阅读说明：本技术 一种三维肝脏类器官复苏液和三维肝脏类器官的复苏方法 (Three-dimensional liver organoid resuscitation solution and three-dimensional liver organoid resuscitation method ) 是由 高毅 黎少 欧楚澎 彭青 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种三维肝脏类器官复苏液和三维肝脏类器官的复苏方法,涉及细胞复苏液技术领域。该三维肝脏类器官复苏液的组分包括：烟酰胺50-200mmol/L、胰岛素1-50μg/ml、肝细胞生长因子1-50ng/ml、表皮生长因子1-50ng/ml、地塞米松1-100μmol/L、胎牛血清8-12wt%、亚麻酸1-50μg/ml和极性溶剂0.01-0.1wt%以及余量的基础培养基。本申请提供的复苏液能够提供足够的营养供类器官内肝细胞维持活性和生长培养。其成分简单,组容易获取、价格相对低廉,并且基础培养基有多种选择,可以适用于各种肝脏类器官,应用范围广,本申请提供的复苏方法简单,可操作性强。(The invention discloses a three-dimensional liver organoid resuscitation solution and a resuscitation method of three-dimensional liver organoids, and relates to the technical field of cell resuscitation solutions. The three-dimensional liver organoid resuscitation solution comprises the following components: 50-200mmol/L nicotinamide, 1-50 mug/ml insulin, 1-50ng/ml hepatocyte growth factor, 1-50ng/ml epidermal growth factor, 1-100 mug/L dexamethasone, 8-12wt% fetal calf serum, 1-50 mug/ml linolenic acid, 0.01-0.1wt% polar solvent and the balance of basal medium. The resuscitation solution provided by the application can provide enough nutrition for the maintenance activity and growth culture of liver cells in organoid. The resuscitation medium has the advantages of simple components, easy group acquisition, relatively low price, multiple choices of basic culture media, suitability for various liver organs, wide application range, simple resuscitation method and strong operability.)

1. The three-dimensional liver organoid resuscitation fluid is characterized by comprising the following components: 50-200mmol/L nicotinamide, 1-50 mug/ml insulin, 1-50ng/ml hepatocyte growth factor, 1-50ng/ml epidermal growth factor, 1-100 mug/L dexamethasone, 8-12wt% fetal calf serum, 1-50 mug/ml linolenic acid, 0.01-0.1wt% polar solvent and the balance of basal medium.

2. The three-dimensional liver organoid resuscitation fluid of claim 1, comprising: 80-120mmol/L nicotinamide, 20-30 mug/ml insulin, 20-30ng/ml hepatocyte growth factor, 20-30ng/ml epidermal growth factor, 40-60 mug/L dexamethasone, 9-11 wt% fetal calf serum, 10-25 mug/ml linolenic acid, 0.05-0.08 wt% polar solvent and the balance of basal medium.

3. The three-dimensional liver organoid resuscitation fluid of claim 1, wherein said nicotinamide and said dexamethasone are dissolved in said polar solvent prior to addition to said basal medium.

4. The three-dimensional liver organoid resuscitation fluid of claim 1, wherein the polar solvent is DMSO.

5. The three-dimensional liver organoid resuscitation fluid of claim 1, wherein said basal medium is selected from the group consisting of DMEM/F12 cell culture medium, MEM cell culture medium, William's E cell culture medium, and DMEM high glucose cell culture medium.

6. A method of resuscitating a three-dimensional liver organoid, comprising: performing continuous resuscitation culture on the cryopreserved three-dimensional liver organoid by using the three-dimensional liver organoid resuscitation fluid of any one of claims 1-5.

7. The method for resuscitating a three-dimensional liver organoid of claim 6, wherein the number of target hepatocytes in the cryopreserved three-dimensional liver organoid is greater than 10⁶；

The target liver cell is selected from a liver cancer cell line or a normal human liver cell line.

8. The method for resuscitating a three-dimensional liver organoid of claim 6, wherein the three-dimensional liver organoid comprises a spheroid, a suspended liver organoid, or a hydrogel organoid.

