CN110684712A - Preparation method of tissue engineering liver model - Google Patents

Preparation method of tissue engineering liver model Download PDF

Info

Publication number
CN110684712A
CN110684712A CN201810730524.4A CN201810730524A CN110684712A CN 110684712 A CN110684712 A CN 110684712A CN 201810730524 A CN201810730524 A CN 201810730524A CN 110684712 A CN110684712 A CN 110684712A
Authority
CN
China
Prior art keywords
cells
model
liver
culture medium
culturing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN201810730524.4A
Other languages
Chinese (zh)
Inventor
李润芝
张勇杰
金岩
胡成虎
卢永波
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangdong Bo Xi Biotechnology Co Ltd
Original Assignee
Guangdong Bo Xi Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangdong Bo Xi Biotechnology Co Ltd filed Critical Guangdong Bo Xi Biotechnology Co Ltd
Priority to CN201810730524.4A priority Critical patent/CN110684712A/en
Publication of CN110684712A publication Critical patent/CN110684712A/en
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/237—Oncostatin M [OSM]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/85—Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C12N2501/855—Corticotropin [ACTH]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/28—Vascular endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to a preparation method of a tissue engineering liver model, which comprises the following steps: the method comprises the following steps: culturing and amplifying seed cells; step two: preparing a model-built liquid and a matrix; step three: collecting cells; step four: and (5) carrying out model construction. The tissue engineering liver model consists of primary hepatocytes, endothelial cells and hepatic stellate cells, forms a bile duct-like structure similar to natural liver tissues, has the functions of albumin secretion and bile acid transport, and has strong cytochrome P450 activity and high mitochondrial function; compared with the liver cells cultured independently, the expression level of albumin and urea of the liver tissue model constructed by the invention is obviously improved, the tissue activity can be maintained in vitro for 28 days, the long-term drug exposure can be realized, and a more sensitive and accurate test model is provided for hepatotoxicity and drug effect evaluation based on the in vitro cells.

