CN111394299B - In-vitro construction method and application of liver organoid - Google Patents
In-vitro construction method and application of liver organoid Download PDFInfo
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Abstract
The invention discloses an in vitro construction method of liver organoid and application thereof, which comprises extracting mouse liver cells to induce and construct liver organoid, observing the shape under a mirror, and identifying the dry and epithelial cell attributes by immunofluorescence and PCR. Establishing a 70% post-hepatectomy acute liver failure mouse model, evaluating the liver functions at 1 st, 4 th and 7 th days after liver organoid transplantation, comparing the liver-body ratio, and verifying the treatment effect of the liver organoids on the acute liver failure through HE staining and immunohistochemistry. Inflammation and proliferation indexes such as HE staining, ki-67 staining and the like show that the proliferation of the transplanted liver organoid is obviously enhanced. Liver organoids have strong drying and proliferation functions; can effectively relieve the liver function of 70 percent of mice with acute liver failure after hepatectomy, increase the liver-to-body ratio, and promote the liver regeneration and repair of 70 percent of mice with acute liver failure after hepatectomy by relieving infiltration of liver inflammation.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to an in-vitro construction method and application of liver organoids.
Background
Acute Liver Failure (ALF) is a clinical syndrome of massive hepatocyte necrosis and severe liver injury in a short time caused by factors such as viruses, drugs, toxins or alcohol, and often causes complications such as jaundice, hepatic encephalopathy, multiple organ failure, and the like, and the death rate is high. Liver transplantation is currently the only effective treatment, however, the shortage of liver supplies is far from meeting the clinical need, and many patients die while waiting for liver supplies. Furthermore, medical treatment is only a symptomatic support treatment and does not improve patient prognosis, so there is an urgent need to develop an alternative treatment.
Stem cells have unique functions in self-renewal and differentiation potential and are considered the most promising therapeutic approaches for tissue repair and regeneration. Compared with liver transplantation, stem cell transplantation has the advantages of small invasiveness, repeatable transplantation treatment and the like, but simultaneously faces the problems of low implantation efficiency, short survival time of cells after implantation and the like. Research shows that the 3D stereo structure is favorable to the proliferation and differentiation of cells. The 3D culture can simulate the in vivo extracellular environment, and realizes efficient energy transfer and various molecular signal exchanges between cells and the environment in long-term culture. Compared with the traditional 2D cell culture, the urea secretion and albumin synthesis capacity of the Induced Pluripotent Stem Cells (iPSCs) cultured by 3D is obviously improved. Organoids are clusters of cells constructed in vitro based on 3D culture systems, possess the ability to be automatically renewed and assembled, and have partial functions of target organs in vivo. The existing research proves that the liver function maintaining capability of liver organoid with stem cell source implanted into pig and mouse is obviously enhanced. Human liver samples are separated into EPCAM and epithelial cells by Huch and the like, liver organoids are constructed in vitro, the chromosome structure and the gene composition of the liver organoids are amplified to keep stable, the liver organoids have the potential of being differentiated into hepatocytes and bile duct cells, and the liver organoids can be successfully differentiated into functional hepatocytes after being transplanted into nude mice. Through a 3D culture mode, the in vitro construction of organoid grafts is a development direction of future replacement therapy of acute liver failure.
Liver disease treatment methods based on cell transplantation have been developed extensively, ranging from embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells to hepatic progenitor cells. Although hepatic-like stem cells can be induced from pluripotent stem cells, clinical application is still limited due to the problems of differentiation efficiency of cells, immune rejection, ethical problems, teratogenic risk, and the like. And pure cell transplantation has a certain curative effect, but lacks an ideal cell source, the curative effect is not exact and unstable, and the clinical application has the risk of infiltration of other organs, thereby limiting the large-scale popularization of the pure cell transplantation. At present, the combination of cells and a tissue engineering technology is a hotspot of current research, organoids are cell clusters constructed in vitro based on a 3D culture system, compared with the traditional culture mode, the signal action between cells, between cells and the environment is further enhanced, and the organoids have the capability of automatic updating and assembling, thereby realizing partial functions of original organs in vivo. Current studies have demonstrated that hepatic progenitors mediate liver regeneration in chronic liver diseases, while liver organoids have utility in protecting hepatocytes and promoting liver regeneration. There is no report on whether the liver organoid with the properties similar to hepatic progenitor cells is exogenously implanted to promote liver regeneration after acute liver injury such as partial hepatectomy.
