CN103509751A - Co-culturing method of human primary hepatocytes and liver nonparenchymal cells - Google Patents
Co-culturing method of human primary hepatocytes and liver nonparenchymal cells Download PDFInfo
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Abstract
The invention discloses a co-culturing method of human primary hepatocytes and liver nonparenchymal cells. The co-culturing method comprises following steps: fresh hepatic tissue is washed thoroughly with exposed blood vessel perfusate I and perfusate II until elasticity of the hepatic tissue is lost, and complete digestion is realized; the hepatic tissue is delivered into a culture dish filled with a cell washing liquor, and is screened so as to obtain a single-cell suspension; a culture plate or a culture dish coated with collagen is inoculated with the single-cell suspension with a low inoculum density so as to obtain monolayer co-cultured cells with a fusion degree of 100% in the presence of growth factors; and a maintaining culture medium containing 2% DMSO is added into the culture plate or the culture dish for culturing, and then a liver island structure is formed by accumulation of hepatic cells, wherein the liver island structure is surrounded and invaded by liver nonparenchymal cells. Routine hepatocyte separation technology is employed in the co-culturing method, in vitro long-term culturing of hepatocytes as long as 120 days are realized, and HBV susceptibility is maintained for as long as 72 days. In addition, the co-culturing method can be used for screening and evaluating anti-HBV medicines, and possesses significant importance on researches of antiviral medicines.
Description
Technical field
The present invention relates to the cultural method of cell, be specifically related to a kind of easy, can long term maintenance human primary hepatocyte characteristic and liver cell and the liver non-parenchymal cell co-culture method of HBV susceptibility.
Background technology
Liver cell can be divided into hepatic parenchymal cells and liver non-parenchymal cell in functional classification, and hepatic parenchymal cells is liver cell, fulfils the most of function of liver as digestive function, energy metabolism, removing toxic substances etc.; And liver non-parenchymal cell comprises stellate cells, hole shape endotheliocyte, liver epithelial cell, Kupffer cell etc., the main functions such as auxiliary liver cell, Liver immunity and liver structure support of exercising.So far, traditional co-culture method is by pure primary hepatocyte and pure liver non-parenchymal cell mixed culture by a certain percentage, in common culturing process, liver cell and liver non-parenchymal cell have recovered the natural communication between part cell and cell, therefore, the morphological specificity of primary hepatocyte and physiological function can be maintained to greatest extent.Yet this traditional co-culture system has critical defect, for example limited cell survival, complicated loaded down with trivial details purification step and vital co-cultured cell ratio are difficult to grasp.Therefore, be badly in need of a kind of method easily that can make in vitro hepatocyte differentiation characteristic be able to long term maintenance of exploitation.Utilize primary hepatocyte and liver non-parenchymal cell mixture through the co-culture method of low density bed board, can form that liver cell piles up, liver non-parenchymal cell is around the island structure of, invasion growth, the method is relatively simple, do not need loaded down with trivial details liver cell and nonparenchymal cell purge process, the differentiation characteristic that has greatly extended primary hepatocyte is held time, and the longest holding time reaches 120 days.
It is a kind of common disease that hepatitis B virus (english abbreviation is HBV) infects, and approximately there are 1.7 hundred million HBVer, the patient approximately 1,000,000 who dies from every year relative disease in China.Due to long-term lacking suitable anti-HBV drug screening and evaluation platform, therapeutic strategy and the method for HBV relatively lag behind.Cell model for anti-HBV drug screening and evaluation is confined to hepatoma cell line for a long time as HepG2, HepG2.2.15, Huh7 etc.Because these clones are dedifferented seriously, compare on differentiation state and differ greatly with liver cell in body, so can not reflect the real antiviral effect of medicine and cytotoxicity when screening and the anti-HBV medicine of evaluation.These clones do not support HBV to infect worse, can not be for anti HBV infecting medicine as researchs such as little peptide, antibody.Human primary hepatocyte is that HBV studies ideal cell infection model, a large amount of research shows that primary hepatocyte can be infected by HBV, but when cultivating in vitro, it loses very soon differentiation characteristic, the susceptibility of HBV was also lost completely conventionally in 1 week, and this has seriously restricted the foundation to the cell model of the research of HBV infection mechanism, drug screening and evaluation.Method described in patent of the present invention, by the common cultivation of human primary hepatocyte and liver non-parenchymal cell, the liver cell characteristic of primary hepatocyte is able to long term maintenance as morphological specificity, albuminous expression, the susceptibility of HBV is maintained and can reach 72 days, greatly promoted the using value of human primary hepatocyte for anti-HBV drug screening and evaluation.
A large amount of researchs show, hepatocyte transplantation is distinguished to liver failure patient's result for the treatment of, can greatly alleviate patient to the demand for liver, are a kind of very promising therapeutic strategies.Because Hepatocytes culture in vetro is relatively backward, the long term maintenance that realizes hepatocellular amplification in vitro and hepatocyte differentiation characteristic remains the difficult problem of pendulum in face of medical worker.
