CN113817667A - Culture solution for hepatocyte hypoxia culture, preparation method and application thereof - Google Patents
Culture solution for hepatocyte hypoxia culture, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of hepatocyte hypoxia culture, in particular to a culture solution for hepatocyte hypoxia culture, a preparation method and application thereof. The culture solution for the hypoxic culture of the liver cells comprises a liver cell culture medium, resveratrol and a mesenchymal stem cell conditioned medium, wherein the volume ratio of the mesenchymal stem cell conditioned medium to the liver cell culture medium is 1:1-2, and the concentration of the resveratrol in the culture solution is 10-20 mu mol/L. The culture solution can reduce hypoxia injury of liver cells, has good protection effect on liver cells, and can be used for treating liver injury, especially liver injury caused by anoxia.
Description
Technical Field
The invention relates to the technical field of hepatocyte hypoxia culture, in particular to a culture solution for hepatocyte hypoxia culture, a preparation method and application thereof.
Background
Liver is an important metabolism and detoxification organ of human body, however, various factors can cause the hypoxic damage of liver cells, which is commonly seen in liver ischemia reperfusion in liver surgery and clinical liver transplantation. Ischemia reperfusion injury largely affects the success or failure of surgery and liver transplantation. Therefore, how to effectively prevent the liver cells from generating hypoxia injury is a problem to be solved urgently in clinic.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a culture solution for low-oxygen culture of liver cells, a preparation method and application thereof. The culture solution provided by the embodiment of the invention can reduce hypoxia injury of liver cells, has good protection effect on the liver cells, and can further treat the liver injury, particularly the liver injury caused by hypoxia.
The invention is realized by the following steps:
in a first aspect, the invention provides a culture solution for hepatocyte hypoxia culture, which comprises a hepatocyte culture medium, resveratrol and a mesenchymal stem cell conditioned medium, wherein the volume ratio of the mesenchymal stem cell conditioned medium to the hepatocyte culture medium is 1:1-2, and the concentration of the resveratrol in the culture solution is 10-20 μmol/L.
In alternative embodiments, the mesenchymal stem cell conditioned medium is a cell-free supernatant isolated from mesenchymal stem cells after culturing;
preferably, the mesenchymal stem cell conditioned medium is cell-free supernatant separated after the mesenchymal stem cells are subjected to hypoxic culture;
preferably, the oxygen concentration in the hypoxic culture is less than 6%, preferably 1%.
In alternative embodiments, the mesenchymal stem cells comprise umbilical cord mesenchymal stem cells.
In alternative embodiments, the hepatocyte culture medium is DMEM medium.
In a second aspect, the present invention provides a method for preparing a culture solution for hypoxic culture of hepatocytes, according to the foregoing embodiment, including: mixing the hepatocyte culture medium, resveratrol and mesenchymal stem cell conditioned medium.
In alternative embodiments, the preparation of the mesenchymal stem cell conditioned medium comprises: culturing mesenchymal stem cells by using a special culture medium for stem cells, replacing the special culture medium for stem cells with a hepatocyte culture medium when the fusion degree of the cultured mesenchymal stem cells reaches 75-85%, then carrying out hypoxia culture, and then separating to obtain cell-free supernatant.
In alternative embodiments, the conditions of the hypoxic culture include: the oxygen concentration is lower than 6%, preferably 1%, and the culture time is 36-48 hours;
preferably, the mesenchymal stem cell is a mesenchymal stem cell of any of 2-10 generations.
In an alternative embodiment, the step of separating comprises: after the hypoxic culture was completed, the cell supernatant was collected by a first centrifugation, and then the cell supernatant was subjected to a second centrifugation, followed by filtration.
In an alternative embodiment, the conditions of the first centrifugation include: the speed is 250-350g, and the time is 10-15 minutes;
conditions for the second centrifugation included: the speed is 2000-3000g, and the time is 10-15 minutes;
the filter membrane used in the filtration is a filter membrane with the pore diameter of 0.22-0.45 μm.
