1Institute of Biomaterials and Biomedical Engineering / Department of Chemical Engineering and Applied Chemistry, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto
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Chamberlain, M. D., Butler, M. J., Ciucurel, E. C., Fitzpatrick, L. E., Khan, O. F., Leung, B. M., et al. Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells. J. Vis. Exp. (46), e2177, doi:10.3791/2177 (2010).
1) Preparation of Tubing
2) Neutralization of Collagen
Note: 1 ml of collagen fills about 2 m of polyethylene tubing.
3) Embedding of Cells (Optional)
Note: Depending on the cell type the cells can be embedded at a concentration of between 1 million cells per mL to 20 million cells per mL. Use the medium required for the cells that you want to embed.
4) Gelling of Collagen
5) Cutting of Tubing Containing the Gelled Collagen
6) Removing Collagen Modules from Tubing
7) Coating the modules with endothelial cells
8) Representative Results:
The modules will look cylindrical when they first are removed from the tube. If the modules contain embedded cells and/or are coated with endothelial cells they can contract up to 50% in volume and develop an oval shape (Figure 1). The modules will also become denser and more opaque when viewed by light microscopy (Figure 1). Also when the endothelial cells are confluent on the surface of the modules there is a formation of tight junctions which can be seen by immunofluorescence staining of VE-Cadherin (Figure 2). Mass transfer analysis has demonstrated that modules are capable of supporting high cell densities (8x107 cells/cm3) without developing a necrotic core due to inadequate oxygen transport, which is often problematic in larger tissues (e.g. modules of large diameter, D = 1.4mm) (Figure 3).
Figure 1: Module fabrication and contraction. Module contraction occurred during the three days following HUVEC-C (endothelial cell line) seeding results significantly smaller module diameter and length (p < 0.001) (A). Embedded HepG2 cells were uniformly distributed within modules at time of fabrication and retained high viability (B). [Live cells green; dead cells red] [adapted from Corstorphine 2010]
Figure 2: VE-Cadherin immunofluorescent staining of EC tight cell junctions 10 days after RAEC where coated on the surface of a module. Scale bar = 50 μm.
Figure 3: Masson's trichrome staining of typical modules (0.76 mm initial diameter) and large modules (1.4 mm initial diameter) demonstrate the effect of oxygen diffusion limitations. Seven days following fabrication, a large number of dead cells had formed within the core of the large modules (lower right panel), leaving only a thin rim (~200μm thick) of viable cells. Conversely, the small modules retained a uniform and high distribution of live cells. [Modules embedded with HepG2 cells and coated with endothelial cells.] [Taken from Corstorphine 2010]
Figure 4: (A) HMEC-1 seeded on poloxamine - collagen modules. After Day 1 of seeding the poloxamine-collagen modules retain their cylindrical shape and there is limited cell attachment properties compared to collagen only modules. Scale bar = 200 μm. (B) Light microscope image of a collagen module containing PLGA-based biodegradable microspheres. Scale bar = 500 μm.
Figure 5: Examples of in vitro assays. (A) Angiogenesis assay: capillary-like formations on the module seeded with endothelial cells can be easily detected and quantified after 5 days of incubation with adipose-derived stem cells. (B) Confocal microscopy images of a live/dead assay on modules where the staining for live cells is green and for dead cells is red.
Figure 6: Collagen modules containing embedded mesenchymal stem cells and a surface layer of endothelial cells. Modules were exposed to shear stress (~0.64 dyn/cm2) for 7 days in a microfluidic chamber and begin to show fusion at points of contact. Endothelial cells are stained with vWF (brown) and mesenchymal stem cell nuclei appear blue (H&E). Scale bar = 100μm.
Figure 7: Hundreds of collagen modules containing Adipose-Derived Stem cells (ASC) and coated with Human Microvascular Endothelial Cells (HMEC) are implanted under mouse skin to study ASC/HMEC interaction in vivo for fat regeneration applications.
We have fabricated several different micro-tissues using modules embedded with different cell types1-11. We have successfully embedded primary cardiomyocytes, islets, adipose stem cells and mesenchymal stromal cells as well as several cell lines including HepG2, NIH 3T3 and clone 9 liver cells. We have coated the modules with several types of endothelial cells including rat aortic endothelial cells, human umbilical vein endothelial cells and human microvascular endothelial cells. Modules have also been produced with a mixture of collagen and a poloxamine polymer or containing drug eluting microspheres (Figure 4). Several in vitro assays have been shown to be compatible with modules including immunofluorescence, western blot, angiogenesis, alamar blue proliferation and live/dead assays (Figure 5). They have also been used in microfluidic chambers (Figure 6) and implanted into mice and rats (Figure 7) to study the interaction between different cell types and how the micro-tissues are remodeled by the host response. Modules have many applications and can be used for the study of the effect of 3D tissue culture on cells, interaction of different cells in a 3D conformation, remodeling of micro-tissues in a microfluidic chamber and in vivo remodeling of micro-tissues by a host response.
No conflicts of interest declared.
|Intramedic tubing PE60||BD Biosciences||427416||Different diameter of tubing can be used to change the diameter of the modules|
|Phosphate-buffered saline (PBS)||GIBCO, by Life Technologies||20012-027|
|Trypsin-EDTA||GIBCO, by Life Technologies||25200-072|
|Purcol acidificed collagen, 3 mg/mL||Cedarlane Labs||5005-B|
|20G needle||BD Biosciences||305175||Diameter of the needle needs to be similar to the diameter of the tubing|
|3 mL syringe||BD Biosciences||309585|
|15 mL tube||BD Biosciences||352096|
|50 mL tube||BD Biosciences||352070|
|Convertors Self-Seal Pouch 7 1/2” x 13”||Cardinal Health||92713|
|10 ml Wide Tip serological pipet||BD Biosciences||357504|
|10 ml serological pipet||BD Biosciences||357551|
|5 ml serological pipet||BD Biosciences||357543|