Optimization of Hepatocyte Spheroid Formation for Hepatic Tissue Engineering on Three-Dimensional Biodegradable Polymer within a Flow Bioreactor prior to Implantation

Török, É; Pollok, Joerg M.; Ma, Peter; Kaufmann, Peter; Dandri, Maura; Petersen, J.; Burda, Martin R.; Kluth, Dietrich; Perner, F.; Rogiers, Xavier

doi:10.1159/000047858

Cited by 56 publications

(15 citation statements)

References 31 publications

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“…These spheroid-like structures in the 3D cultures did not exceed a diameter of 150 lm which should allow sufficient sustenance of the inner cells. 20 The cluster area of the highest concentrated cells increased slightly within the first week of cultivation but the following week did not yield further augmentation which is rather likely due to reduced proliferation caused by contact inhibition. 21 A similar behavior was published previously about HepG2 cells grown as an organotypic culture in spheroid-like structures.…”

Section: Discussionmentioning

confidence: 94%

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

et al. 2015

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The in vitro study of liver functions and liver cell specific responses to external stimuli deals with the problem to preserve the in vivo functions of primary hepatocytes. In this study, we used the biochip OrganoPlate TM (MIMETAS) that combines different advantages for the cultivation of hepatocytes in vitro: (1) the perfusion flow is achieved without a pump allowing easy handling and placement in the incubator; (2) the phaseguides allow plating of matrix-embedded cells in lanes adjacent to the perfusion flow without physical barrier; and (3) the matrix-embedding ensures indirect contact of the cells to the flow. In order to evaluate the applicability of this biochip for the study of hepatocyte's functions, Matrigel TM -embedded HepG2 cells were cultured over three weeks in this biochip and compared to a static Matrigel culture (3D) and a monolayer culture (2D). Chip-cultured cells grew in spheroid-like structures and were characterized by the formation of bile canaliculi and a high viability over 14 days. Hepatocyte-specific physiology was achieved as determined by an increase in albumin production. Improved detoxification metabolism was demonstrated by strongly increased cytochrome P450 activity and urea production. Additionally, chip-cultured cells displayed increased sensitivity to acetaminophen. Altogether, the OrganoPlate seems to be a very useful alternative for the cultivation of hepatocytes, as their behavior was strongly improved over 2D and static 3D cultures and the results were largely comparable and partly superior to the previous reports on biochip-cultured hepatocytes. As for the low technical needs, this platform has the appearance of being highly applicable for further studies of hepatocytes' responses to external stimuli. V C 2015 AIP Publishing LLC. [http://dx

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Section: Discussionmentioning

confidence: 94%

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

et al. 2015

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“…24,25 In the pulsatile flow bioreactor culture, primary rat hepatocytes preserved viability, morphology, and differentiated function as demonstrated by functional and structural analysis. We presume that the formation of spheroids is an advantageous configuration in improving the posttransplantation viability of hepatocytes.…”

Section: Discussionmentioning

confidence: 97%

“…24,26,27 Polymers were manufactured to dimensions of 18 mm diameter and 1 mm thickness, with a pore size of 200 to 400 mm, and then cold-gas sterilized with ethylene oxide (H.W. Anderson Products, Chapel Hill, NC) before use.…”

Section: Polymer Fabricationmentioning

confidence: 99%

Morphological and Functional Analysis of Rat Hepatocyte Spheroids Generated on Poly(L-lactic acid) Polymer in a Pulsatile Flow Bioreactor

et al. 2006

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Liver neo-tissue suitable for transplantation has not been established. Primary rat hepatocytes were cultured on three-dimensional biodegradable polymer matrices in a pulsatile flow bioreactor with the intention of inducing tissue formation and improving cell survival. Functional and structural analysis of the hepatocytes forming liver neo-tissue was performed. Biodegradable poly(L-lactic acid) (PLLA) polymer discs were seeded with 4 x 10(6) primary rat hepatocytes each, were exposed to a pulsatile medium flow of 24 mL/min for 1, 2, 4, or 6 days and were investigated for monoethylglycinexylidine (MEGX) formation, ammonia detoxification, Cytokeratin 18 (CK18) expression, and preserved glycogen storage. Fine structural details were obtained using scanning and transmission electron microscopy. Spheroids of viable hepatocytes were formed. MEGX-specific production was maintained and ammonia removal capacity remained high during the entire flow-culture period of 6 days. CK18 distribution was normal. Periodic-acid- Schiff reaction demonstrated homogenous glycogen storage. The hepatocytes reassembled to form intercellular junctions and bile canaliculi. Functional and morphological analysis of rat hepatocytes forming spheroids in a pulsatile flow bioreactor indicated preserved and intact hepatocyte morphology and specific function. Pulsatile flow culture on PLLA scaffolds is a promising new method of hepatic tissue engineering leading to liver neo-tissue formation.

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“…In a previous study we had found that the optimal flow culture conditions for rat hepatocytes on highly porous PLLA were a seeding concentration of 10 × 10 6 cells/ml with a flow rate of 24 ml/min [58]. The optimal outcome of flow culture was defined by the highest density of spheroids within the polymer and the highest ratio of aggregated versus nonaggregated hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

Hepatic Tissue Engineering on 3-Dimensional Biodegradable Polymers within a Pulsatile Flow Bioreactor

Török

Pollok

et al. 2001

Dig Surg

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Background: An optimal method for hepatocyte transplantation is not yet determined. With the principles of tissue engineering in vitro conditioning of hepatocytes on biodegradable polymer in a flow bioreactor before implantation forming spheroids may achieve increased cell mass and function to replace lost organ function in vivo. Methods: Biodegradable poly-L-lactic (PLLA) polymer discs were seeded with rat hepatocytes in a concentration of 10 × 10⁶ cells per ml and exposed to a medium flow of 24 ml/min for 1, 2, 4 and 6 days. The number and diameter of spheroidal aggregates was measured by phase-contrast microscopy. H&E histology was performed. Albumin production as hepatocyte specific function was determined by ELISA. Results: Spheroids of viable hepatocytes of 50–200 µm in diameter were formed. Both the number and diameter of the spheroids increased during the first 2 days and then remained constant until day 6. Albumin production was maintained throughout the culture period. Conclusion: Short (2– 3 days) pre-transplant conditioning of hepatocytes in a flow bioreactor on biodegradable PLLA resulted in formation of spheroids with a liver-like morphology and preserved specific metabolic function. Tissue engineered hepatocyte spheroids on polymer may represent a functionally active and easy transplantable neotissue and may serve as an in vivo substitute for lost liver function.

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Optimization of Hepatocyte Spheroid Formation for Hepatic Tissue Engineering on Three-Dimensional Biodegradable Polymer within a Flow Bioreactor prior to Implantation

Cited by 56 publications

References 31 publications

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

Morphological and Functional Analysis of Rat Hepatocyte Spheroids Generated on Poly(L-lactic acid) Polymer in a Pulsatile Flow Bioreactor

Hepatic Tissue Engineering on 3-Dimensional Biodegradable Polymers within a Pulsatile Flow Bioreactor

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