1Department of Biology, University of Victoria, 2Department of Mechanical Engineering, Division of Medical Sciences, University of Victoria
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Kolehmainen, K., Willerth, S. M. Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications. J. Vis. Exp. (61), e3641, doi:10.3791/3641 (2012).
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.
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 400 μl fibrin scaffolds in each well.
1. Day One: Making fibrinogen solutions and overnight dialysis
2. Day 2: Polymerization of fibrin scaffolds and seeding of stem cells into scaffolds
All steps described for this process should be performed in a sterile tissue culture hood as these scaffolds will be seeded with stem cell cultures.
3. Representative Results
Figure 1 shows a schematic of the side view of an individual well containing the 3D fibrin scaffold culture system seeded with an embryoid body. Figure 2A shows representative images of mouse induced pluripotent stem cells being cultured on mouse embryonic fibroblast feeder layers. These cells are then induced to form embryoid bodies, aggregates of cells containing neural progenitors, using an 8 day retinoic acid treatment protocol (Figure 2B) as previously described 14 while Figure 3 shows the appearance of an iPS-derived embryoid body after 3 days of culture inside of 3D fibrin scaffolds. Similar results have been obtained previously using mouse embryonic stem cells 7. Additionally, this method has been used to culture iPS-derived embryoid bodies produced using a different protocol involving retinoic acid and purmorphamine 15 inside of 3D fibrin scaffolds, demonstrating the versatility of this culture system.
Figure 1. Schematic showing the side view of an individual well containing a 3D fibrin scaffold seeded with an embryoid body.
Figure 2. Mouse induced pluripotent stem (iPS) cell culture and differentiation . To differentiate these cells into neural phenotypes, the iPS cells are cultured in suspension to produce aggregates of cells called embryoid bodies (EBs). These EBs are then treated with retinoic acid to induce neural differentiation and this protocol was previously used to induce neural differentiation of embryonic stem cells. A) Undifferentiated mouse iPS cell colony cultured on a mouse embryonic fibroblast feeder layer. B) Embryoid bodies derived from mouse iPS cells in suspension culture taken on day 8 after treatment with retinoic acid to induce neural differentiation. Scale bar is 100 μm.
Figure 3. Example results of a mouse iPS embryoid body after 3 days of culture inside of a 3D fibrin scaffold. Cells have begun to migrate and differentiate inside of the fibrin scaffold. Scale bar is 500 μm.
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 remain for 2 weeks in vitro before becoming completely degraded. To further increase the stability of these scaffolds, aprotinin (a protease inhibitor) can be used to slow degradation through addition to the cell culture media.7,16 These scaffolds have also been used successfully for neural tissue engineering applications, specifically the generation of tissue similar to that found in the central nervous system17. While other groups have combined iPS cells with fibrin glue for treating Ischemic stroke18, this work represents the first known report of combining iPS cells with fibrin scaffolds for neural tissue engineering applications. This work also served as a starting point for combining a range of stem cell types with fibrin scaffolds for other tissue engineering applications.
The authors have nothing to disclose.
The authors would like to acknowledge NSERC Discovery Grant 402462 "Tissue engineered scaffolds for controlling induced pluripotent stem cell behavior".
Tissue culture incubator (37°C, 5% CO2)
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)
5.0 μm syringe filters
Individually wrapped sterile 0.22 μm syringe filters
24 well tissue culture plates