CN113897332A - Microgel liver carrier and preparation method thereof - Google Patents
Microgel liver carrier and preparation method thereof Download PDFInfo
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Abstract
The application provides a microgel liver carrier and a preparation method thereof, wherein the microgel liver carrier comprises a biological gel formed by crosslinking type I collagen and a matrix gel, the biological gel carries liver cells and growth factors, and the liver cells comprise one or a combination of two of liver stem cells, liver non-parenchymal cells and liver tumor cells. The microgel liver carrier can provide a good culture medium for liver cells, carries growth factors required by proliferation and survival of the liver cells, can realize loading of different combination modes of the liver cells, provides a platform for unlimited rapid proliferation of the cells, is suitable for in-vitro long-term cell culture of the liver cells, and is favorable for drug screening and morphogenesis research and application.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a microgel liver carrier and a preparation method thereof.
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. For example, two-dimensional cultured cells are thought to be more susceptible to drug action. In addition, cell culture on a two-dimensional plane results in inhibition of cell differentiation due to limited cell-cell interactions. Therefore, a more suitable cell culture environment can improve the prediction of drug sensitivity and help to understand tissue morphogenesis.
In recent years, different biomaterials, such as nanofiber scaffolds, porous sponge scaffolds, hydrogels and microcapsules, have been the focus of research for three-dimensional cell culture. Among the existing collagens, particularly type I collagen has high use frequency, and the collagens have the obvious advantages of simulating tissues rich in collagen in vivo and being simple and easy to operate. The ability to assemble three-dimensional hydrogels allows for the production of 3D gel carriers with different stiffness by polymerization of collagen solutions of different concentrations, which allows for different carrier pore sizes for cell migration, however, collagen matrix is single in component and lacks other important components of extracellular matrix. Further, it is widely believed that three-dimensional culture provides a platform for rapid proliferation of cells in an unlimited manner, but three-dimensional culture cells rapidly proliferate without limitation, and central cell necrosis is likely to occur after long-term culture.
Disclosure of Invention
The main purpose of the present application is to provide a microgel liver vector which provides a rapid proliferation platform for liver cells, and is suitable for long-term in vitro culture of liver cells.
Another object of the present application is to provide a method for preparing the microgel-like liver carrier.
In order to achieve the above object, the present application provides the following technical solutions:
in a first aspect, the present application relates to a microgel-based liver vector, comprising a biogel formed by cross-linking type I collagen and a matrix gel, wherein the biogel carries liver cells and growth factors, and the liver cells comprise one or a combination of liver stem cells, liver parenchymal cells and liver tumor cells.
Further setting: the mass ratio of liver non-parenchymal cells in the liver cell combination is 5-10%, and the mass ratio of liver stem cells or liver tumor cells is 90-95%.
Further setting: the type I collagen is rat tail type I collagen, and the mixing mass ratio of the rat tail type I collagen, the matrigel and the growth factor is 1:1: 1.
further setting: the growth factor comprises a hepatocyte growth factor or an epidermal growth factor, and the final concentration of the growth factor is 10-30 mug/mL.
As a second aspect, the present application relates to a method for preparing a microgel based liver carrier, as described above, comprising the steps of:
mixing the pre-cooled type I collagen, the matrigel and the growth factor according to the mass ratio of 1:1:1, adding the liver cells with the cell number larger than the preset number, and uniformly mixing;
adding the uniformly mixed material into a cell pore plate, heating to a preset temperature, and gelatinizing the type I collagen and the matrigel;
centrifuging the gelled material at low speed to form a gel-like liver carrier;
incubating the gel-like liver vector free-floating in a culture medium until the size of the gel-like liver vector is substantially constant.
Further setting: and placing the gel liver vector in a cell culture medium, incubating to a state of constant size, and updating the cell culture medium at intervals of 1-2 days.
Further setting: when the cell culture medium is renewed, the primary cell culture medium supernatant is discarded, the underlying material is washed with phosphate buffer solution, and then fresh cell culture solution is added.
Further setting: the precooling temperature before mixing the type I collagen, the matrigel and the growth factors is 0-10 ℃.
Further setting: the liver cells were first incubated in an incubator with a volume percentage of 5% CO2 at 37 ℃ with the medium being changed every 24 hours.
Further setting: the gelled material was placed at the minimum centrifugation radius of the centrifuge and centrifuged at a centrifugation speed of 300rpm for 10 min.