In a third aspect, the present invention provides a method for culturing a hepatocyte in a hypoxic condition, comprising: culturing the hepatocytes by using the culture solution for the hypoxic culture of the hepatocytes according to the foregoing embodiment;
preferably, hypoxic culture is performed.
In a fourth aspect, the present invention provides a use of the culture solution for hypoxic culture of hepatocytes according to the foregoing embodiment in the preparation of a medicament for treating liver injury;
preferably, the liver injury comprises hepatic ischemia reperfusion;
preferably, the liver injury comprises hypoxic injury to the liver caused by a hypoxic environment.
The invention has the following beneficial effects: according to the culture solution provided by the embodiment of the invention, rich nutrients in the hepatocyte culture medium and the mesenchymal stem cell conditioned medium are matched with resveratrol, so that the oxidative stress of hepatocytes under the anoxic condition is relieved, meanwhile, the hypoxia injury of the hepatocytes is prevented, the good protection effect on the hepatocytes is achieved, the hepatic injury can be treated, and particularly, the hepatic injury caused by the hypoxia is treated.
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 cell diagram of umbilical cord mesenchymal stem cells with culture passage number P4, provided in example 1 of the present invention;
FIG. 2 is a graph showing the results of Oil Red O staining provided in example 1 of the present invention;
FIG. 3 is a graph showing alizarin red staining results provided in example 1 of the present invention;
FIG. 4 is a graph showing the results of the cell activities of the HepaRG cells provided in Experimental example 1 of the present invention;
FIG. 5 is a graph showing the results of the cell activity of L-02 cells provided in Experimental example 1 of the present invention;
FIG. 6 is a graph showing the cell TUNEL staining results of Hepall cells according to Experimental example 2 of the present invention;
FIG. 7 is a graph showing the TUNEL staining results of L-02 cells according to Experimental example 2 of the present invention.
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 invention provides a preparation method of a culture solution for low-oxygen culture of liver cells, which comprises the following steps:
s1, preparing a mesenchymal stem cell conditioned medium;
and culturing the mesenchymal stem cells of any generation in 2-10 generations by using the special culture medium for the stem cells. The medium for exclusive use of stem cells used at this time is a conventional medium for exclusive use of stem cells, and the culture conditions at this time are conventionally known conventional culture conditions.
Wherein the mesenchymal stem cells comprise umbilical cord mesenchymal stem cells.
And after culturing until the fusion degree of the mesenchymal stem cells reaches 75-85%, replacing the special culture medium for the stem cells with a hepatocyte culture medium, and then performing hypoxia culture. The hepatocyte culture medium used herein is a conventional hepatocyte culture medium, such as DMEM.
Further, the conditions of the hypoxic culture include an oxygen concentration of less than 6%, preferably 1%, and a culture time of 48 hours; specifically, the oxygen concentration is less than 6%, the carbon dioxide concentration is 5%, and the rest of the gas is supplemented with nitrogen.
Further, after the hypoxia culture is finished, separating the culture system to obtain cell-free supernatant, namely the mesenchymal stem cell conditioned medium. Specifically, after the completion of the hypoxic culture, a first centrifugation is performed to collect the cell supernatant, and then a second centrifugation is performed on the cell supernatant, followed by filtration. Wherein the conditions of the first centrifugation comprise: a speed of 250-300g, e.g., any value between 250-300g such as 250g, 260 g, 270 g, 280 g, 290 g, and 300g, for a time of 10-15 minutes, e.g., any value between 10-15 minutes such as 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, and 15 minutes; conditions for the second centrifugation included: a speed of 2000-3000g, e.g., 2000 g, 2100 g, 2150 g, 2300 g, 2450 g, 2500 g, 2600 g, 2750 g, 2850 g, 2900 g, 3000g, and any value between 2000-3000g, a time of 10-15 minutes, e.g., 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, and 15 minutes, and any value between 10-15 minutes; the filter membrane used in the filtration is a filter membrane with a pore size of 0.22-0.45 μm, preferably 0.22 μm.
And (4) storing the mesenchymal stem cell conditioned medium obtained after filtration at-80 ℃ and taking out when in use.
