CN118526521A - Application of hepatocytes cultured under hypoxia condition by using cordycepin in preparation of drugs for treating liver injury - Google Patents

Abstract

The present application is an application of hepatocytes cultured under hypoxic conditions using cordycepin in the preparation of a drug for treating liver damage, wherein the culture fluid of the hepatocytes includes a basal culture medium, 5-15 μmol/L cordycepin, 0.5-4 μg/mL of mesenchymal stem cell conditioned exosomes, and the culture conditions are 1% to 6% oxygen concentration, 5% carbon dioxide concentration, and the remaining gas is nitrogen, and the culture time is 24-36h. The application of this application uses hepatocytes cultured under hypoxic conditions, which can be stably obtained through the improvement of the culture fluid and the improvement of the culture conditions, so that the possibility of clinical application is increased.

Description

Application of hepatocytes cultured under hypoxic conditions using cordycepin in the preparation of drugs for treating liver damage

Technical Field

The present application relates to the technical field of liver treatment, and in particular, to the use of hepatocytes cultured under hypoxic conditions using cordycepin in the preparation of a drug for treating liver damage.

Background Art

The liver is an important metabolic and detoxification organ in the human body. However, a variety of factors can cause hypoxic damage to liver cells, which is often seen in liver ischemia-reperfusion during liver surgery and clinical liver transplantation. Ischemia-reperfusion injury greatly affects the success or failure of surgery and liver transplantation. Therefore, how to effectively prevent hypoxic damage to liver cells is a problem that needs to be solved urgently in clinical practice.

Mesenchymal stem cell (MSC)-based therapy has emerged as a promising strategy for the treatment of liver diseases through tissue repair and immunomodulation. However, the use of MSCs has some disadvantages, such as the need for a continuous supply of cells that stably express the phenotype, which is time-consuming and costly to produce and process. In addition, ectopic tissue formation, infusion toxicity caused by cell retention in the lung microvasculature, or cell rejection have also been reported. Studies have shown that exosomes secreted by MSCs can participate in the therapeutic effects of MSCs by mediating intercellular microcommunication and transporting paracrine factors. MSC-derived exosomes have produced good results in a variety of liver disease models. Compared with MSCs, exosomes have many advantages: they are smaller than cells and have simpler components, so they are easier to produce and store. Therefore, MSC-derived exosomes may become an ideal tool for liver diseases in the near future. However, the low yield of exosomes limits their clinical use.

Summary of the invention

In view of the shortcomings of the existing methods, the present application proposes an application of hepatocytes cultured under hypoxic conditions using cordycepin in the preparation of drugs for treating liver damage, so as to solve the technical problem that mesenchymal stem cells and their exosomes are difficult to be clinically used in the treatment of liver damage in related technologies.

The present application provides an application of hepatocytes cultured under hypoxic conditions using cordycepin in the preparation of a drug for treating liver damage, wherein the culture medium of the hepatocytes comprises a basal culture medium, 5-15 μmol/L cordycepin, 0.5-4 μg/mL of mesenchymal stem cell conditioned exosomes, the culture conditions are an oxygen concentration of 1% to 6%, a carbon dioxide concentration of 5%, and the remaining gas is nitrogen, and the culture time is 24-36 hours.

Alternatively, the method for preparing mesenchymal stem cell conditioned exosomes comprises the following steps:

Use a special medium for mesenchymal stem cells to culture the mesenchymal stem cells until the confluence reaches 75-85%;

The mesenchymal stem cells were cultured in a three-dimensional hypoxic environment and the culture medium was replaced with a hepatocyte culture medium;

The mesenchymal stem cell conditioned exosomes are separated and obtained by centrifugation.

Furthermore, the mesenchymal stem cells are mesenchymal stem cells cultured in vitro for 2 to 8 generations.

Further optionally, culturing the mesenchymal hepatocytes in a three-dimensional mode comprises inoculating the mesenchymal hepatocytes into a three-dimensional culture mold for culturing.