Compared with the prior art, the scheme of the application has the following advantages:
in the microgel liver carrier, a good culture medium can be provided for liver cells, growth factors required by proliferation and survival of the liver cells are carried, loading of the liver cells in different combination modes can be realized, a platform is provided for rapid proliferation without limitation of the cells, and the liver cells are not easy to cause central cell necrosis after long-time culture, so that the microgel liver carrier is suitable for in-vitro long-term cell culture of the liver cells, and is favorable for drug screening and morphogenesis research and application. And the preparation method of the microgel liver carrier is simple and convenient to operate and suitable for popularization and use.
Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present application.
Drawings
The foregoing and/or additional aspects and advantages of the present application will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a morphological observation result of HepRG cells and C3A cells of the microgel based liver vector of the present application after 12 days of culture;
FIG. 2 is a microscopic observation of three-dimensional morphology of HepRG cells cultured with microgel-based liver vectors at day 12 according to example one of the present application;
FIG. 3 is the observation result of microscopic observation of three-dimensional morphology of cells on day 12 of culture of C3A cells containing microgel-based liver vectors according to example two of the present application;
FIG. 4 is a result of observing viable cell staining under a fluorescent microscope on day 12 of HepRG cell culture of microgel based liver vectors in example one of the present application;
FIG. 5 is a result of observing viable staining of cells under a fluorescent microscope at day 12 of culturing the microgel based liver vector C3A in example two of the present application;
FIG. 6 is a result of observing the expression of cellular markers under a fluorescent microscope on day 12 of HepRG cell culture of microgel-like liver vectors in example one of the present application;
FIG. 7 is the results of observing the expression of cell markers under a fluorescent microscope on day 12 of culture of C3A cells containing microgel-like liver vectors in example II of the present application.
Detailed Description
Reference will now be made in detail to embodiments of the present application, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are exemplary only for the purpose of explaining the present application and are not to be construed as limiting the present application.
In order to solve the problem that the conventional three-dimensional cultured cells can cause central cell necrosis after long-term culture due to unlimited rapid proliferation, the application provides the microgel liver carrier capable of providing a platform for rapid proliferation of liver cells and the preparation method thereof, and the microgel liver carrier is suitable for in-vitro long-term liver cell culture, drug screening and morphogenesis research and application.
The microgel liver carrier comprises biological gel formed by crosslinking I type collagen and matrigel, wherein the I type collagen is particularly rat tail I type collagen, the rat tail I type collagen is a natural culture medium and a natural adhesive, can be used for preparing three-dimensional collagen gel and constructing a three-dimensional space for cell growth, and the matrigel is polymerized to form a three-dimensional matrix with biological activity, can simulate the structure, composition, physical characteristics and functions of an in-vivo cell basement membrane, is favorable for culturing and differentiating in-vitro cells, and can be used for researching cell morphology, biochemical function, migration, infection, gene expression and the like. The three-dimensional scaffold for culturing the in-vitro liver cells is formed by crosslinking the type I collagen and the matrigel, the biological gel formed by crosslinking the type I collagen and the matrigel is mild in property, the survival of the liver cells can be supported, the liver cells can be induced to form a solid form under the specific growth condition, and the live liver cells interact with the biological gel, so that the volume, the size, the carrier density, the cell density, the mechanical property and the stability of the liver cells can be changed.
The biological gel also carries liver cells and growth factors, the liver cells comprise one or two combinations of liver stem cells, liver parenchymal cells and liver tumor cells, in this embodiment, a combination mode of the liver stem cells, the liver parenchymal cells and the liver tumor cells and the liver parenchymal cells is preferably adopted, wherein the mass ratio of the liver stem cells to the liver tumor cells in the two cell combination modes is 90-95%, and the mass ratio of the liver parenchymal cells is 5-10%.
The growth factor is a cytokine for stimulating the growth activity of liver cells, comprises a hepatocyte growth factor or an epidermal growth factor, and can be used for promoting the generation, survival and regeneration of liver cells and inhibiting the apoptosis of the liver cells. In addition, the mass ratio of the mixture of the type I collagen, the matrigel and the growth factor in the embodiment is 1:1:1, and the final concentration of the growth factor is 10-30 mug/mL.
In addition, the application also relates to a method for preparing the microgel liver carrier by adopting the material, which comprises the following steps:
firstly, pre-cooled type I collagen, matrigel and growth factor are prepared and mixed according to the mass ratio of 1:1:1, then a preset number of liver cells are added and mixed uniformly, and the liver cells are placed in 5% CO by volume2Incubation was carried out in an incubator at 37 ℃ until a preset amount, and the medium in the incubator was changed every 24 hours during the incubation period. The predetermined number of liver cells in this example is 1X 106The above.
