Name: preparation of tumor antigen-loaded mature dendritic cells for immunotherapy
Uploaded: 2013-08-01T12:00:00
Description: Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

लेखकों पाली ICLC के उपहार के लिए एन्ड्रेस Salazar (Oncovir, इंक) को धन्यवाद देना चाहूंगा.

1. PBMCs 4 के अलगाव और Cryopreservation <ol><li> Aseptically कील एक प्लाज्मा हस्तांतरण सेट का उपयोग कर leukapheresis बैग में उपयोग बंदरगाहों में से एक है. एक 60 मिलीलीटर सिरिंज का प्रयोग, एक बाँझ 500 मिलीलीटर की बोतल में रोगियों से प्राप्त le...

<ol>
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F., Oosterwijk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1-polarizing+capacity+of+clinical-grade+dendritic+cells+is+triggered+by+Ribomunyl+but+is+compromised+by+PGE2:+the+importance+of+maturation+cocktails.">Th1-polarizing capacity of clinical-grade dendritic cells is triggered by Ribomunyl but is compromised by PGE2: the importance of maturation cocktails.</a> J. Immunother. 28, 480-487 (2005).</li><li>Krause, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E2+is+a+key+factor+for+monocyte-derived+dendritic+cell+maturation:+enhanced+T+cell+stimulatory+capacity+despite+IDO.">Prostaglandin E2 is a key factor for monocyte-derived dendritic cell maturation: enhanced T cell stimulatory capacity despite IDO.</a> J. Leukoc. Biol. 82, 1106-1114 (2007).</li><li>Morelli, A. E., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+under+the+spell+of+prostaglandins.">Dendritic cells under the spell of prostaglandins.</a> Trends Immunol. 24, 108-111 (2003).</li><li>Adams, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+(DC)+based+therapy+for+cervical+cancer:+use+of+DC+pulsed+with+tumour+lysate+and+matured+with+a+novel+synthetic+clinically+non-toxic+double+stranded+RNA+analogue+poly+[I]:poly+[C(12)U]+(Ampligen+R).">Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R).</a> Vaccine. 21 (12), 787-790 (2003).</li><li>Colombo, M. 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प्रथम चरण और द्वितीय monocyte व्युत्पन्न DCs की क्लिनिकल परीक्षण वे रोगियों लेकिन नैदानिक ​​सफलता 1 सीमित किया गया है में प्रतिरक्षा प्रतिक्रिया को प्रेरित दिखाया है. यह ट्यूमर immunotherapeutic उपयोग के लिए इष्टत?...

<table border="1">
<tbody>
 <tr>
 <td fo:width="2in">Reagent/Supplies/Equipment</td>
 <td>Manufacturer</td>
 <td>Catalog No.</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI-1640 medium with L-glutamine</td>
 <td>BioWhittaker</td>
 <td>12-702F</td>
 <td></td>
 </tr>
 <tr>
 <td>1M HEPES buffered saline</td>
 <td>BioWhittaker</td>
 <td>17-737E</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>BioWhittaker</td>
 <td>17-516F</td>
 <td></td>
 </tr>
 <tr>
 <td>Human albumin, 25% solution USP</td>
 <td>Aventis Behring</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ficoll-Hypaque PREMIUM</td>
 <td>GE Healthcare</td>
 <td>17-5442-03</td>
 <td></td>
 </tr>
 <tr>
 <td>Human AB serum</td>
 <td>Valley Biomedical</td>
 <td>HP1022</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile saline USP</td>
 <td>Hospira</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CryoMACS DMSO </td>
 <td>Miltenyi Biotec</td>
 <td>170-076-303</td>
 <td></td>
 </tr>
 <tr>
 <td>Leukine GM-CSF, 0.5 mg/ml</td>
 <td>Berlex</td>
 <td>A02266</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS GMP IL-4</td>
 <td>Miltenyi Biotec</td>
 <td>170-076-101</td>
 <td></td>
 </tr>
 <tr>
 <td>Hiltonol, Poly-ICLC, 2 mg/ml</td>
 <td>Oncovir</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>VACMUNE KLH</td>
 <td>Biosyn</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>225 sq cm EasyFlasks</td>
 <td>Nalgene Nunc</td>
 <td>159934</td>
 <td></td>
 </tr>
 <tr>
 <td>Falcon 6-well tissue culture plates</td>
 <td>Becton Dickinson</td>
 <td>353046</td>
 <td></td>
 </tr>
 <tr>
 <td>1.8 ml CryoTube vials</td>
 <td>Nalgene Nunc</td>
 <td>377267</td>
 <td></td>
 </tr>
 <tr>
 <td>Controlled Rate Freezer</td>
 <td>Thermo</td>
 <td>CryoMed</td>
 <td></td>
 </tr>
 </tbody>
</table>