Description

Preparation method of tissue engineering liver model
Technical Field
The invention belongs to the technical field of regenerative medicine and tissue engineering, and particularly relates to a preparation method of a tissue engineering liver model.
Background
The liver, as the largest internal organ of the human body, is responsible for many important physiological functions, such as the metabolism of biotransformation sugar, protein, lipid, vitamin and hormone, synthesis and secretion of bile, etc., and is known as the "metabolic plant" of the human body; the liver is also the 'detoxification factory' of the human body and is the organ showing the toxicity of the most important drugs and metabolites thereof in the human body, so the liver becomes an important target organ for drug toxicity and metabolism research. Hepatotoxicity is a significant cause of failure in the development of new drugs, and about two-thirds of drugs entering phase iii clinics have to be stopped due to their hepatotoxicity, which is also a major cause of the recovery of marketed drugs. Therefore, an effective, reliable, accurate and inexpensive tool for detecting hepatotoxicity is highly desirable.
At present, models for drug hepatotoxicity evaluation comprise an in vivo model and an in vitro model, the in vivo evaluation model usually uses various experimental animals, but animal experiments are long in period, cost is high, and species differences exist between the models and human beings, the in vitro evaluation model comprises a primary hepatocyte model, an in vitro liver model, a liver slice model, a hepatocyte line model and the like.
The organ chip is a new research direction appearing in recent years, mainly simulates the environment in an organism on a microfluidic chip to culture cells and tissues, and truly reflects the interaction of cells-cells, cells-matrix and cells-environment to a certain extent. The construction of liver chips with biological functions is of great significance for drug evaluation, however, the current chip liver is mainly to simply culture liver cells in the chip, and the chip liver is far away from the final target.
The 3D printing technology is a research hotspot at present, and a certain progress is made in 3D organ printing, so that the 3D printer filled with cells is used by the Organovo corporation of California of America to print mini livers with the depth of 0.5mm and the width of 4mm, the mini livers have many functions the same as those of real livers, and the organ chip has more real organ functions and more credible detection results. However, the major obstacle faced by this technology is still the problem of the fabrication of organ microvessels, many viable cells lose their viability after 3D printing is completed, becoming dead cells.
Disclosure of Invention
The invention aims to overcome the defect that the existing liver model can not maintain the structure and the function of the liver in vitro for a long time, thereby better performing the function detection of drug metabolism, toxicity evaluation and the like.
Therefore, the invention provides a preparation method of a tissue engineering liver model, which comprises the following steps:
the method comprises the following steps: culturing and amplifying seed cells;
step two: preparing a model-built liquid and a matrix;
step three: collecting cells;
step four: and (5) carrying out model construction.
The first step is as follows: the seed cell culture and amplification are carried out, and the method comprises the following steps:
step 1, Matrigel preparation, namely freezing and thawing a Matrigel matrix on ice at 4 ~ 8 ℃ overnight, uniformly mixing the Matrigel matrix with a pre-cooled pipette tip to be in a homogenate shape, adopting pre-cooled pure DMEM (diluting the Matrigel matrix according to the ratio of 1:3 ~ 30), coating the diluted Matrigel matrix in a required culture vessel, wherein the coating amount at least covers the growth surface of the whole culture vessel, and removing unbound Matrigel after incubating for 1 ~ 3 hours at room temperature;
step 2, extracting and culturing primary hepatocytes: rinsing the liver tissue with Hank's buffer solution to remove blood stain, and mechanically cutting the liver tissue into piecesDigesting collagenase 0.01 ~ 0.1.1% in volume percentage at 37 deg.C under stirring for 30 ~ 60 minutes, then digesting with pancreatin 0.1 ~ 0.25.25% in volume percentage at 37 deg.C for 30 ~ 60 minutes, filtering with a screen to obtain a single cell suspension, counting living cells, inoculating to a culture dish coated with Matrigel matrix, adding hepatocyte culture medium at 37 deg.C, and 5% CO2Culturing, and changing liquid every 24 hr, wherein the hepatocyte culture medium is prepared by adding 5 ~ 15% Fetal Bovine Serum (FBS) 0.1 ~ 1.0.0 × 10 per 500ml HBM basic culture medium6mol insulin, 5 ~ 15ng hEGF, 0.05 ~ 1.0.0X 106mol hydrocortisone and 10 ~ 50ng hepatocyte growth factor.
