Preparation method of liver organoid
The application is a divisional application of a patent application 'method for reprogramming umbilical cord mesenchymal stem cells into liver cells and prepared liver organoids', wherein the application date of the original application is 8-28-2020-s, the application number is 2020108879718, and the publication number is CN 112111444A.
Technical Field
The application relates to a cell culture technology, in particular to a preparation method of liver organoids.
Background
Liver tumors usually require major resection of the liver, the remaining liver will be regenerative-compensated, and post-surgery can easily lead to post-hepatectomy liver failure (PHLF), one of the most alarming complications, which must be treated by transplantation and life-long immunosuppression. The shortage of donor livers, high cost and immunosuppression limit the use of this treatment modality. Treatment of liver failure can also be improved by temporary liver support methods. Such as bioartificial liver, continuous hemodiafiltration by plasmapheresis and albumin dialysis can improve certain liver function parameters, but it has not been proven that it can improve patient survival. Moreover, this technique is limited by the lack of a safe source of hepatocytes. Potential alternative sources of hepatocytes include porcine hepatocytes, immortalized human hepatocytes, ES cell-derived hepatocytes and various adult stem cells, but still do not address the lack of primary hepatocytes in current artificial liver therapy procedures. Further, it has been shown that transplantation of Mesenchymal Stem Cells (MSCs) or hepatocyte-like cells derived from MSCs improves liver function in rodents or patients suffering from liver damage. However, several conventional methods used so far have been of little effect, and these hepatocyte-like cells only show a part of the functions of markers and primary hepatocytes.
Disclosure of Invention
The method aims to overcome the defects in the prior art, provide a simple and easily-repeated method for reprogramming umbilical cord mesenchymal stem cells into liver cells, and prepare liver organoids by using the obtained liver cells, wherein the method is used for clinically obtaining a large number of functional liver cells.
In order to achieve the technical purpose, the technical scheme adopted by the application is as follows:
firstly, a method for reprogramming umbilical cord mesenchymal stem cells into liver cells comprises the following steps:
obtaining umbilical cord mesenchymal stem cells;
pre-treating the umbilical cord mesenchymal stem cells for 2 d;
performing hepatogenic induction on the pretreated umbilical cord mesenchymal stem cells by using the hepatogenic induction composition for 7 d;
performing primary maturation induction on the umbilical cord mesenchymal stem cells subjected to the liver formation induction treatment for 7d by using the first maturation induction composition to obtain liver cells;
and performing secondary maturation induction on the liver cells for 12-20 days by using a secondary maturation induction composition to obtain functional liver cells.
Preferably, the umbilical cord mesenchymal stem cells are cultured in vitro to a logarithmic growth phase before being subjected to pretreatment.
Further preferably, the umbilical cord mesenchymal stem cells are cultured in vitro under serum-free conditions before being pretreated.
Specifically, the pre-treated reagent comprises 20ng/mL of epidermal growth factor and 10ng/mL of basic fibroblast growth factor;
the hepatogenic inducing composition comprises 30ng/mL basic fibroblast growth factor, 20ng/mL hepatocyte growth factor and 0.61g/L nicotinamide;
the first maturation inducing composition comprises a 40ng/mL mixture of ITS Premix, 10ng/mL recombinant human osteoprotegerin-M and 1umol/L dexamethasone;
the second maturation inducing composition comprises ITS + Premix at 40ng/mL, recombinant human osteoprotegerin-M at 40ng/mL, dexamethasone at 1umol/L, human gamma secretase complex inhibitor at 10umol/L, and small molecule inhibitor SB431542 at 10 umol/L.
Under an optical microscope, the functional liver cells are irregular circles or polygons, and the boundary of nucleoli is clear.
The liver-specific gene expression rate of the functional liver cells is higher than that of the liver cells.
In particular, the liver-specific genes include alpha-fetoprotein, albumin, and cytokeratin.
Preferably, the functional liver cell is positive in glycogen accumulation function identification.
