Method of culturing proliferative hepatocytes

A proliferative, primary hepatocyte technology, applied in the field of cell culture, 3D hepatocyte culture

Pending Publication Date: 2021-03-16
INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM) +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as with cancer cell lines and immortalized cell lines, rodent hepatocyte cultures do not constitute the best in vitro model to recapitulate physiological human liver biology
[0008] Therefore, studying and exploiting the proliferative capacity of hepatocytes for pharmaceutical or therapeutic applications by using in vitro models remains a major challenge

Method used

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  • Method of culturing proliferative hepatocytes
  • Method of culturing proliferative hepatocytes
  • Method of culturing proliferative hepatocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0357] Materials and Method

[0358] material

[0359] cell

[0360] Human liver samples were obtained from patients due to primary hepatocellular carcinoma or liver metastasis due to primary hepatocellular carcinoma or liver metastasis due to primary hepatocellular carcinoma or liver metastasis due to primary hepatocellular carcinoma or liver transfer. The research program was carried out under the French legal guidelines and the local institutional Ethics Committee. The clinical features of human liver samples are detailed in Table 1 below.

[0361] Table 1: Clinical features from their liver samples

[0362]

[0363] Human hepatocytes were separated by two-step collagenase perfusion procedure, and substantive cells were maintained in improved William E medium (referred to as WE HH (human liver cells)), which contain penicillin (100u), streptomycin. (100 μg / ml), insulin (15 μg / mL), glutamine (2 mM), albumin (0.1% (w / v)), transferronin (5.5 μg / mL), sodium selenate (5μ...

Embodiment 2

[0428] Materials and Method

[0429] material

[0430] cell

[0431] Constitutional hepatocytes (PHH) were obtained as described above.

[0432] method

[0433] Primary human hepatocyte culture

[0434] The primary human hepatocytes 3d cultures were established as described above (see Example 1). In short, PHH was first cultured in a low attachment plate. Then, the aggregate of the obtained pHH is then embedded in the collagen matrix and is further cultured in the collagen matrix.

[0435] Medium

[0436] PHH was cultured in the WE HH medium defined above (see Example 1).

[0437] immunochemistry

[0438] Immunohistochemical staining is performed as described above to evaluate the proliferation of PHH.

[0439] result

[0440] Further studies the importance of growth factor stimulation for the proliferation of PHH in accordance with the 3D culture of the present invention. The pHH in the cultured collagen matrix as described above is deprived of EGF, HGF, and / or ITS (insul...

Embodiment 3

[0453] Materials and Method

[0454] material

[0455] cell

[0456] Sente people hepatocytes (PHH) were obtained as described above (see Example 1).

[0457] method

[0458] Primary human hepatocyte culture

[0459] The primary human hepatocytes 2D and 3D cultures in the collagen matrix are established as described above (see Example 1). PHH was cultured in WE HH medium as described above.

[0460] Comet Determination (Comet Assay)

[0461] The PHH (3D) of (3D) (3d) (3d) (3d) (see Table 7) is incubated with indication concentration (see Table 7) in the control 2D culture (2d) or in a collagen matrix. . After 9 days, PHH was extracted from the collagen matrix by the effect of purified collagenase. The cell precipitate was resuspended in 0.5% low melting agarose and paved on a conventional microscope slide covered with conventional agarose. The electrophoresis migration treatment was transferred and at least 100 images were obtained using a fluorescent microscope after dyeing...

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Abstract

The present invention relates to a method of culturing animal cells, preferably primary hepatocytes, comprising a first step of culturing the animal cells in non-adherent culture vessel, preferably alow or ultra-low attachment culture vessel, a second step of embedding the animal cells in a collagen matrix or in a gelatin matrix, and a third step of culturing the animal cells embedded in the collagen matrix or in the gelatin matrix, thereby obtaining 3D animal cell structures comprising proliferative animal cells, preferably spheroids comprising proliferative primary hepatocytes. The invention also relates to a spheroid comprising proliferative primary hepatocytes and the uses thereof for engineering an artificial liver model or an artificial liver organ, and for assessing in vitro the liver toxicity, genotoxicity and/or the effects of a drug or a compound.

Description

Technical field [0001] The present invention relates to cell culture, preferably hepatocytes culture, and in particular 3D cell culture, preferably 3D hepatocytes cultured. [0002] Inventory background [0003] Hepatocytes are the main substantive cells of the liver and account for up to 70-85% of liver quality. Hepatic cells are highly differentiated cells that achieve most of the liver function, especially related to metabolism, detoxification and systemic steady state. In the liver, hepatocytes are typically longevity still cells. However, after damage or loss of function block, hepatocytes can proliferate, thus regenerating the liver. [0004] In vitro cultures for establishing hepatocytes have been pursued for a long time, the purpose is to develop in vitro models that can faithfully reproduce key liver function. Such models can be used in research to understand and study normal liver function. They can also be used to understand and study liver pathology, such as viral hepa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0671C12N2501/727C12N2500/30G01N33/5067
Inventor G·柏菲特S·朗圭特F·伊赞S·罗斯M·库维利埃
Owner INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)
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