Optionally, the hepatocyte culture medium comprises growth factors, wherein the growth factors include FGF-2 at a concentration of 5-15 ng/mL, SDF-1 at a concentration of 5-15 ng/mL, and IGF-1 at a concentration of 1-4 ng/mL.

Furthermore, the separation of the mesenchymal stem cell conditioned exosomes by centrifugation comprises the following steps:

Performing a first centrifugation to collect the cell supernatant, wherein the conditions of the first centrifugation include: speed 250-350g, time 10-15min;

The cell supernatant collected by the first centrifugation is subjected to a second centrifugation to collect the supernatant, and the conditions of the second centrifugation include: speed 2000-3000g, time 10-15min;

The cell supernatant collected by the second centrifugation is subjected to a third centrifugation to collect the supernatant, wherein the conditions of the third centrifugation include 10000g-12000g, and the time is 30-35min;

The cell supernatant collected by the third centrifugation is filtered and then the precipitate is collected by the fourth centrifugation to obtain the mesenchymal stem cell conditioned exosomes; the filter membrane used for filtration has a pore size of 0.22-0.45 μm; the conditions of the fourth centrifugation include 10000g-12000g, time 2-2.5h.

Optionally, the basal culture medium comprises DMEM medium.

Furthermore, the liver damage includes damage caused by liver ischemia-reperfusion, or liver hypoxia damage caused by a hypoxic environment.

Alternatively, the drug for treating liver damage comprises the culture of hepatocytes cultured under hypoxic conditions using cordycepin for co-culture with primary hepatocytes.

Furthermore, the drug for treating liver damage is used to prevent liver cell damage, maintain liver cell activity and/or reduce the level of liver cell apoptosis.

The beneficial technical effects brought about by the technical solution provided by the embodiments of the present application include:

(1) The application of the present application uses hepatocytes cultured under hypoxic conditions. The hepatocytes can be stably obtained by improving the culture medium and the culture conditions, thereby increasing the possibility of clinical application.

(2) The mesenchymal stem cell conditioned exosomes used to prepare the culture medium in the application of the present application are stable and reliable, and the enrichment degree of the exosomes is high, which is conducive to clinical application.

(3) The culture medium used in the present application includes cordycepin, which is nutritious and non-toxic, and can provide sufficient nutrition for hepatocytes cultured under hypoxic conditions and maintain the physiological state of hepatocytes under hypoxic conditions.

(4) The application of this application uses cordycepin to co-culture hepatocytes cultured under hypoxic conditions with primary hepatocytes to achieve protection of primary hepatocytes, which has the prospect of cell therapy for the liver.

Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

FIG1 is a cell diagram of umbilical cord mesenchymal stem cells at a culture generation of P4 provided in an embodiment of the present application;

FIG2 is a diagram of Oil Red O staining results provided in an embodiment of the present application;

FIG3 is a diagram showing the results of alizarin red staining provided in an embodiment of the present application;

FIG4 is a diagram showing the 3D mold ball forming result provided in an embodiment of the present application;

FIG5 is an electron micrograph of exosomes provided in an embodiment of the present application;

FIG6 is a diagram of the particle size of exosomes provided in an embodiment of the present application;

FIG. 7 is a cell of mouse primary hepatocytes cultured at generation P0 provided in an embodiment of the present application;

FIG8 is a graph showing the cell activity results of the normal human liver cell line L-02 provided in Experimental Example 1 of the present application;

FIG9 is a graph showing the cell activity results of primary normal mouse liver cells provided in Experimental Example 1 of the present application;

FIG10 is a diagram showing the results of TUNEL staining of normal human liver cell line L-02 cells provided in Experimental Example 2 of the present application;

FIG. 11 is a diagram showing the results of TUNEL staining of primary normal mouse liver cells provided in Experimental Example 2 of the present application.

DETAILED DESCRIPTION

The embodiments of the present application are described below in conjunction with the drawings in the present application. It should be understood that the implementation methods described below in conjunction with the drawings are exemplary descriptions for explaining the technical solutions of the embodiments of the present application and do not constitute a limitation on the technical solutions of the embodiments of the present application.