Step 3, culturing human umbilical vein endothelial cells: the human umbilical vein endothelial cells were inoculated into a Matrigel matrix-coated culture dish, and endothelial cell culture medium was added thereto at 37 ℃ with 5% CO2Culturing, changing the culture solution every 48 hours, and avoiding overhigh cell plating rate in the culturing process, wherein the endothelial cell culture medium is prepared by adding 5 ~ 15% FBS, 1 ~ 5 mu g hEGF, 0.01 ~ 0.5.5 mg hydrocortisone and 0.1 ~ 4% endothelial cell growth factor (ECGS) into every 500ml of EBM basal medium.
Step 4, hepatic stellate cell culture: inoculating hepatic stellate cells into uncoated culture flask, adding culture medium, and standing at 37 deg.C under 5% CO2Culturing under the condition of changing liquid every 24 hours, wherein the culture medium is DMEM basal medium every 500ml, and 5 ~ 15% FBS and 1 mM L-glutamine are added.
The second step is as follows: preparing a model building fluid and matrix comprising the steps of:
a. mixing the liver cell culture medium and the endothelial cell culture medium according to the volume ratio of 1:1 ~ 3, and adding 0 ~ 50mg/ml transferrin, 0.02 ~ 0.1.1 mu M/ml ascorbic acid, 20 ~ 50 mu g/ml fatty acid free from BSA (BSA-FAF) and 20ng/ml oncostatin M;
b. mixing Matrigel matrix and human umbilical vein endothelial cell culture medium at a volume ratio of 1:1 ~ 3, adding into 24-well culture plate at a concentration of 400 ul/well and 37 deg.C with 5% CO2The incubator was incubated for 20 ~ 40 minutes.
The third step is that: collecting cells; the specific operation is as follows: primary hepatocytes, human umbilical vein endothelial cells, and hepatic stellate cells were individually deleted, centrifuged, and then the cells were resuspended and counted in respective media, and the cell concentrations were adjusted to the same concentrations.
The fourth step is that: model construction is carried out, and the concrete operation is as follows: and (3) performing primary hepatocyte, human umbilical vein endothelial cell and hepatic stellate cell obtained in the step three according to the ratio of 10: 7: 2, then centrifuging, removing supernatant, then resuspending with a construction liquid, inoculating into a well plate with matrix glue laid in advance, adding 1ml of model construction culture medium into the well plate, and performing 5% CO at 37 ℃ to obtain a suspension2Culturing, changing liquid every 24 hours, culturing for 48 hours to form aggregate, and performing experiment after 5 days.
The invention has the beneficial effects that: according to the preparation method of the tissue engineering liver model provided by the invention, the constructed liver tissue model consists of primary hepatocytes, endothelial cells and hepatic stellate cells, forms a bile duct-like structure similar to a natural liver tissue, has albumin secretion and bile acid transport functions, and is strong in cytochrome P450 activity and high in mitochondrial function. Compared with the liver cells cultured independently, the expression level of albumin and urea of the liver tissue model constructed by the invention is obviously improved, the tissue activity can be maintained in vitro for 28 days, the long-term drug exposure can be realized, and a more sensitive and accurate test model is provided for hepatotoxicity and drug effect evaluation based on the in vitro cells.
The present invention will be described in further detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of preparation of a tissue engineering liver model.
FIG. 2a is a schematic diagram of albumin expression levels of hepatocytes cultured alone compared with a tissue-engineered liver model.
FIG. 2b is a schematic diagram showing the comparison of urea expression levels of hepatocytes cultured alone and a tissue-engineered liver model.
FIG. 3 is a schematic diagram of tissue viability level maintenance during long-term in vitro culture of a tissue-engineered liver model.
Detailed Description
To further explain the technical means and effects of the present invention adopted to achieve the intended purpose, the embodiments, the structural features and the effects of the present invention are described in detail in the following with reference to the accompanying drawings and examples.
