Influence of Alginate Gel Entrapment and Cryopreservation on Survival and Xenobiotic Metabolism Capacity of Rat Hepatocytes

doi:10.1006/taap.1996.0299

Toxicology and Applied Pharmacology

Volume 141, Issue 2, December 1996, Pages 349-356

https://doi.org/10.1006/taap.1996.0299 Get rights and content

Abstract

Rat hepatocytes immobilized in calcium–alginate beads were tested for their ability to survive and functionin vitroby comparison with hepatocyte monolayer cultures before and after cryopreservation. The freeze–thaw protocol previously designed for suspended hepatocytes was used; the freezing medium was the Leibovitz medium containing 10% fetal calf serum and 16% dimethylsulfoxide. The functions investigated included protein, lipid, and urea synthesis, albumin secretion, glycogen content, and various phase I and phase II enzyme activities. Cell viability was not altered by immobilization and the freeze–thaw procedure. Most functions tested were expressed at similar levels in unfrozen immobilized hepatocytes and corresponding hepatocyte monolayers. Only protein neosynthesis and some phase II drug metabolizing enzyme activities were decreased. The freeze–thaw process had no detrimental effect, whatever the function investigated. In addition, cryopreserved immobilized hepatocytes remained responsive to inducers: ethoxyresorufinO-deethylase activity was increased 11–12 times by 3-methylcholanthrene treatment in both fresh and cryopreserved alginate-entrapped hepatocytes. These results clearly show that hepatocytes immobilized in calcium–alginate beads remain functional even after cryopreservation indicating that they represent a promising approach for xenobiotic metabolism and toxicity studies.

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Cited by (91)

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Citation Excerpt :
Thus, in order to understand and facilitate the future research on the slow freezing of microencapsulated cells, the existing studies in the area will be summarized below. Different studies have determined the hydrogels cryoprotective effect in the cryopreservation of microencapsulated mammalian cells [93–99], and in plant cryopreservation [100]. Different mechanisms have been related to post-thaw cell function improvement on the cryopreservation of microencapsulated cells: the influence of the polymeric matrix on extracellular ice formation [101], initiation of intracellular ice formation [102,103], buffering the CPA diffusion or protection against apoptosis [104].
Over the past few decades, the use of cell microencapsulation technology has been promoted for a wide range of applications as sustained drug delivery systems or as cells containing biosystems for regenerative medicine. However, difficulty in their preservation and storage has limited their availability to healthcare centers. Because the preservation in cryogenic temperatures poses many biological and biophysical challenges and that the technology has not been well understood, the slow cooling cryopreservation, which is the most used technique worldwide, has not given full measure of its full potential application yet. This review will discuss the different steps that should be understood and taken into account to preserve microencapsulated cells by slow freezing in a successful and simple manner. Moreover, it will review the slow freezing preservation of alginate-based microencapsulated cells and discuss some recommendations that the research community may pursue to optimize the preservation of microencapsulated cells, enabling the therapy translate from bench to the clinic.
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In vitro metabolism of bisphenol A (BPA), an weak estrogen, was studied with cryopreserved hepatocytes from rat, monkey and human, and was compared with in vivo metabolism reported. The metabolites identified include a major metabolite, BPA glucuronide (BPAG) and BPA sulfate (BPAS). The metabolic rates of bisphenol A at 20 μM by the hepatocytes (BPAG plus BPAS, nmol/10⁶ cells/h) followed the order of rats (48 + 12) > monkeys (18 + 4) > humans (8.6 + 0.8), respectively. The rate of BPAG formation was much higher than that of BPAS formation in all these species. For the BPAG formation, we have determined the apparent K_m (μM) of rats (3), monkeys (7), and humans (5). V_max (nmol/10⁶ cells/h) in hepatocytes followed the order of rats (55) > monkeys (22) > humans (11). The total CL_H for the hepatic formation of BPAG plus BPAS (L/h/kg BW) estimated by well-stirred model with low f_B value followed the order of rats (3.0) > monkeys (0.68) > humans (0.27), correlating well with in vivo studies of BPA subcutaneously injected rats and monkeys. This study showed that the cryopreserved hepatocytes could be a useful tool for assessing BPA metabolism and predicting systemic exposure of BPA.
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Hepatitis B-virucidal testing of biocides in quantitative suspension tests using duck hepatitis B virus (DHBV) requires primary duck embryonic hepatocytes for viral propagation. To improve the test system and availability of these cells, commercial culture plates with different growth surfaces were tested for cell cultivation and different approaches for cryopreservation of hepatocyte suspension were examined. After 12 days of culture, the largest amounts of hepatocytes were grown in CellBIND and TTP plates and CellBIND surface showed the lowest tendency of monolayer detachment nearly comparable with collagen 1-coated CELLCOAT plates. For cryopreservation of hepatocyte suspension, the use of growth medium supplemented with fetal calf serum (FCS) and dimethyl sulfoxide (ME₂SO), FCS supplemented with ME₂SO or cryosafe-1 as cryoprotective agents provided the highest rates of surviving cells after thawing. The freezing–thawing process did not significantly reduce the susceptibility of hepatocytes to infection with DHBV. In conclusion, plates without collagen 1 such as CellBIND are recommended for cultivation of primary duck embryonic hepatocytes in infectivity experiments of DHBV for virucidal testing of biocides. The use of cryopreserved hepatocytes is possible when freshly isolated cells from the liver of duck embryos are not available.
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Regular ArticleInfluence of Alginate Gel Entrapment and Cryopreservation on Survival and Xenobiotic Metabolism Capacity of Rat Hepatocytes

Abstract

Regular Article
Influence of Alginate Gel Entrapment and Cryopreservation on Survival and Xenobiotic Metabolism Capacity of Rat Hepatocytes