Disclosure of Invention
The invention aims to provide an in-vitro construction method of a liver organoid and application thereof aiming at the problems in the prior art, which comprises the steps of extracting mouse liver cells to induce and construct the liver organoid, observing the shape under a mirror, identifying the dryness and epithelial cell attributes by immunofluorescence and PCR, establishing a 70% liver failure mouse model after hepatectomy, evaluating the liver functions after liver organoid transplantation for 1, 4 and 7 days after operation, comparing the liver body ratio, and verifying the treatment effect of the liver organoid on the acute liver failure by HE dyeing and immunohistochemistry.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
an in vitro construction method of liver organoids comprises the following steps:
s1: preparing tissue suspension, taking the liver of a normal mouse under an aseptic condition, rinsing the liver, and shearing to obtain the tissue suspension;
s2: preparing cell suspension, adding liver tissue digestive juice into the tissue suspension prepared in the step S1, then transferring the mixture into a water bath pot for standing and digesting, mechanically blowing the mixture after digesting, and collecting supernatant serving as the cell suspension after cell precipitation;
s3: plating, filtering, centrifuging and cracking the cell suspension obtained in the step S2 to obtain cell sediment, adopting matrigel to re-suspend the cell sediment, planting the cell sediment at the central position of a 24-pore plate, after the matrigel is fixed, adding a liver organoid induction culture medium to the wall of the cell sediment for culture to obtain an initial organoid;
s4: and (5) carrying out passage on the initial organoids obtained in the step S3, and selecting 3-5 generations as finally prepared liver organoids.
In order to optimize the technical scheme, the specific measures adopted further comprise:
in step S1, the liver was first rinsed in pre-cooled 30mL DMEM/F-12 medium to remove blood residues, and then minced to 1mm in fresh 30mL DMEM/F-12 medium.
The specific steps of step S2 include: transferring the tissue suspension into a normal-temperature 50mL centrifuge tube by using a pipette, standing for 1 minute to remove a supernatant, adding 10mL of liver digestive juice, transferring into a 37-degree water bath pot, standing for digesting for 20 minutes, mechanically blowing and then standing for 1 minute, removing the supernatant when the cells are precipitated at the bottom, continuously adding 10mL of liver digestive juice, transferring into the 37-degree water bath pot, standing for digesting for 20 minutes, mechanically blowing and then standing for 1 minute, depositing the cells at the bottom, collecting the supernatant in a 50mL precooled centrifuge tube, continuously repeating the step of digesting and collecting for 4 times, and collecting 50mL of cell supernatant as a cell suspension.
The liver tissue digestive juice is prepared by the following steps: 7.5mL of neutral protease and 7.5mL of collagenase type IV were added to 45mL of DMEM/F12 medium.
The step S3 specifically includes:
s31: filtering the collected cell suspension by using a 100um cell screen to leave cells with the diameter smaller than the 100um screen, filtering the filtrate by using a 30um cell screen again, discarding the filtrate, inverting the 30um screen on a 50mL centrifuge tube, washing by using a DMEM/F-12 culture medium, and collecting the cells intercepted on the 30um screen;
s32: centrifuging at 4 ℃ and 300g for 5 minutes, and removing supernatant;
s33: adding erythrocyte lysate with the volume 3-5 times that of the cells, gently blowing and beating, cracking for 4-5 minutes at 4 degrees, centrifuging for 5 minutes at 100g, and removing supernatant to obtain cell sediment;
s34: resuspending the cell sediment by adopting 240uL matrigel, blowing the sediment up and down uniformly to avoid generating bubbles, and planting the sediment at the position of 8 holes in the center of a 24-hole plate;
s35: and (3) standing the cell culture box at the temperature of 37 ℃ for 10 minutes, adding 750uL of liver organoid induction culture medium into the cell culture box adherent to the cell culture box after matrigel is fixed, and replacing the induction culture medium every 2-3 days according to the culture condition to obtain the initial organoid.
The step S4 specifically includes:
s41: removing the induction culture medium, slightly rinsing with PBS, removing PBS, adding DMEM/12 culture medium, standing for digestion for 1 min, and mechanically blowing and beating the initial organoid;
s42: centrifuging at 4 ℃ and 300g for 5 minutes, and removing supernatant;
s43: precooling a gun head, resuspending cell precipitation by adopting 240uL matrigel, blowing and beating uniformly up and down to avoid generating bubbles, and planting the bubbles at the position of 8 holes in the center of a 24-hole plate;
s44: and (3) standing the cell culture box at the temperature of 37 ℃ for 10 minutes, adding 750uL liver organoid induction culture medium into the cell culture box in an adherent manner after matrigel is fixed, and carrying out passage according to the ratio of 1: 4.
The specific preparation of the liver organoid induction culture medium is as follows: mixing the basic culture medium of the liver organoid and the additive of the basic culture medium of the liver organoid according to the volume ratio of 10:1, adding an anti-mycoplasma reagent, subpackaging after mixing uniformly, and storing at the temperature of-20 ℃.
The invention also protects the application of the liver organoid prepared by the method in treating acute liver failure.
The invention has the beneficial effects that:
1. the mouse liver organoid prepared by the method of the invention is cultured in vitro for 3 days, the diameter of the cell mass is expanded from 20um cystic structure to about 100um, the liver sternness genes EPCAM, SOX9 and CK19 are obviously up-regulated, and EPCAM, SOX9, CK19, TBX3, AFP, SOX17, FOXA2, HNF4A, CEBPA and CEBPB are stable before and after passage.
