Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells
Summary
Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.
Abstract
This protocol describes the fabrication of a type of micro-tissues called modules. The module approach generates uniform, scalable and vascularized tissues. The modules can be made of collagen as well as other gelable or crosslinkable materials. They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells. The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable. These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues. There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.
Protocol
1) Preparation of Tubing Cut 2 to 3 m lengths of polyethylene tubing (0.76 mm I.D. x 1.22 mm O.D.). Thread a 20 G1 needle into one end of the tubing. Coil the tubing and place in a convertors self-seal pouch (7 1/2″ x 13″). The two ends of the tubing should be placed at the unsealed end of the bag and taped in place with gas sterilization tape. Seal bag and gas sterilize. 2) Neutralization of Collagen Note: 1 m…
Discussion
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 prod…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Funding was provided by the National Institutes of Health (EB 001013), Natural Sciences and Engineering Research Council of Canada and Canadian Institutes of Health Research. We thank Dr. A. P. McGuigan for her expertise and help in development of modules.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Intramedic tubing PE60 | Becton Dickson | 427416 | Different diameter of tubing can be used to change the diameter of the modules | |
| Phosphate-buffered saline (PBS) | Gibco | 20012-027 | ||
| Trypsin-EDTA | Gibco | 25200-072 | ||
| Purcol acidificed collagen, 3 mg/mL | Cedarlane | 5005-B | ||
| Sodium bicarbonate | Sigma-Aldrich | S5761 | ||
| 20G needle | Becton Dickson | 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 Falcon | 357504 | ||
| 10 ml serological pipet | BD Falcon | 357551 | ||
| 5 ml serological pipet | BD Falcon | 357543 |
References
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Cite This Article
Chamberlain, M. D., Butler, M. J., Ciucurel, E. C., Fitzpatrick, L. E., Khan, O. F., Leung, B. M., Lo, C., Patel, R., Velchinskaya, A., Voice, D. N., Sefton, M. V. Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells. J. Vis. Exp. (46), e2177, doi:10.3791/2177 (2010).