CN109337860A

CN109337860A - A method for constructing a 3D model of liver fibrosis in vitro

Info

Publication number: CN109337860A
Application number: CN201811267050.0A
Authority: CN
Inventors: 张彬; 冒祖明; 许珂; 陈维维
Original assignee: Wonderful (shanghai) Biotechnology Co Ltd
Current assignee: Wonderful (shanghai) Biotechnology Co Ltd
Priority date: 2018-10-29
Filing date: 2018-10-29
Publication date: 2019-02-15
Anticipated expiration: 2038-10-29
Also published as: CN109337860B

Abstract

The present invention relates to cytobiology technologies and application field, and in particular to a kind of hepatic fibrosis in vitro 3D model building method.This method step are as follows: mouse primary hepatic parenchymal cells, hepatic stellate cells and Kupffer Cell are obtained using enzyme digestion-cell culture method respectively; each cell is mixed with magnetic suspension particle and carries out digestion after incubation and obtains that the hepatic parenchymal cells containing magnetic particle are unicellular, hepatic stellate cells is unicellular and Kupffer Cell is unicellular; and by three kinds of cell co-inoculations containing magnetic particle in 3D culture medium; it is cultivated 15-25 days under the conditions of magnetic drive; obtain 3D co-cultured cell; liver fibrosis inducer is added, carries out induction and obtains liver fibrosis 3D model.This method can be good at simulating the intracorporal time of day of each cell, while can be quickly obtained liver fibrosis 3D model, improve efficiency, and save time cost, easily operated, is suitable for large-scale medicine and screens.

Description

A kind of hepatic fibrosis in vitro 3D model building method

Technical field

The present invention relates to cytobiology technologies and application field, and in particular to a kind of hepatic fibrosis in vitro 3D model construction Method.

Background technique

Liver fibrosis be liver to chronic injury caused by the various causes of disease respond as a result, its essence is tissue damage Afterwards in repair process extracellular matrix synthesis, degradation and deposition.Hepatic parenchymal cells and hepatic stellate cells are that fibrosis occurs, sends out Most important cell during exhibition, hepatic parenchymal cells damage are the important starting factor of liver fibrosis, the activation of hepatic stellate cells It is the key link that liver fibrosis occurs.

With deepening continuously for people's research, people have deeper understanding to the mechanism of liver fibrosis, in recent years A possibility that reverse of liver fibrosis is as treatment cirrhosis provides a kind of new by the extensive concern of domestic and foreign scholars Therapeutic strategy.The main research model of liver fibrosis at present is the animal model based on mouse and rat, utilizes four chlorinations The reagents such as carbon, dimethyl nitramine are administered animal, and successive administration 6 weeks in the case where normal diet raising, it is fine to obtain liver Dimensionization animal model.

It is long that above-mentioned hepatic fibrosis animal model establishes the period, and since every kind of intracorporal pathogenic factor is different, occurs fine The specific mechanism of dimensionization is different, and stability, the poor repeatability of experimental model, it is impossible to meet the demands of new drug development.

Summary of the invention

For the deficiencies in the prior art, the present invention provides a kind of hepatic fibrosis in vitro 3D model building methods. Specific steps are as follows:

Mouse primary hepatic parenchymal cells, hepatic stellate cells and withered no thin are obtained using enzyme digestion-cell culture method respectively Born of the same parents；

The mouse primary hepatic parenchymal cells, hepatic stellate cells and the Kupffer Cell that culture density is 75%-85% are chosen, It is each that magnetic suspension particle is added, it is uniformly mixed and is placed in incubation 10-12h in incubator；

Pancreas enzyme -EDTA-digestive juice is respectively added in hepatic parenchymal cells, hepatic stellate cells and Kupffer Cell after taking the incubation Digestion 2-4min is carried out, adds serum mixing, centrifugation 5-8min obtains that the hepatic parenchymal cells containing magnetic particle are unicellular, liver star Shape cell is unicellular and Kupffer Cell is unicellular.

The hepatic parenchymal cells containing magnetic particle are unicellular, hepatic stellate cells is unicellular and Kupffer Cell is unicellular connects It on kind to 3D culture medium, is cultivated 15-25 days under the conditions of magnetic drive, obtains 3D co-cultured cell；

Liver fibrosis inducer is added in the 3D co-cultured cell, carries out induction and obtains liver fibrosis 3D model.

The revolving speed of the centrifugal process is 300-400rpm.

It is described the hepatic parenchymal cells containing magnetic particle are unicellular, hepatic stellate cells is unicellular and Kupffer Cell is slender Born of the same parents are seeded to during 3D culture medium, and hepatic parenchymal cells are unicellular, hepatic stellate cells is unicellular and Kupffer Cell single cells population Ratio be 1: 0.8-1.2: 0.8-1.2, the cell number of each culture medium inoculated is 4000-6000.