Those skilled in the art will appreciate that, unless expressly stated, the "said" and "the" used herein may also include plural forms. It should be further understood that the term "including" used in the specification of the present application refers to the presence of the features, integers, steps, operations, but does not exclude the implementation of other features, information, data, steps, operations, elements, components and/or combinations thereof supported by the technical field. The term "and/or" used herein refers to at least one of the items defined by the term, for example, "A and/or B" may be implemented as "A", or as "B", or as "A and B".

In order to make the objectives, technical solutions and advantages of the present application more clear, the implementation methods of the present application will be further described in detail below with reference to the accompanying drawings.

The present application provides an application of hepatocytes cultured under hypoxic conditions using cordycepin in the preparation of a drug for treating liver damage. Optionally, the hepatocytes use a specified culture medium and form a culture under hypoxic conditions for the treatment of liver damage.

Specifically, the method for preparing the specified culture solution comprises:

S1. Preparation of mesenchymal stem cell exosomes;

The mesenchymal stem cells of any generation from 2 to 8 are cultured using a stem cell-specific culture medium, wherein the stem cell-specific culture medium used at this time is a conventional stem cell-specific culture medium, and the culture conditions at this time are conventional culture conditions known in the art.

Among them, mesenchymal stem cells include umbilical cord mesenchymal stem cells.

After the mesenchymal stem cells are cultured into spheres using a 3D mold, the mesenchymal stem cell-specific culture medium is replaced with a hepatocyte culture medium, and a growth factor mixture is added and then hypoxic culture is performed, wherein the growth factor mixture includes FGF-2 at a concentration of 5-15 ng/mL, SDF-1 at a concentration of 5-15 ng/mL, and IGF-1 at a concentration of 1-4 ng/mL, preferably FGF-2 at a concentration of 10 ng/mL, SDF-1 at a concentration of 10 ng/mL, and IGF-1 at a concentration of 2 ng/mL, and then the obtained mesenchymal stem cell conditioned exosomes are separated by ultracentrifugation. The hepatocyte culture medium used here is a conventional hepatocyte culture medium, such as DMEM.

Furthermore, the conditions for hypoxic culture include an oxygen concentration lower than 6%, preferably 1%, and a culture time of 48 hours; specifically, an oxygen concentration lower than 6%, a carbon dioxide concentration of 5%, and the remaining gases are supplemented with nitrogen.

Furthermore, after the hypoxic culture is completed, the culture system is subjected to ultracentrifugation to separate exosomes, namely, mesenchymal stem cell conditional exosomes. Specifically, after the hypoxic culture is completed, the cell supernatant is collected by the first centrifugation, and then the cell supernatant is collected by the second centrifugation, and the supernatant is collected by the third centrifugation, and then filtered, and finally the fourth centrifugation is performed, the supernatant is discarded, and the exosomes are collected. Wherein, the conditions for the first centrifugation include: speed 250-300g, for example, any value between 250-300g such as 250g, 260g, 270g, 280g, 290g and 300g, and time 10-15min, for example, any value between 10min, 11min, 12min, 13min, 14min and 15min; the conditions for the second centrifugation include: speed 2000-3000g, for example, 2000g, 2100g, 2150g, 2300g, 2450g, 2500g, 2600g, 2750g, 2850g, 2900g and 3000g. g, time 10-15 min, for example, any value between 10 min, 11 min, 12 min, 13 min, 14 min and 15 min; the conditions for the third centrifugation include: speed 10000-12000 g, for example, any value between 10000 g, 10250 g, 10500 g, 10750 g, 11000 g, 11025 g, 11050 g, 11075 g, and 12000 g, time 30-35 min, for example, any value between 30 min, 31 min, 32 min, 33 min, 34 min and 35 min; the filter membrane used in filtration is a filter membrane with a pore size of 0.22-0.45 μm, preferably a filter membrane of 0.22 μm. The conditions for the fourth centrifugation include: a speed of 10000-12000 g, for example, 10000 g, 10250 g, 10500 g, 10750 g, 11000 g, 11025 g, 11050 g, 11075 g, and 12000 g, and a time of 2-2.5 h, for example, any value between 2-2.5 such as 2 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, and 2.5 h.