9. The method for resuscitation of three-dimensional liver organoids according to claim 6, wherein said continuous resuscitation culture comprises seeding said three-dimensional liver organoids into a low-adhesion well plate or a microsphere plate, placing in CO containing said three-dimensional liver organoid resuscitation fluid₂Performing static culture in a cell culture box at 35-37 deg.C for 1-5 days;

replacing the three-dimensional liver organoid resuscitation solution every 20-30 hours within the time of the continuous resuscitation culture;

replacing the three-dimensional liver organoid resuscitation fluid comprises: the supernatant in the culture vessel was discarded, washed twice with buffer solution, and then fresh three-dimensional liver organoid resuscitating solution was added.

10. The method for resuscitating a three-dimensional liver organoid according to any of claims 6-9, further comprising pre-treating the cryopreserved three-dimensional liver organoid before culturing the cryopreserved three-dimensional liver organoid by continuous resuscitation with the three-dimensional liver organoid resuscitation fluid, wherein the pre-treating comprises thawing the cryopreserved three-dimensional liver organoid at 35-37 ℃, centrifuging at 500 rpm for 3-5min after thawing, discarding the cryopreserved fluid, and washing with phosphate buffer solution for 2-3 times.

Technical Field

The invention relates to the technical field of cell resuscitation solutions, in particular to a three-dimensional liver organoid resuscitation solution and a three-dimensional liver organoid resuscitation method.

Background

Unlike the surface of two-dimensional cells, three-dimensional culture requires cells to adapt to their morphology, phenotype, etc., and is a culture system with good proliferation conditions and induction of functional activity. Existing two-dimensional cell culture models are often not ideal for studying cell growth in vitro because they are unable to form more natural tissue-like structures, which has a significant impact on cell performance.

More and more researches show that more developments of three-dimensional culture make liver organoids become research and development tools for liver disease research and drug metabolism. Liver organoids are aggregates that are cultured in vitro and can form a biomimetic liver in terms of function and structure. Because key structure and functional characteristics in a bionic body can be formed in vitro, the method has more advantages compared with the traditional two-dimensional cell culture, and especially has important research significance in the aspects of drug toxicity detection, liver cancer phenotype analysis, liver tissue engineering research and the like.

The liver organoids which can be frozen completely and in a fragment form can be rapidly recovered after a long-term freezing state at low temperature, and have important research and application values for in vitro long-term application.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a three-dimensional liver organoid resuscitation solution and a three-dimensional liver organoid resuscitation method.

The invention is realized by the following steps:

in a first aspect, the invention provides a three-dimensional liver organoid resuscitation fluid, which comprises the following components: 50-200mmol/L nicotinamide, 1-50 mug/ml insulin, 1-50ng/ml hepatocyte growth factor, 1-50ng/ml epidermal growth factor, 1-100 mug/L dexamethasone, 8-12wt% fetal calf serum, 1-50 mug/ml linolenic acid, 0.01-0.1wt% polar solvent and the balance of basal medium.

In an alternative embodiment, the components thereof comprise: 80-120mmol/L nicotinamide, 20-30 mug/ml insulin, 20-30ng/ml hepatocyte growth factor, 20-30ng/ml epidermal growth factor, 40-60 mug/L dexamethasone, 9-11 wt% fetal calf serum, 10-25 mug/ml linolenic acid, 0.05-0.08 wt% polar solvent and the balance of basal medium.

In an alternative embodiment, said nicotinamide and said dexamethasone are dissolved in said polar solvent before being added to said basal medium.

In alternative embodiments, the polar solvent is DMSO.

In alternative embodiments, the basal medium is selected from one of DMEM/F12 cell culture medium, MEM cell culture medium, William "s E cell culture medium, and DMEM high glucose cell culture medium.

In a second aspect, the present invention provides a method of resuscitating a three-dimensional liver organoid, comprising: performing continuous resuscitation culture on the cryopreserved three-dimensional liver organoids by using the three-dimensional liver organoid resuscitation fluid according to any one of the preceding embodiments.

In alternative embodiments, the target hepatocytes in the three-dimensional liver organoid cryopreserved are greater than 10⁶；

The target liver cell is selected from a liver cancer cell line or a normal human liver cell line.

In alternative embodiments, the three-dimensional liver organoid comprises a spheroid, a suspended liver organoid, or a hydrogel organoid.