preparation of tumor antigen-loaded mature dendritic cells for immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

10 के बीच - शुरू PBMCs के 20% संस्कृति अवधि के अंत में डीसी में अंतर. परिपक्व DCs CD11c हैं + CD14-CD83 + CD40 +, और CCR7 + (चित्रा 1). वे MHC वर्ग मैं और द्वितीय अणुओं और costimulatory अणुओं CD80 और CD86 के उच्च स्तर को व्यक्त करते हैं. अन्य TLR agonists के <su...

Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

ट्यूमर प्रतिरक्षा चिकित्सा में उपयोग के लिए ऑटोलॉगस वृक्ष के समान कोशिकाओं की बड़ी संख्या (डीसी) पैदा करने के लिए सबसे अधिक इस्तेमाल किया विधि वर्णित है. विधि monocytes से डीसी अंतर करने के लिए आईएल -4 और जीएम-CSF का उपयोग करता है. वे रोगी में वापस इंजेक्शन हैं पहले अपरिपक्व डीसी परिपक्व और फिर एंटीजन के साथ लोड करने के लिए प्रेरित कर रहे हैं.

Immunotherapy के लिए ट्यूमर एंटीजन से भरी हुई परिपक्व वृक्ष के समान कोशिकाओं की तैयारी

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Research

JoVE Journal

Immunology and Infection

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बी कोशिकाओं की CD40 सक्रियण के murine मॉडल

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

सेल परिपक्वता के बिना वृक्ष के समान कोशिकाओं के कुशल अभिकर्मक के लिए अनुकूलित प्रोटोकॉल

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Isolation of Mouse Lung Dendritic Cells

माउस फेफड़े वृक्ष के समान कोशिकाओं का अलगाव

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

मानव थाइमस से माइलॉयड वृक्ष के समान कोशिकाओं और उपकला कोशिकाओं का अलगाव

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

देखभाल के ग्लियोब्लास्टोमा मानक के संदर्भ में दत्तक थेरेपी के लिए कार टी कोशिकाओं की पीढ़ी

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

मेटाबोलिक phenotypes भिन्न के साथ अपरिपक्व, परिपक्व और Tolerogenic वृक्ष के समान कोशिकाओं की पीढ़ी

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

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Draining लिम्फ नोड्स में Murine त्वचा वृक्ष के समान कोशिकाओं के प्रवास का अध्ययन करने के साथ संक्रमण के दौरान एक CFSE आधारित परख<em&gt; माइकोबैक्टीरियम बोविस</em&gt; Bacille Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

दुर्लभ प्रतिजन-विशिष्ट बी कोशिकाओं से रक्त में परिचालित में भेदभावपूर्ण मानव मोनोक्लोनल एंटीबॉडी की पीढ़ी

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

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विकास और एक Ultrasensitive एकल अणु सरणी डिजिटल एंजाइम के सत्यापन-मानव इंटरफेरॉन के लिए Immunosorbent परख से जुड़े-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

फिजियोलॉजिकल इम्यूनोजेनिक डेन्ड्रिटिक कोशिकाओं के सृजन के लिए उपन्यास प्रोटोकॉल

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...