The 3D culture medium is 10%-15% fetal calf serum/DMEMF12.

The liver fibrosis inducer is paracetamol, carbon tetrachloride, any one in dimethyl nitramine, induction Time be 20-30h.

Technical solution provided by the invention include it is following the utility model has the advantages that

By using magnetic suspension 3D culture systems to cell category main in liver -- hepatic parenchymal cells, Kupffer Cell with Sternzellen has carried out 3D co-cultivation, on the one hand can preferably simulate hepatic parenchymal cells, Kupffer Cell and sternzellen and exist Intracorporal time of day；On the other hand, it can be quickly obtained liver fibrosis 3D model, improved efficiency, time cost is saved, be easy to Operation is suitable for large-scale medicine and screens.

Detailed description of the invention

Attached drawing 1 is 3D co-culture model schematic device.

Attached drawing 2 is that the collagen of 3D co-cultured cell dyes.

Attached drawing 3 is that co-cultured cell uses paracetamol collagen expression quantity comparison before and after the processing.

Attached drawing 4 is the 3D co-cultured cell under microscope.

Specific embodiment

In order to more fairly set out technology contents of the invention, it is described in detail in conjunction with specific embodiments herein, it is clear that Cited embodiment is the preferred embodiment of the technical program, and those skilled in the art can be according to disclosed skill The other technologies scheme that art content is apparent from still falls within protection scope of the present invention.

Embodiment:

Mouse primary hepatic parenchymal cells, hepatic stellate cells and withered no thin are obtained using enzyme digestion-cell culture method respectively Born of the same parents.

The method for obtaining mouse hepatocytes, specifically includes the following steps:

Using appropriate isoflurane anesthetized mice, mouse web portion is fixed on workbench upwards, pin fixing limbs；

Thoroughly cleaning abdomen and chest area, are carried out disinfection, scissors and friction clamp remove abdominal incision, cut off with 75% ethyl alcohol Epidermis and muscle layer continue straight line and cut off mouse skin and muscle layer until liver, portal vein and inferior caval vein sufficiently expose；

Start peristaltic pump, after trochar leading portion has liquid outflow, trochar is placed in portal vein rapidly, can be seen at this time Bleach rapidly to liver, cuts inferior caval vein immediately；

After cutting inferior caval vein, perfusion rate is increased to 7ml/min immediately, allows all HBSS preheated in advance (Hank ' s Balanced Salt Solution, Hank ' s balanced salt solution) passes through liver perfusion；

When the liquid in containers for holding HBSS will exhaust, 70ml IV Collagenase Type is poured into container and (is purchased from Worthington, 9001-12-1) final concentration of 100CDU/mL digestive juice, remaining about 20ml digestive juice is poured into Ware lid is covered in 10cm culture dish and rapidly；

It with the progress of digestion process, can gradually observe that liver slowly expands, stop digestion after 1 minute, close and wriggle It pumps and carefully removes casing；

It carefully makes each lobe of the liver, and the central part of exposure liver in order with straight forceps, finds the fibre bundle tie point of each lobe of the liver, It uses curved tweezers gripper folder instead and liver is pulled to oneself slightly, exposure thoracic cavity and the various connective tissues for connecting liver.Cut off to careful These connections, and remove gall-bladder；

Liver is immersed immediately and is filled in the 10cm culture dish of digestive juice, close the lid and be transferred in Biohazard Safety Equipment by Requirement according to rigorous aseptic continues subsequent operation；

Lobe of the liver is torn using two secondary sterilized new tweezers, the fibre bundle tie point of liver is clamped with tweezers, is gently shaken To disperse remaining cell, suspension is blown and beaten three times using in Pasteur's Guan Yuan 10cm culture dish, it is thin by 70 μm of strainer filterings Born of the same parents' suspension；

Filtered cell suspending liquid is centrifuged using pre-cooling centrifuge, 4 DEG C are centrifuged 2 minutes；

It is inhaled with sterile Pasteur pipe and abandons supernatant, 25mL pre-cooling growth medium (10% fetal calf serum/DMEMF12) is added, gently Featheriness is beaten several times to dissolve the cell mass of bottom, and is resuspended；

The repetition above-mentioned washing process of step, totally 3 times.Second operate after supernatant should close to transparent, after third time operation on It clearly should be completely limpid；

After the completion of last time is centrifuged, inhales and abandon supernatant and the cold culture medium of 25mL is added.Pasteur's pipe is soft to be resuspended cell.With shifting Liquid device draws the cell suspension after 80uL is resuspended and is used for Trypan Blue and counting；

It is 3x10 that 2ml cell density is added into 12 orifice plates⁵The cell suspension of a/ml shakes gently so that cell distribution Uniformly；

Cell inoculation is placed on be incubated for 60 minutes, 5 hours in incubator after be changed to serum free medium (DMEM/F12)；

Next day replaces fresh growth medium, every other day replaces fresh growth medium later, and culture obtains Mouse Liver Parenchyma.