The mesenchymal stem cell conditioned exosomes obtained after discarding the supernatant were stored at -80°C and taken out when used.

The formed mesenchymal stem cell conditioned exosomes contain rich nutrients, such as growth factors and other cytokines, which not only retain the function of MSCs, but also have the advantages of precise and efficient delivery methods and easy storage and regulation, which can prevent hypoxic damage to liver cells. At the same time, it is easy to obtain, making mesenchymal stem cell conditioned medium a potential treatment option for liver damage, with great application prospects.

S2, forming a culture solution;

The mesenchymal stem cell conditioned exosomes prepared above are mixed with the basal culture medium of hepatocytes, and the concentration of the exosomes in the culture medium is 0.5-4 μg/mL, for example, 0.5 μg/mL, 1 μg/mL, 1.5 μg/mL, 2 μg/mL, 2.5 μg/mL, 3 μg/mL, 3.5 μg/mL and 4 μg/mL, etc., any value between 0.5-4 μg/mL.

Then, cordycepin is added to the mixed culture medium formed by mixing the above-mentioned mesenchymal stem cell exosomes and the hepatocyte culture medium to form a culture solution, and the concentration of cordycepin in the culture solution is 5-10 μmol/L, for example, 5 μmol/L, 6 μmol/L, 7 μmol/L, 8 μmol/L, 9 μmol/L, 10 μmol/L, 11 μmol/L, 12 μmol/L, 13 μmol/L, 14 μmol/L and 15 μmol/L, etc., any value between 5-15 μmol/L.

By using hepatocyte culture medium, mesenchymal stem cell conditioned exosomes and cordycepin, and limiting their content, the oxidative stress response of hepatocytes under hypoxic conditions can be reduced. At the same time, hypoxic damage to hepatocytes can be prevented, and the cell survival rate of hepatocytes under hypoxic conditions can be significantly improved, thereby treating liver damage, especially liver damage caused by hypoxia.

The hepatocyte basal culture medium used herein may be a hepatocyte culture medium in the prior art, such as DMEM culture medium.

Therefore, the culture medium for hypoxic culture of hepatocytes provided in an embodiment of the present application includes a hepatocyte culture medium, mesenchymal stem cell conditioned exosomes and cordycepin, wherein the concentration of the mesenchymal stem cell exosomes in the culture medium is 0.5-4 μg/mL, and the concentration of the cordycepin in the culture medium is 5-15 μmol/L.

Mesenchymal stem cell conditioned exosomes are exosomes separated by ultracentrifugation after mesenchymal stem cells are cultured (e.g., hypoxic culture). The hepatocyte culture medium is DMEM culture medium.

Furthermore, the effect of the drug in treating liver damage was investigated using hepatocytes cultured under hypoxic conditions.

The features and performance of the present application are further described in detail below in conjunction with the embodiments.

1. Isolation and culture of mesenchymal stem cells

Umbilical cord mesenchymal stem cells were isolated by direct tissue block adhesion method: the blood in the umbilical cord was rinsed with PBS several times under a sterile operating table, and the umbilical cord was cut into pieces of 1-2 cm in length. The blood vessels (one vein and two arteries) and the outer amniotic membrane of the umbilical cord were peeled off with tissue scissors and vascular forceps to prevent endothelial cell contamination. The Wharton's jelly in the umbilical cord was cut into small pieces with a volume of 0.5-1 cm ³ , and the cut small pieces of umbilical cord were evenly spread on the bottom of a 10 cm culture dish, with an interval of 0.5 cm. The umbilical cord was covered with 10 mL of mesenchymal stem cell-specific culture medium containing 0.1% penicillin-streptomycin double antibody and 10% FBS, and the culture dish was placed in a 5% CO ₂ , 37°C incubator for culture. The edge of the umbilical cord was observed under an inverted microscope every day for cells to swim out, and the medium was changed every 4 days. When a large number of cells swam out around the umbilical cord block, the umbilical cord block was removed and continued to be cultured.

