Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Summary

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Abstract

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels^1,2.

Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable^3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size.

The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)³. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels⁵. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries⁶. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models^3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)⁸. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Protocol

Autoclave

Assemble and autoclave the tubing for the flow system and bioreactor components (bioreactor itself and the silicone stopper lid) as instructed in Figure 1 and Figure 2. Feeding tube has a male connector on one end and an open end on the other side. Three short tubing segments are inserted through a silicone cap for gas exchange.

1. Sewing PGA Mesh

1. Cut PGA mesh to 1.1cm x ~8cm sheet (dependent on bioreactor size).
2. Clean silicone tubing (3mm inner diameter) with distilled water (dH20) and air dry before usage.
3. Use Dexon 6.0 suture to sew PGA mesh around the clean silicone tubi....

Discussion

The quality of engineered vessels is in large part dictated by the quality of the SMCs used in tissue culture. The critical aspects of SMC phenotype include contractile morphology, low passage number, and the ability to proliferate inside the bioreactor. We recommend that the passage number be no greater than P3 at the time of cell seeding onto the polymer scaffold. Moreover, it is crucial to confirm that the SMC sources are mycoplasma free prior to use. We have observed that mycoplasma-contaminated cells lead to substan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FBS (Fetal Bovine Serum) Heat-Inactivated	Hyclone	SH30071
DMEM	GIBCO, by Life Technologies	11885
rhFGF-basic	R&D Systems	234-FSE
rrPDGF-BB	R&D Systems	520-BB
Penicilin G	Sigma-Aldrich	PENNA
Copper(II) Sulfate	Sigma-Aldrich	C8027
Gylcine	Sigma-Aldrich	C8790
L-Alanine	Sigma-Aldrich	A7469-25G
L-Proline	Sigma-Aldrich	P5607-25G
Ascorbic Acid	Sigma-Aldrich	A4544-25G
HEPES	Sigma-Aldrich	H3375-100G
Silicone Stopper	Cole-Parmer	06298-24
Masterflex tubes L/S	Cole-Parmer	06508-16, 06508-18
Masterflex pump	Cole-Parmer	7553-80
Dacron cuff	Maquet	174406
PGA felt	Concordia	MO000877-01
4-0 1.5 metric Surgipro II suture	Syneture	VP-557-X
6-0 0.7 metric Dexon suture	Syneture	7538-11
0.22μm PTFE filters	Whatman, GE Healthcare	6780-2502
Three Way Stop-cock	Edwards Lifesciences	593WSC
Pressure Transducer	Edwards Lifesciences	PX212
IV bags	Baxter Internationl Inc.	R4R2110
Saline dilution set	Arrow International	W20030
Silicone tubing	Saint-Gobain	F05027