The method for obtaining mouse primary hepatic stellate cells, specifically includes the following steps:

Using appropriate isoflurane anesthetized mice, abdominal cavity is opened after skin routine disinfection, so that liver, portal vein and cavity of resorption are quiet Arteries and veins sufficiently exposes；

Make portal catheterization with No. 16 trochars, 37 DEG C of perfusion D-Hanks liquid, 10ml/min is full to liver, cuts off down It is ligatured after vena cave bloodletting.Thoracic cavity is opened immediately, is made superior vena cava intubation with No. 16 trochars, is formed portal vein-superior vena cava Circulation；

With 0.1% pronase e 5mL/min perfusion 3 minutes, 0.03%II Collagenase Type 5ml/min was perfused 3 minutes；

Liver is taken out, Glisson's capsule is removed, is shredded with eye scissors.Pronase e is added to final concentration of 0.006%, DNA enzymatic To final concentration of 10ug/ml.37 DEG C, 5%CO₂Incubator digested 30 minutes；

200 mesh screens are crossed, culture solution is washed 4 times, cell is laid in the cell Separating tube for preparing gradient；

4 DEG C of 800rpm horizontal rotors are centrifuged 30 minutes, draw the upper cell of 1.040 density layers, and culture solution is washed 2 times；

Serum-free RPMII640 culture solution is added, in 37 DEG C of 5%CO₂Incubator is incubated for 15 minutes: being drawn cell suspension and is counted Number, with 2x10⁶The density of a/ml is inoculated with, every 3 days replacement fresh growth mediums (10% fetal calf serum/DMEM) of follow-up cultivation, Culture obtains mouse primary hepatic stellate cells.

The method for obtaining mouse Kupffer Cell, specifically includes the following steps:

Mouse liver is taken out, is moved into superclean bench, is weighed and record, shredded liver to about 1mm using eye scissors³ The RMPI1640 liquid 10mL for containing 0.05% pronase e and 0.005%DNase l, room temperature are added according to every gram of liver for size Digestion 10 minutes；

200 mesh net filtrations 2 times obtain hepatic tissue liquid, and 4 DEG C of 1000rpm are centrifuged 7 minutes；

Supernatant is abandoned, and 5mL tri-distilled water is added into pipe, is incubated for 10 seconds and adds 5mL 1.8%NaCl, 4 DEG C of 1000rpm Centrifugation 7 minutes；

Cell is laid in the cell Separating tube for preparing gradient, 4 DEG C centrifugation 1000rpm gradient centrifugation 15 minutes, take Between confluent monolayer cells；

Cell count and according to 10⁶The cell density of a/mL is inoculated with, and 10% fetal calf serum of subsequent use/ RPMI 1640 is cultivated, and replaces fresh growth medium every three days, obtains mouse Kupffer Cell.

Magnetic suspension particle is removed from refrigerator, and in thaw at RT 15min.Choose the mouse that culture density is about 80% Primary hepatic parenchymal cells, hepatic stellate cells and Kupffer Cell magnetic suspension particle are added into culture bottle or culture dish, according to culture The bottle ware floor space magnetic suspension particle every square centimeter that 8ul is added, the light culture bottle that shakes make magnetic suspension particle be evenly distributed on training It supports in base.

Culture bottle is put back in incubator, so that cell is incubated for magnetic suspension particle and in conjunction with magnetic suspension particle, is incubated for 12h discards the culture medium in culture bottle or culture dish, is disappeared using pancreas enzyme -EDTA digestive juice (being purchased from Corning, 25-053-Cl) Change cell 2 minutes, when cell falls off from culture dish or culture bottle bottom, is added into culture bottle or culture dish and is four times in pancreas The serum of enzyme-EDTA digestive juice volume, mixes and is transferred in centrifuge tube, and 300rpm is centrifuged 5 minutes, while by magnetic driven device, 96 Orifice plate and Cover Gasket with 70% alcohol spray disinfectant and are put into Biohazard Safety Equipment.

Cell is resuspended with 3D co-cultured cell growth medium, mixes and the cell in centrifuge tube is counted.