2. According to the invention, the CK8, Desmin, AFP and PCNA are positive through immunofluorescence, the liver-to-body ratio is obviously increased after liver organoid transplantation, and the liver functions such as ALT, AST, ALB and TG are recovered on day 4. Inflammation and proliferation indexes such as HE staining, ki-67 staining and the like show that the proliferation of the transplanted liver organoid is obviously enhanced.
3. The liver organoid of the invention has stronger drying and proliferation functions; can effectively relieve the liver function of 70 percent of mice with acute liver failure after hepatectomy, increase the liver-to-body ratio, and promote the liver regeneration and repair of 70 percent of mice with acute liver failure after hepatectomy by relieving infiltration of liver inflammation.
Drawings
FIG. 1 is a diagram showing organoid morphology in 1, 2 and 3 days of culture under an optical microscope,
Figure 2 is a diameter statistical plot of the organoids shown in figure 1.
FIG. 3 is a comparison chart of gene detection of first generation liver organoids, third generation liver organoids and liver tissues.
FIG. 4 is a schematic diagram of organoid appreciation power detection.
FIG. 5 is a schematic diagram showing the liver body ratio of the control group to the treated group in application example 1.
FIG. 6 is a graph showing the comparison between the change in glutamic pyruvic transaminase of the control group and the treatment group in application example 1.
FIG. 7 is a graph showing the comparison between the changes in aspartate aminotransferase in the control group and the treatment group in application example 1.
FIG. 8 is a graph showing the comparison of the change in hepatic albumin between the control group and the treatment group in application example 1.
FIG. 9 is a graph showing the results of reducing the inflammatory response of acute liver failure after 70% hepatectomy by liver organoid transplantation and promoting liver regeneration.
Detailed Description
The invention is further illustrated by the following figures and examples. The specific conditions are not indicated in the examples, and are carried out according to conventional conditions well known in the art or recommended by the manufacturer, and the equipment or reagents used are all conventional products commercially available, see in particular the following table.
In this example, the experimental animals were selected from 6-week-old C57B/6 mice, and were allowed to freely ingest and drink water at a temperature of 22 ℃ during a 12h/12h light/dark cycle (06:00-18: 00). The animal experiment center is purchased from the laboratory animal center of Nanjing medical university, affiliated Drum building Hospital, of Nanjing medical college. The raising place is managed by the experimental animal center of the affiliated drum hospital of the medical college of Nanjing university, and all animal experiments are carried out by the experimental animal center of the affiliated drum hospital of the medical college of Nanjing university. All animal experiments were approved by the ethical committee on animal experiments in the drum hospital affiliated with the medical college of Nanjing university (approval No.: 2018010017).
Example 1
Organoid in vitro construction
1. Preparation of the formulation
1) Organoid induction medium: selecting HepatiCuLtTMOGM Mouse Basal Medium is used as liver organoid basic culture Medium, HepatiCuLt is selectedTMOGM Mouse Supplement is used as a liver organoid basic culture medium additive, and Plasmocin is selectedTMthe treatment reagent is used as an anti-mycoplasma reagent. Mixing HepatiCuLtTMOGM Mouse basic Medium and HeapatiCuLtTMOGM Mouse Supplement is prepared by mixing the following components in a volume ratio of 10:1 mixing, then adding 200uL of PlasmocinTMAnd (5) treating the mixture by using a treatment agent, uniformly mixing, subpackaging, and storing at-20 ℃.
2) Liver tissue digestive juice: 45mL of Advanced DMEM/F12 was added with 7.5mL of Dispase and 7.5mL of collagen type IV and mixed well for use.
2. Taking materials
In a super clean bench, selecting a C57BL/6 normal mouse with the age of 6-8 weeks, and killing the mouse by dislocation of cervical vertebrae after inhalation anesthesia. The four limbs were fixed on an operating table, and the abdomen was sterilized with an alcohol cotton ball. The liver was taken under sterile conditions, rinsed in 30mL of DMEM/F-12 medium, first 1 time rinsed in pre-cooled 30mL of DMEM/F-12 medium to remove residual blood, and then minced to 1mm in fresh 30mL of DMEM/F-12 medium.
3. Digestion of
Transferring the tissue suspension into a normal-temperature 50mL centrifuge tube by using a pipette, standing for 1 minute to remove a supernatant, adding 10mL of liver digestive juice, transferring into a 37-degree water bath pot, standing for digestion for 20 minutes, mechanically blowing and then standing for 1 minute, removing the supernatant when the cells are precipitated at the bottom, continuously adding 10mL of liver digestive juice, transferring into the 37-degree water bath pot, standing for digestion for 20 minutes, mechanically blowing and then standing for 1 minute, collecting the supernatant in a 50-mL precooled centrifuge tube when the cells are precipitated at the bottom, continuously repeating the steps of digestion and collection for 4 times, and collecting about 50mL of cell supernatant.
