Perfused multiwell plate for 3D liver tissue engineering

Domanský, Karel; Inman, W. R.; Serdy, James; Dash, Ajit; Lim, M. H. M.; Griffith, Linda G.

doi:10.1039/b913221j

Cited by 398 publications

(396 citation statements)

References 32 publications

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“…Our data are consistent with another study showing that 3-dimensional cultures under constant perfusion can maintain the hepatocytes function. 20 Our data also demonstrated that DSM provide comparable environment for hepatocytes survive and function with DLM, since they had the similar mechanism.…”

Section: Discussionsupporting

confidence: 51%

Hepatocyte Culture in Autologous Decellularized Spleen Matrix

Gao¹,

Xiang³

et al. 2015

Organogenesis

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ABSTRACT. Background and Aims: Using decellularized scaffold to reengineer liver tissue is a promising alternative therapy for end-stage liver diseases. Though the decellularized human liver matrix is the ideal scaffold for reconstruction of the liver theoretically, the shortage of liver donors is still an obstacle for potential clinical application. Therefore, an appropriate alternative scaffold is needed. In the present study, we used a tissue engineering approach to prepare a rat decellularized spleen matrix (DSM) and evaluate the effectiveness of this DSM for primary rat hepatocytes culture. Methods: Rat decellularized spleen matrix (DSM) was prepared by perfusion of a series of detergents through spleen vasculature. DSM was characterized by residual DNA and specific extracellular matrix distribution. Thereafter, primary rat hepatocytes were cultured in the DSM in a 3-dimensional dynamic culture system, and liver cell survival and biological functions were evaluated by comparison with 3-dimensional sandwich culture and also with cultured in decellularized liver matrix (DLM). Results: Our research found that DSM did not exhibit any cellular components, but preserved the main extracellular matrix and the intact vasculature evaluated by DNA detection, histology, immunohistochemical staining, vessel corrosion cast and upright metallurgical microscope. *Correspondence to: Yi Lv; Email: luyi169@126.com Received July 7, 2014; Revised September 10, 2014; Accepted January 18, 2015.16 Organogenesis, 11:16-29, 2015 Ó 2015 Taylor & Francis Group, LLC ISSN: 1547-6278 print / 1555-8592 online DOI: 10.1080/15476278.2015 Moreover, the method of DSM preparation procedure was relatively simple with high success rate (100%). After seeding primary hepatocytes in DSM, the cultured hepatocytes survived inside DSM with albumin synthesis and urea secretion within 10 d. Additionally, hepatocytes in dynamic culture medium had better biological functions at day 10 than that in sandwich culture. Albumin synthesis was 85.67 § 6.34 mg/10 7 cell/24h in dynamic culture in DSM compared to 62.43 § 4.59 mg/10 7 cell/ 24h in sandwich culture (P < 0.01) and to 87.54 § 5.25 mg/10 7 cell/24h in DLM culture (P > 0.05); urea release was 32.14 § 8.62 mg/10 7 cell/24h in dynamic culture in DSM compared to 20.47 § 4.98 mg/10 7 cell/24h in sandwich culture (P < 0.05) and to 37.38 § 7.29 mg/10 7 cell/24h cultured in DLM (P > 0.05). Conclusion: The present study demonstrates that DSM can be prepared successfully using a tissue engineering approach. The DSM is an appropriate scaffold for primary hepatocytes culture.

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Section: Discussionsupporting

confidence: 51%

Hepatocyte Culture in Autologous Decellularized Spleen Matrix

Gao¹,

Xiang³

et al. 2015

Organogenesis

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“…Linda Griffith, a bioengineer at MIT, has built silicon scaffolds less than 2 centimetres across and filled them with wells that allow liver cells to grow in three dimensions 7 . These structures are placed inside multiwell plates.…”

Section: The Real Mccoymentioning

confidence: 99%

A living system on a chip

Baker

2011

Nature

186

142

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“…[29][30][31][32][33][34][35][36][37][38] Several strategies have previously been proposed to culture LSECs while retaining their phenotype. [37][38][39][40][41][42][43] LSECs, cultured in media supplemented with lipids and oleic acid, in particular, was shown to maintain their metabolic and endocytotic activity for up to 5 days by influencing Akt/PKB and ERK signaling pathways. 39 Another study with culturing LSECs and hepatocytes in a three-dimensional (3D) microreactor shows retention of LSEC phenotype expression for 13 days, primarily through modulation of vascular endothelial growth factor in the culture media.…”

mentioning

confidence: 99%

Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells

Bale

Golberg

Jindal

et al. 2015

Tissue Engineering Part C: Methods

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Hepatocytes and their in vitro models are essential tools for preclinical screening studies for drugs that affect the liver. Most of the current models primarily focus on hepatocytes alone and lack the contribution of nonparenchymal cells (NPCs), which are significant through both molecular and the response of the NPCs themselves. Models that incorporate NPCs alongside hepatocytes hold the power to enable more realistic recapitulation and elucidation of cell interactions and cumulative drug response. Hepatocytes and liver sinusoidal endothelial cells (LSECs) account for *80% of the liver mass where the LSECs line the walls of blood vessels, and act as a barrier between hepatocytes and blood. Culturing LSECs with hepatocytes to generate multicellular physiologically relevant in vitro liver models has been a major hurdle since LSECs lose their phenotype rapidly after isolation. To this end, we describe the application of collagen gel (1) in a sandwich and (2) as an intervening extracellular matrix layer to coculture hepatocytes with LSECs for extended periods. These coculture configurations provide environments wherein hepatocyte and LSECs, through cell-cell contacts and/or secretion factors, lead to enhanced function and stability of the cocultures. Our results show that in these configurations, hepatocytes and LSECs maintained their phenotypes when cultured together as a mixture, and showed stable secretion and metabolic activity for up to 4 weeks. Immunostaining for sinusoidal endothelial 1 (SE-1) antibody demonstrated retention of LSEC phenotype during the culture period. In addition, LSECs cultured alone maintained high viability and SE-1 expression when cultured within a collagen sandwich configuration up to 4 weeks. Albumin production of the cocultures was 10-15 times higher when LSECs were cultured as a bottom layer (with an intervening collagen layer) and as a mixture in a sandwich configuration, and native CYP 1A1/2 activity was at least 20 times higher than monoculture controls. Together, these data suggest that collagen gel-based hepatocyte-LSEC cocultures are highly suitable models for stabilization and long-term culture of both cell types. In summary, these results indicate that collagen gel-based hepatocyte-LSEC coculture models are promising for in vitro toxicity testing, and liver model development studies.

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Perfused multiwell plate for 3D liver tissue engineering

Cited by 398 publications

References 32 publications

Hepatocyte Culture in Autologous Decellularized Spleen Matrix

Hepatocyte Culture in Autologous Decellularized Spleen Matrix

A living system on a chip

Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells

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