CN111206013A

CN111206013A - Culture method for maintaining functional state of human primary hepatocytes for long time

Info

Publication number: CN111206013A
Application number: CN202010090593.0A
Authority: CN
Inventors: 鄢和新; 陈依
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Shanghai Celliver Biotechnology Co Ltd
Priority date: 2020-02-13
Filing date: 2020-02-13
Publication date: 2020-05-29

Abstract

The invention relates to the technical field of medicines, in particular to a culture method for maintaining the functional state of human primary hepatocytes for a long time, which is 3 multiplied by 10⁴/cm²Inoculating density, and culturing the human primary hepatocytes in a hepatocyte amplification medium. According to the invention, by high-density culture in the hepatocyte amplification culture medium, the function of the primary hepatocyte (PHH) can be maintained within at least 30 days, the long-time in vitro culture and function maintenance of the primary human hepatocyte can be realized, and a basic guarantee is provided for drug metabolism evaluation and hepatocyte treatment.

Description

Culture method for maintaining functional state of human primary hepatocytes for long time

Technical Field

The invention relates to the technical field of medicines, in particular to a culture method for maintaining the functional state of human primary hepatocytes for a long time.

Background

Primary Human Hepatocytes (PHH) are considered to be the most ideal cells for hepatocyte therapy, and more the gold standard cells for drug metabolism assessment and modeling studies. However, the use of PHH is hampered by the extremely low proliferation rate and lack of metabolic function during the in vitro culture of PHH. The important reason for this is that the stabilization of the function of terminally differentiated cells in vivo is highly dependent on the precise spatio-temporal regulation of microenvironment signals, whereas isolated primary cells often lose their core gene expression characteristics and specific functions. To overcome this problem, researchers have proposed many methods for long-term in vitro maintenance of hepatocyte function, including 3D spheronization, collagen-gel sandwich culture, co-culture with non-parenchymal cells, epigenetic regulation of hepatic genes, and optimization of culture microenvironment by increasing oxygen supply. However, these culture methods require special culture equipment and complicated operation techniques, which somewhat limits the options for PHH analysis methods for evaluating drug effectiveness and applicability in high-throughput sequencing. (references: G Lolo mez-Lech Lolo n M J, Tolosa L, Conde I, et al. Complementary of differential Cell modules to prediction human hepatoxic drugs. expert Optin Drug though Toxicol,2014,10(11): 1553. Cell 1568. Xu J, Du Y, Deng H. direct Linear modification: Strategies, Mechanisms, and applications. Cell Stem, 2015,16(2): 119. mu. 134.)

Deng found in the study by using 5 small molecules (5C medium): forskolin, SB431542, DAPT, IWP2 and LDN193189, can maintain the liver function of PHH stored at low temperature and freshly isolated for up to 30 days, and 5C-cultured hepatocytes have CYP metabolic activity and the ability to infect hepatitis b virus. (reference: Xiaong C, Du Y, Meng G, et. Long-term functional main of primary human machinery in science,2019,364(6438):399-

Disclosure of Invention

The invention aims to provide a culture method for maintaining the functional state of human primary hepatocytes for a long time. Given that long-term maintenance of cellular function in vitro requires complex signal-regulatory networks, we have invented a chemical strategy that uses a combination of small molecules to achieve modulation of specific cellular targets by modulating the intrinsic signal network. The method for maintaining the number and the function of the hepatocytes for a long time through small molecule combination in vitro has certain application value in drug metabolism evaluation and in vitro modeling, and provides basic guarantee for drug metabolism evaluation and hepatocyte treatment.

In a first aspect of the invention, there is provided a culture method for maintaining the functional state of human primary hepatocytes in vitro for an extended period of time, said method being performed at 2 × 10⁴-3×10⁴/cm²And (2) inoculating density, and culturing the human primary hepatocytes in a hepatocyte amplification culture medium (below the density, the primary hepatocytes can be reversibly transformed and transformed into hepatocyte precursor cells, and above the density, the primary hepatocytes can have overhigh cell density and accelerate the aging and death process of the hepatocytes).

