Fisher Healthcare 13-678-27E Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets.
MACSmix tube rotator Miltenyi 130-090-753
100um Cell strainer Falcon 352360
Density gradient medium: OptiPrep Axis-Shield
Name Company Catalog Number Comments
2. Cell sorting
5mL Polypropylene round-bottom tube Falcon 352063
Anti-mouse CD16/CD32 (Fc Block) BD Biosciences 553142 Use as undiluted, 2uL per sample
Anti-mouse CD45-PercpCy5.5 eBioscience 45-0451-80 Use at 1:150, 10uL per sample
Anti-mouse CD326 (EpCAM)-PE eBioscience 12-5791-82 Use at 1:100, 10uL per sample
BD Influx BD Biosciences
Single cell analysis software FlowJo
Name Company Catalog Number Comments
2. EAK gel assembly
Anti-His-Tag AnaSpec 29673 "anti-His-Tag IgG"
Purified anti-mouse CD326 (EpCAM) BioLegend 118202 "anti-EpCAM IgG"
Recombinant protein A/G Pierce Biotechnology
1.5mL Safe-Lock Tubes, Biopur, Sterile Fisher Healthcare 05-402-24B referred as "1.5mL microcentrifuge tube" 
96-well, Tissue culture plate, Round-bottom with low evaporation lid BD Falcon 353917
Rocking platform: Nutator Mixer no.1105 BD Clay Adams
10% sucrose Sigma S0389 Prepare with sterile distilled water
EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH2) American Peptide Company  custom synthesized, 10mg/mL
EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH2) American Peptide Company  custom synthesized, 7.5mg/mL
Complete medium RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5mM HEPES, 50uM 2-Mercaptoethanol
RPMI-1640  Gibco 11879-020
FBS (Fetal Bovine Serum) Atlanta Biologicals S11150 Heat inactivated before use
Pen/Strep Gibco 15140-122
L-glutamine 200mM (100x) Gibco 25030-081
NEAA (non-essential amino acid) 100x Gibco 11140-050
1M HEPES BioWhittaker 17-737E
2-Mercaptoethanol (100X) Millipore ES-007-E
Platform shaker: The Belly Dancer Stovall Life Sciences Inc. model: USBDbo

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