Summary of the invention
Technical problem to be solved by this invention is just to provide a kind of human primary hepatocyte and liver non-parenchymal cell co-culture method, human primary hepatocyte and liver non-parenchymal cell mixture in vitro, through low density bed board with through cultivating, formed that liver cell piles up, liver non-parenchymal cell is around the liver island structure of, invasion growth, realized reach 120 days the external long-term cultivation of liver cell with reach 72 days and keep HBV susceptibility.
For solving the problems of the technologies described above, a kind of human primary hepatocyte of the present invention and liver non-parenchymal cell co-culture method, comprise the following steps:
1) blood vessel of fresh hepatic tissue through exposing pours into approximately 30 minutes with primer solution I, until blood is rinsed totally in hepatic tissue;
2) hepatic tissue of rinsing well in the primer solution II perfusion step 1) with 37 ℃ of preheatings, until hepatic tissue follows the string, digestion is completely;
3) by step 2) digest hepatic tissue completely and be transferred in the culture dish that contains cell washing lotion, the cell of carefully shaking off to have digested, forms cell suspension;
4) by cell suspension in step 3), the cell by 70 μ m sieves, and obtains single cell suspension;
5) single cell suspension step 4) being obtained under 50 * g and 4 ℃ of conditions, centrifugal 5 minutes sedimentation liver cells and liver non-parenchymal cell, resuspended by above-mentioned cell washing lotion, repeated centrifugation three times, then utilizes bed board nutrient solution re-suspended cell;
6) utilize the blue staining calculation procedure 5 of platform phenol) in cell density and the cell viability of re-suspended cell;
7) by single cell suspension in step 6) with≤8 * 10
4viable cell/cm
2cell density be inoculated in collagen coated culture plate or culture dish, the coated object of collagen is to promote maintaining of cell attachment and liver cell characteristic, adds bed board nutrient solution, shakes up, at 37 ℃, 5% CO
2in incubator, cultivate fully to allow it adherent; After 4 to 6 hours, add grown cultures liquid to cultivate, within 3 to 4 days, change 1 secondary growth nutrient solution;
8) cultivate 28 to 35 days, add the maintain liquid that contains 2%DMSO, within 3 to 4 days, change 1 time maintain liquid;
9) cultivate 30 to 40 days, liver cell pile up to form liver non-parenchymal cell around the liver island structure of, invasion growth;
Wherein, described primer solution I for specifically filling a prescription is: 8.00g/l sodium-chlor, 0.40g/l Repone K, 3.57g/l hydroxyethyl piperazine ethanesulfonic acid, 0.06g/l phosphate dihydrate disodium hydrogen, 0.06g/l potassium primary phosphate, 1g/l glucose, 1mM pyruvic acid are received, 2mM ethyleneglycolbis(2-aminoethylether)tetraacetic acid, adjusts pH to 7.4;
The concrete formula of described primer solution II is: 8.00g/l sodium-chlor, 0.40g/l Repone K, 3.57g/l hydroxyethyl piperazine ethanesulfonic acid, 0.06g/l phosphate dihydrate disodium hydrogen, 0.06g/l potassium primary phosphate, 1g/l glucose, 1mM pyruvic acid are received, 5mM calcium chloride, 0.3g/l type Ⅳ collagenase, adjusts pH to 7.4;
Described cell washing lotion is: the DMEM nutrient solution (Gou Zhi U.S. GIBCO company) of buying in commercialization adds 10% v/v foetal calf serum, 100units/ml penicillin, 100mg/ml Streptomycin sulphate before using;
Described bed board nutrient solution is: William ' the s E medium(buying in commercialization is called for short WE, Gou Zhi U.S. GIBCO company) before use, add 10mM nicotinamide, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10
-7m dexamethasone, ITS+premix, 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 5% v/v foetal calf serum; Wherein, ITS+premix contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid;
Described grown cultures liquid is: the WE buying in commercialization adds 10mM nicotinamide, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10 before using
-7m dexamethasone, ITS+premix, 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 20ng/ml Urogastron; Wherein, ITS+premix contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid;
Maintain liquid is: the WE buying in commercialization adds 10mM nicotinamide, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10 before using
-7m dexamethasone, ITS+premix, 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 2% v/v DMSO; Wherein, ITS+premix contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid.
As preferred version, in described step 6), the method of determining cell density and cell viability is: the volume ratio by the blue staining fluid of cell suspension and 0.4% m/v platform phenol with 1 ︰ 1 mixes, rapidly 20 μ l mixed solutions are joined in tally and by this tally and inserted in corresponding calculating instrument, instrument is read corresponding cell density and cell viability, the blue staining fluid specific configuration of 0.4% phenol method is: the blue solid of 0.4g platform phenol is dissolved in the phosphoric acid buffer liquor of 100ml (english abbreviation is PBS) completely, concrete formula is: 8.0g/l sodium-chlor, 0.2g/l Repone K, 3.58g/l disodium hydrogen phosphate, 0.24g/l potassium primary phosphate, pH7.2 to 7.4, high pressure steam sterilization, 4 ℃ of maintenances are standby.