The formed mesenchymal stem cell conditioned medium contains abundant nutrient components, such as: growth factors and other cytokines and the like, not only retain the function of the MSCs, but also have the advantages of accurate and efficient transmission mode and easy storage and regulation, and further can prevent the hypoxic damage of the liver cells. Meanwhile, the mesenchymal stem cell conditioned medium is easy to obtain, becomes a potential treatment option for liver injury, and has great application prospect.
S2, forming a culture solution;
mixing the prepared mesenchymal stem cell conditioned medium and the hepatocyte culture medium, wherein the volume ratio of the mesenchymal stem cell conditioned medium to the hepatocyte culture medium is 1:1-2, for example, 1:1, 1: 1.1, 1: 1.2, 1: 1.3, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.7, 1: 1.8, 1: 1.9, 1: 2, etc. in any number between 1:1 and 2.
Then adding the resveratrol into a mixed culture medium formed by mixing the mesenchymal stem cell conditioned medium and the hepatocyte culture medium to form a culture solution, wherein the concentration of the resveratrol in the culture solution is 10-20 mu mol/L, such as any value between 10-20 mu mol/L, such as 10 mu mol/L, 11 mu mol/L, 12 mu mol/L, 13 mu mol/L, 14 mu mol/L, 15 mu mol/L, 16 mu mol/L, 17 mu mol/L, 18 mu mol/L, 19 mu mol/L and 20 mu mol/L.
By adopting the hepatocyte culture medium, the resveratrol and mesenchymal stem cell conditioned medium and limiting the content of the resveratrol and mesenchymal stem cell conditioned medium, the oxidative stress reaction of the hepatocytes under the anoxic condition can be relieved, meanwhile, the hypoxic damage of the hepatocytes is prevented, the cell survival rate of the hepatocytes under the anoxic condition is obviously improved, and then the hepatic damage, especially the hepatic damage caused by the hypoxia can be treated.
The hepatocyte culture medium used therein may be a hepatocyte culture medium of the prior art, such as DMEM medium.
In a second aspect, the embodiment of the invention provides the culture solution for the hypoxic culture of the hepatocyte, which comprises a hepatocyte culture medium, resveratrol and a mesenchymal stem cell conditioned medium, wherein the volume ratio of the mesenchymal stem cell conditioned medium to the hepatocyte culture medium is 1:1-2, and the concentration of the resveratrol in the culture solution is 10-20 μmol/L.
The mesenchymal stem cell conditioned medium is a cell-free supernatant isolated from mesenchymal stem cells after culturing (e.g., hypoxic culturing). The hepatocyte culture medium is DMEM culture medium.
In a third aspect, the present invention provides a method for culturing a hepatocyte in a hypoxic condition, comprising: culturing the hepatocytes by using the culture solution for the hypoxic culture of the hepatocytes according to the foregoing embodiment;
preferably, hypoxic culture is performed.
In a fourth aspect, the present invention provides a use of the culture solution for hypoxic culture of hepatocytes according to the foregoing embodiment in the preparation of a medicament for treating liver injury;
preferably, the liver injury comprises hepatic ischemia reperfusion;
preferably, the liver injury comprises hypoxic injury to the liver caused by a hypoxic environment.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
1. Isolation and culture of mesenchymal stem cells
Separating umbilical cord mesenchymal stem cells by adopting a tissue block direct wall pasting method: the cord was cut into 1-2cm length pieces by rinsing the blood in the cord with PBS several times under a sterile operating table. The vessels of the umbilical cord (one vein and two arteries) and the outer amniotic membrane were dissected using tissue scissors and vascular clamps to prevent endothelial cell contamination. Cutting the Wharton jelly part in the umbilical cord into small pieces with a volume of 0.5-1cm3The cut small umbilical cords are evenly spread on the bottom of a culture dish of 10cm, and the interval is preferably 0.5 cm. Covering the umbilical cord with 10ml of a mesenchymal stem cell special culture medium containing 0.1% streptomycin double antibody and 10% FBS, and placing the culture dish in 5% CO2And cultured in an incubator at 37 ℃. Observing whether cells are effused from the edge of the umbilical cord under an inverted microscope every day, changing the liquid every 4 days until a large number of cells are effused from the periphery of the umbilical cord massWhen the umbilical cord mass is taken out, the culture is continued.