Umbilical cord mesenchymal stem cells were passaged to 2-8 generations for preparing the mesenchymal stem cell conditional exosomes (denoted as: MSC-CM-exo. After passage, the cell growth accelerated, the cells were arranged regularly, mainly in a spindle shape, and grew evenly, as shown in Figure 1, which shows umbilical cord mesenchymal stem cells at the culture generation of P4.

2. Differentiation of mesenchymal stem cells into adipocytes

The above-mentioned umbilical cord mesenchymal stem cells were seeded in a 6-well plate at 1×10 ⁵ cells/well, and adipogenic induction medium was added. The adipogenic induction medium consisted of low-glucose DMEM containing 10% FBS, 2mM IBMX and 5μg/mL insulin. The adipogenic induction medium was replaced every 3-4 days. The induction time was 3W, and Oil Red O staining was used to observe whether fat droplets were produced.

See Figure 2 . In Figure 2 , fat droplets are produced in umbilical cord mesenchymal stem cells of generation P4 (left side of Figure 2 ) and P6 (right side of Figure 2 ) after 3 weeks of adipogenic induction, indicating that umbilical cord mesenchymal stem cells have the potential to differentiate into adipocytes.

3. Differentiation of mesenchymal stem cells into osteocytes

Umbilical cord mesenchymal stem cells were seeded in a 6-well plate at 1×10 ⁵ cells/well, and chondrogenic induction medium was added. The chondrogenic induction medium was A100701 StemPro Chondro DIFF kit from Gibco. The chondrogenic induction medium was replaced every 3-4 days. The induction time was 3 weeks. After induction, Alizarin red staining was used to determine whether osteoblasts were produced.

Referring to FIG. 3 , in FIG. 3 , both P4 (left side of FIG. 3 ) and P6 (right side of FIG. 3 ) generation umbilical cord mesenchymal stem cells were positively stained with Alizarin Red after 3 weeks of osteogenic induction, indicating that umbilical cord mesenchymal stem cells have the potential to differentiate into osteoblasts.

FIG2 and FIG3 show that umbilical cord mesenchymal stem cells have multidirectional differentiation potential, and further prove that umbilical cord mesenchymal stem cells are prepared in the examples of the present application.

4. Preparation of mesenchymal stem cell conditioned exosomes (MSC-CM-exo)

The P4 generation umbilical cord mesenchymal stem cells were cultured with stem cell-specific medium until the confluence reached 80%, the stem cell-specific medium was discarded, the cells were washed twice with PBS, 2 ml of trypsin was added for digestion, and 4 ml of serum-free protease stop solution was added to terminate the digestion after the cell outline shrank. After centrifugation, the MSC cells were planted on the 3D mold, as shown in Figure 4. After the MSCs were spherical in the 3D mold, the stem cell-specific medium was replaced with an equal volume of hepatocyte medium (DMEM), and a growth factor mixture (10 ng/mL FGF-2, 10 ng/mL SDF-1 and 2 ng/mL IGF-1) was added. Then, the umbilical cord mesenchymal stem cells were cultured for 36 hours in a hypoxic incubator (1% O ₂ ), the gas composition of the hypoxic incubator was 1% O ₂ , 5% CO ₂ , and the remaining gas was supplemented by N ₂ . After culturing for 36 hours, the cell culture system was added to the centrifuge tube and the centrifuge speed was adjusted to 300g for 10 minutes, and then the cell supernatant was collected. Then, the centrifuge speed was adjusted to 3000g for 10 minutes, and then the centrifuge speed was adjusted to 10000g for 30 minutes; then, the supernatant was filtered through a 0.22μm filter membrane, and finally the centrifuge speed was adjusted to 10000g for 2 hours, and the supernatant was discarded to obtain mesenchymal stem cell conditional exosomes (MSC-CM-exo). Figure 5 is an electron micrograph of the extracted exosomes, and Figure 6 is a particle size distribution diagram of the extracted exosomes. Among them, the mesenchymal stem cell conditional exosomes (MSC-CM-exo) can be stored at -80°C and thawed when needed.