Further, the inoculation density is 3 multiplied by 10⁴/cm²。

Further, the hepatocyte expansion culture medium is:

the liquid basal medium is DMEM/F12, 1% insulin-transferrin-sodium selenite mixed solution, 50ng/ml epidermal growth factor, 20ng/ml interleukin-6, 20ng/ml hepatocyte growth factor, 20ng/ml fibroblast growth factor-2, 1 mu M CHIR9902, 10 mu M Y-27632, 1.25 mu M Acetylcysteine and 1 mu M A83-01.

In one embodiment of the present invention, the culture method for maintaining the functional state of human primary hepatocytes in vitro for a long time specifically comprises:

A) in a biological safety cabinet, adding 10ml of culture medium into a 15ml centrifuge tube, and preheating for 20 minutes in a constant-temperature water bath kettle at 37 ℃;

B) rapidly transferring the primary hepatocytes subjected to cryopreservation from liquid nitrogen to a constant-temperature water bath kettle at 37 ℃, and carrying out clockwise rotation thawing in water for about 90-120 seconds until only small pieces of crushed ice in a cryopreservation tube float;

C) sucking out the cells by using a blunt gun head, transferring the cells into a 15ml centrifuge tube containing 10ml of preheated hepatocyte amplification culture medium in a dropwise manner, slightly reversing the cells from top to bottom for 2-3 times, and uniformly mixing;

D) after standing at room temperature for 30-45 minutes (mixing by gently inverting every 15 minutes), low-speed centrifugation (50 Xg, normal temperature centrifugation for 5 minutes) is carried out again;

E) removing supernatant, resuspending cells with 5ml of hepatocyte amplification medium, and determining survival rate and total amount of cells by trypan blue exclusion method;

F) 2X 10 in terms of number of living cells⁴-3×10⁴/cm²(preferably 3X 10)⁴/cm²) Density, inoculating to collagen coated culture plate, shaking, and culturing in 37 deg.C/5% carbon dioxide incubator;

G) after culturing for 6-8 hours, observing the adherent state, and changing the liquid; the solution was changed every 2 days thereafter.

In a second aspect of the present invention, there is provided an application of the primary hepatocytes cultured by the above-mentioned culture method for maintaining the functional state of human primary hepatocytes in vitro for a long time in drug development.

The invention has the advantages that:

the invention can maintain the function of the primary hepatocyte (PHH) within at least 30 days by high-density culture in the hepatocyte amplification culture medium, is used for drug development of the primary hepatocyte, and provides a new method for long-time culture and function maintenance of the primary hepatocyte in vitro.

Drawings

FIG. 1 Primary hepatocyte morphology;

fig. 2 primary hepatocyte function.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example (b):

first, experimental material

Recombinant protein HGF used in this example was purchased from Peprotech; while the remaining small molecule compounds such as Y27632, A83-01 and CHIR99021 are all available from TargetMol; the primary cells used were purchased from the company BiorecamationIVT and cultured using a specific cell culture medium (i.e., hepatocyte expansion medium); penicillin and streptomycin double-combination antibiotics and a basic culture medium for a primary hepatocyte maintenance experiment are purchased from Yuanpehi Biotech company; fetal Bovine Serum (FBS) was purchased from Gibco; matrigel used for cultured primary hepatocytes was purchased from BD.

Second, Experimental methods

1. Cell recovery and seeding

Taking out the frozen cells from the liquid nitrogen tank, quickly shaking in a water bath at 37 ℃ until the cells are completely melted, centrifuging at 4 ℃ and 700rpm for 3 minutes, discarding the supernatant, adding 1ml of fresh culture medium to resuspend the cell precipitate, and preparing into single cell suspension. Diluting the prepared single cell suspension according to a certain proportion, taking 20ul of cell suspension to be slightly close to the inclined plane of the cell counting chamber, observing and counting the total number of cells in the four squares under a microscope after the cell suspension is siphoned into the cell counting chamber, and calculating the cell concentration according to the following formula: cell concentration equals cell number/ml stock suspension equals four big grid cell total number × 10000 × dilution multiple/4. According to 2X 10⁴-3×10⁴/cm²Inoculating at the inoculation density, culturing in a carbon dioxide cell incubator at 37 deg.C, and changing the culture medium every 2-3 days.

