3 डी आतंच की तैयारी स्टेम सेल संस्कृति अनुप्रयोगों के लिए scaffolds
Summary
यह काम 3 डी आतंच की तैयारी विवरण के संवर्धन और plutipotent स्टेम कोशिकाओं फर्क करने के लिए scaffolds. इस तरह के scaffolds के स्टेम सेल के व्यवहार पर विभिन्न जैविक यौगिकों के प्रभाव स्क्रीन के रूप में अच्छी तरह के रूप में दवा वितरण प्रणाली को शामिल करने के लिए संशोधित करने के लिए इस्तेमाल किया जा सकता है.
Abstract
Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications – demonstrating the versatility of this approach 8-12.
Protocol
Discussion
इस प्रोटोकॉल के ऊपर विस्तृत pluripotent स्टेम सेल संस्कृति के लिए 3 डी आतंच scaffolds के सृजन, माउस भ्रूण और प्रेरित pluripotent स्टेम सेल के लिए विशेष रूप से करने के लिए एक तरीका प्रदान करता है. इस 3 डी biomaterial आधारित संस्कृति प्?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए प्रेरित pluripotent स्टेम सेल के व्यवहार को नियंत्रित करने के लिए ऊतक इंजीनियर scaffolds के NSERC डिस्कवरी अनुदान 402,462 स्वीकार करते हैं करना चाहते हैं.
Materials
Equipment needed:
Analytical balance
pH meter
Tissue culture incubator (37°C, 5% CO2)
Stir plate
Spectrophotometer
Sterile tissue culture hood
Tris buffered saline (pH 7.4) (Need 4 L plus enough for dissolving fibrinogen)
50 mM calcium chloride solution
Sterile conical tubes (15 or 50 mL)
35 mm Petri dishes
Dialysis tubing (7,000 MW cutoff)
Dialysis clips
5.0 μm syringe filters
Individually wrapped sterile 0.22 μm syringe filters
Syringe
24 well tissue culture plates
| Name of the reagent | Company | Catalogue number | Comments |
| Fibrinogen (human) | Calbiochem | 341578 | |
| Thrombin (human) | Sigma | T7009 |
References
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