Use of a method as hereinbefore described for the preparation of a liver organoid.
Secondly, a liver organoid comprising: an acellular matrix hydrogel, stem cells and liver cells, the stem cells and liver cells being distributed in the acellular matrix hydrogel.
Specifically, the acellular matrix hydrogel is prepared from umbilical cord tissue; the stem cells are totipotent stem cells, pluripotent stem cells or unipotent stem cells; the liver cell is obtained by inducing umbilical cord mesenchymal stem cells to differentiate, or the liver cell is derived from a primary cultured liver cell or a subculture liver cell line.
The preparation method of the liver organoid comprises the following steps:
uniformly mixing the acellular matrix hydrogel diluent, the stem cells and the liver cells by using a serum-free culture medium;
performing static culture in a culture vessel until the acellular matrix hydrogel is solidified;
and (3) after determining that the cells in the solidified acellular matrix hydrogel survive, continuing culturing to obtain the liver organoid.
Preferably, the pH of the diluted solution of the acellular matrix hydrogel is adjusted to 7-7.5 before mixing.
Specifically, the mixing ratio of the acellular matrix hydrogel diluent, the stem cells and the liver cells is as follows: 1X 107 stem cells, 1X 106 liver cells and 3ml of acellular matrix hydrogel diluent were added to 12ml of serum-free medium.
Alternatively, the survival of the reprogrammed liver cells and normal liver cells in the decellularized matrix hydrogel is determined by a staining method.
Further, after the cells in the acellular matrix hydrogel survive, continuing to culture for 6-7 days to obtain the liver organoid.
Compared with the prior art, the method has the following advantages:
(1) according to the preparation method of the liver organoid, cells derived from umbilical cord mesenchymal stem cells and acellular matrix hydrogel are used, so that the obtained liver organoid has ultralow immunogenicity and the possibility of cross-species transplantation, and experiments prove that the human liver organoid can be maintained and grown in a rat body.
(2) According to the preparation method of the liver organoid, the used adult liver inducing composition, the first maturation inducing composition and the second maturation inducing composition only contain factors and small molecular compounds, and the preparation method is simple in formula and easy to prepare.
(3) According to the preparation method of the liver organoid, the step of secondary maturation induction is added, the culture time is prolonged, the induction efficiency is obviously improved, and particularly the albumin expression of the obtained functional liver cells is obviously improved.
(4) According to the preparation method of the liver organoid, the inhibitor is added in the secondary maturation inducing composition, and the signal conduction of bypass is inhibited, so that the mesenchymal stem cells are promoted to be differentiated into the liver cells.
Drawings
Fig. 1 is a morphological diagram under an optical microscope of umbilical cord mesenchymal stem cells obtained by the present application.
Fig. 2 is a morphological view under an optical microscope of various stages of differentiation of umbilical cord mesenchymal stem cells into functional liver cells using the method for reprogramming umbilical cord mesenchymal stem cells into liver cells of the present application.
FIG. 3 is a graph showing the results of immunofluorescence expression of functional liver cells obtained in the present application.
FIG. 4 is a graph showing a comparison between the expression levels of liver-specific genes in liver cells and functional liver cells obtained in the present application.
FIG. 5 is a graphical representation of glycogen staining identification of functional liver cells obtained in the present application.
FIG. 6 is a graph identifying the survival of cells in liver organoids obtained in the present application.
FIG. 7 is a morphological diagram under an electrical microscope of a liver organoid obtained by the present application.
FIG. 8 is a graph showing the maintenance of liver organoids transplanted in rats obtained by the present application.
Detailed Description
The present application is described in further detail below with reference to the attached drawings and the detailed description.
Example one
Method for reprogramming umbilical cord mesenchymal stem cells into liver cells
The umbilical cord is from human, but is generally treated as medical waste, has wide sources, does not relate to ethical problems, and is an excellent tissue source for obtaining mesenchymal stem cells in large quantities in clinic and research. Umbilical cord mesenchymal stem cells (UC-MSCs) have strong proliferation activity and are used for the treatment and treatment research of various diseases.