In an alternative embodiment, the continuous resuscitation culture comprises seeding the three-dimensional liver organoid into a low-adhesion well plate or a microspherical plate, placing in CO containing the three-dimensional liver organoid resuscitation fluid₂Performing static culture in a cell culture box at 35-37 deg.C for 1-5 days;

replacing the three-dimensional liver organoid resuscitation solution every 20-30 hours within the time of the continuous resuscitation culture;

replacing the three-dimensional liver organoid resuscitation fluid comprises: the supernatant in the culture vessel was discarded, washed twice with buffer solution, and then fresh three-dimensional liver organoid resuscitating solution was added.

In an optional embodiment, before the three-dimensional liver organoid resuscitation fluid is used for performing continuous resuscitation culture on the cryopreserved three-dimensional liver organoid, the method further comprises pretreating the cryopreserved three-dimensional liver organoid, wherein the pretreating comprises thawing the cryopreserved three-dimensional liver organoid at 35-37 ℃, centrifuging the three-dimensional liver organoid at 500 rpm for 3-5min after thawing, discarding the cryopreservation fluid, and washing with a phosphoric acid buffer solution.

The invention has the following beneficial effects: the three-dimensional liver organoid resuscitation solution provided by the application can provide energy for resuscitation and culture of liver organoids and promote differentiation of liver organoids by adding nicotinamide, dexamethasone and linolenic acid into a basic culture medium, and can be added with insulin, hepatocyte growth factor and epidermal growth factor to promote proliferation of cells, so that sufficient nutrients can be further provided for growth of liver organoids by adding fetal calf serum. The three-dimensional liver organoid resuscitation solution provided by the application can provide enough nutrition for maintaining activity and growth culture of liver cells in organoids. The components are simple, the components are easy to obtain, the price is relatively low, and the preparation cost of the resuscitation solution can not be greatly improved; the recovery liquid can be prepared by using various basic culture media, can adapt to hepatocytes of different cell lines or cell lines, can also adapt to hepatocytes of different maturity, and provides sufficient nutrition for rapid recovery and activity maintenance of various hepatocytes; the application provides a three-dimensional liver organoid resuscitation liquid is through carrying out the continuous culture of presetting the day number with target liver organoid, can make multiple type cryopreserved liver organoid survival rate and function resume, easy operation, the application is strong, can realize that more liver organoids are preserved and use fast.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a morphological light mirror image of liver cancer cells HepG2 cultured by resuscitating the three-dimensional liver organoid resuscitating solution provided in example 1 for 5 days;

FIG. 2 is a morphological light microscope image of liver cancer cells cultured by resuscitating C3A hydrogel organoids in the three-dimensional liver organoid resuscitating fluid provided in example 1 for 5 days;

FIG. 3 is a morphological photomicrograph of the liver stem cell Heparg three-dimensional organoid cultured by resuscitation with the three-dimensional liver organoid resuscitation solution provided in example 1 after 5 days;

FIG. 4 is a graph showing the statistics of cell numbers after 5 days of three-dimensional organoid resuscitation culture of hepatic stem cells HepaRG and HepG2 using the three-dimensional liver organoid resuscitation solution provided in example 1;

FIG. 5 is a light-microscopic image of the dead and alive staining results of cells after 5 days of resuscitating and culturing a C3A three-dimensional organoid with the three-dimensional liver organoid resuscitating fluid provided in comparative examples 1 to 6, example 1 and example 5;

FIG. 6 is a graph showing the statistical results of ELISA expression on cell supernatants after 5 days of resuscitating and culturing C3A three-dimensional organoids using the three-dimensional liver organoid resuscitating fluid provided in comparative examples 1 to 6, example 1 and example 5.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment of the application provides a three-dimensional liver organoid resuscitation solution, which comprises the following components: 50-200mmol/L Nicotinamide (Nicotinamide), 1-50 mu g/ml Insulin (Insulin), 1-50ng/ml Hepatocyte Growth Factor (HGF), 1-50ng/ml Epidermal Growth Factor (EGF), 1-100 mu mol/L dexamethasone, 8-12wt% fetal bovine serum, 1-50 mu g/ml linolenic acid, 0.01-0.1wt% polar solvent and the balance of basal medium.

In the application, nicotinamide is used as a nutrient component of a culture medium to provide energy for the recovery and culture of liver organoids.