By mouse primary liver parenchyma it is unicellular, withered it is no it is unicellular, it is starlike it is unicellular be inoculated with according to 1: 1: 1 ratio, So that the every hole cell number of 96 orifice plates is about 5000, liquid volume 75ul shakes gently 96 orifice plates and makes the culture medium being added It is uniformly distributed in hole.

Cover Gasket, magnetic driven device and 96 orifice plate lids are successively covered on 96 orifice plates, attached drawing 1 is shown in specific setting, by 96 holes Culture plate is put into incubator and cultivates, and every other day replaces fresh growth medium, final to obtain 3D co-cultured cell.

Wherein the replacing options of culture medium specifically include: 96 orifice plates being removed from incubator, and are sprayed with 70% alcohol Orifice plate outer surface moves into Biohazard Safety Equipment；It successively opens 96 orifice plate lids, magnetic driven device and Cover Gasket and overturns placement；It will Magnetic driven device is placed in 96 orifice plate bottoms, and 3D cell coculture should be attracted by magnetic force and be moved to orifice plate bottom at this time, light to shake 96 orifice plates make 3D coculture offset bore center, and removing culture medium at this time prevents the damage of 3D coculture, and new training is added Base is supported, the magnetic driven device of 96 orifice plate bottoms is removed, Cover Gasket, magnetic driven device and 96 orifice plate lids is successively covered on 96 orifice plates, It is put into incubator and cultivates.

Liver fibrosis inducer 20mM paracetamol, acetparaminosalol are added to the 3D co-cultured cell in 96 orifice plates Phenol processing obtains liver fibrosis 3D model afterwards for 24 hours.

Comparative example:

Liver fibrosis inducer paracetamol is not added, other steps are identical as embodiment.

The performance test of liver fibrosis 3D model:

Degree of hepatic fibrosis observation:

The liver fibrosis 3D model obtained to above-described embodiment and comparative example carries out dyeing processing, obtains liver fibrosis journey Degree dyes specific steps are as follows: the culture medium for removing 3D co-cultured cell is changed to PBS (phosphate buffer saline) Phosphate buffered saline solution, then it is primary with PBS rinse, PBS is removed, 4%PFA paraformaldehyde is added into hole, successively covers Cover Gasket, Magnetic driven device and 96 orifice plate lids after room temperature fixes 15 minutes, remove PFA in hole and are changed to PBS, use PBS rinse again Once, PBS is removed；3D co-cultured cell is made as 5 microns of paraffin sections；It is contaminated using collagen specific antibody Color, DAB method (cytochrome oxidase diaminobenzidine explicit representation) color development, and redyed using hematoxylin eosin staining method, it ties Fruit is observed with the white field of Leica microscope, and result is detailed in attached drawing 2.

The measurement of collagen mrna expression amount:

The total serum IgE of 3D co-cultured cell is extracted using RNA Miniprep kit, and uses ThermoScript RT- PCR system carries out reverse transcription.By resulting cDNA SYBR-Green Master PCR Mix (Applied Biosystem) It is triplicate to carry out qPCR, obtain embodiment and comparative example co-cultured cell collagen mrna expression amount, result such as attached drawing 3 It is shown.

3D co-cultured cell form observation method:

3D co-cultivation is carried out to mouse primary hepatic parenchymal cells, Kupffer Cell, sternzellen using magnetic suspension system, according to The density is seeded in 96 orifice plates, and culture is observed after 24 hours, opens microscope power supply, 96 orifice plates are placed in loading On platform, alignment needs the cell observed, and is adjusted to white field mode, adjusts rough quasi-coil under mirror, so that getting a clear view；Further Using thin quasi- burnt adjustable diameter and screw adjusting focal length, shot using microscope photographing software.3D co-cultured cell form such as 4 institute of attached drawing Show.

According to above-mentioned every detection test it is found that carrying out the liver that liver fibrosis induction obtains using 20mM paracetamol Fibrosis model degree of fibrosis is high, and collagen mrna expression amount is big, can be quickly obtained using magnetic suspension 3D culture systems 3D model, improves efficiency, and saves time cost, and magnetic suspension particle has no adverse effects to sensitive liver cell, does not change Cell viability, it is easily operated.

Embodiment described above is merely preferred embodiments of the present invention, but protection scope of the present invention not office Be limited to this, anyone skilled in the art within the technical scope of the present invention, according to the technique and scheme of the present invention And its design is subject to equivalent substitution or change, should be covered by the scope of protection of the present invention.

