Influence of preculture on the prefreeze and postthaw characteristics of hepatocytes

Hubel, Allison; Conroy, Mary Margaret; Darr, T. B.

doi:10.1002/1097-0290(2000)71:3<173::aid-bit1007>3.0.co;2-2

Cited by 20 publications

(5 citation statements)

References 41 publications

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“…After cell isolation and freezing preparation shown in Figure 1, a controlled freezing rate program for freezing cryovials was established to maximize cell survival. The freezing program was modified after Hubel et al (16), tailored for our cell population, and can be seen with the chamber and sample temperatures with respect to time in Figure 2. A sharp drop in temperature when the chamber reaches −8°C minimizes liquid–ice phase entropy seen within samples during freezing.…”

Section: Resultsmentioning

confidence: 99%

Hyaluronan-Supplemented Buffers Preserve Adhesion Mechanisms Facilitating Cryopreservation of Human Hepatic Stem/Progenitor Cells

et al. 2012

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The supply of human hepatic stem cells (hHpSCs) and other hepatic progenitors has been constrained by the limited availability of liver tissues from surgical resections, the rejected organs from organ donation programs, and the need to use cells immediately. To facilitate accessibility to these precious tissue resources, we have established an effective method for serum-free cryopreservation of the cells, allowing them to be stockpiled and stored for use as an off-the-shelf product for experimental or clinical programs. The method involves use of buffers, some serum-free, designed for cryopreservation and further supplemented with hyaluronans (HA) that preserve adhesion mechanisms facilitating postthaw culturing of the cells and preservation of functions. Multiple cryopreservation buffers were found to yield high viabilities (80–90%) of cells on thawing of the progenitor cells. Serum-free CS10 supplemented with 0.05% hyaluronan proved the most effective, both in terms of viabilities of cells on thawing and in yielding cell attachment and formation of expanding colonies of cells that stably maintain the stem/progenitor cell phenotype. Buffers to which 0.05 or 0.1% HAs were added showed cells postthaw to be phenotypically stable as stem/progenitors, as well as having a high efficiency of attachment and expansion in culture. Success correlated with improved expression of adhesion molecules, particularly CD44, the hyaluronan receptor, E-cadherin, β4 integrin in hHpSCs, and β1 integrins in hepatoblasts. The improved methods in cryopreservation offer more efficient strategies for stem cell banking in both research and potential therapy applications.

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Section: Resultsmentioning

confidence: 99%

Hyaluronan-Supplemented Buffers Preserve Adhesion Mechanisms Facilitating Cryopreservation of Human Hepatic Stem/Progenitor Cells

et al. 2012

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“…However, these studies have not been carried out so far. There is also evidence by a few research groups that shows that a pre-incubation of hepatocytes as a suspension culture after their isolation greatly improves their recovery after cryopreservation (Darr and Hubel 2001; Hubel et al 2000; Gomez-Lechón et al 2006). Furthermore, several groups investigated the beneficial effect of anti-oxidants within this pre-incubation medium and concluded that the presence of anti-oxidant agents greatly helps the recovery of hepatocytes following thawing (Gomez-Lechón et al 2008; Terry et al 2006; Silva et al 1999).…”

Section: Cryopreservation Of Hepatocytes and Recent Developmentsmentioning

confidence: 99%

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Hewitt²,

et al. 2013

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This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

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“…Freezing of HS has been studied toxic and at the same time protect cells against intra-and extracellular ice formation. Vitrification solutions can be comprehensively by Hubel and coworkers (5,15,16). As the result of thoughtful investigations, several factors made up of penetrating and/or nonpenetrating cryoprotectants.…”

Section: Cooling-warming Processmentioning

confidence: 99%

Vitrification Successfully Preserves Hepatocyte Spheroids

et al. 2008

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This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time-and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (−196°C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 ± 14.39 to 70.38 ± 11.29 µg/10 6 cells and from 56.84 ± 14.48 to 71.79 ± 16.65 µg/10 6 cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.

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Influence of preculture on the prefreeze and postthaw characteristics of hepatocytes

Cited by 20 publications

References 41 publications

Hyaluronan-Supplemented Buffers Preserve Adhesion Mechanisms Facilitating Cryopreservation of Human Hepatic Stem/Progenitor Cells

Hyaluronan-Supplemented Buffers Preserve Adhesion Mechanisms Facilitating Cryopreservation of Human Hepatic Stem/Progenitor Cells

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Vitrification Successfully Preserves Hepatocyte Spheroids

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