4. Plate (sterile operation)
1) The collected cell suspension was first filtered through a 100um cell screen, leaving cells with a diameter <100um screen. The filtrate was filtered through a 30um cell sieve again and the filtrate was discarded. The 30um filter screen is placed on a 50mL centrifuge tube in an inverted mode, the centrifuge tube is washed by Advanced DMEM/F12, and cells trapped on the 30um filter screen are collected;
2) centrifuging at 4 ℃ and 300g for 5 minutes, and removing supernatant;
3) adding erythrocyte lysate with the volume 3-5 times that of the cells, gently blowing and beating, cracking for 4-5 minutes at 4 degrees, centrifuging for 5 minutes at 100g, and removing supernatant to obtain precipitate; sterilizing the erythrocyte lysate with a disposable syringe needle filter before use, and operating in a dark place;
4) resuspending the cell pellet with 240uL matrigel (ice operation), blowing up and down uniformly, and planting in the position of 8 holes in the center of a 24-hole plate with care to avoid air bubbles;
5) standing for 10 minutes in a 37-degree cell culture box, adding 750uL of liver organoid induction culture medium into the cell culture box after matrigel is fixed, and replacing the induction culture medium according to the culture condition for 2-3 days.
5. Passage of culture
1) Removing the induction culture medium, gently rinsing with PBS, removing PBS, adding Advanced DMEM/F12, standing for digestion for 1 min, and mechanically blowing out organoids;
2) centrifuging at 4 ℃ and 300g for 5 minutes, and removing supernatant;
3) precooling a gun head, adopting 240uL matrigel to resuspend cell sediment (operating on ice), blowing and beating uniformly up and down, and planting in the position of 8 holes in the center of a 24-hole plate by taking care to avoid generating bubbles;
4) standing in a cell culture box for 10 minutes, adding 750uL organoid induction culture medium into the cell culture box for adherence after matrigel is fixed, carrying out passage according to the ratio of 1:4, and selecting 3-5 generations as liver organoids.
Referring to FIGS. 1-2, it can be seen that liver organoids are highly clonal and can be rapidly expanded from a small number of starting cells under specific culture conditions, and that organoids are assembled from day 1 cystic structures into day 3 spheroids that grow in diameter from 20um to greater than 100 um.
Example 2
Phenotypic characterization of liver organoids
1. Gene identification:
1) cellular RNA extraction
a) Removing culture medium from organoid, rinsing with PBS for 2 times to ensure that residual culture medium does not affect RNA purity, adding 500uL Trizol into one well of 24-well plate, repeatedly blowing for cracking, standing at room temperature for 10 min to separate protein-nucleic acid complex, and transferring to enzyme-free 1.5ml EP tube;
b) adding chloroform according to the proportion of Trizol to chloroform of 5:1, shaking up and down vigorously and mixing uniformly for 10s, standing for 5 minutes at room temperature to naturally split phases,
c) after centrifugation at 12000g for 15 minutes at 4 degrees, the upper aqueous phase was carefully removed and transferred to another new 1.5mL EP tube;
d) adding equal volume of isopropanol into the supernatant, mixing, standing at-20 ℃ for 1 hour to increase RNA precipitation;
e) centrifuge at 12000g for 10 min at 4 ℃ and discard the supernatant, and the RNA settles at the bottom of the tube.
f) Adding 1mL of 75% ethanol (prepared by DEPC water) into the RNA precipitate, mixing uniformly, gently oscillating the centrifugal tube, and suspending the precipitate;
g) centrifuge at 7500g for 5 minutes at 4 ℃ and carefully pipette off the supernatant.
h) Air drying at room temperature for 5-10 min to volatilize small amount of ethanol.
i) The RNA precipitate was dissolved in 20uL of DEPC water and the RNA solution was stored at-80 ℃.
2) Determination of RNA concentration
a) Zeroing the nucleic acid protein analyzer by using ddH2O, and sucking 1uLRNA to determine the purity, wherein OD260/OD280 is between 1.8 and 2.0, and the concentration is preferably 500 ng/uL;
b) the RNA concentration was adjusted to 500ng/uL with DEPC water, and each sample was wiped clean with a piece of paper to avoid interaction between samples.
3) Reverse transcription of RNA into cDNA
Using Chinese Shanghai assist saint science and technology BioCorp
Ⅱ 1st Strand cThe DNAsynthesis Supermix for qPCR reverse transcription kit comprises the specific steps of operating according to the instruction.
a) Removal of residual genomic DNA: the following mixture was prepared in an RNase free EP tube and gently pipetted and mixed. Incubate at 42 ℃ for 2 minutes. The following reaction system was prepared in RNase free EP tube:
b) preparation of reverse transcription reaction system (20. mu.L system): adding 2 XHifair II Supermix plus directly into the reaction tube in the step a), and gently pipetting and mixing the mixture. The following amounts were used:
c) reverse transcription program setup: the mixture was subjected to reverse transcription at 25 degrees (5 minutes), 42 degrees (30 minutes) and 85 degrees (5 minutes), and the reverse transcription product was stored at-20 ℃.