As preferred version, the culture plate that described collagen is coated or the preparation method of culture dish: 172 μ l Glacial acetic acid are joined in 100ml deionized water, then to add final concentration be the IV type mouse tail collagen of 50 μ g/ml, under aseptic condition, after 0.22 μ m membrane filtration degerming, 4 ℃ of maintenances are stand-by; By this working fluid with 5 μ g/cm
2package amount join and need in the coated culture plate or culture dish of processing, normal temperature is hatched; After 1 hour, by the sucking-off of collagen working fluid, naturally dry 4 ℃ of preservations of rear sealing; With front, the culture plate that collagen is coated or culture dish need clean once with PBS, to remove residual acetic acid.
Beneficial effect of the present invention is:
One, the present invention utilizes conventional liver cell isolation technique, be separated to the mixture of liver cell and liver non-parenchymal cell, with low-density mode bed board, through factors stimulated growth, finally form the monolayer cell culture of 100% degrees of fusion, realized the amplification in vitro of human primary hepatocyte and liver non-parenchymal cell, be expected to develop into the cell derived of new hepatocyte transplantation;
Its two, the present invention is easier with respect to conventional co-culture method, does not need the blockade operation of purifying liver cell and liver non-parenchymal cell;
They are three years old, this co-culture system is under the induction of DMSO, and liver cell is piled into liver non-parenchymal cell around the liver island structure of, invasion, and the liver cell on island can long term maintenance primary hepatocyte proterties, the longest culture cycle can reach 120 days, has overcome short defect of primary hepatocyte life-span;
They are four years old, liver cell on the island that the present invention obtains is under the maintaining of DMSO, the susceptibility of HBV can maintain and reach 72 days, this has greatly overcome the defect that liver primary cell is lost in 1 week the susceptibility of HBV, tentatively set up novel anti-HBV and infected the In vitro cell model of drug screening and evaluation, this co-culture method is that the amplification in vitro in medical liver cell source maintains possibility is provided with long-term liver cell characteristic, and this system can develop into new anti-HBV drug screening and evaluation platform, the research of enantiopathy cytotoxic drug has important value.
Accompanying drawing explanation
Fig. 1 is for being total to culture hepatocyte at the morphological observation of different incubation time points;
Fig. 2 is that morphological observation and the immunofluorescence of liver island structure detects;
Fig. 3 is the liver cell that HBV infects long-term cultivation;
Fig. 4 is that liver island structure forms and HBV successfully infects mode chart.
Embodiment
In order to explain better the present invention, below in conjunction with specific embodiment, further illustrate main contents of the present invention, but content of the present invention is not only confined to following examples.
1. liang separated human primary hepatocyte of step collagenase perfusion method and liver non-parenchymal cell:
The two step collagenase perfusion method the first steps: the blood vessel of fresh hepatic tissue material through exposing pours into approximately 30 minutes with primer solution I, until blood is rinsed totally in hepatic tissue.The concrete formula of primer solution I is: 8.00g/l sodium-chlor, 0.40g/l Repone K, 3.57g/l hydroxyethyl piperazine ethanesulfonic acid, 0.06g/l phosphate dihydrate disodium hydrogen, 0.06g/l potassium primary phosphate, 1g/l glucose, 1mM pyruvic acid are received, 2mM ethyleneglycolbis(2-aminoethylether)tetraacetic acid, adjust pH to 7.4, with 0.22 μ m millipore filter filtration sterilization, 4 ℃ of storages.
Two step collagenase perfusion method second steps: pour into until hepatic tissue follows the string with the primer solution II of 37 ℃ of preheatings.The concrete formula of primer solution II is: 8.00g/l sodium-chlor, 0.40g/l Repone K, 3.57g/l hydroxyethyl piperazine ethanesulfonic acid, 0.06g/l phosphate dihydrate disodium hydrogen, 0.06g/l potassium primary phosphate, 1g/l glucose, 1mM pyruvic acid are received, 5mM calcium chloride, 0.3g/l type Ⅳ collagenase (Gou Zhi U.S. Sigma company), adjust pH to 7.4, with 0.22 μ m millipore filter filtration sterilization, 4 ℃ of storages.