5. Preparation of culture medium for hypoxic culture of hepatocytes

The mesenchymal stem cell conditioned exosomes (MSC-CM-exo) prepared above were mixed with hepatocyte culture medium (DMEM) at a final concentration of 2 μg/mL, and then cordycepin was added at a final concentration of 10 μmol/L.

6. Extraction of primary mouse hepatocytes

The two-step perfusion method was used to isolate primary mouse hepatocytes: perfusion fluid I (0.5mmol/L EGTA) and perfusion fluid II (0.04g/mL type IV collagenase) were perfused from the portal vein of the mouse, and the perfusion was terminated when the liver became light yellow and soft. The liver was cut off and transferred to the clean bench. After the surface was washed with 4℃ DMEM, the liver capsule was torn off, and after the cells flowed out, they were poured into a 200-mesh cell sieve for filtration. After filtration, high-glucose DMEM medium was added and centrifuged twice, and then DMEM complete medium was added and placed in an incubator. The medium was changed for 4 hours, and then cultured. Figure 7 is a bright field image of primary hepatocytes of P0 mice. It can be seen that most cells have typical hepatocyte morphology, the cells are polygonal, stretched out, with clear boundaries, transparent cell bodies, round nuclei, and most cells are binuclear or multinuclear.

7. Hepatocyte hypoxia injury model

The hepatocyte hypoxia model is to cause hepatocyte damage by placing hepatocytes in a hypoxic (1% O ₂ ) incubator. In this experiment, L-02 cells and mouse primary hepatocytes were first cultured in a normoxic incubator (20% O ₂ ) with complete culture medium (DMEM+10% FBS) until the cell confluence reached 70%, then the complete culture medium was discarded, PBS was washed twice, and the culture medium was replaced with hepatocyte culture medium (DMEM). The cells were placed in the hypoxic incubator and cultured for 36 hours. As the oxygen concentration dropped from 20% to 1%, the mitochondrial respiration in the cells was limited by the oxygen supply, and the ATP level decreased. Since the cellular processes necessary to maintain the balance of the intracellular environment could not be maintained, hypoxic cell damage would occur.

Experimental Example 1

Cell CCK-8 detection

Cell viability was detected by CCK-8 kit. Changes in cell activity can reflect the damage of liver cells under hypoxic environment.

Methods: L-02 cells and mouse primary hepatocytes were seeded in a 96-well plate at a density of 5000 cells/well. 100 μL of complete medium (DMEM+10% FBS) was added and cultured in a normoxic incubator (20%) for 24 h. The complete medium was then discarded and the plates were washed twice with PBS.

The cells were divided into the following groups: a normoxic cell culture control group (the first column from left to right in Figures 8 and 9 ), a hypoxic cell culture control group (the second column from left to right in Figures 8 and 9 ), a hypoxic cell culture + MSC-CM-exo experimental group (the third column from left to right in Figures 8 and 9 ), and a hypoxic cell culture + hepatocyte hypoxic culture medium experimental group (the fourth column from left to right in Figures 8 and 9 ). Among them, the normoxic culture control group refers to the cells added with 100 μL of hepatocyte culture medium (DMEM) and cultured in a normoxic incubator, the cell hypoxia culture control group refers to the cells added with 100 μL of hepatocyte culture medium (DMEM) and cultured in a hypoxic incubator, the cell hypoxia culture + MSC-CM-exo experimental group refers to the cells added with 100 μL of hepatocyte culture medium (DMEM) containing stem cell conditioned exosomes (MSC-CM-exo) with a final concentration of 2 μg/mL and cultured in a hypoxic incubator, and the cell hypoxia culture + hepatocyte hypoxia culture fluid experimental group refers to the cells added with hepatocyte hypoxia culture fluid and cultured in a hypoxic incubator.

After the cells were treated with normoxia (oxygen concentration of 20%) and hypoxia (oxygen concentration of 1%) for 36 hours, the culture medium was discarded, 10 μL of CCK8 solution was added to each well, incubated at 37°C for 2 hours, and the absorbance at 450 nm was measured using a microplate reader.