4) Real-time fluorescent quantitative PCR
Using Chinese Shanghai assist saint science and technology BioCorp
qPCR SYBR Green Master Mix (High Rox Plus) kit, performed as described.
a) Is configured as the following reaction system (operation in dark on ice)
The PCR reaction was carried out under the following conditions
b) Taking beta-actin as an internal reference, calculating 2(-△△CT)And comparing the relative expression amount of the target gene. The sequences of the primers of the genes of interest used are shown in the following table.
Referring to fig. 3, the first generation of liver organoids and the third generation of liver organoids were used to extract RNA, and compared with the liver tissue by gene level detection, wherein the expression of liver dryness genes EPCAM, SOX9 and CK19 was significantly up-regulated compared with the liver tissue, and EPCAM, SOX9, CK19, TBX3, AFP, SOX17, FOXA2, HNF4A, CEBPA and CEBPB were not significantly changed before and after passage, and hepatocytes were induced into dry liver organoids with strong proliferative capacity.
2. Immunofluorescence
1) Air-drying the frozen section in a ventilated place for 5 minutes;
2) precooling 4% paraformaldehyde, and fixing for 20 minutes at room temperature;
3) washing with 1 XPBS for 3 times, 5 min/time, and discarding PBS;
4)1mL0.5 percent TritonX-100 breaks membranes at room temperature for 10 minutes;
5) washing with 1 × PBS for 3 times, and discarding PBS;
6) PBS containing 2% BSA was blocked for 1 hour at room temperature;
7) the area was delineated with an immunofluorescent pen.
8) Adding primary antibody incubation (CK8, AFP, Des min, PCNA) with proper dilution ratio, dripping primary antibody dilution liquid into negative control, putting into a wet box, and standing overnight at 4 ℃;
9) recovering primary antibody, washing with 1X PBST for 3 times, 5 min/time, and discarding PBST;
10) fluorescent secondary antibodies (goat anti-mouse IgG H & L and goat anti-rabbit IgG H & L) were incubated in the dark for 30 min;
11) washing with 1X PBST for 3 times, 5 min/time, and discarding PBST;
12) hoest nuclear staining and incubation in dark for 5 minutes;
13) washing with 1X PBST for 3 times, 5 min/time, and discarding PBST;
14) glycerol was added dropwise, coverslips were lightly covered, coverslips were fixed, and observed under a fluorescence microscope (Leica, usa).
Referring to FIG. 4, it shows that Desmin, AFP, PCNA, CK8 are positively expressed, indicating that hepatocytes are induced into dry liver organoids with strong proliferative capacity.
Application example 1
Liver organoid transplantation for improving liver function of acute liver failure after 70% hepatectomy
Constructing an animal model: the mice 6-8 weeks old and C57BL/6 are randomly divided into 3 groups, each group comprises 3 mice, one group is not treated, namely a blank group, and the rest two groups construct an acute liver failure model after 70% hepatectomy: one group of postoperative liver capsule injection 200uL PBS as control group (i.e. control group), one group of postoperative liver capsule transplantation 200uL organoid suspension (1X 10)6Individual cells) as a treatment group (i.e., treatment group). The control group and the treatment group were sacrificed on days 1, 4, and 7, and blood was collected from the orbit of the mouse, and the liver was weighed and fixed with 10% neutral formaldehyde.
The specific steps of constructing the mouse model of the 70% acute liver failure after the hepatectomy are as follows: the diet is forbidden before the operation for 1 day, the anesthesia parameters of the anesthesia machine for the small animals are set according to the weight of the mice, the mice are placed into an anesthesia box, and an anesthesia channel is opened. After the mouse is anesthetized, the far ends of the four limbs are fixed by an adhesive tape, the mouse is backed on a mouse plate, and a breathing mask is arranged. The surgical site was disinfected with iodophor (ranging up to the level of the axillary wiring and down to the level of the inguinal superior wiring). The connecting line of the lower edge of the rib arch at the junction with the abdominal cord is used as an operation incision: a small incision of about 0.5cm is cut, the abdominal wall arteries on both sides are clamped by hemostatic forceps, and the abdominal white lines continue to extend upwards and downwards to a proper length. The ligamentum hepaticum and the mesenterium between the caudal lobe, the gastric and diaphragm muscles and the left lobe of the liver were cut off with the aid of a wet cotton swab. Completely dissociating each liver lobe, pre-wetting 3-0 surgical thread, ligating the root of the left lobe until the color becomes dark, cutting off the lobe, and removing residual blood. The 3-0 operation line is matched with a wet cotton swab to continue ligaturing the liver middle lobe, and after the color becomes dark, the lobe is cut off to remove residual blood. The vital signs of the mice were observed, with or without extensive bleeding.
The control group was injected subcontracted with 200uL PBS and hemostasis was applied by compression with dry cotton balls. The treatment groups were injected with 200uL of liver organoid suspension under the liver capsule (slow bolus injection) and stopped bleeding by gentle pressure with a dry cotton ball. Removing residual blood in abdominal cavity, suturing abdominal muscle layer and skin hair layer by layer, and sterilizing wound. And (3) after operation, putting the mouse into an incubator for resuscitation, observing the activity of the mouse, and prolonging the resuscitation time if necessary.