In the application, the UC-MSCs are cultured under the serum-free condition to 6 minutes, namely the UC-MSCs are cultured in vitro until the UC-MSCs enter the logarithmic growth phase, as shown in figure 1, when the UC-MSCs are cultured in vitro until the third generation, the UC-MSCs are uniform in shape and grow in a long fusiform spiral fossa shape, and the UC-MSCs are subjected to cell surface marking identification and differentiation capacity identification subsequently, so that the extracted UC-MSCs are proved to meet the requirements of the subsequent reprogramming step.
Subsequently, the induced differentiation treatment is started, and the UC-MSCs are induced to be reprogrammed by adding the following reagents into a serum-free culture medium in sequence:
a pretreatment stage: epidermal Growth Factor (EGF)20ng/mL (Gibco; PHG0315), basic fibroblast growth factor (bFGF)10ng/mL (PeproTech; 100-31-25), pretreatment 2 d;
liver-forming inducing composition: 30ng/mL of basic fibroblast growth factor (bFGF), 20ng/mL of Hepatocyte Growth Factor (HGF) (PeproTech; 100-39-10), 0.61g/L of nicotinamide (nicotinamide) (Sigma; N0636), 7d of induction;
first maturation inducing composition: ITS + Premix40 ng/mL (Gibco; 51500056), recombinant human osteoprotegerin-M (OSM)10ng/mL (PeproTech; 300-10-2), Dexamethasone (Dexamethane) 1umol/L (Invitrogen; D1383), Induction 7D;
second maturation inducing composition: ITS + Premix40 ng/mL, recombinant human osteoprotegerin-M (OSM)10ng/mL, Dexamethasone (Dexamethane) 1umol/L, human-derived gamma secretase complex inhibitor (gamma-secretase inhibitor or DAPT for short) 10umol/L (MCE; HY-13027), small molecule inhibitor SB 43154210 umol/L (abcam; ab146590), and induction for 12-20 days.
The identification of reprogrammed cells during induction of UC-MSCs resulted in the following:
1. cell morphology was observed under an inverted microscope: the cells grow well in the induction reprogramming process, and after the pretreatment (day1-day2) and the liver formation induction stage (day3-day9), the MSCs become more slender, the liver cell maturation stage (day10-day16), the UC-MSCs are differentiated into liver cells, the cell morphology gradually becomes flat and round from long spindle, the liver cells obtained by reprogramming in the liver cell secondary maturation stage (day17-day32) further mature into functional liver cells, at the moment, the cells turn into irregular round or polygonal shapes, the nucleolus is obvious, and apoptosis is not seen (as shown in figure 2).
2. Immunofluorescence was used to identify whether liver-specific genes were expressed: albumin (ALB, using the abcam kit: 207327), alpha-fetoprotein (AFP, abcam kit: ab169552) and cytokeratin (CK19, abcam kit: ab52625) were identified as AFP (+) (see first column panel in FIG. 3), ALB (+) (see first column panel in FIG. 3), CK19(+) (see second column panel in FIG. 3). Further, statistical analysis was performed on the fluorescence values, and the AFP, ALB, and CK19 expression rates were significantly increased when the induction phase was compared between day32 and day16 (fig. 4), indicating that the reprogrammed liver cells were further mature and more closely related to liver cells with liver function.
3. Glycogen accumulation function assay (using Solarbio kit: G285): as shown in FIG. 5, staining of glycogen was observed in the hepatocytes, and the result of functional identification of glycogen accumulation was positive.
In conclusion, functional liver cells reprogrammed and differentiated by UC-MSCs are successfully obtained through the processes of pretreatment, liver formation induction and twice maturation induction.
