Promover 3-D agregação de células FACS purificada epiteliais tímicos com EAK 16-II / EAKIIH6 de auto-montagem Hidrogel
Summary
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.
Abstract
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.
Introduction
O timo é o órgão linfóide primário responsável pela geração de uma população diversa de células, T auto-tolerante patógeno-reactivo que é essencial à função do sistema imune adquirida. Ele é um órgão dinâmico onde timócitos em desenvolvimento, emigraram da medula óssea como células de linfócitos progenitoras, migram através da tridimensional (3-D) de matriz semelhante a esponja do estroma tímico, submeter a linhagem de especificação e diferenciação, e, eventualmente, emigrar quanto madura T-células. O êxito deste processo bem programado depende em grande parte da conversa cruzada entre os timócitos migram e as células epiteliais tímicas residenciais (TEC), a população predominante do estroma tímico que são essenciais para o estabelecimento e a manutenção da integridade do microambiente timo.
Com base na sua localização anatômica e função original, TEC pode ser dividido em dois subconjuntos: os TEC no córtex (CTECs), que são responsável por selecionando auto-MHC (complexo principal de histocompatibilidade) restringiu as células T (seleção positiva), e os TEC na medula (mTECs) que são essenciais para a eliminação de células T auto-reativas (seleção negativa) 1,2. Muitos fatores (por exemplo, envelhecimento, infecção, irradiação, tratamentos de drogas) pode causar danos irreversíveis ao epitélio do timo, resultando na imunidade adaptativa comprometida. Apesar de numerosas tentativas, restaurando a função do timo tem sido difícil devido à dificuldade em reproduzir o microambiente do timo. Notavelmente, a configuração do timo tridimensional (3-D) é crítico para a sobrevivência e função de TEC, enquanto TEC cultivadas num ambiente de 2-D diminuir rapidamente a expressão de genes críticos para a timopoiese 3,4.
EAK16-II (AEAEKAKAEAEAKAK) e o seu análogo histidinylated C-terminal EAKIIH6 (AEAEKAKAEAEAKAKHHHHHH) são de baixo peso molecular, oligopéptidos anfifílicas que são solúveis até o final desionizadaR, mas que são submetidos a gelificação para formar p-fibrilas quando expostos à força iónica mais elevada do que 20 mM de NaCl (concentração normal de sal no fluido corporal humano é de 154 mM). Esta propriedade responsiva ambiental torna-os blocos de construção versáteis para formar estruturas 3D. A His-tag em EAKII-H6 proporciona um mecanismo de encaixe, pelo que os anti-His IgG / Fc-proteína de ligação recombinante A / G (αH6: PA / g) complexos podem servir como um adaptador para ancorar fármacos de proteína e outras biomoléculas ( por exemplo, anticorpos) no composto 5-8 hidrogel.
Foi anteriormente demonstrado que as moléculas de IgG marcado com fluorescentes ancoradas no hidrogel pode ser mantida no local de injecção para até 13 dias 9. Além disso, quando os αH6: adaptadores pA / G ancorados com anti-CD4 IgG foram adicionados ao hidrogel EAK16-II / EAKIIH6 (EAK), células T CD4 + foram especificamente capturado 10. Utilizando uma técnica semelhante, temos demonstrado recentemente que o 3-D agregação de TEC could ser promovida em EAK hidrogel com complexos de adaptadores que transportam os anticorpos anti-EpCAM específico-TEC (αH6: PA / G: αEpCAM). Quando transplantadas sob as cápsulas dos rins de ratinhos nude, os aglomerados TEC incorporados no hidrogel EAK pode efectivamente suportar o desenvolvimento de células T funcionais in vivo 11,12.
Aqui, ilustramos nosso método para purificar rapidamente e eficazmente com TEC de células activadas por fluorescência (FACS) e gerar 3-D agregados TEC com o sistema de EAK hidrogel.
Protocol
Representative Results
Discussion
Enquanto TEC são a população predominante do estroma tímico e desempenham papéis essenciais para a estrutura e a função das glândulas de timo, eles representam apenas cerca de 0,1-0,5% da celularidade do timo total. Eles também são células frágeis como elevadas percentagens de morte celular são ocasionalmente observados após a digestão de colagenase, ou seja, o tratamento de TEC dissociar a partir da matriz extracelular (ECM). Sua raridade (~ 200.000 por timo mouse) e fragilidade tornou uma taref…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Este trabalho foi financiado em parte pelos Institutos Nacionais de Saúde concede R21 AI113000 (WSM) e R01 AI123392 (YF).
Materials
| 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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