Liver function detection: the levels of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), and liver ALBumin (ALBumin, ALB) were determined using an automated biochemical analyzer (mai, china).
Referring to FIG. 5, differences in liver size were counted and it was found that the treated mice significantly increased on days 1 and 7 after 70% hepatectomy.
Referring to fig. 6-8, alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) were reduced in the treatment group on the fourth day compared to the control group, and liver ALBumin (ALBumin, ALB) synthesis was also significantly increased, indicating recovery of liver function after organoid transplantation.
Application example 2
The liver organoid transplantation relieves the inflammatory reaction of acute liver failure after 70 percent of hepatectomy and promotes liver regeneration.
HE staining, comprising the following steps:
1) material taking: after inhalation anesthesia, fixing a mouse and an operating table, opening an abdominal cavity, shearing a heart to kill the mouse, shearing a mesentery between a liver caudal lobe, a stomach, a diaphragm and a left outer lobe of the liver and a sickle-shaped ligament between a liver and stomach ligament and a liver middle lobe and the diaphragm, stripping off complete liver tissues, rinsing by PBS, putting the mouse into 10% neutral formaldehyde, and fixing the morphological structure of cells;
2) washing: end of fixation, ddH2Flushing for several hours;
3) and (3) dehydrating: dehydrating the tissue with 70%, 80% and 90% ethanol for 3 times (30 min each time), and dehydrating with 95% and 100% ethanol for 2 times (20 min each time);
4) and (3) transparency: preparing mixed solution (in a ratio of 1: 1) by using xylene and absolute alcohol, placing the tissue in the mixed solution for 15 minutes, and then respectively placing the tissue in xylene I and xylene II for 15 minutes until the tissue is transparent;
5) wax penetration: preparing a mixed solution (1:1 ratio) of paraffin and xylene, placing the tissue in the mixed solution for 15 minutes, and respectively carrying out wax penetration on paraffin I and paraffin II for 1 hour;
6) embedding: preheating a paraffin mould by using an alcohol lamp, placing the paraffin mould on a horizontal table, taking out a wax cup from an incubator, pouring a proper amount of paraffin, placing tissues in the paraffin mould with the section facing downwards by using heated tweezers, arranging the tissues in order, placing the tissues in an embedding box, and pouring molten wax;
7) slicing: taking out the wax block, loading the wax block on a clamping table of a slicing machine according to the instruction, installing a blade, starting to push the spiral wax block cutting with the upward edge, adjusting the angle and the position between the wax block and the edge if the wax block is close to the edge but not beyond the edge, and adjusting the thickness adjuster to a proper gear. Placing the wax block on a horizontal desktop by using a writing brush, and checking that the section meets the standard;
8) exhibition of film and pasting of film: adjusting the water temperature of a water bath kettle to 40 ℃, taking a clean glass slide, placing a tissue slice in the middle of the glass slide, marking by using a mark pen, and placing on a slice rack;
9) dewaxing and rehydration: adjusting the water temperature of the water bath to 60 ℃, putting the slices into a dry dyeing cylinder together with a shelf, putting the slices into the water bath, and sealing the cover for 30 minutes until the wax is melted. The paraffin sections were then dewaxed with xylene I and II for 15 minutes each, then placed in 100%, 95%, 90%, 80%, 70% alcohol solutions for 5 minutes each, and then ddH was added2Flushing in O for 3 minutes;
10) dyeing: the section is placed in hematoxylin staining solution for staining for 5 minutes;
11) washing with water: by ddH2Flushing for 15 minutes until the color of the slices turns blue;
12) differentiation: placing the slices in 1% hydrochloric acid ethanol, and fading and turning red the slices;
13) anti-blue: slicing in ddH2O, it returns blue;
14) and (3) dehydrating: the slices were placed in ethanol 3 times at 50%, 70%, 80% concentration for 5 minutes each;
15) counterdyeing: staining with eosin ethanol solution (0.5%) for 2 min;
16) and (3) dehydrating: putting the slices into ethanol with concentration gradient of 50%, 70%, 80%, 90%, 95% and 100% in sequence, each for 5 minutes;
17) and (3) transparency: putting slices into dimethylbenzenes I and II for 5 minutes respectively;
18) sealing: after blocking with neutral gum, place in a 37 degree oven and bake the pieces overnight. The stained liver tissue sections were analyzed under an inverted microscope (Leica, usa) and photographed.