And after the materials are completely mixed, adding the mixed materials into a cell pore plate, and keeping for a period of time after the operation temperature is increased to the preset temperature so that the biological material is accelerated to be gelatinized, but the cell morphology is not changed.
Preferably, in the above steps, the precooling temperature is between 0 ℃ and 10 ℃, the thermal stability of the type I collagen is poor, and the matrigel can be gelled at a temperature above 10 ℃, so that the biomaterial of the present application is precooled first, the type I collagen, the matrigel and the growth factor can be smoothly mixed, then the temperature is increased to a preset temperature of 25 ℃ to 37 ℃, the type I collagen and the matrigel are gelled to form the biogel, and the liver cells at the temperature of 25 ℃ to 37 ℃ can generate a large amount of mitochondria and albumin, the biological activity of the liver cells is high, and the inactivation of the cells caused by overhigh temperature is avoided.
And then, putting the gelated material into a centrifuge for centrifugation, specifically, putting the gelated material at the minimum centrifugation radius of the centrifuge for centrifugation with minimum mechanical force, and centrifuging at the centrifugation speed of 300rpm for 10min, thereby rapidly gelling to form the gel-type liver carrier.
And then placing the gel liver-like carrier in a cell culture medium to be incubated to a state of constant size, wherein the gel liver-like carrier automatically floats in the culture medium in the incubation process. Specifically, the gel-type liver carrier can be kept floating freely for 2-12 days at a temperature of about 37 ℃, and the culture medium needs to be renewed every 1-2 days, when the culture medium is replaced, the supernatant of the original culture medium needs to be discarded, the lower-layer precipitated substances (containing the gel-type liver carrier) are washed by using a phosphate buffer solution, and then, fresh cell culture solution is added.
The following is a specific example for preparing microgel-like liver carriers of the present application.
Example one
Taking hepatic stem cells (HepaRG cells) as an example, the preparation method of the gel-like hepatic vector comprises the following steps:
(1) preparing a biological material: rat tail type I collagen, matrigel and Hepatocyte Growth Factor (HGF), and storing the above materials at 0-10 deg.C.
(2) Liver cell selection of hepatic Stem cells (HepaRG cells) and hepatic non-parenchymal cellsCulturing the liver cells in a volume percentage of 5% CO2Culturing at 37 deg.C in incubator until the number reaches 1 × 106Of the order of magnitude or more, and requires medium replacement every 24 hours during cell culture.
(3) Preparing and mixing rat tail type I collagen, matrigel and hepatocyte growth factor according to the mass ratio of 1:1:1, wherein the final concentration of the hepatocyte growth factor is 10 mug/mL.
(4) After the rat tail type I collagen, the matrigel and the hepatocyte growth factor are mixed and prepared, the liver cells are added and mixed evenly, after the whole mixture is mixed evenly, the mixed material is added into a cell pore plate, the temperature is raised to 25-37 ℃, in the process of temperature rise, the accelerated phase change of the biological material gelation is realized, then the gelation deformation of the biological material is completed after a period of time, and in the gelation process, the shape of the liver cells is not changed.
(5) Placing the gelled biomaterial at the minimum centrifugation radius of a centrifuge, centrifuging with minimum mechanical force, and centrifuging at 300rpm for 10min, such that the gelled biomaterial can be rapidly gelled to form a gel-like liver vector.
(6) Incubating the gel-like liver vector in a medium at a temperature of about 37 ℃ for 2-12 days while maintaining free floating until the size of the gel-like liver vector is substantially constant. During the incubation period of the gel liver carrier, the cell culture medium needs to be replaced every 1-2 days, the supernatant of the culture medium is discarded, the sediment substances at the lower layer are washed twice by using a phosphoric acid buffer solution, and then a fresh culture solution is added.
Example two
Taking liver tumor cells (C3A cells) as an example, the preparation method of the gel-like liver carrier comprises the following steps:
(1) preparing a biological material: rat tail type I collagen, matrigel and Hepatocyte Growth Factor (HGF), and storing the above materials at 0-10 deg.C.
(2) Liver cell selection of liver tumor cell (C3A cell) and liver non-parenchymal cellLiver cells in 5% CO by volume2Culturing at 37 deg.C in incubator until the number reaches 1 × 106Of the order of magnitude or more, and requires medium replacement every 24 hours during cell culture.