2. Morphological observation of cells

Firstly, the color and clarity of a culture medium for culturing cells are preliminarily observed by naked eyes, then the morphology, the density and the growth state of the cells are carefully observed under a microscope low-power and high-power lens, and a representative visual field is selected for photographing and recording.

3. Cell secretion ALB, AAT assay

Maintaining the state of the primary hepatocytes by using a conditioned medium, reserving cell supernatants once every 3 days, replacing fresh medium 24 hours before reserving the supernatants, collecting the supernatants every other day, and freezing and storing in a refrigerator at-80 ℃ for later use. Quantitative detection was performed according to ALB and AAT ELISA kits.

4. Urea synthesis and ammonia removal detection

3mM NH₄The Cl solution was added to the cell culture wells, after 24h of reaction, the reacted supernatant was pipetted 1mL into a 1.5mL EP tube, centrifuged at 3000rpm for 5min to remove the precipitate, 800ul of the supernatant was pipetted into a new 1.5mL EP tube sample for use, the cells were washed 2 times with PBS, digested for 5min with 1mL of 0.25% T/E, and counted on a Counterstar cytometer. And (4) carrying out sample detection according to the urea analysis kit, and substituting the obtained OD value into a standard curve formula to obtain the corresponding urea concentration.

5. Statistical analysis

The corresponding experimental data were analyzed using Graphpad7 biometric software and the differences between two or more different treatments were compared using the two-tailed unpaired t-test and one-way ANOVA analysis of variance. All statistical data, using at least three independent samples or replicates, are expressed as means ± s.e.m., where ns is insignificant and P <0.05 is considered statistically different, where P, P <0.01, P < 0.0001.

Third, experimental results

1. Morphology of primary hepatocytes

The status of primary hepatocytes was maintained in hepatocyte expansion medium, and the cell morphology was observed under the microscope and photographed on days 0, 3, 7, 14, 21, 24, and 28 of cell culture, respectively.

The experimental results are as follows: referring to fig. 1, it was observed that the hepatocytes were able to maintain polygonal morphology over 28 days of culture, substantially identical to primary hepatocytes morphology.

2. Primary hepatocyte function

And respectively reserving supernatants of the cultured cells on days 0, 3, 7, 10, 14, 21 and 28 of cell culture, and respectively detecting ALB and AAT secretion levels of the cells by an ELISA method, detecting urea synthesis capacity of the cells by a urea detection kit and detecting detoxification capacity of the liver cells by an ammonia removal kit.

The experimental results are as follows: referring to fig. 2, the hepatocyte growth medium can effectively maintain the ALB and AAT secretion ability of hepatocytes within one month, while the urea synthesis and ammonia scavenging ability are maintained at the same level as primary cells, as measured by ELISA.

In summary, by high density culture in hepatocyte amplification medium, primary hepatocyte (PHH) function can be maintained for at least 30 days, providing a new method for long time culture and function maintenance of primary cells in vitro.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Claims

1. A culture method for maintaining the functional state of human primary hepatocytes in vitro for a long time, characterized in that the method is performed at 2X 10⁴-3×10⁴/cm²Inoculating density, and culturing the human primary hepatocytes in a hepatocyte amplification medium.

2. Culture method for maintaining the functional state of human primary hepatocytes in vitro for a prolonged period of time according to claim 1, which comprisesCharacterized in that the inoculation density is 3 multiplied by 10⁴/cm²。

3. The method according to claim 1, wherein the hepatocyte expansion medium is:

4. Use of primary hepatocytes cultured by the method according to any one of claims 1 to 3 for long-term maintenance of the functional state of human primary hepatocytes in vitro in the development of a medicament.