Example 1
In order to overcome the defect that the existing liver model can not maintain the structure and the function of the liver in vitro for a long time, so that the function detection such as drug metabolism and toxicity evaluation is carried out; the invention provides a preparation method of a tissue engineering liver model shown in figure 1, which comprises the following steps:
the method comprises the following steps: culturing and amplifying seed cells;
step two: preparing a model-built liquid and a matrix;
step three: collecting cells;
step four: and (5) carrying out model construction.
The first step is as follows: the seed cell culture and amplification are carried out, and the method comprises the following steps:
step 1, Matrigel preparation, namely freezing and thawing a Matrigel matrix on ice at 4 ~ 8 ℃ overnight, uniformly mixing the Matrigel matrix with a pre-cooled pipette tip to be in a homogenate shape, adopting pre-cooled pure DMEM (diluting the Matrigel matrix according to the ratio of 1:3 ~ 30), coating the diluted Matrigel matrix in a required culture vessel, wherein the coating amount at least covers the growth surface of the whole culture vessel, and removing unbound Matrigel after incubating for 1 ~ 3 hours at room temperature;
step 2, primary hepatocyte extraction and culture, wherein Hank's buffer solution is used for rinsing liver tissues to remove blood stains, then the liver tissues are mechanically cut into pieces, collagenase with the volume percentage of 0.01 ~ 0.1.1% is used for digesting the liver tissues for 30 ~ 60 minutes at 37 ℃ under stirring, pancreatin with the volume percentage of 0.1 ~ 0.25.25% is used for digesting the liver tissues for 30 ~ 60 minutes at 37 ℃, a single cell suspension is prepared after filtering the liver cells by a screen mesh, the live cells are counted and then inoculated into a culture dish coated by Matrigel matrix, a hepatocyte culture medium is added, the temperature is 37 ℃, and 5% CO is used2Culturing, and changing liquid every 24 hr, wherein the hepatocyte culture medium is prepared by adding 5 ~ 15% Fetal Bovine Serum (FBS) 0.1 ~ 1.0.0 × 10 per 500ml HBM basic culture medium6mol insulin, 5 ~ 15nghEGF，0.05~1.0×106mol hydrocortisone and 10 ~ 50ng hepatocyte growth factor.
Step 3, culturing human umbilical vein endothelial cells: the human umbilical vein endothelial cells were inoculated into a Matrigel matrix-coated culture dish, and endothelial cell culture medium was added thereto at 37 ℃ with 5% CO2Culturing, changing the culture solution every 48 hours, and avoiding overhigh cell plating rate in the culturing process, wherein the endothelial cell culture medium is prepared by adding 5 ~ 15% FBS, 1 ~ 5 mu g hEGF, 0.01 ~ 0.5.5 mg hydrocortisone and 0.1 ~ 4% endothelial cell growth factor (ECGS) into every 500ml of EBM basal medium.
Step 4, hepatic stellate cell culture: inoculating hepatic stellate cells into uncoated culture flask, adding culture medium, and standing at 37 deg.C under 5% CO2Culturing under the condition of changing liquid every 24 hours, wherein the culture medium is DMEM basal medium every 500ml, and 5 ~ 15% FBS and 1 mM L-glutamine are added.
The second step is as follows: preparing a model building fluid and matrix comprising the steps of:
a. mixing the liver cell culture medium and the endothelial cell culture medium according to the volume ratio of 1:1 ~ 3, and adding 0 ~ 50mg/ml transferrin, 0.02 ~ 0.1.1 uM/ml ascorbic acid, 20 ~ 50 μ g/ml fatty acid free from BSA (BSA-FAF) and 20ng/ml oncostatin M;
b. mixing Matrigel matrix and human umbilical vein endothelial cell culture medium at a volume ratio of 1:1 ~ 3, adding into 24-well culture plate at a concentration of 400 ul/well and 37 deg.C with 5% CO2The incubator was incubated for 20 ~ 40 minutes.
The third step is that: collecting cells; the specific operation is as follows: primary hepatocytes, human umbilical vein endothelial cells, and hepatic stellate cells were individually deleted, centrifuged, and then the cells were resuspended and counted in respective media, and the cell concentrations were adjusted to the same concentrations.
The fourth step is that: model construction is carried out, and the concrete operation is as follows: and (3) performing primary hepatocyte, human umbilical vein endothelial cell and hepatic stellate cell obtained in the step three according to the ratio of 10: 7: 2, then centrifuging, removing the supernatant, resuspending with a construction liquid, and inoculating to a pre-paved mediumAdding 1ml model building culture medium into the wells of the plate, and culturing at 37 deg.C with 5% CO2Culturing, changing liquid every 24 hours, culturing for 48 hours to form aggregate, and performing experiment after 5 days.
