Design and Performance of an Optically Accessible, Low-Volume, Mechanobioreactor for Long-Term Study of Living Constructs

Abstract

Currently available bioreactor systems used by tissue engineers permit either direct, high-magnification observation of cell behavior or application of mechanical loads to growing tissue constructs, but not both simultaneously. Further, in most loading bioreactors, the volume of the dead space is not minimized to reduce the cost associated with perfusion media, exogenous stimulatory/inhibitory agents, proteases, and label. We have designed, developed, and tested a bioreactor that simultaneously satisfies the combined requirements of providing (i) controlled tensile mechanical stimulation, (ii) direct high-magnification imaging capability, and (iii) low dead-space volume. This novel mechanostimulatory (uniaxial tensile loading) bioreactor operates on an inverted microscope and permits continuous optical access (up to 600 · ) to a loaded, growing construct for extended periods of time (weeks). The reactor employs an adjustable reaction chamber in which the dead space can be reduced to < 2 mL. The device has been used to cultivate our human primary corneal fibroblastderived, tissue-engineered system for up to 14 days. Using the instrument we have successfully recorded (i) the process of fibroblasts populating, growing to confluence, and stratifying on different substrates; (ii) recorded complex and organized cell sheet motions; and (iii) recorded the behavior of a subpopulation of what appear to be degradative/catabolic cells within our fibroblast culture. The device is capable of providing detailed, long-term, dynamic images of mechanically stimulated cell/matrix interaction that have not been observed previously.

Collagen network strengthening following cyclic tensile loading

“…The force was measured using a 500 g load cell (Honeywell, Morristown, NJ) at a sampling rate of 1 Hz. The DDCS was mounted between two spring-loaded grips [45], and preloaded to 0.002 N. The reference length and width of the sample were measured, and the specimen was subjected to uniaxial tension cyclic loading. The length and width of the specimen was measured again following cyclic loading, and the specimens were subjected to a final loading to failure.…”

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

“…In this method, fibroblast explants are positioned on a fibrin gel in different geometric patterns. The gels are maintained in an environmentally-controlled, microscope-mounted bioreactor 18 , and the process of compaction and fiber realignment is monitored with time-lapse differential interference contrast (DIC) microscopy. Displacement fields are quantified with custom algorithms.…”

Human Corneal Fibroblast Pattern Evolution and Matrix Synthesis on Mechanically Biased Substrates

“…One million (1000 cells/mL) PHCFs, extracted from a 12-year-old donor cornea, suspended in cell culture medium were injected into the bioreactor through one of the two inlet ports. The cell culture medium was continuously supplied to the bioreactor at a rate of 8 mL/min, as described in Paten et al 28 On day 3, 0.05 mg/mL ascorbic acid was added to the medium.…”

“…The mechanical properties of the DDCS have been previously characterized. 28,29 Extraction of PHCFs. PHCFs were extracted from a 12-year-old donor cornea using our standard cell extraction protocol.…”

“…27 PHCF colonies cultured in a scaffold-free, unloaded tissue engineering model have been shown to synthesize a thick, locally organized, corneal stromal analog. 25 In this investigation, we have combined a PHCF culture system with a custom mechanobioreactor 28 to open a remarkable series of windows into the minute-by-minute behavior of human fibroblasts while they initially populate common culture substrates, self-organize, and then synthesize ECM. The device (Fig.…”