Example two
Liver organoid and its preparing process
Organoids belong to three-dimensional cell cultures, which contain some key properties that represent the organ. Organoids are also an in vitro culture system comprising a self-renewing stem cell population that can differentiate into a plurality of organ-specific cell types, having similar spatial organization as the corresponding organ and being capable of reproducing a part of the function of the corresponding organ, thereby providing a highly physiologically relevant system. Organoid cultures have been used in a variety of tissues including the gut, liver, pancreas, kidney, prostate, lung, optic cup, and brain. Organoids are also tools that find application in developmental biology, disease pathology, cell biology, regenerative mechanisms, precision medicine, and drug toxicity and efficacy testing, among others. For these and other applications, organoid culture enables a high information content complementation of existing two-dimensional culture methods and animal model systems.
In addition to their use in clinical liver failure transplantation, liver organoids can be used as a first aid bridge for the transition from liver failure to liver regeneration, or to supplement extensive hepatectomy and temporary maintenance of liver function during the waiting period for transplantation.
The liver organoid is prepared by the method for reprogramming umbilical cord mesenchymal stem cells into liver cells and the obtained functional liver cells, and specifically, the liver organoid construction method comprises the following steps:
(1) 3ml of umbilical cord acellular matrix pre-gel is taken on ice and is trimmed with 300ul of 10 XPBS to form hydrogel;
(2) adjusting the pH value to 7-7.5 by NaOH for later use;
(3) adding 1X 10 to 12ml of serum-free medium7UC-MSCs, 1X 106Fully blowing, beating and uniformly mixing liver cells and 3ml of balanced hydrogel;
(4) adding 2.5ml of the above mixed liquid into each well of 6-well plate, standing in incubator for 2-4 hr, and adding 2ml of serum-free culture medium after gelling. Observing the growth condition of the cells in the hydrogel under a microscope, and carrying out validity identification on the cultured cells in the hydrogel: the 1d, 3d and 7d were cultured and stained to identify the survival of the cells.
(5) The formed "3D-hOs" (liver organoids, see FIG. 7) was obtained in 6-7 days.
The umbilical cord acellular matrix hydrogel is prepared from umbilical cord tissues, so that the low immunogenicity relative to a human body is maintained, the integrity of an internal structure of an ECM (extracellular matrix) is maintained to the maximum extent, and the proliferation and survival of stem cells and liver cells in the hydrogel are facilitated, even after the umbilical cord acellular matrix hydrogel is transplanted into a human body. As shown in FIG. 6, the cells in the hydrogel were cultured at 1d, 3d and 7d, and the density of the living cells was higher, which proves that the cells grew well in the gaps of the hydrogel.
The UC-MSCs can also be replaced by other types of stem cells, and from the development potential, totipotent stem cells, pluripotent stem cells and unipotent stem cells can have the potential of forming liver organoids.
The liver cell can be obtained by inducing umbilical cord mesenchymal stem cells to differentiate by the method, or the liver cell is derived from a primary cultured liver cell or a subcultured liver cell line.
The whole preparation process of the liver organoid uses a serum-free and xeno-free culture medium, the preparation method accords with GMP standard, all cells and tissues are obtained by human, and the preparation method is easy to obtain in large quantity and does not relate to medical ethics. Due to the immunoregulation property of the MSCs, the umbilical cord decellularized ECM hydrogel has ultralow immunogenicity, the constructed 3D-hOs cannot cause immune reaction when transplanted among different individuals, even can be transplanted across species, has low cost and wide sources, can be produced in large scale and batch, is easy to reproduce after system maturation, has stable effect, and lays a foundation for future clinical tests. Further, the liver organoid was transplanted into the rat, and the rat was dissected 10 days after the transplantation, and it was seen that the graft migrated to the liver surface and the blood vessels were covered above the 3D-hOs (fig. 8), indicating that the human 3D-hOs could be maintained and grown in the rat without causing immune rejection.
In summary, the liver organoid of the present application uses umbilical cord mesenchymal stem cell-derived cells and acellular matrix hydrogel, so that the obtained liver organoid has ultra-low immunogenicity and has the possibility of cross-species transplantation.
The above embodiments are only preferred embodiments of the present application, but not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present application should be construed as equivalents and are included in the scope of the present application.