To sum up, according to the preparation method of the tissue engineering liver model, the constructed liver tissue model consists of primary hepatocytes, endothelial cells and hepatic stellate cells, forms a bile duct-like structure similar to a natural liver tissue, and has the functions of albumin secretion and bile acid transport, strong cytochrome P450 activity and high mitochondrial function. Compared with the liver cells cultured independently, the expression levels of albumin and urea of the liver tissue model constructed by the invention are obviously improved, as shown in fig. 2a and 2 b; and the tissue viability can be maintained in vitro for 28 days, as shown in fig. 3, long-term drug exposure can be realized, and a more sensitive and accurate test model is provided for hepatotoxicity and drug effect evaluation based on in vitro cells.
Example 2
Preparing a matrigel: the Matrigel matrix was freeze-thawed in a refrigerator at 4 ℃ overnight and then mixed with a pre-chilled pipette tip to a homogenate. 50ml of pre-cooled EP tube was added to 28ml of pre-cooled pure DMEM medium (serum free, additive free and antibiotic free) and diluted with 2ml of Matrigel matrix, all on ice. Taking out the culture vessel to be coated, placing on ice, and coating the diluted Matrigel matrix in the culture vessel to cover at least the whole growth surface of the vessel. Incubate at room temperature for 1 hour. Unbound Matrigel matrix was removed and gently rinsed with pure DMEM medium.
Extracting and culturing primary hepatocytes: weighing a certain amount of liver tissue, mechanically cutting, digesting with 0.1% collagenase at 37 deg.C under stirring for 30 min, digesting with 0.125% pancreatin at 37 deg.C for 30 min, filtering with 40 mesh screen to obtain single cell suspension, collecting 10 μ l cell suspension, counting trypan blue stained viable cells, and counting by 5 × 104cells/cm2Inoculating into a Matrigel-coated culture dish, adding hepatocyte culture medium, 37 deg.C, and 5% CO2Incubate and then change the liquid every 24 hours.
The above hepatocyte culture methodThe culture medium is prepared by adding 5 ~ 15% Fetal Bovine Serum (FBS) and 0.1 ~ 1.0.0 × 10 per 500ml HBM basal medium6mol insulin, 5 ~ 15ng hEGF, 0.05 ~ 1.0.0X 106mol hydrocortisone and 10 ~ 50ng hepatocyte growth factor.
Human umbilical vein endothelial cell culture: human umbilical vein endothelial cells at 6 × 103cells/cm2Inoculating into a culture dish coated with Matrigel medium, adding endothelial cell culture medium, 37 deg.C, and 5% CO2Culturing, changing the culture solution 24 hours after inoculation, and then changing the culture solution every 48 hours, wherein the cell plating rate is prevented from being higher than 80% in the culture process.
The endothelial cell culture medium is prepared by adding 5 ~ 15% FBS, 1 ~ 5 μ g hEGF, 0.01 ~ 0.5mg hydrocortisone, and 0.1 ~ 4% endothelial cell growth factor (ECGS) into 500ml EBM basal medium.
Culturing hepatic stellate cells: hepatic stellate cell size 5X 105cells were inoculated into uncoated T75 flasks, medium was added, and the mixture was incubated at 37 ℃ with 5% CO2Culturing under the condition, and changing the culture solution every 24 hours.
The culture medium is prepared by adding 5 ~ 15% FBS and 1 mM L-glutamine into 500ml DMEM basal medium.
Preparing a tissue engineering liver model:
preparing a culture medium:
model construction liquid system preparation, the above-mentioned hepatocyte culture medium and endothelial cell culture medium are mixed according to the volume ratio of 1:1 ~ 3, and 0 ~ 50mg/ml transferrin, 0.02 ~ 0.1uM/ml ascorbic acid, 20 ~ 50 μ g/ml fatty acid free from BSA (BSA-FAF), and 20ng/ml oncostatin M are added.
Preparing matrigel:
mixing Matrigel and a precooled human umbilical vein endothelial cell culture medium in a volume ratio of 1:2, adding the mixture into a 24-well culture plate at a volume ratio of 400 mu l/well, and performing all operations on ice; after the plate is hardened, the 24-hole plate is placed at 37 ℃ and 5% CO2Incubate for 30 minutes.
Cell collection:
primary hepatocytes, human umbilical vein endothelial cells and hepatic stellate cells were separately digested, centrifuged, and then the cells were resuspended and counted using the respective media, and the respective cell concentrations were adjusted to the same concentrations.
Co-culturing:
