Towards layer-by-layer manufacturing of engineered tissues

McFee, Matthew C. (Chemical and Biological Engineering, The University of British Columbia)
Vaez Ghaemi, Roza (Biomedical Engineering, The University of British Columbia)
Yadav, Vikramadytia G. (Chemical and Biological Engineering, The University of British Columbia)

Introduction

Fibrin is a natural biodegradable, non-toxic and non-immunogenic polymer [1]. It has been widely used in tissue engineering and cell culture to test different cell types attachments. In particular, it is greatly haptotactic for endothelial cells [2]. In this study, the attachment of the transformed mouse endothelial cell line (bEnd.3), on fibrin beads were investigated. Fibrin beads of size 1 mm can be used as a model to represent the 3D structure of organiod and examine the attachment behavior of these cells. 

Materials and Methods

BEnd.3 cells have been shown to have barrier characteristics equivalent to primary brain microvascular endothelial cells (BEMC) [3]. Fibrin beads of 0.8-1 mm in diameter were synthesized via synchronous polymerization of fibrinogen and alginate (figure 1) [4]. The particle size was measured by light-field microscopy and the beads with diameter range of 0.8-1 mm were sieved and were then seeded with bEnd.3 cells. A day after seeding, the unattached cells were removed and then the attachment kinetics of bEnd.3 cells to the beads were determined by CellTitre 96Aqeous colorimetric assay (MTS assay) and fluorescent microscopy.

Results

The barrier functionality of Bend.3 cells has been shown to be maximum after 10 days in culture,  The cells were cultured for 10days on the beads and the restrictive features of the bEnd3 cells were assessed using transendothelial electrical resistance (TEER) as well as horseradish peroxidase (HRP) permeability assays. The tight junctions protein expression and F-actin distribution were analyzed by western blot and direct fluorescent staining before and after seeding on fibrin beads.

Our results shows 100% confluency of the bEnd.3 cells on the surface of the fibrin/alginate beads. The cells formed an uniform layer of 10-20 µm on the bead surface. TEER analysis of the cells after the prolonged culture on the beads was almost identical to the bEnd.3 cells monolayer, indicating no significant effect from the beads.

Discussion and Conclusion

The results of this study, lays the foundation for layer-by-layer manufacturing of engineered tissues.  The layer-by-layer tissue engineering mainly depends on the potential of the cells to attach to the existing 3D structure and maintain their functionality. This can be ideally achieved by using fibrin beads to investigate the kinetics of the attachment and evaluate the properties and functions of the cell after seeding on the beads. This can be a preliminary model for attachment behavior and characteristics of the cells on organoids.


Schematic of fibrin/alginate synchronous polymerization to get beads with sizes ranging 0.8-1.2 mm.

References

1. Ahmed, Tamer AE, Emma V. Dare, and Max Hincke. "Fibrin: a versatile scaffold for tissue engineering applications." Tissue Engineering Part B: Reviews 14.2 (2008): 199-215.

2. Gorodetsky, Raphael, et al. "Fibrin microbeads (FMB) as biodegradable carriers for culturing cells and for accelerating wound healing." Journal of investigative dermatology 112.6 (1999): 866-872.

3. Joubin, Katherine, et al. "The Endothelial Cell Line bEnd. 3 Maintains Human Pluripotent Stem Cells." Stem cells and development 21.12 (2012): 2312-2321.

4. Perka, Carsten, et al. "Matrix‐mixed culture: New methodology for chondrocyte culture and preparation of cartilage transplants." Journal of Biomedical Materials Research Part A 49.3 (2000): 305-311.

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