(3) Preparing and mixing rat tail type I collagen, matrigel and hepatocyte growth factor according to the mass ratio of 1:1:1, wherein the final concentration of the hepatocyte growth factor is 10 mug/mL.
(4) After the rat tail type I collagen, the matrigel and the hepatocyte growth factor are mixed and prepared, the liver cells are added and mixed evenly, after the mixture is mixed evenly, the mixed material is added into a cell pore plate, the temperature is raised to 25-37 ℃, then the biological material is subjected to gelation accelerated phase change within a period of time, and the liver cell morphology is not changed within the gelation time of the biological material.
(5) Placing the gelled biomaterial at the minimum centrifugation radius of a centrifuge, centrifuging with minimum mechanical force, and centrifuging at 300rpm for 10min, such that the gelled biomaterial can be rapidly gelled to form a gel-like liver vector.
(6) Incubating the gel-like liver vector in a medium at a temperature of about 37 ℃ for 2-12 days while maintaining free floating until the size of the gel-like liver vector is substantially constant. During the incubation period of the gel liver carrier, the cell culture medium needs to be replaced every 1-2 days, the supernatant of the culture medium is discarded, the sediment substances at the lower layer are washed twice by using a phosphoric acid buffer solution, and then a fresh culture solution is added.
In addition, the microgel liver vector of the present application was subjected to a relevant test, and the results showed that:
(1) fig. 1 shows the morphological observation results of HepRG cells and C3A cells of the microgel liver vectors in the first and second examples of the present application after 12 days of culture, respectively, and the HepRG cells and C3A cells in the microgel liver vectors both exhibit a circular microgel morphology and are structurally stable.
(2) Fig. 2 shows observation results of three-dimensional morphology of cells observed under a microscope at day 12 of culture of the HepRG cells of the microgel liver vector in example one of the present application, which shows that in the microgel liver vector, the HepRG cells are different from a flat structure in a two-dimensional plane, and appear as a three-dimensional structure of elongated cells in a three-dimensional space.
(3) Fig. 3 shows the observation result of observing the three-dimensional morphology of the cells under a microscope at day 12 of culturing the C3A cells of the microgel-based liver vector in example two of the present application, and the result shows that in the microgel-based liver vector, the C3A cells are different from the flat-shaped structure in the two-dimensional plane, and present a circular three-dimensional structure state in the three-dimensional space.
(4) Fig. 4 shows the results of observation of cell survival staining under a fluorescent microscope on day 12 of HepRG cell culture of the microgel-based liver vector in example one of the present application, which shows that the number of green fluorescent cells (viable cells) is large in the microgel-based liver vector, and that central necrosis does not occur.
(5) Fig. 5 shows the observation result of cell survival staining observed under a fluorescent microscope at day 12 of the culture of C3A cells in the microgel-based liver carrier in example two of the present application, which shows that the number of green fluorescent cells (viable cells) is large in the microgel-based liver carrier, and that central necrosis does not occur.
(6) Fig. 6 shows the results of observing the expression of cell markers under a fluorescent microscope at day 12 of the culture of the HepRG cells of the microgel liver vector in example one of the present application, in which 1 is directed to the nucleus, 2 is directed to albumin, and 3 is directed to alpha-fetoprotein, and the results show that the HepRG cells are rich in the expression of albumin and alpha-fetoprotein in the microgel liver vector.
(7) Fig. 7 shows the observation results of cell marker expression under a fluorescent microscope at day 12 of the culture of C3A cells of the microgel-based liver vector in example two of the present application, in which 1 is directed to the nucleus, 2 is directed to albumin, and 3 is directed to alpha-fetoprotein, and the results show that the microgel-based liver vector is rich in the expression of albumin and alpha-fetoprotein in C3A cells.
In conclusion, the microgel liver carrier prepared by the preparation method can provide a good culture medium for liver cells, carries growth factors required by proliferation and survival of the liver cells, can realize loading of the liver cells in different combination modes, provides a platform for unlimited rapid proliferation of the cells, is not easy to cause central cell necrosis after long-time culture of the liver cells, is suitable for in-vitro long-term cell culture of the liver cells, and is beneficial to drug screening and morphogenesis research and application. And the preparation method of the microgel liver carrier is simple and convenient to operate and suitable for popularization and use.
The foregoing is only a partial embodiment of the present application, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the principle of the present application, and these modifications and decorations should also be regarded as the protection scope of the present application.
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Application publication date: 20220107 |