Insulin can act on an insulin receptor on the surface of a liver organoid, enhance the energy intake and utilization of the liver organoid, promote the synthesis of RNA, protein and fatty acid in the liver organoid, inhibit apoptosis and enhance the activity and function of the liver organoid.

The hepatocyte growth factor and the epidermal growth factor can promote the proliferation of the hepatocyte and regulate the function of the hepatocyte.

Dexamethasone is an artificially synthesized corticosteroid, can resist inflammation and toxicity, can induce liver organoid differentiation, and provides hormone for the liver organoid.

Fetal bovine serum is rich in nutrients essential for cell growth.

Linolenic acid is an essential nutrient that can be added to cell culture to promote cell function and growth.

Polar solvents are used to dissolve nicotinamide and dexamethasone. Since nicotinamide is insoluble in water, dexamethasone cannot be dissolved directly in a basal medium in water as solvent, by adding a polar solvent, in this application DMSO (dimethyl sulfoxide) is preferred. DMSO is known as a "universal solvent" and is commonly used to dissolve various additives added to liquid or solid media, but DMSO is weakly toxic to cells, and thus the amount of DMSO used is controlled within a biologically acceptable range in this application. In this example, the final concentration of DMSO is no more than 0.1%, preferably 0.01-0.1%, more preferably 0.05-0.08%. In the application, both the nicotinamide and the dexamethasone are dissolved by using the polar solvent, so that the control of the addition amounts of the nicotinamide and the dexamethasone is facilitated.

The basal medium is selected from one of DMEM/F12 cell culture medium, MEM cell culture medium, William's E cell culture medium and DMEM high-glucose cell culture medium.

Preferably, the three-dimensional liver organoid resuscitation fluid provided by the present application comprises the following components: 80-120mmol/L nicotinamide, 20-30 mug/ml insulin, 20-30ng/ml hepatocyte growth factor, 20-30ng/ml epidermal growth factor, 40-60 mug/L dexamethasone, 9-11 wt% fetal calf serum, 10-25 mug/ml linolenic acid, 0.05-0.08 wt% polar solvent and the balance of basal medium.

In addition, the embodiment of the application also provides a resuscitation method of the three-dimensional liver organoid, which comprises the following steps:

s1, selecting the three-dimensional liver organoid in the frozen storage form.

Wherein the three-dimensional liver organoids comprise spheroids, suspended liver organoids or hydrogel organoids. Greater than 10 target hepatocytes in cryopreserved three-dimensional liver organoids⁶(ii) a Preferably, the target liver cell is selected from a liver cancer cell line or a normal human liver cell line.

And S2, preprocessing the frozen three-dimensional liver organoids.

Thawing the frozen three-dimensional liver organoid at 35-37 deg.C, and centrifuging at 500 rpm for 3-5 min; the frozen stock solution is discarded and washed 2-3 times with phosphoric acid buffer solution.

And S3, utilizing the three-dimensional liver organoid resuscitation solution to perform continuous resuscitation culture on the cryopreserved three-dimensional liver organoids.

The continuous recovery culture comprises inoculating three-dimensional liver organoid into low-adhesion pore plate or microsphere plate, and placing in CO containing three-dimensional liver organoid recovery liquid₂Performing static culture in a cell culture box at 35-37 deg.C for 1-5 days; replacing the three-dimensional liver organoid resuscitation solution every 20-30 hours within the time of continuous resuscitation culture; specifically, the replacement of a three-dimensional liver organoid resuscitation fluid comprises: the supernatant in the culture vessel was discarded, washed twice with buffer, and then fresh three-dimensional liver organoid resuscitating solution was added.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a three-dimensional liver organoid resuscitation fluid which comprises a basic culture medium and an additive, wherein the basic culture medium in the embodiment is DMEM/F12 cell culture medium, and the additive comprises nicotinamide with a final concentration of 100 mmol/L, insulin with a final concentration of 25 mu g/ml, hepatocyte growth factor with a final concentration of 25 ng/ml, epidermal growth factor with a final concentration of 25 ng/ml, dexamethasone with a final concentration of 50 mu mol/L, FBS with a concentration of 10 percent, linolenic acid with a final concentration of 10 mu g/ml and DMSO with a final concentration of 0.1 percent.

The preparation method comprises the following steps:

(1) diluting nicotinamide and dexamethasone by DMSO;

(2) to DMEM/F12 cell culture medium was added DMSO dissolved nicotinamide and dexamethasone, insulin, hepatocyte growth factor, as well as epidermal growth factor and linolenic acid.