Umbilical cord mesenchymal stem cells were passed on to 4-10 surrogate for preparing the mesenchymal stem cell conditioned medium (noted: MSC-CM). After passage, the growth of the cells is accelerated, the cells are arranged regularly, mainly in a fusiform shape and grow uniformly, and the figure 1 shows that the umbilical cord mesenchymal stem cells with the culture passage number of P4.
2. Differentiation of mesenchymal stem cells into adipocytes
The umbilical cord mesenchymal stem cells are prepared into the form of 1 x 105Each cell/well was inoculated into a 6-well plate, and a fat-inducing medium consisting of low-sugar DMEM containing 10% FBS, 2mM IBMX and 5. mu.g/mL insulin was added thereto, the medium was changed every 3 to 4 days with an induction time limit of 3W, and the presence or absence of fat droplets was observed by Oil Red O staining.
Referring to fig. 2, fat droplets were generated from both the passage P4 (left side of fig. 2) and the passage P6 (right side of fig. 2) of the umbilical cord mesenchymal stem cells in fig. 2 through the adipogenic induction of 3W, indicating the potential of the umbilical cord mesenchymal stem cells to differentiate into adipocytes.
3. Differentiation of mesenchymal stem cells into osteocytes
Umbilical cord mesenchymal stem cells are treated according to 1 x 105Inoculating each cell/well into a 6-well plate, adding a cartilage induction culture medium, wherein the cartilage induction culture medium is an A100701 StemPro Chondro DIFF kit of Gibco company, changing the liquid with the cartilage induction culture medium every 3-4 days, the induction time limit is 3W, and alizarin red staining after induction has no osteoblast generation.
Referring to fig. 3, the umbilical cord mesenchymal stem cells of P4 (left side of fig. 3) and P6 (right side of fig. 3) generation in fig. 3 are positive in alizarin red staining after osteogenic induction of 3W, which indicates the potential of the umbilical cord mesenchymal stem cells to differentiate into osteoblasts.
Fig. 2 and fig. 3 show that the umbilical cord mesenchymal stem cells have a multipotentiality, and then prove that the umbilical cord mesenchymal stem cells are prepared by the embodiment of the invention.
4. Preparation of mesenchymal Stem cell conditioned Medium (MSC-CM)
Culturing umbilical cord mesenchymal stem cells of P4 generation by using a special culture medium for the stem cells until the fusion degree is reached80 percent, absorbing and removing the special culture medium for the stem cells, washing the cells twice by PBS, and replacing the cells with the hepatocyte culture medium (DMEM) with the same volume. Then, umbilical cord mesenchymal stem cells were placed in a hypoxic incubator (1% O)2) Continuing to culture for 48h, wherein the gas composition of the hypoxia culture box is 1% O2，5%CO2The rest gas is composed of N2And (4) supplementing. After culturing for 48h, adding the cell culture system into a centrifuge tube, regulating the rotating speed of the centrifuge to 300g, centrifuging for 10min, collecting cell supernatant, regulating the rotating speed of the centrifuge to 3000g, centrifuging for 10min, and collecting supernatant; filtering the supernatant with a 0.22 mu m filter membrane to obtain a mesenchymal stem cell conditioned medium (MSC-CM), wherein the mesenchymal stem cell conditioned medium (MSC-CM) can be stored at-80 ℃ and is unfrozen for use when in need.
5. Preparation of culture solution for hypoxic culture of liver cells
Mixing the prepared mesenchymal stem cell conditioned medium (MSC-CM) and hepatocyte culture medium (DMEM) according to the volume of 1-2, and adding resveratrol with final concentration of 10 μmol/L.