The hepatic tissue having digested is transferred in the culture dish that contains cell washing lotion, the cell of carefully shaking off to have digested, forms cell suspension.Cell washing lotion is the DMEM nutrient solution (Gou Zhi U.S. GIBCO company) that commercialization is bought, and adds 10% v/v foetal calf serum (Gou Zhi U.S. GIBCO company), 100units/ml penicillin, 100mg/ml Streptomycin sulphate before use.By this cell suspension, the cell by 70 μ m sieves again, to obtain single cell suspension.Single cell suspension is transferred in whizzer centrifugal with sedimentation liver cell and liver non-parenchymal cell, centrifugal condition is: 50 * g, 4 ℃, 5 minutes.Resuspended by cell washing lotion, repeat above centrifugal three times.Finally, utilize bed board nutrient solution re-suspended cell, the blue staining of recycling platform phenol is calculated cell density and cell viability.The bed board nutrient solution of re-suspended cell is that William ' the s E medium(that commercialization is bought is called for short WE, Gou Zhi U.S. GIBCO company), before use, add 10mM nicotinamide, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10
-7m dexamethasone, ITS+premix(contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid), 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 5% v/v foetal calf serum (Gou Zhi U.S. GIBCO company).
2. the detection of cell concn and cell viability:
Volume ratio by the blue staining fluid of cell suspension and 0.4% m/g platform phenol with 1 ︰ 1 mixes, rapidly 20 μ l mixed solutions are joined in tally to (Gou Zhi U.S. Counterstar company) and this tally is inserted in corresponding calculating instrument, instrument is read corresponding cell density and cell viability.0.4% the blue staining fluid specific configuration of phenol method is that in the phosphoric acid buffer liquor of 100ml, (english abbreviation is PBS to the blue dissolution of solid of 0.4g platform phenol, concrete formula is: 8.0g/l sodium-chlor, 0.2g/l Repone K, 3.58g/l disodium hydrogen phosphate, 0.24g/l potassium primary phosphate, pH7.2 to 7.4, high pressure steam sterilization, 4 ℃ of maintenances are standby).
3. human primary hepatocyte and liver non-parenchymal cell mixture low density bed board and cultivation:
The single cell suspension obtaining through method (1) separation, to be less than or equal to 8 * 10
4viable cell/cm
2density be inoculated in collagen coated culture plate or culture dish, add appropriate bed board nutrient solution, shake up, at 37 ℃, 5% CO
2in incubator, cultivate fully to allow cell attachment.Bed board nutrient solution is the WE that commercialization is bought, and adds 10mM nicotinamide, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10 before use
-7m dexamethasone, ITS+premix(contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid), 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 5%v/v foetal calf serum (Gou Zhi U.S. GIBCO company).After 4 to 6 hours, by the sucking-off of bed board nutrient solution, add fresh grown cultures liquid, within every 3 to 4 days, change 1 time fresh medium.Grown cultures liquid is the WE that commercialization is bought, and adds 10mM nicotinamide, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10 before use
-7m dexamethasone, ITS+premix(contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid), 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 20ng/ml Urogastron (purchasing to U.S. company BD).Cultivate approximately after one month, typical monolayer culture forms, and adds the maintain liquid containing 2% v/v DMSO, within 3 to 4 days, changes one time fresh medium.Maintain liquid is the WE that commercialization is bought, and adds 10mM nicotinamide, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10 before use
-7m dexamethasone, ITS+premix(contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid), 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 2% v/v DMSO.Cultivate approximately after one month, liver cell is piled up and forms liver non-parenchymal cell around the liver island structure of, invasion growth.This co-culture system generally also can be cultivated and reach two months.
Detect and analyze
1. pair this co-culture system carries out liver cell Characteristics Detection:
On the one hand, the liver cell form at different incubation time points with phase microscope morphological observation; On the other hand, utilize the method for immunofluorescence to identify albuminous expression.
The concrete grammar of albumin immunofluorescence is: take a hole of six orifice plates is example, during cell cultures to two month, suck old nutrient solution, with normal temperature PBS, wash once, with the concrete formula of 4% ice-cold paraformaldehyde 1ml(, be immediately: take 4g paraformaldehyde, be placed in Erlenmeyer flask, add 80ml deionized water, put into 37 ℃ of constant water bath box, every 1 to 2 hour, rock and mix, after 16 to 24 hours, paraformaldehyde can dissolve completely, supplements deionized water to 100ml, regulate pH value to 7.2), normal temperature is fixed 10 to 15 minutes; With PBS, on horizontal shaking table, wash 3 times each 5 minutes; For strengthening the combination of antibody and intracellular protein, use 1ml to process 45 minutes containing the PBS solution normal temperature of 0.25% v/v Triton100; With PBS, on horizontal shaking table, wash 3 times each 5 minutes; Sealing, adds appropriate confining liquid (containing 5% v/v goat normal serum, the PBS solution of 2% m/v BSA), and normal-temperature water yawing bed is hatched at least 1 hour; Utilize the PBS solution dilution antibody containing 3% v/v goat normal serum, how anti-(the Gou Zhi U.S. Proteintech company) Dilution ratio of albumin rabbit is 400,4 ℃ of shaking table overnight incubation of 1 ︰; With the PBS solution containing 3% goat normal serum, on horizontal shaking table, wash 3 times again, each 10 minutes, to remove the antibody of non-specific binding; Two resist for goat antirabbit lgG coupling DyLight-488(Gou Zhi U.S. Thermo Fisher Scientific Inc company), with 1 ︰ 400, be diluted to containing the PBS solution ，Yu dark place lucifuge normal temperature of 3% goat normal serum and hatch 1 hour, after this operate same lucifuge; Again with washing 2 times containing the PBS solution of 3% goat normal serum, each 10 minutes, while cleaning for the third time, add the DAPI(Gou Zhi Switzerland Roche company of 1 ︰ 1000 on horizontal shaking table) for nucleus dyeing, normal temperature is hatched 10 minutes; Clean once to wash residual DAPI again 10 to 20 minutes; Add the appropriate PBS solution containing 3% goat normal serum to prevent that cell is because of drying distortions when observing.Finally, utilize Leica DFC425C fluorescent microscope (purchase to German Leica company) to observe fluorescent signal.