the obtained primary hepatocytes, human umbilical vein endothelial cells and hepatic stellate cells were expressed as (1X 10)6：7×105：1×105) The above-mentioned components were mixed, centrifuged, and the supernatant was removed, then resuspended in 500ul of a construction liquid, inoculated into a well plate previously plated with matrigel, and 1ml of a medium was added to the well plate at 37 ℃ and 5% CO2Culturing, changing liquid every 24 hours, culturing for 48 hours to form aggregate, and performing experiment after 5 days.
Example 3
Preparing a matrigel: the Matrigel matrix was freeze-thawed in a refrigerator at 4 ℃ overnight and then mixed with a pre-chilled pipette tip to a homogenate. 50ml of pre-cooled EP tube was added to 29ml of pre-cooled pure DMEM medium (serum free, additive free and antibiotic free) and diluted with 1ml of Matrigel matrix, all on ice. The vessel to be coated is taken out, placed on ice, and the diluted Matrigel matrix is coated in the required culture vessel in an amount at least covering the whole growth surface of the vessel. Incubate at room temperature for 1 hour. Unbound Matrigel matrix was removed and gently rinsed with pure DMEM medium.
Extracting and culturing primary hepatocytes: weighing a certain amount of liver tissue, mechanically cutting, digesting with 0.1% collagenase at 37 deg.C under stirring for 30 min, digesting with 0.125% pancreatin at 37 deg.C for 30 min, filtering with 40 mesh screen to obtain single cell suspension, collecting 10ul cell suspension, counting trypan blue stained viable cells, and counting at 5 × 104cells/cm2Inoculating into a Matrigel coated culture dish, adding hepatocyte culture medium, and adding 5% CO at 37 deg.C2Incubate and then change the liquid every 24 hours.
The hepatocyte culture medium is prepared by adding 5 ~ 15% Fetal Bovine Serum (FBS) and 0.1 ~ 1.0.0 × 10 to 500ml HBM basic culture medium6mol insulin, 5 ~ 15ng hEGF, 0.05 ~ 1.0.0X 106mol hydrocortisone and 10 ~ 50ng hepatocyte growth factor.
Human umbilical vein endothelial cell culture: human umbilical vein endotheliumCells were arranged at 6X 103cells/cm2Inoculating into a Matrigel matrix coated culture dish, adding endothelial cell culture medium, 37 deg.C, and 5% CO2Culturing, changing the culture solution 24 hours after inoculation, and then changing the culture solution every 48 hours, wherein the cell plating rate is prevented from being higher than 80% in the culture process.
The endothelial cell culture medium is prepared by adding 5 ~ 15% FBS, 1 ~ 5 μ g hEGF, 0.01 ~ 0.5mg hydrocortisone, and 0.1 ~ 4% endothelial cell growth factor (ECGS) into 500ml EBM basal medium.
Culturing hepatic stellate cells: hepatic stellate cell size 5X 105cells were inoculated into uncoated T75 flasks, medium was added, and the mixture was incubated at 37 ℃ with 5% CO2Culturing under the condition, and changing the culture solution every 24 hours.
The culture medium is prepared by adding 5 ~ 15% FBS and 1 mM L-glutamine into 500ml DMEM basal medium.
Preparing a tissue engineering liver model:
preparing a culture medium:
model construction liquid system preparation, the above-mentioned hepatocyte culture medium and endothelial cell culture medium are mixed according to the volume ratio of 1:1 ~ 3, and 0 ~ 50mg/ml transferrin, 0.02 ~ 0.1uM/ml ascorbic acid, 20 ~ 50 μ g/ml fatty acid free from BSA (BSA-FAF), and 20ng/ml oncostatin M are added.
Preparing matrigel:
mixing a Matrigel matrix and a precooled human umbilical vein endothelial cell culture medium in a volume ratio of 1:3, adding the mixture into a 96-well culture plate at a concentration of 65 mu l/well, and performing all operations on ice; after the plates were hardened, the 96-well plate was set at 37 ℃ and 5% CO2Incubate for 30 minutes.
Cell collection:
primary hepatocytes, human umbilical vein endothelial cells and hepatic stellate cells were separately digested, centrifuged, and then the cells were resuspended and counted using the respective media, and the respective cell concentrations were adjusted to the same concentrations.
Co-culturing:
the obtained primary hepatocytes, human umbilical vein endothelial cells and hepatic stellate cells were expressed as (1X 10)4：7×103：1×103) The amount of the mixture was mixed, after removing the supernatant, resuspended with 100. mu.l of the construction liquid, inoculated into a well plate previously plated with matrigel, 100. mu.l of the culture medium was added to the well plate at 37 ℃ with 5% CO2Culturing, changing liquid every 24 hours, culturing for 48 hours to form aggregate, and performing experiment after 5 days.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (5)

