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Novel cell culture method, cell culture system and uses thereof

A cell culture, cell line technology, applied in the new cell culture, cell culture system and its application field

Inactive Publication Date: 2019-01-04
艾伯特李
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main challenge lies in the in vivo translation of events observed in hepatocyte cultures, since the properties of human hepatocytes maintained in artificial media are known to be different from those of the liver in vivo

Method used

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  • Novel cell culture method, cell culture system and uses thereof
  • Novel cell culture method, cell culture system and uses thereof
  • Novel cell culture method, cell culture system and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Cultivation of Human Hepatocytes in Complete (100%) Human Plasma

[0083] In the human body, plasma nourishes all cells. Therefore, human plasma should be the most suitable medium for culturing human cells.

[0084] Pre-cryopreserved 100% confluent plateable human hepatocytes (HH1053 / 57 / 62; three donors) were cultured in 100% human plasma (slightly modified human plasma pooled from five donors) and William E William E medium is a reduced serum-supplemented medium for long-term cell culture of adult rat liver epithelial cells but can also be used for culturing human hepatocytes. Hepatocytes were cultured in 96-well plates for a total of 29 days with media changes every Monday, Wednesday and Friday.

[0085] The cell morphology of the cultured hepatocytes was evaluated on days 7, 14, 24 and 29, where human hepatocytes cultured in William E medium started to disintegrate on day 14 and continued to disintegrate until the end of the experiment on day 29 . In c...

Embodiment 2

[0087] Example 2: Cytochrome P450 induction

[0088] Hepatocytes from human donors (pre-cryopreserved) were plated in 100% human plasma (slightly modified human plasma medium pooled from donor; HPZ-A) and protein-free hepatocyte medium (HIM), total Lasts 29 days. Hepatocytes were plated in 96-well collagen-coated tissue culture plates using Universal Cryopreservation Plating Medium (UCPM). After 4 hours of attachment, the medium was replaced with HPZ-A and HIM containing 0.25 mg / mL Matrigel. The medium was changed to HPZ-A without Matrigel on the second day, and the medium was changed every 2-3 days thereafter, and the culture time was 29 days.

[0089] image 3 Morphology of human hepatocytes cultured in human plasma medium compared to HIM at days 7, 14, 24 and 29 is shown. Human hepatocytes cultured in human plasma medium remained viable and had normal epithelial cell morphology at day 29. Thus, 100% Human Plasma Medium (HPZ-A) can be successfully used to culture human ...

Embodiment 3

[0095] Example 3: Cytochrome P450 3A4 induction

[0096] CYP3A4 is a member of the cytochrome P450 superfamily of enzymes.

[0097] Hepatocytes (previously cryopreserved) from two human donors were plated and cultured as disclosed in Example 2. For comparison of CYP3A4 induction, multiple batches of human hepatocytes (HH1051 and HH1053) were used. For hepatocytes cultured in HIM, rifampicin (concentration range from 1 μM to 20 μM) was added on day 2 after plating for 3 days, and for hepatocytes cultured in human plasma medium, rifampicin was added on day 2 after plating. Rifampicin (concentration range from 10 μM to 200 μM) was added for 5 days and the treatment was continued for 3 days. Gene expression by RT-PCR was used to determine the induction of CYP3A4 by rifampicin.

[0098] Figure 6 and 7 Shows dose-dependent induction of CYP3A4 gene expression by rifampicin. At the concentrations evaluated, the maximum fold induction in hepatocytes cultured in human plasma medi...

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Abstract

A method is disclosed wherein human organ cells (e.g. hepatocytes) are cultured in human plasma medium (e.g., 70% to 100% human plasma) in place of the routinely used non-human plasma cell culture growth medium. This culture method allows the human organ cells to be in an environment that closely resembles that of the in vivo environment, where the cells are bathed in human plasma. Also, disclosedherein are cell culture systems containing the human organ cells and human plasma medium in a cell culture vessel. Uses of the cell culture system includes application of cultured human organ cells to evaluate test compound properties, including pharmacological, pharmacokinetic, and toxicological effects of drugs, organ disease progression, such as liver disease progression including hepatitis Binfection or hepatitis C infection, or cell biology process, such as gene expression, protein synthesis or response to hormones, that can be directly translated to human in vivo.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 340,702, filed May 24, 2016, the contents of which are hereby incorporated by reference in their entirety. field of invention [0003] The invention of the present disclosure generally relates to cell culture methods, cell culture systems and methods for evaluating pharmacological activity of drugs, drug metabolism, drug-drug interactions, and drug toxicity using cultured cells. Background of the invention [0004] The present invention relates to methods for cell culture of organ cells, such as hepatocytes, in human plasma, and the use of these cultured cells for evaluating drug pharmacological activity, drug metabolism, drug-drug interaction and drug toxicity. [0005] In vitro cell culture experimental systems are commonly used in biomedical research. The main difference between cell culture and in vivo mammalian organisms is that cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/12A61P31/12C12N5/071C12Q1/02
CPCA61K35/12C12N5/067C12N2500/84C12N2502/11C12N2533/54A61K35/407G01N33/5008G01N33/5014G01N2800/085A61P31/12Y02A50/30G01N33/50C12Q1/025G01N2500/10
Inventor 艾伯特·李
Owner 艾伯特李