Interpretation of result
A. altogether culture hepatocyte in the morphological observation analysis of different incubation time points:
The unicellular mixture that utilizes above-mentioned two step collagenase perfusion method separation to obtain comprises maxicell (mature hepatocytes), minicell (prematurity liver cell), stellate cells, liver epidermic cell, hole shape endotheliocyte etc.For fear of the contact inhibition of primary cell, this mixture (is less than or equal to 8 * 10 with low density
4viable cell/cm
2) mode bed board, under the cultivation of growth medium, the fast breeding of cell cultured supernatant and liver non-parenchymal cell so greatly.As shown in Fig. 1 (A), in 10Tian Shi liver cell district (large arrow) by nonparenchymal cell around, even it is not tight that liver cell contacts with liver non-parenchymal cell, there is certain acellular district, now cell still needs continued growth, bile canaliculus clear in structure visible (small arrow) between liver cell, and be mainly stellate cells (pentagram) around what grow; After 30 days (as B figure), cell tightness degree is deepened, and particularly nonparenchymal cell district, is now the terminal of grown cultures, and it is unfavorable to long-term cultivation to continue to cultivate in growth medium; After this, cell is being cultivated containing in the maintain base of 2%DMSO, and in the time of the 60th day, this is cultivated noticeable change has occurred altogether, and liver cell assembles projection, and bile canaliculus structure disappears, and the boundary line of liver cell and nonparenchymal cell is highlighted obviously, forms typical liver island structure; Ensuing cultivation, the 80th day (as D figure), and when the 100th day (as E figure), cell state does not have considerable change, from cellular form liver cell, still maintains true hepatocyte differentiation state; But the 120th day (as F figure), liver cell state is fair, and there is apoptosis and catabiosis in liver non-parenchymal cell, and there are collective's phenomena of mortality in indivedual regions, and we are defined as this time point at the terminal of common cultivation.Picture scale is 100 microns.
B. the morphological observation of liver island structure and immunofluorescence detect:
In order further to understand liver island structure and hepatocellular state, first utilize phase microscope under the same visual field, to observe same liver island structure, in different focal planes, take pictures, as Fig. 2 (A, B, C) shown in, liver cell is piled into the length of clustering, and on horizontal plane, liver cell grows higher than nonparenchymal cell; In order further to understand fully whether the liver cell whether liver non-parenchymal cell intrudes on bottom growth Ji Gan island, liver island has liver cell characteristic, we utilize the method for immunofluorescence to detect hepatocellular Expression of Albumin and whether liver cell nuclear is overlapping with nonparenchymal cell, as Fig. 2 (D: green, albumin; E: blueness, nucleus; F: albumin and nucleus overlay chart under the same visual field), liver cell expression albumin on liver island, illustrate that this liver cell not yet loses liver cell characteristic, moreover the nucleus of liver cell and liver non-parenchymal cell partly overlaps, illustrate that liver non-parenchymal cell intrudes into hepatocellular bottom, form the feeder layer structure in a kind of similar cytobiology.This structure is that liver cell is able to the key point that long term maintenance is cultivated.Picture scale is 100 μ m.
2.HBV infects the liver cell of long-term cultivation:
After bed board 42 days to 72 days, carried out the investigation of HBV susceptibility.The collocation method that infects liquid is: in WE, add 2% v/v DMSO, 4% m/v(mass volume ratio) (virus titer is 2 * 10 for the patients serum of PEG 8000, the high virus titer of 10% v/v
9copy/ml).The infection liquid configuring joins in cell to be infected, and hatches 16 to 24 as a child, removes infection liquid, with phosphoric acid buffer, cleans 3 times, adds the fresh maintain liquid containing 2%DMSO, within 2 days, changes fresh nutrient solution 1 time; Collect old nutrient solution as enzyme linked immunosorbent assay, analyze the secretion level of hepatitis B virus surface antigen (english abbreviation is HBsAg), HBeAg (english abbreviation is HBeAg).In the later stage of cultivating, after cell is fixing, by the method for immunofluorescence, detect the expression of intracellular nucleic heart antigen (english abbreviation is HBcAg).