1. A preparation method of a tissue engineering liver model is characterized by comprising the following steps:
the method comprises the following steps: culturing and amplifying seed cells;
step two: preparing a model-built liquid and a matrix;
step three: collecting cells;
step four: and (5) carrying out model construction.
2. The method for preparing a liver model for tissue engineering according to claim 1, wherein the first step is: the seed cell culture and amplification are carried out, and the method comprises the following steps:
step 1, Matrigel preparation, namely freezing and thawing a Matrigel matrix on ice at 4 ~ 8 ℃ overnight, uniformly mixing the Matrigel matrix with a pre-cooled pipette tip to be in a homogenate shape, adopting pre-cooled pure DMEM (diluting the Matrigel matrix according to the ratio of 1:3 ~ 30), coating the diluted Matrigel matrix in a required culture vessel, wherein the coating amount at least covers the growth surface of the whole culture vessel, and removing unbound Matrigel after incubating for 1 ~ 3 hours at room temperature;
step 2, primary hepatocyte extraction and culture, wherein Hank's buffer solution is used for rinsing liver tissues to remove blood stains, then the liver tissues are mechanically cut into pieces, and collagenase 3 with the volume percentage of 0.01 ~ 0.1.1 percent is firstly usedDigesting at 7 deg.C under stirring for 30 ~ 60 min, digesting at 37 deg.C for 30 ~ 60 min with pancreatin 0.1 ~ 0.25.25% by volume, filtering with screen to obtain single cell suspension, counting living cells, inoculating to a culture dish coated with Matrigel matrix, adding hepatocyte culture medium at 37 deg.C and 5% CO2Culturing, and changing liquid every 24 hr, wherein the hepatocyte culture medium is prepared by adding 5 ~ 15% Fetal Bovine Serum (FBS) 0.1 ~ 1.0.0 × 10 per 500ml HBM basic culture medium6mol insulin, 5 ~ 15ng hEGF, 0.05 ~ 1.0.0X 106Culturing hydrocortisone mol and 10 ~ 50ng Hepatocyte Growth Factor (HGF);
step 3, culturing human umbilical vein endothelial cells: the human umbilical vein endothelial cells were inoculated into a Matrigel matrix-coated culture dish, and endothelial cell culture medium was added thereto at 37 ℃ with 5% CO2Culturing, changing the culture solution every 48 hours, and avoiding overhigh cell plating rate in the culture process, wherein the endothelial cell culture medium is prepared by adding 5 ~ 15% FBS, 1 ~ 5 mug hEGF, 0.01 ~ 0.5.5 mg hydrocortisone and 0.1 ~ 4% endothelial cell growth factor (ECGS) into every 500ml of EBM basal medium;
step 4, hepatic stellate cell culture: inoculating hepatic stellate cells into uncoated culture flask, adding culture medium, and standing at 37 deg.C under 5% CO2Culturing under the condition of changing liquid every 24 hours, wherein the culture medium is DMEM basal medium every 500ml, and 5 ~ 15% FBS and 1 mM L-glutamine are added.
3. The method for preparing a liver model for tissue engineering according to claim 1, wherein the second step: preparing a model building fluid and matrix comprising the steps of:
a. mixing the liver cell culture medium and the endothelial cell culture medium according to the volume ratio of 1:1 ~ 3, and adding 0 ~ 50mg/ml transferrin, 0.02 ~ 0.1.1 mu M/ml ascorbic acid, 20 ~ 50 mu g/ml fatty acid free from BSA (BSA-FAF) and 20ng/ml oncostatin M;
b. mixing Matrigel matrix and human umbilical vein endothelial cell culture medium at a volume ratio of 1:1 ~ 3, adding into 24-well culture plate at a concentration of 400 ul/well and 37 deg.C with 5% CO2Incubator hatchIncubate for 20 ~ 40 minutes.
4. The method for preparing a liver model for tissue engineering according to claim 1, wherein the step three: collecting cells; the specific operation is as follows: primary hepatocytes, human umbilical vein endothelial cells, and hepatic stellate cells were individually deleted, centrifuged, and then the cells were resuspended and counted in respective media, and the cell concentrations were adjusted to the same concentrations.
5. The method for preparing a liver model for tissue engineering according to claim 1, wherein the fourth step: model construction is carried out, and the concrete operation is as follows: and (3) performing primary hepatocyte, human umbilical vein endothelial cell and hepatic stellate cell obtained in the step three according to the ratio of 10: 7: 2, then centrifuging, removing supernatant, then resuspending with a construction liquid, inoculating into a well plate with matrix glue laid in advance, adding 1ml of model construction culture medium into the well plate, and performing 5% CO at 37 ℃ to obtain a suspension2Culturing, changing liquid every 24 hours, culturing for 48 hours to form aggregate, and performing experiment after 5 days.
CN201810730524.4A 2018-07-05 2018-07-05 Preparation method of tissue engineering liver model Withdrawn CN110684712A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810730524.4A CN110684712A (en) 2018-07-05 2018-07-05 Preparation method of tissue engineering liver model