Ki67 immunohistochemistry
The tissue acquisition and the paraffin section preparation are the same as above, and the composition comprises the following specific steps:
1) dewaxing: sequentially dewaxing the paraffin sections by dimethylbenzenes I and II for 15 minutes respectively, then sequentially placing the paraffin sections into alcohol solutions with concentration gradients of 100 percent, 95 percent, 90 percent, 80 percent and 70 percent respectively for 5 minutes each time, and finally washing the paraffin sections for 3 minutes by using distilled water;
2) inactivation: the slices were treated with 3% hydrogen peroxide for 10 minutes at room temperature;
3) antigen retrieval: placing the slices in sodium citrate buffer (0.01M), heating to 100 ℃, cooling for 5 minutes, and repeating for 2 times;
4) washing: cooling the to-be-sliced pieces to room temperature, and washing the to-be-sliced pieces for 3 times with PBS (phosphate buffer solution) for 5 minutes each time;
5) and (3) sealing: sections were blocked with BSA (3%), 37 degrees for 1 hour;
6) primary antibody binding: binding pre-diluted Ki67 primary antibody at the appropriate concentration, 4 degrees overnight;
7) rewarming: putting the slices into a 37-DEG C oven for rewarming for 1 hour;
8) washing: the sections were washed 10 times with PBS for 2 minutes each;
9) and (3) binding of a secondary antibody: combining pre-diluted secondary antibodies with proper concentration and corresponding species at 37 ℃ for 1 hour;
10) washing: the sections were washed 10 times with PBS for 2 minutes each;
11) adding SABC: dropping SABC on the slices at 37 ℃ for 30 minutes;
12) washing: sections were washed 5 times with PBS for 2 minutes each;
13) color development: dripping the prepared DAB color developing solution on the slices, standing at room temperature, observing the reaction time (generally 1 to 5 minutes) under a mirror, and flushing with PBS to stop color development after the optimal effect is achieved;
15) dyeing: the slices are placed into hematoxylin staining solution for counterstaining for 1 minute, and PBS is fully washed;
16) and (3) dehydrating: placing the slices in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, anhydrous ethanol I and anhydrous ethanol II for 1 minute respectively;
17) and (3) transparency: placing the slices in dimethylbenzenes I and II for 5 minutes respectively;
18) sealing: after mounting with neutral gum, the pieces were baked overnight in a 37 degree oven, and the finished liver tissue sections were stained, analyzed under an inverted microscope for observation and photographed.
Referring to fig. 9, it can be seen from the graph a that the hepatocytes of the normal group of mice are in a regular cord shape, the spaces between the liver blood sinuses are clear, and there is no infiltration of inflammatory cells; the liver cells were normal in morphology and size and free of fat or ballooning. On the 1 st day after operation, the liver cord structure of the mice in the treatment group and the control group disappears, the cells are disorderly arranged and have different sizes, inflammatory cells infiltrate and have less clear shapes, a large amount of fat vacuoles exist, and the liver fat vacuoles in the treatment group are more serious; on the 4 th day after operation, the control mice had dark staining of liver cell nucleoli, enlarged spaces of liver blood sinuses, inflammatory cell infiltration, and liver cell necrosis zones in partial areas. The shape and size of the liver cells of the mice in the treatment group are basically recovered to be normal, the shape is clear, and the mice are free from inflammatory cell infiltration, fat or balloon-like deformation; as can be seen from the graph B, after 70% hepatectomy, on day 1, the proliferation of the control group mice was significantly stronger than that of the treated group, on day 4, both the control group and the treated group showed proliferation status, while the regeneration of the treated group was significantly higher than that of the control group, showing vigorous proliferation.
In conclusion, it is demonstrated that organoid transplantation can treat acute liver failure by alleviating liver inflammatory reaction and promoting liver regeneration.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (3)
1. An in vitro construction method of liver organoids is characterized by comprising the following steps:
s1: preparing tissue suspension, taking the liver of a normal mouse under an aseptic condition, rinsing the liver, and shearing to obtain the tissue suspension;
s2: preparing cell suspension, adding liver tissue digestive juice into the tissue suspension prepared in the step S1, then transferring the tissue suspension into a water bath pot for standing and digesting, mechanically blowing the tissue suspension after digestion, and collecting supernatant serving as the cell suspension after cell precipitation, wherein the specific steps comprise: transferring the tissue suspension into a normal-temperature 50mL centrifuge tube by using a pipette gun, standing for 1 minute to remove a supernatant, adding 10mL of liver tissue digestive juice, transferring into a 37-degree water bath pot, standing for digesting for 20 minutes, mechanically blowing and standing for 1 minute, removing the supernatant when the cells are precipitated at the bottom, continuously adding 10mL of liver tissue digestive juice, transferring into the 37-degree water bath pot, standing for digesting for 20 minutes, mechanically blowing and standing for 1 minute, collecting the supernatant into a 50mL precooled centrifuge tube when the cells are precipitated at the bottom, continuously repeating the step of digesting and collecting for 4 times, and collecting 50mL of cell supernatant as a cell suspension; the liver tissue digestive juice is prepared by the following steps: adding 7.5mL of neutral protease and 7.5mL of collagenase IV into every 45mL of DMEM/F12 culture medium;