Claims

1. an in vitro liver fibrosis 3D model construction method, is characterized in that, described method comprises the steps:

The primary mouse hepatocytes, hepatic stellate cells and Kupffer cells were obtained by enzymatic digestion method and cell culture method;

Select the mouse primary hepatocytes, hepatic stellate cells and Kupffer cells with a culture density of 75%-85%, respectively add magnetically suspended particles, mix them evenly, and incubate in an incubator for 10-12 hours;

Take the incubated hepatocytes, hepatic stellate cells and Kupffer cells, respectively add trypsin-EDTA-digestion solution for digestion for 2-4 minutes, then add serum to mix well, and centrifuge for 5-8 minutes to obtain liver cells containing magnetic particles. Parenchymal single cell, hepatic stellate single cell and Kupffer cell single cell;

Inoculating the magnetic particle-containing hepatocyte single cell, hepatic stellate cell single cell and Kupffer cell single cell onto a 3D medium, and culturing for 15-25 days under magnetic driving conditions to obtain 3D co-cultured cells;

A liver fibrosis inducer is added to the 3D co-cultured cells, and induction is performed to obtain a 3D model of liver fibrosis.

2. The method for constructing a 3D model of liver fibrosis in vitro according to claim 1, wherein the step of obtaining the mouse primary liver parenchyma cells by an enzymatic digestion method-cell culture method specifically comprises:

The mouse liver was perfused with HBSS solution and perfused with type IV collagenase to digest the tissue to obtain liver I;

The liver I was moved into a petri dish containing the digestive solution, the liver lobe was torn and the connection point of the liver lobe fiber bundle was gently shaken to obtain the hepatocyte suspension I;

The hepatocyte suspension I was filtered, centrifuged and resuspended, the cells were washed three times with a growth medium, the washed hepatocyte suspension I was resuspended with a pre-cooled growth medium, and the cells were inoculated and cultured. Primary mouse hepatocytes were obtained.

3. The method for constructing a 3D model of hepatic fibrosis in vitro according to claim 1, wherein the step of obtaining mouse primary hepatic stellate cells by enzymatic digestion method-cell culture method specifically comprises:

The mouse liver was perfused with D-Hanks, and perfused with pronase E and type II collagenase, respectively, to obtain liver II;

The liver II was removed from the liver envelope and cut into pieces, added with pronase E and DNase I, and placed in an incubator to digest to obtain hepatocyte suspension II;

Said hepatocyte suspension II was sieved and washed with growth medium, centrifuged and the upper cells were aspirated;

The upper layer cells were washed with growth medium and resuspended in pre-cooled growth medium, and then inoculated and cultured to obtain mouse primary hepatic stellate cells.

4. The method for constructing an in vitro 3D model of liver fibrosis according to claim 1, wherein the step of obtaining the mouse primary Kupffer cells by an enzymatic digestion method-cell culture method specifically comprises:

The mouse liver was perfused with D-Hanks to obtain liver III;

The liver III is cut into pieces, and pronase E and DNase 1 are added to digest to obtain liver tissue filtrate;

The liver tissue filtrate was filtered to obtain liver tissue fluid, the liver tissue fluid was centrifuged to remove the lower layer of cell fluid, incubated with tri-distilled water for 10-15s, then added with 1.8% sodium chloride, centrifuged to obtain cell suspension III, and inoculated and cultured to obtain cell suspension III. Murine primary Kupffer cells.

5 . The method for constructing a 3D model of liver fibrosis in vitro according to claim 1 , wherein the rotational speed of the centrifugation process is 300-400 rpm. 6 .

6 . The method for constructing a 3D model of liver fibrosis in vitro according to claim 1 , wherein the magnetic particles-containing single cells of hepatocytes, single cells of hepatic stellate cells and single cells of Kupffer cells are inoculated. 7 . In the process of reaching the 3D medium, the ratio of the number of single cells of hepatocytes, single cells of hepatic stellate cells and single cells of Kupffer cells was 1:0.8-1.2:0.8-1.2.

7. The method for constructing a 3D model of liver fibrosis in vitro according to claim 1, wherein the magnetic particle-containing single cells of hepatocytes, single cells of hepatic stellate cells and single cells of Kupffer cells are inoculated To 3D medium, the number of cells seeded per medium is 4000-6000.

8. The method for constructing a 3D model of liver fibrosis in vitro according to claim 1, wherein the 3D medium is 10%-15% fetal bovine serum/DMEMF12.

9. The method for constructing a 3D model of in vitro hepatic fibrosis according to claim 1, wherein the hepatic fibrosis inducing agent is any one of acetaminophen, carbon tetrachloride and dimethylnitramine .

10 . The method for constructing a 3D model of liver fibrosis in vitro according to claim 1 , wherein the induction time is 20-30 h. 11 .