Fabrication of Myogenic Engineered Tissue Constructs

Summary

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block^[1].

Abstract

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols^{[2, 3]}. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel™, and NaHCO₃. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size^{[1, 4, 5]}. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures^[6]. Once the tissue has solidified at 37°C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Protocol

Part 1: Assemble construct casting molds

1. Use a razor blade to halve silicone tubing (VWR) and cut it into 3 cm long pieces.
2. Place a drop of implant-grade RTV silicone adhesive (Rhodia) on the inside of each end of the tubing.
3. Quickly place a small piece (1 cm x 1 cm) of polyester mesh (McMaster-Carr) on the silicone adhesive drop and align it with the end of the tubing. This will provide a slightly raised and flat surface for construct attachment. Repeat for the other end.
4. Allow the mold to dry at room temperature for 3 days. It is helpful to make 20 to 30 molds at a time, as these are essentially disposable.
5. Once th....

Discussion

The molds in which the tissue construct will be cast can be made in any shape and size; however, there needs to be at least two points of attachment. Otherwise, the matrix and cells form a spherical structure and the cells die. In the present protocol, we describe the use of a polyester mesh for this purpose, yet we have also successfully used stainless steel mesh. Obviously, larger molds will require more cells and a larger volume of the other ingredients. When making the molds, it is important to minimize the amount of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silicone tubing	VWR international	60985-724
Silicone adhesive	Rhodia Silicones	MED ADH 4300 RTV
Polyester Mesh	McMaster-Carr	93185T17
Laminin	Sigma-Aldrich	L2020
Nutrient Mixture F-10 HAM	Sigma-Aldrich	N6908
Fetal Bovine Serum	Atlanta Biologicals	S11550
Penicillin/Streptomycin	Invitrogen	15140
Fungizone	Invitrogen	15290-018
Dispase-2	Roche Group	10295825001
Collagenase 2	Worthington Biochemical	46H8863
Basic Fibroblast Growth Factor	Promega Corp.	G5071
150 mm tissue culture dishes	BD Biosciences	353025
0.05% (1X) Trypsin-EDTA	GIBCO, by Life Technologies	25300
1X Hanks Balanced Salt Solution	Invitrogen	14170-112
7.5% NaHCO3	GIBCO, by Life Technologies	25080-094
70 μm cell strainer	BD Biosciences	352350
6-well plates	BD Biosciences	353046
50 mL Conical Vial	BD Biosciences	352098
15 mL Conical Vial	BD Biosciences	352099
0.2 μm filter	Nalge Nunc international	194-2520