s3: the plate specifically comprises the following steps:
s31: collecting the cell suspension obtained in the step S2, firstly filtering with a 100um cell screen to leave cells with the diameter smaller than the 100um screen, then filtering the filtrate again with a 30um cell screen, discarding the filtrate, inverting the 30um screen on a 50mL centrifuge tube, washing with a DMEM/F-12 culture medium, and collecting the cells trapped on the 30um screen;
s32: centrifuging at 4 ℃ and 300g for 5 minutes, and removing supernatant;
s33: adding erythrocyte lysate with the volume 3-5 times that of the cells, gently blowing and beating, cracking for 4-5 minutes at 4 degrees, centrifuging for 5 minutes at 100g, and removing supernatant to obtain cell sediment;
s34: resuspending the cell sediment by adopting 240uL matrigel, blowing the sediment up and down uniformly to avoid generating bubbles, and planting the sediment at the position of 8 holes in the center of a 24-hole plate;
s35: standing the cell culture box at the temperature of 37 ℃ for 10 minutes, adding 750uL liver organoid induction culture medium into the cell culture box in an adherent manner after matrigel is fixed, and replacing the liver organoid induction culture medium every 2-3 days according to the culture condition to obtain an initial organoid;
the specific preparation of the liver organoid induction culture medium is as follows: mixing the basic culture medium of liver organoid with the additive of basic culture medium of liver organoid at a volume ratio of 10:1, adding anti-mycoplasma reagent, mixing, packaging, and storing at-20 deg.C, wherein the basic culture medium of liver organoid is selected from HepatiCuLtTMOGM Mouse Basal Medium, liver organoid Basal Medium additive selection hepaticuLtTMOGM Mouse Supplement, anti-mycoplasma reagent selection PlasmocinTM treatment；
S4: and (5) carrying out passage on the initial organoids obtained in the step (S3), and selecting 3-5 generations as finally prepared liver organoids, wherein the passage specifically comprises:
s41: removing the induction culture medium, slightly rinsing with PBS, removing PBS, adding DMEM/12 culture medium, standing for digestion for 1 min, and mechanically blowing and beating the initial organoid;
s42: centrifuging at 4 ℃ and 300g for 5 minutes, and removing supernatant;
s43: precooling a gun head, resuspending cell precipitation by adopting 240uL matrigel, blowing and beating uniformly up and down to avoid generating bubbles, and planting the bubbles at the position of 8 holes in the center of a 24-hole plate;
s44: and (3) standing the cell culture box at the temperature of 37 ℃ for 10 minutes, adding 750uL liver organoid induction culture medium into the cell culture box in an adherent manner after matrigel is fixed, and carrying out passage according to the ratio of 1: 4.
2. The method of claim 1, wherein in step S1, the liver is first rinsed in pre-cooled 30mL DMEM/F-12 medium to remove residual blood, and then minced to 1mm in new 30mL DMEM/F-12 medium.
3. Use of the liver organoid prepared by the method of claim 1 in the preparation of a medicament for treating acute liver failure in a mouse.
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| CN111979183B (en) * | 2020-08-10 | 2021-05-28 | 创芯国际生物科技(广州)有限公司 | Drug hepatotoxicity evaluation method based on liver organoid model |
| CN111961642B (en) * | 2020-08-10 | 2021-05-14 | 创芯国际生物科技(广州)有限公司 | Mouse liver organoid model and establishing method and application thereof |
| CN111979179B (en) * | 2020-08-24 | 2022-12-27 | 扬州大学 | Pig liver tissue organoid model and in-vitro construction method thereof |
| CN112410301A (en) * | 2020-11-26 | 2021-02-26 | 邹冬玲 | Method for accurately predicting drug administration of ovarian cancer patient through in-vitro construction |
| CN112553161A (en) * | 2020-12-11 | 2021-03-26 | 邹冬玲 | Method for amplifying ovarian cancer organoid |
| CN112538457B (en) * | 2020-12-23 | 2023-03-28 | 中国科学院亚热带农业生态研究所 | Pig liver and spleen duct stem cell separation and three-dimensional organoid culture method |
| CN115537395A (en) * | 2021-11-04 | 2022-12-30 | 上海万何圆生物科技有限公司 | Treatment method for co-culture of liver cancer organoid and TILs (tumor necrosis factor-associated stem cells) and application thereof |
| CN115521912A (en) * | 2021-11-04 | 2022-12-27 | 上海万何圆生物科技有限公司 | Immune cell treatment method by co-culture of organoid and T cell and application |
| CN115521898A (en) * | 2021-11-04 | 2022-12-27 | 上海万何圆生物科技有限公司 | Immune cell treatment method for co-culture of liver cancer organs and NK cells and application |
| US20230220353A1 (en) * | 2022-01-11 | 2023-07-13 | Hai Bing YUE | In vitro liver organoids and mini-bile duct models of biliary artresia and applications thereof |
| CN114149961B (en) * | 2022-02-09 | 2022-04-22 | 天九再生医学(天津)科技有限公司 | Multi-lineage liver organoid and construction method and application thereof |
| CN114736866A (en) * | 2022-03-21 | 2022-07-12 | 深圳大学总医院 | Culture medium and culture method for bile duct cancer organoid |
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