Method for preparing spheroids of human primary hepatocytes
A technology of primary hepatocytes and hepatocytes, applied in the direction of artificial cell constructs, hepatocytes, biochemical equipment and methods, etc., which can solve problems such as removal of bad bile
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Embodiment 1
[0063] Example 1 : Isolation of Primary Hepatocytes
[0064] Primary human hepatocytes were isolated from the livers of 3 individuals with metabolic disease who had undergone liver transplantation. Two patients suffered from urea cycle disorders, while one patient had primary hyperoxalatosis. More details about the donor data are shown in Table 1.
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[0066] Table 1 : Details of the patient from whom primary human hepatocytes were obtained for in vitro testing
[0067] Immediately after removal, use cold (4°C) HTK solution (F. Dr. Chemie, Bensheim, Germany) the liver tissue was perfused and stored at 4°C until the time of hepatocyte isolation. Samples of native tissue were snap frozen and stored at -80°C before starting the cell isolation procedure. A two-step collagenase digestion was used to obtain liver single cell suspensions as previously described by Dandri et al. (2001), Hepatology 33, 981. Liver tissue was placed in sterile glass jars in a 37°C wa...
Embodiment 2
[0069] Example 2 : Cell Seeding and Culture
[0070] Alginate scaffolds in 24-well plates (AlgiMatrix TM 3D culture system) was purchased from Invitrogen (Carlsbad, California (CA), USA, catalog number 12684-023). Scaffolds do not contain animal-derived compounds and have a pore size of 50-200 μm, according to the manufacturer. Immediately before use, the scaffolds were transferred to 24-well culture plates (Corning, Lowell, USA) with a specific ultra-low attachment surface to minimize cell attachment to the culture plates.
[0071] Hepatocytes were resuspended in medium to obtain a single cell suspension. Use 200 μl of a defined volume of cell suspension to evenly inoculate the alginate scaffolds so that each scaffold contains 1 × 10 6 liver cells. After inoculation, 400 μl of medium was added to each well. Hepatocytes on alginate scaffolds were cultured in the previously described (Bierwolf et al. (2011) Biotechnol Bioeng 108, 141) supplemented with Williams' Medium ...
Embodiment 3
[0075] Example 3 : Lactate dehydrogenase assay
[0076] Lactate dehydrogenase (LDH) release from damaged cells was monitored to determine cell viability and to screen for toxic effects caused by scaffolds and their degradation products. As with other biochemical assays described in Examples 4 to 6 below, LDH release measurements were performed every other day during cell culture using cell-free culture supernatant (i.e., medium) that Contact with cultured spheroids for at least 24 h. LDH activity was determined using the Cytotoxicity Detection Kit Plus (Roche, Basel, Switzerland) based on colorimetric measurements. Follow exactly the incubation time in the dark. LDH standard curve was established via LDH solution from porcine muscle (Roche). The color response at 490nm and 690nm was monitored and the amount of LDH was calculated relative to a standard curve, taking dilution and background into account.
[0077] result : LDH release from damaged cells decreased from 6.05±...
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