Model of hepatocyte hypoxia injury
The model of hepatocyte hypoxia is obtained by placing hepatocytes under hypoxia (1% O)2) The incubator thus causes damage to the hepatocytes. The experiment was first performed by placing HepaRG cells and L-O2 cells in an aerobic incubator (20% O) using complete medium (DMEM +10% FBS)2) After the medium culture until the cell confluence reaches 70%, the complete medium is aspirated, washed twice with PBS, and the medium is changed to hepatocyte medium (DMEM). The cells were placed in the hypoxic incubator for a further 48 h. As the oxygen concentration is reduced from 20% to 1%, intracellular mitochondrial respiration is limited by the supply of oxygen, ATP levels are reduced, and hypoxic cell damage results because cellular processes necessary to maintain intracellular homeostasis cannot be maintained.
Experimental example 1
Cellular CCK-8 detection
Cell viability was determined by CCK-8 kit. The change of the cell activity can reflect the damage condition of the liver cells under the hypoxia environment.
The method comprises the following steps: HepaRG cells and L-O2 cells were seeded at a density of 5000 cells/well in a 96-well plate, and after adding 100. mu.L of complete medium (DMEM +10% FBS) to an aerobic incubator (20%) and culturing for 24 hours, the complete medium was aspirated and washed twice with PBS.
Cells were divided into the following groups: a cell normoxic culture control group (the first column from left to right in fig. 4 and 5), a cell hypoxic culture control group (the second column from left to right in fig. 4 and 5), a cell hypoxic culture + MSC-CM experimental group (the third column from left to right in fig. 4 and 5), and a cell hypoxic culture + hepatocyte hypoxic culture broth experimental group (the fourth column from left to right in fig. 4 and 5). The cell hypoxia culture + MSC-CM experimental group is characterized in that 100 mu L of stem cell conditioned medium (MSC-CM) is added into cells to be cultured in a hypoxia culture box, and the cell hypoxia culture + hepatocyte hypoxia culture solution experimental group is characterized in that hepatocyte hypoxia culture solution and the hypoxia culture box are added into the cells to be cultured.
After the cells were treated with normoxic (oxygen concentration of 20%) and hypoxic (oxygen concentration of 1%) for 48 hours, the culture broth was aspirated. 10ul of CCK8 solution was added to each well and incubated at 37 ℃ for 2h before measuring absorbance at 450nm with a microplate reader.
See fig. 4 and 5 for results. FIG. 4 is a graph showing the results of the cell activities of the Hepall cells, and it can be seen from FIG. 4 that the stem cell conditioned medium (MSC-CM) group can significantly improve the cell activities of the Hepall cells compared to the Hepall cell hypoxic group, and the hepatocyte hypoxic medium can prevent the Hepall cells from hypoxic damage to a higher degree compared to the stem cell conditioned medium (MSC-CM). FIG. 5 is a graph showing the results of cell activities of L-02 cells, and it can be seen from FIG. 5 that the cell activities of L-02 cells were significantly improved in the stem cell conditioned medium (MSC-CM) group and that the hypoxic injury of L-02 cells was prevented to a higher degree in the hepatocyte hypoxic culture medium than in the L-02 cell hypoxic group.
Experimental example 2
Cell TUNEL staining
Cells were apoptosis stained by TUNEL staining kit. TUNEL staining may reflect apoptosis in cells under hypoxic conditions, and thus damage to cells.
The method comprises the following steps: HepagG cells and L-O2 cells were treated with 2 x 105The density of individual cells/well was plated in 6-well plates, and after 2mL of complete medium (DMEM +10% FBS) was added and cultured in an normoxic incubator (20%) for 24h, the complete medium was aspirated and washed twice with PBS.
Cells were divided into the following groups: a cell normoxic culture control group (the first graph from left to right in fig. 6 and fig. 7), a cell hypoxic culture control group (the second graph from left to right in fig. 6 and fig. 7), a cell hypoxic culture + MSC-CM experimental group (the third graph from left to right in fig. 6 and fig. 7), and a cell hypoxic culture + hepatocyte hypoxic culture medium experimental group (the fourth graph from left to right in fig. 6 and fig. 7). The groups were grouped in agreement with the CCK-8 experiment, with the amount of broth added only increasing from 100. mu.L to 2 mL. After the cells are treated by normal oxygen and hypoxia for 48 hours, the cells are dyed according to the operation of a kit, and the cells are observed under a fluorescence microscope with the excitation wavelength range of 450-565 nm (green fluorescence), wherein blue is cell nucleus and green is apoptotic cell.
