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

1. Cut 2 to 3 m lengths of polyethylene tubing (0.76 mm I.D. x 1.22 mm O.D.).
2. Thread a 20 G1 needle into one end of the tubing.
3. 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.
4. Seal bag and gas sterilize.

2) Neutralization of Collagen

Note: 1 ml of collagen fills about 2 m of polyethylene tubing.

1. Place the necessary amount of collagen needed for the desired length of tubing i....

Discussion

We have fabricated several different micro-tissues using modules embedded with different cell types^1-11. 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Sodium bicarbonate	Sigma-Aldrich	S5761
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