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810730524.4A CN110684712A (en) 2018-07-05 2018-07-05 Preparation method of tissue engineering liver model

Publications (1)

Publication Number Publication Date
CN110684712A true CN110684712A (en) 2020-01-14

Family

ID=69107273

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810730524.4A Withdrawn CN110684712A (en) 2018-07-05 2018-07-05 Preparation method of tissue engineering liver model

Country Status (1)

Country Link
CN (1) CN110684712A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113293127A (en) * 2021-05-24 2021-08-24 江南大学 Construction and application of multi-cell co-culture three-dimensional liver microsphere model
CN114456936A (en) * 2022-04-12 2022-05-10 北京大橡科技有限公司 Chip, organoid model, construction method and construction device of organoid model and application of organoid model

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113293127A (en) * 2021-05-24 2021-08-24 江南大学 Construction and application of multi-cell co-culture three-dimensional liver microsphere model
CN114456936A (en) * 2022-04-12 2022-05-10 北京大橡科技有限公司 Chip, organoid model, construction method and construction device of organoid model and application of organoid model

Similar Documents

Publication Publication Date Title
Török et al. Primary human hepatocytes on biodegradable poly (l‐lactic acid) matrices: A promising model for improving transplantation efficiency with tissue engineering
CN105154386B (en) Human liver cell maintains for a long time and the special culture media and cultural method of propagation secondary culture
RU2009102643A (en) DIFFERENTIATION OF STEM CELLS FROM THE URBINE MATRIX TO CELLS OF THE HEPATOCYTES LINE
CN110878286B (en) Culture medium for culturing liver cancer organoid cell balls
CN110684712A (en) Preparation method of tissue engineering liver model
CN105010307A (en) Cryopreservation solution and cryopreservation resuscitation method for liver primary cells
Strain Isolated hepatocytes: use in experimental and clinical hepatology.
Yoffe et al. Cultures of human liver cells in simulated microgravity environment
CN103842497A (en) Bioartificial proximal tubule systems and methods of use
CN110904026B (en) Preparation method and application of hepatic precursor-like cells from different sources
US20080280357A1 (en) Cryopreservation of Hepatocytes
CN113943755B (en) Method for constructing in-situ primary esophageal cancer animal model
CN109576308B (en) Method for improving detoxification function of human stem cell-derived liver-like cells and application thereof
Watanabe et al. Differentiation of pancreatic acinar carcinoma cells cultured on rat testicular seminiferous tubular basement membranes
CN105695392A (en) Culturing method for improving in-vitro differentiation phenotype and function of hepatic cells
CN114214282B (en) Method for culturing lung tumor organoids
CN110093305B (en) Method for inducing hepatocyte in-vitro amplification
CN114369573B (en) Method for constructing in-situ primary nasopharyngeal carcinoma animal model
CN101368170A (en) In vitro together culture technique for hepatocyte and kupffer cell
US20220135942A1 (en) Cultured tissue and method for producing same
CN103305467B (en) A kind of human liver cancer cell system HLCZ02 and application thereof
Maekawa et al. Long-term culture of rat hepatocytes using human amniotic membrane as a culture substrate
CN113564098A (en) Culture method for enhancing hepatocyte functionality and hepatocyte culture solution used in culture method
CN109022348A (en) A kind of isolation and culture method of primary porcine hepatocyte
Poole et al. Preparation and properties of primary maintenance cultures of adult rabbit hepatocytes

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication
WW01 Invention patent application withdrawn after publication

Application publication date: 20200114