Examples 2 to 4

Examples 2-4 provide three-dimensional liver organoid resuscitation fluids with the same supplements as example 1, but different basal media:

in example 2, the basal medium was MEM cell culture medium;

in example 3, the basal medium was William's E cell culture medium;

in example 4, the basal medium was DMEM high glucose cell culture medium.

Example 5

The embodiment provides a three-dimensional liver organoid resuscitation fluid which comprises a basic culture medium and an additive, wherein the basic culture medium is DMEM/F12 cell culture medium, and the additive comprises nicotinamide with a final concentration of 150 mmol/L, insulin with a final concentration of 40 mu g/ml, hepatocyte growth factor with a final concentration of 40 ng/ml, epidermal growth factor with a final concentration of 40 ng/ml, dexamethasone with a final concentration of 65 mu mol/L, FBS with a concentration of 12%, linolenic acid with a final concentration of 20 mu g/ml and DMSO with a final concentration of 0.1%.

Comparative example 1

This comparative example is different from example 1 in that the three-dimensional organoids were subjected to resuscitation culture using only a basal medium (DMEM cell culture medium) without adding additives to the basal medium.

Comparative example 2

This comparative example differs from example 2 in that the three-dimensional organoids were subjected to resuscitation culture using only a basal medium (MEM cell culture medium) without adding additives to the basal medium.

Comparative example 3

This comparative example differs from example 3 in that the three-dimensional organoids were subjected to resuscitation culture using only a basal medium (William's E cell culture medium) without adding additives to the basal medium.

Comparative example 4

This comparative example is different from example 4 in that the three-dimensional organoids were subjected to resuscitation culture using only a basal medium (DMEM/F12 high glucose cell culture medium) without adding additives to the basal medium.

Comparative example 5

Niacinamide in example 1 was omitted.

Comparative example 6

Dexamethasone in example 1 was omitted.

Examples of the experiments

The three-dimensional liver organoid resuscitation solutions of the examples 1 to 5 and the comparative examples 1 to 6 are used for resuscitating and culturing three-dimensional liver organoids corresponding to the examples or the comparative examples in the following table, and the method specifically comprises the following steps:

obtaining three-dimensional liver organoids in a frozen state, quickly thawing at 37 ℃, centrifuging at 500 rpm for 3-5 minutes, discarding frozen stock solution, and washing twice by using a phosphoric acid buffer solution; adding resuscitating solution which has been warm-bathed in advance, continuously culturing for a predetermined number of days in incubator under 5% CO₂At 37 ℃. The specific operation steps refer to the following: inoculating three-dimensional liver organoid into low-adhesion pore plate or microsphere plate, and placing in CO containing three-dimensional liver organoid resuscitation solution₂And (5) performing static culture in the cell incubator. During the continuous culture, the resuscitating fluid/control medium was changed every 24 hours.

As can be seen from the above table and the accompanying drawings 1 to 6, after 5 days of the first addition of the resuscitation fluid provided in examples 1 to 5, the cell morphology and the survival rate in the liver organoids, whether three-dimensional spheroids or hydrogels, were well maintained, and the conventional basal medium provided in comparative examples 1 to 4 was not suitable for resuscitating three-dimensional organoids, and as can be seen from FIGS. 5 and 6, the resuscitation effect obtained by omitting nicotinamide in example 1 in comparative example 5 and dexamethasone in example 1 in comparative example 6 was significantly inferior to that obtained by example 1. To sum up, three-dimensional liver organoid recovery liquid that this application provided promotes liver organoid differentiation simultaneously through adding niacinamide, dexamethasone and linolenic acid in to basal medium for the recovery of liver organoid and culture provide energy, still adds insulin, hepatocyte growth factor and epidermal growth factor simultaneously and is favorable to promoting the proliferation of cell, and fetal calf serum's addition further provides sufficient nutrient substance for the growth of liver organoid. The three-dimensional liver organoid resuscitation solution provided by the application can provide enough nutrition for maintaining activity and growth culture of liver cells in organoids. The composition is simple, the operation is simple and convenient, and the preservation and the rapid application of more liver organs are realized through the application of the rapid resuscitation liquid.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.