See fig. 6 and 7 for results. FIG. 6 is a graph showing the result of TUNEL staining of HepARG cells, and it can be seen from FIG. 6 that the stem cell conditioned medium (MSC-CM) group can significantly reduce the apoptosis of HepARG cells compared with the HepARG cell hypoxic group, and the hepatocyte hypoxic culture medium can reduce the apoptosis level of HepARG cells to a higher degree compared with the stem cell conditioned medium (MSC-CM). FIG. 7 is a graph showing the results of TUNEL staining of L-02 cells, and it can be seen from FIG. 7 that the L-02 cells were significantly less apoptotic in the stem cell conditioned medium (MSC-CM) group and that the L-02 cells were more apoptotic in the hepatocyte hypoxic medium than in the L-02 cells-conditioned medium (MSC-CM).
In summary, the results of fig. 4-7 show that the culture solution for hepatocyte hypoxia culture provided in the embodiments of the present invention can effectively prevent cell damage under hepatocyte hypoxia, maintain cell activity, and can be used as a therapeutic drug for hepatocyte hypoxia damage.
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.
Claims (10)
1. A culture solution for hepatocyte hypoxia culture is characterized by comprising a hepatocyte culture medium, resveratrol and a mesenchymal stem cell conditioned medium, wherein the volume ratio of the mesenchymal stem cell conditioned medium to the hepatocyte culture medium is 1:1-2, and the concentration of the resveratrol in the culture solution is 10-20 mu mol/L.
2. A culture solution for hypoxic culture of liver cells according to claim 1, wherein the mesenchymal stem cell conditioned medium is a cell-free supernatant isolated after mesenchymal stem cells are cultured.
3. A culture broth for hypoxic culture of hepatocytes according to claim 2, wherein the mesenchymal stem cells comprise umbilical cord mesenchymal stem cells.
4. A culture solution for hypoxic culture of hepatocytes according to claim 1, wherein the hepatocyte culture medium is DMEM medium.
5. A method for preparing the culture solution for the hypoxic culture of the liver cells according to claim 1, wherein the method comprises the following steps: mixing the hepatocyte culture medium, resveratrol and mesenchymal stem cell conditioned medium.
6. The method of claim 5, wherein the preparing the mesenchymal stem cell conditioned medium comprises: culturing mesenchymal stem cells by using a special culture medium for stem cells, replacing the special culture medium for stem cells with a hepatocyte culture medium when the fusion degree of the cultured mesenchymal stem cells reaches 75-85%, then carrying out hypoxia culture, and then separating to obtain cell-free supernatant.
7. The method of claim 6, wherein the conditions of the hypoxic culture include: the oxygen concentration is lower than 6%, and the culture time is 36-48 hours;
the mesenchymal stem cell is any generation of mesenchymal stem cell in 2-10 generations.
8. The method of claim 6, wherein the step of separating comprises: after the hypoxic culture is finished, carrying out first centrifugation to collect cell supernatant, then carrying out second centrifugation on the cell supernatant, and then filtering;
wherein the conditions of the first centrifugation comprise: the speed is 250-350g, and the time is 10-15 minutes;
conditions for the second centrifugation included: the speed is 2000-3000g, and the time is 10-15 minutes;
the filter membrane used in the filtration is a filter membrane with the pore diameter of 0.22-0.45 μm.
9. A method for culturing liver cells in a hypoxic condition, which is characterized by comprising the following steps: culturing the hepatocytes by using the culture solution for hypoxic culture of hepatocytes according to claim 1.
10. Use of the culture solution for hypoxic culture of hepatocytes according to claim 1, in the preparation of a medicament for the treatment of liver injury.
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