ELISA and immunofluorescence detect the key index detection method after virus infection:
The hepatitis B virus e antigen diagnostic kit specification sheets providing with reference to Shanghai Xiamen Kehua is provided HBeAg, first, 25 times of concentrated washingss that provide in test kit are diluted to the working concentration of 1 times, concrete grammar is: measure 480ml purified water, add 20ml concentrated cleaning solution, after fully mixing, become 500ml working concentration washings, stand-by.Every hole adds testing sample 50 μ l, and each sample is established three repetitions, establishes each 2 Kong，Mei holes of positive and negative contrast and adds negative control or each 50 μ l of positive control, and establish blank 1 hole; Follow every hole and add enzyme conjugates 50 μ l, fully mix, shrouding, is placed in 37 ℃ and hatches 30 minutes; Then, by hand wash plate, discard liquid hole in, the washings providing with test kit is filled with each hole, and standing 5 seconds, drying, patted dry after repeating five times; Developer A liquid that Xi Banhoumei hole adds test kit to provide, each 50 μ l(of developer B liquid or 1 are provided), fully mix, shrouding, puts 37 ℃ and hatches 15 minutes, every hole adds stop buffer 50 μ l(or 1 afterwards), mix; Finally use microplate reader reading, get wavelength 450nm, first, with blank well school zero, then read each hole OD value.
The hepatitis B virus surface antigen diagnostic kit specification sheets providing with reference to Shanghai Xiamen Kehua is provided HBsAg, first 25 times of concentrated washingss that provide in test kit are diluted to the working concentration of 1 times, concrete grammar is: measure 480ml purified water, add 20ml concentrated cleaning solution, after fully mixing, become 500ml working concentration washings, stand-by; Then add 75 μ l testing samples (three repetitions of each sample) and positive and negative in comparison with (reserved negative control 3 holes, positive control 1 hole, the reserved blank of suggestion 1 hole altogether) in reacting hole, with mounting paper, cover after Sptting plate, Sptting plate is placed in to 37 ℃ and hatches 60 minutes; Take out Sptting plate, tear mounting off, in the hole that adds testing sample and negative, positive control, add 50 μ l enzyme conjugates, in 10 seconds of manual light shaking, with mounting paper, cover after Sptting plate afterwards, Sptting plate is placed in to 37 ℃ and hatches 30 minutes; Take out Sptting plate, tear mounting paper off, washing reaction plate 5 times (washing plate by hand: discard liquid in hole, fill with each hole with the working concentration washings of preparation, standing 30 to 60 seconds, drying, repeated, after 5 times, to pat dry on clean thieving paper); After washing finishes, in institute, add respectively 50 μ l of developer A, developer B that test kit provides in porose immediately, mix, 10 seconds of manual light shaking.Then with mounting, cover after Sptting plate, Sptting plate is put to 37 ℃ and hatch 30 minutes, every hole adds 50 μ l stop buffers afterwards, and concussion reacted for 5 seconds, made it fully to mix.Finally use microplate reader reading, get wavelength 450nm,, first with blank well school zero, then read each hole OD value.
The concrete grammar of analyzing the cAg HBcAg immunofluorescence of HBV is: take a hole of six orifice plates is example, and cell infection, after at least 12 days, sucks old nutrient solution, with normal temperature PBS, washes once, uses immediately 4% ice-cold paraformaldehyde 1ml, and normal temperature is fixed 10 to 15 minutes; With PBS, on horizontal shaking table, wash 3 times each 5 minutes; For strengthening the combination of antibody and intracellular protein, use 1ml to process 45 minutes containing the PBS solution normal temperature of 0.25% v/v Triton100; With PBS, on horizontal shaking table, wash 3 times each 5 minutes; Sealing, adds appropriate confining liquid (containing 5% v/v goat normal serum, the PBS solution of 2% m/v BSA), and normal-temperature water yawing bed is hatched at least 1 hour; Utilize the PBS solution dilution antibody containing 3% goat normal serum, how anti-(the Gou Zhi Denmark Dako company) Dilution ratio of HBcAg rabbit is 400,4 ℃ of shaking table overnight incubation of 1 ︰; With the PBS solution containing 3% goat normal serum, on horizontal shaking table, wash 3 times again, each 10 minutes, to remove the antibody of non-specific binding; Two anti-goat antirabbit lgG coupling DyLight-488(Gou Zhi U.S. Thermo Fisher Scientific Inc companies) with 1 ︰ 400, be diluted to containing the PBS solution ，Yu dark place lucifuge normal temperature of 3% goat normal serum and hatch 1 hour, after this operate same lucifuge; With the PBS solution containing 3% goat normal serum, on horizontal shaking table, wash 2 times again, each 10 minutes, while cleaning for the third time, add the DAPI (Gou Zhi Switzerland Roche company) of 1 ︰ 1000 for nucleus dyeing, normal temperature is hatched 10 minutes; Clean once to wash residual DAPI again 10 to 20 minutes; Add the appropriate PBS solution containing 3% goat normal serum to prevent that cell is because of drying distortions when observing.Finally, utilize Leica DFC425C fluorescent microscope (purchase to German Leica company) to observe fluorescent signal.