The results are shown in Figures 8 and 9. Figure 8 is a graph showing the cell activity of L-02 cells. It can be seen from Figure 8 that compared with the L-02 cell hypoxia group, the stem cell conditioned exosome (MSC-CM-exo) group can significantly improve the cell activity of L-02 cells, and the hepatocyte hypoxia culture fluid can prevent L-02 cell hypoxia damage to a higher degree than the stem cell conditioned exosome (MSC-CM-exo) group. Figure 9 is a graph showing the cell activity of primary mouse hepatocytes. It can be seen from Figure 9 that compared with the primary mouse hepatocyte hypoxia group, the stem cell conditioned exosome (MSC-CM-exo) group can significantly improve the cell activity of primary mouse hepatocytes, and the hepatocyte hypoxia culture fluid can prevent primary mouse cells hypoxia damage to a higher degree than the stem cell conditioned exosome (MSC-CM-exo) group.

Experimental Example 2

Cell TUNEL (TdT-mediated dUTP nick end labeling) staining

Cell apoptosis was stained by TUNEL staining kit. TUNEL staining can reflect the apoptosis of cells under hypoxia, thereby reflecting the damage of cells.

Methods: L-02 cells and mouse primary hepatocytes were seeded in 6-well plates at a density of 2×10 ⁵ cells/well. 2 mL of complete medium (DMEM+10% FBS) was added and cultured in a normoxic incubator (20%) for 24 h. The complete medium was then discarded and the plates were washed twice with PBS.

The cells were divided into the following groups: a cell normoxic culture control group (the first picture from left to right in Figures 10 and 11), a cell hypoxic culture control group (the second picture from left to right in Figures 10 and 11), a cell hypoxic culture + MSC-CM-exo experimental group (the third picture from left to right in Figures 10 and 11), and a cell hypoxic culture + hepatocyte hypoxic culture fluid experimental group (the fourth picture from left to right in Figures 10 and 11). The groups were consistent with the CCK-8 experimental grouping, except that the amount of culture fluid added was increased from 100 μL to 2 mL. After the cells were treated with normoxic and hypoxic treatment for 48 hours, they were stained according to the kit operation, and after staining, they were observed under a fluorescence microscope with an excitation wavelength range of 450-500 nm and an emission wavelength range of 515-565 nm (green fluorescence), where blue was the nucleus and green was the apoptotic cell.

The results are shown in Figures 10 and 11. Figure 10 is a cell TUNEL staining result of L-02 cells. It can be seen from Figure 10 that compared with the L-02 cell hypoxia group, the stem cell conditioned exosome (MSC-CM-exo) group can significantly reduce the apoptosis of L-02 cells, and the hepatocyte hypoxia culture medium can reduce the apoptosis level of L-02 cells to a higher degree than the stem cell conditioned exosome (MSC-CM-exo) group. Figure 11 is a cell TUNEL staining result of mouse primary hepatocytes. It can be seen from Figure 11 that compared with the mouse primary hepatocyte hypoxia group, the stem cell conditioned medium (MSC-CM-exo) group can significantly reduce the apoptosis of mouse primary hepatocytes, and the hepatocyte hypoxia culture medium can reduce the apoptosis level of mouse primary hepatocytes to a higher degree than the stem cell conditioned exosome (MSC-CM-exo) group.

The results of Figures 8 to 11 indicate that the culture medium for hypoxic culture of hepatocytes provided in the embodiments of the present application can effectively prevent cell damage of hepatocytes under hypoxic conditions and maintain cell activity, and can be used as a therapeutic drug for hypoxic injury of hepatocytes, for treating liver damage caused by liver ischemia-reperfusion, or liver hypoxic injury caused by a hypoxic environment.

In summary, the present application is an application of hepatocytes cultured under hypoxic conditions using cordycepin in the preparation of a drug for treating liver damage, wherein the culture fluid of the hepatocytes includes a basal culture medium, 5-15 μmol/L cordycepin, 0.5-4 μg/mL of mesenchymal stem cell conditioned exosomes, the culture conditions are an oxygen concentration of 1% to 6%, a carbon dioxide concentration of 5%, and the remaining gas is nitrogen, and the culture time is 24-36 hours. The application of this application uses hepatocytes cultured under hypoxic conditions, which can be stably obtained through the improvement of the culture fluid and the improvement of the culture conditions, thereby increasing the possibility of clinical application.

