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High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

ABSTRACT: The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

SUBMITTER: Gevaert E

PROVIDER: S-EPMC4136852 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

Gevaert Elien E Dollé Laurent L Billiet Thomas T Dubruel Peter P van Grunsven Leo L van Apeldoorn Aart A Cornelissen Ria R

PloS one 20140818 8

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific ...[more]

PMID: 25133500

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Project description:Development of hydrogel-based tissue engineering constructs is growing at a rapid rate, yet translation to patient use has been sluggish. Years of costly preclinical tests are required to predict clinical performance and safety of these devices. The tests are invasive, destructive to the samples and, in many cases, are not representative of the ultimate in vivo scenario. Biomedical imaging has the potential to facilitate biomaterial development by enabling longitudinal noninvasive device characterization directly in situ. Among the various available imaging modalities, ultrasound stands out as an excellent candidate due to low cost, wide availability, and a favorable safety profile. The overall goal of this work was to demonstrate the utility of clinical ultrasound in longitudinal characterization of 3D hydrogel matrices supporting cell growth. Specifically, we developed a quantitative technique using clinical B-mode ultrasound to differentiate collagen content and fibroblast density within poly(ethylene glycol) (PEG) hydrogels and validated it in an in vitro phantom environment. By manipulating the hydrogel gelation, differences in ultrasound signal intensity were found between gels with collagen fibers and those with non-fiber forming collagen, indicating that the technique was sensitive to the configuration of the protein. At a collagen density of 2.5 mg/mL collagen, fiber forming collagen had a significantly increased signal intensity of 14.90 ± 2.58 × 10(-5) a.u. compared to non-fiber forming intensity at 2.74 ± 0.36 × 10(-5) a.u. Additionally, differences in intensity were found between living and fixed fibroblasts, with an increased signal intensity detected in living cells (5.00 ± 0.80 × 10(-5) a.u. in 1 day live cells compared to 2.26 ± 0.39 × 10(-5) a.u.in fixed cells at a concentration of 1 × 10(6) cells/mL in gels containing collagen). Overall, there was a linear correlation >0.90 for ultrasound intensity with increasing cell density. Results demonstrate the feasibility of using clinical ultrasound for characterization of PEG-based hydrogels in a tissue-mimicking phantom. The approach is clinically-relevant and could, with further validation, be utilized to nondestructively monitor in vivo performance of implanted tissue engineering scaffolds over time in preclinical and clinical settings.

Dataset Information

High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

Publications

High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

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