Interpretation of result
HBV infects the liver cell of long-term cultivation and analyzes
According to world report and wes' the data of not delivering, from primary hepatocyte self-separation, due to dedifferen tiation fast, can be about one week loss totally to the susceptibility of HBV.Yet under our co-culture system, primary hepatocyte was on bed board 42 days and within 72 days, still have HBV susceptibility.As shown in Figure 3, A, B represents respectively this co-culture system HBsAg after bed board infected after 42 days, the secretion level of HBeAg: on the one hand, HBsAg and HBeAg first reduce rear rising, represents that the liver cell under this system has HBV susceptibility; On the other hand, metainfective liver cell still can survive approximately 10 weeks, illustrates with liver non-parenchymal cell and cultivates altogether, can not only keep hepatocellular HBV susceptibility, can also keep HBV to infect rear hepatocellular vigor.The infection (Fig. 3, C, D) of bed board after 72 days, HBsAg, the virus antigen secretion of the secretion level of HBeAg and 42 days is consistent substantially, illustrates that this co-culture system of this time point still has HBV susceptibility.The positive immunofluorescence signal of intracellular virus cAg HBcAg (Fig. 3, E) has more absolutely proved that this liver cell of cultivating altogether for a long time has susceptibility to HBV.
3. liver island structure forms the mode chart structure successfully infecting with HBV:
For embodiment of the present invention are further described simply, we have proposed following mode chart.As shown in Figure 4, liver cell and liver non-parenchymal cell mixture are with low-density mode bed board (A); Under the cultivation of growth medium, liver cell and liver non-parenchymal cell be liver non-parenchymal cell Fast Growth particularly, is paved with whole culture dish (B); Under the maintaining of DMSO, liver cell around, invasion, is formed liver island structure (C) by liver non-parenchymal cell; Liver cell energy long term maintenance liver cell characteristic on this island structure and to the susceptibility of HBV (D).
Claims (3)
1. human primary hepatocyte and liver non-parenchymal cell co-culture method, is characterized in that: comprise the following steps:
1) blood vessel of fresh hepatic tissue through exposing pours into approximately 30 minutes with primer solution I, until blood is rinsed totally in hepatic tissue;
2) hepatic tissue of rinsing well in the primer solution II perfusion step 1) with 37 ℃ of preheatings, until hepatic tissue follows the string, digestion is completely;
3) by step 2) digest hepatic tissue completely and be transferred in the culture dish that contains cell washing lotion, the cell of carefully shaking off to have digested, forms cell suspension;
4) by cell suspension in step 3), the cell by 70 μ m sieves, and obtains single cell suspension;
5) single cell suspension step 4) being obtained under 50 * g and 4 ℃ of conditions, centrifugal 5 minutes sedimentation liver cells and liver non-parenchymal cell, resuspended by above-mentioned cell washing lotion, repeated centrifugation three times, then utilizes bed board nutrient solution re-suspended cell;
6) utilize the blue staining calculation procedure 5 of platform phenol) in cell density and the cell viability of re-suspended cell;
7) by single cell suspension in step 6) with≤8 * 10
4viable cell/cm
2cell density be inoculated in collagen coated culture plate or culture dish, the coated object of collagen is to promote maintaining of cell attachment and liver cell characteristic, adds bed board nutrient solution, shakes up, at 37 ℃, 5% CO
2in incubator, cultivate fully to allow it adherent; After 4 to 6 hours, add grown cultures liquid to cultivate, within 3 to 4 days, change 1 secondary growth nutrient solution;
8) cultivate 28 to 35 days, add the maintain liquid that contains 2%DMSO, within 3 to 4 days, change 1 time maintain liquid;
9) cultivate 30 to 40 days, liver cell pile up to form liver non-parenchymal cell around the liver island structure of, invasion growth;
Wherein, the concrete formula of described primer solution I is: 8.00g/l sodium-chlor, 0.40g/l Repone K, 3.57g/l hydroxyethyl piperazine ethanesulfonic acid, 0.06g/l phosphate dihydrate disodium hydrogen, 0.06g/l potassium primary phosphate, 1g/l glucose, 1mM pyruvic acid are received, 2mM ethyleneglycolbis(2-aminoethylether)tetraacetic acid, adjust pH to 7.4;
The concrete formula of described primer solution II is: 8.00g/l sodium-chlor, 0.40g/l Repone K, 3.57g/l hydroxyethyl piperazine ethanesulfonic acid, 0.06g/l phosphate dihydrate disodium hydrogen, 0.06g/l potassium primary phosphate, 1g/l glucose, 1mM pyruvic acid are received, 5mM calcium chloride, 0.3g/l type Ⅳ collagenase, adjust pH to 7.4;