Those skilled in the art will appreciate that the various operations, methods, steps, measures, and schemes in the processes discussed in this application may be alternated, changed, combined, or deleted. Furthermore, other steps, measures, and schemes in the various operations, methods, and processes discussed in this application may also be alternated, changed, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and schemes in the related art that are similar to those disclosed in this application may also be alternated, changed, rearranged, decomposed, combined, or deleted.

The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, "plurality" means two or more.

In the description of this specification, specific features, structures, materials or characteristics may be combined in an appropriate manner in any one or more embodiments or examples.

The above is only a partial implementation method of the present application. It should be pointed out that for ordinary technicians in this technical field, without departing from the technical concept of the scheme of the present application, other similar implementation methods based on the technical ideas of the present application also fall within the protection scope of the embodiments of the present application.

Claims (10)

1. An application of hepatocytes cultured under hypoxic conditions using cordycepin in the preparation of a drug for treating liver damage, characterized in that the culture fluid of the hepatocytes comprises a basal culture medium, 5-15 μmol/L cordycepin, 0.5-4 μg/mL of mesenchymal stem cell conditioned exosomes, the culture conditions are an oxygen concentration of 1% to 6%, a carbon dioxide concentration of 5%, and the remaining gas is nitrogen, and the culture time is 24-36 hours. 2. The use according to claim 1, characterized in that the method for preparing the mesenchymal stem cell conditioned exosomes comprises the following steps: Use a special medium for mesenchymal stem cells to culture the mesenchymal stem cells until the confluence reaches 75-85%; The mesenchymal stem cells were cultured in a three-dimensional hypoxic environment and the culture medium was replaced with a hepatocyte culture medium; The mesenchymal stem cell conditioned exosomes are separated and obtained by centrifugation. 3. The use according to claim 2, characterized in that the mesenchymal stem cells are mesenchymal stem cells cultured in vitro for 2 to 8 generations. 4. The use according to claim 2, characterized in that culturing the mesenchymal hepatocytes in a three-dimensional mode comprises inoculating the mesenchymal hepatocytes into a three-dimensional culture mold for culturing. 5. The use according to claim 2, characterized in that the hepatocyte culture medium comprises growth factors, and the growth factors include FGF-2 with a concentration of 5-15 ng/mL, SDF-1 with a concentration of 5-15 ng/mL, and IGF-1 with a concentration of 1-4 ng/mL. 6. The use according to claim 2, characterized in that the separation of the mesenchymal stem cell conditioned exosomes by centrifugation comprises the following steps: Performing a first centrifugation to collect the cell supernatant, wherein the conditions of the first centrifugation include: speed 250-350g, time 10-15min; The cell supernatant collected by the first centrifugation is subjected to a second centrifugation to collect the supernatant, and the conditions of the second centrifugation include: speed 2000-3000g, time 10-15min; The cell supernatant collected by the second centrifugation is subjected to a third centrifugation to collect the supernatant, wherein the conditions of the third centrifugation include 10000g-12000g, and the time is 30-35min; The cell supernatant collected by the third centrifugation is filtered and then the precipitate is collected by the fourth centrifugation to obtain the mesenchymal stem cell conditioned exosomes; the filter membrane used for filtration has a pore size of 0.22-0.45 μm; the conditions of the fourth centrifugation include 10000g-12000g, time 2-2.5h. 7. The use according to claim 1, characterized in that the basal culture medium comprises DMEM culture medium. 8. The use according to claim 1, characterized in that the liver damage includes damage caused by liver ischemia-reperfusion, or liver hypoxia damage caused by a hypoxic environment. 9. The use according to claim 1, characterized in that the drug for treating liver damage comprises a culture of hepatocytes cultured under hypoxic conditions using cordycepin, which is used for co-culture with primary hepatocytes. 10. The use according to claim 1, characterized in that the drug for treating liver damage is used to prevent liver cell damage, maintain liver cell activity and/or reduce the level of liver cell apoptosis.