Hepatocyte serum-free culture medium

A technology of serum-free medium and hepatocytes, which is applied in the field of animal cell culture, can solve the problems of the influence of experimental results, irreversible damage of cells, and easy carrying of prions in serum.

Active Publication Date: 2015-11-25
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The main disadvantages of the traditional serum-containing medium are 1. Due to the species specificity, the serum of heterogeneous animals has immune rejection and sensitization; 2. The serum is easy to carry prions, etc., and is easily contaminated by pathogenic microorganisms; 3. Different batches There may be differences between times, which may easily lead to unrepeatable experimental results; 4. Some protein components in serum may af

Method used

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  • Hepatocyte serum-free culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Concentration screening of each component of the hepatocyte serum-free medium of the present invention

[0031] 1. Orthogonal optimization design screens the concentration of each component in the medium:

[0032] Orthogonal test scheme: This study includes 4 factors including sericin (A), dexamethasone (B), hepatocyte growth factor (HGF (C)) and epidermal growth factor (EGF (D)). 3 levels (see Table 1), so choose L9 (3 4 ) orthogonal table, the test scheme is shown in Table 2. The study selected DEME high-glucose type as the basal medium, added insulin 5μg / ml, transferrin 5μg / ml, sodium selenite 10μg / L, linoleic acid 11ng / ml, HEPES10mmol / L, penicillin and streptomycin 100U / ml , and according to the experimental scheme in Table 2, the corresponding doses of 4 components were added, and mixed to prepare 9 groups of culture media.

[0033] Table 1 Orthogonal design factor level table

[0034]

[0035] Table 2 Orthogonal test scheme table

[0036]

[0037] 2. In...

Embodiment 2

[0039] Hepatocyte serum-free culture medium of the present invention is to the culture effect of hepatocyte

[0040] 1. Detection methods of various indicators

[0041] (1) Method for detecting cell viability: take logarithmic phase cells and make 2×10 4 / ml cell suspension, seeded in 96-well plate (100μl / well), in 5% CO 2 and cultured at 37°C for 24 hours, take 5 multiple wells every day, add 10 μl CCK-8 reagent to each well, incubate in the above environment for 1 hour, measure the absorbance at 450nm wavelength with a microplate reader, and draw the absorbance-time curve , take the maximum value of the absorbance of each group.

[0042] (2) Method for cell function detection: Inoculate 1×10 cells per well in a 6-well plate 5 cells, and add 3ml medium, replace the medium every 24h, in 5% CO 2 and 37°C environment, the cell culture supernatant was collected every day, the albumin content was measured by ELISA method, and the AST, LDH and urea content were measured by micr...

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Abstract

The invention discloses a hepatocyte serum-free culture medium. The hepatocyte serum-free culture medium comprises the following components: 500mL of basal culture medium, 0.05-0.5% of sericin protein, 0.1-1000nmol/mL of dexamethasone, 5-20ng.mL of hepatocyte growth factors, 10-50ng/mL of epidermal growth factors and 100U/mL of mycillin. The hepatocyte serum-free culture medium is suitable for growth and function exertion of hepatocyte, and can be used for preparing biological type artificial livers.

Description

technical field [0001] The invention relates to an animal cell culture technology, in particular to a hepatocyte serum-free medium. Background technique [0002] The bioartificial liver support system (BALSS) is based on liver cells with certain functions, and can perform a device similar to the function of the liver to the human body to a certain extent. On the one hand, it can provide short-term support for patients with reversible liver damage. Smooth transition to liver function recovery, on the other hand, can support patients with irreversible liver failure while waiting for liver transplantation, and act as a bridge between liver failure and liver transplantation. Hepatocytes are the core part of BALSS, so cultivating hepatocytes in vitro and making them play their original physiological roles has become the focus of BALSS research. In the past, hepatocytes were mostly cultured with human or animal serum, but due to their shortcomings such as immune rejection, sensit...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 黄耘高毅
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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