The concrete formula of described cell washing lotion is: in DMEM nutrient solution, add 10% v/v foetal calf serum, 100units/ml penicillin, 100mg/ml Streptomycin sulphate;
Described bed board nutrient solution is: William ' s E medium adds 10mM nicotinamide before use, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10
-7m dexamethasone, ITS+premix, 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 5% v/v foetal calf serum; Wherein, ITS+premix contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid;
Described grown cultures liquid is: William ' s E medium adds 10mM nicotinamide before use, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10
-7m dexamethasone, ITS+premix, 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 20ng/ml Urogastron; Wherein, ITS+premix contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid;
Maintain liquid containing 2% v/v DMSO is: William ' s E medium adds 10mM nicotinamide before use, 20mM hydroxyethyl piperazine ethanesulfonic acid, 17mM sodium bicarbonate, 550mg/l Sodium.alpha.-ketopropionate, 0.2mM xitix-2-phosphoric acid, 14mM glucose, 2mM glutamine, 10
-7m dexamethasone, ITS+premix, 100units/ml penicillin, 100mg/ml Streptomycin sulphate, 2% v/v DMSO; Wherein, ITS+premix contains 6.25 μ g/ml Regular Insulin, 6.25 μ g/ml Transferrins,iron complexess, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, 5.35 μ g/ml linolic acid.
2. human primary hepatocyte according to claim 1 and liver non-parenchymal cell co-culture method, it is characterized in that: in described step 6), cell density and cell viability method are: the volume ratio by the blue staining fluid of cell suspension and 0.4% m/v platform phenol with 1 ︰ 1 mixes, rapidly 20 μ l mixed solutions are joined in tally and by this tally and inserted in corresponding calculating instrument, instrument is read corresponding cell density and cell viability, and 0.4% concrete compound method of the blue staining fluid of phenol is: the blue solid of 0.4g platform phenol is dissolved in the PBS solution of 100ml completely.
3. human primary hepatocyte according to claim 1 and liver non-parenchymal cell co-culture method, it is characterized in that: the culture plate that described collagen is coated or the preparation method of culture dish: under aseptic condition, 172 μ l Glacial acetic acid are joined in 100ml deionized water, adding final concentration is the IV type mouse tail collagen of 50 μ g/ml again, after 0.22 μ m membrane filtration degerming, 4 ℃ of maintenances are stand-by; By this working fluid with 5 μ g/cm
2package amount join and need in the coated culture plate or culture dish of processing, normal temperature is hatched; After 1 hour, by the sucking-off of collagen working fluid, naturally dry 4 ℃ of maintenances of rear sealing; With front, the culture plate that collagen is coated or culture dish need clean once with PBS, to remove residual acetic acid.
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| CN104293731A (en) * | 2014-10-13 | 2015-01-21 | 中国水产科学研究院淡水渔业研究中心 | Separation culture method of primary hepatocyte of jian carp |
| CN107988260A (en) * | 2017-11-29 | 2018-05-04 | 立沃生物科技（深圳）有限公司 | A kind of method for establishing hepatitis B virus infection cell model using pig primary hepatocyte and hNTCP recombinant slow virus |
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| WO2018144284A1 (en) * | 2017-02-01 | 2018-08-09 | Phoenixsongs Biologicals, Inc. | Compositions and methods comprising co-culture of hepatocytes |
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| CN104293731A (en) * | 2014-10-13 | 2015-01-21 | 中国水产科学研究院淡水渔业研究中心 | Separation culture method of primary hepatocyte of jian carp |
| CN107988260A (en) * | 2017-11-29 | 2018-05-04 | 立沃生物科技（深圳）有限公司 | A kind of method for establishing hepatitis B virus infection cell model using pig primary hepatocyte and hNTCP recombinant slow virus |
| CN107988260B (en) * | 2017-11-29 | 2021-02-09 | 立沃生物科技（深圳）有限公司 | Method for establishing hepatitis B virus infected cell model by using pig primary hepatocytes and hNTCP recombinant lentiviruses |
| CN109385394A (en) * | 2018-11-02 | 2019-02-26 | 大连理工大学 | A kind of method of extracting and developing and the androgynous xenogenesis liver primary cell of culture |
| CN109609443A (en) * | 2019-01-14 | 2019-04-12 | 上海银海圣生物科技有限公司 | A kind of mouse liver primitive cell culture base, cultural method and incubator |
| CN110904026A (en) * | 2019-11-18 | 2020-03-24 | 中国人民解放军第二军医大学 | Preparation method and application of hepatic precursor-like cells from different sources |
| CN113293127A (en) * | 2021-05-24 | 2021-08-24 | 江南大学 | Construction and application of multi-cell co-culture three-dimensional liver microsphere model |
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