Name: preparation of 3d fibrin scaffolds for stem cell culture applications
Uploaded: 2012-03-02T12:00:00
Description: Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

The authors would like to acknowledge NSERC Discovery Grant 402462 "Tissue engineered scaffolds for controlling induced pluripotent stem cell behavior".

Notes before starting protocol: Fibrinogen is a blood derived protein and thus appropriate safety training must be completed before handling. This protocol requires 2 days to complete so time the desired stem cultures appropriately to ensure they are ready for seeding. In terms of calculating how much fibrinogen to weigh out, three 35 mm petri dishes of tris buffered saline (TBS, pH 7.4) containing 110-130 mg of fibrinogen dissolved in 3 mL of TBS will be sufficient to produce 1 24 well plate containing ...

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This protocol detailed above provides a method for generating 3D fibrin scaffolds for pluripotent stem cell culture, specifically for mouse embryonic and induced pluripotent stem cells. This 3D biomaterial based culture system more accurately mimics the stem cell niche found in vivo and as a result, it can be used to screen biological cues to determine their effects on stem cell differentiation 6. Our observations have shown that these scaffolds when seeded with stem cell derived embryoid bodies remai...

Equipment needed:
	Analytical balance 
pH meter 
Tissue culture incubator (37&deg;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 &mu;m syringe filters 
Individually wrapped sterile 0.22 &mu;m syringe filters 
Syringe 
24 well tissue culture plates

 <table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Fibrinogen (human)</td>
 <td>Calbiochem</td>
 <td>341578 </td>
 <td></td>
 </tr>
 <tr>
 <td>Thrombin (human)</td>
 <td>Sigma</td>
 <td>T7009</td>
 <td></td>
 </tr>
	</tbody>
 </table>

preparation of 3d fibrin scaffolds for stem cell culture applications

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.

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

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 ...

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