CN105132359A - Method for repolarizing primary hepatocytes in vitro - Google Patents
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Abstract
The invention relates to a method for repolarizing primary hepatocytes in vitro. The method is characterized by comprising the following steps of planting a primary hepatocyte suspension into a cell culture insert in a perfusion planting mode; meanwhile, applying a compact force to the primary hepatocytes for enabling cell density in a unit volume to reach over 90 percent; then performing perfusion culture by using a serum-free medium with a hepatocyte phenotype. According to the method disclosed by the invention, a primary mature hepatocyte population is planted in a compact mechanical planting mode, and the repolarization of the hepatocytes in structural and functional mechanics are both formed early at twelfth hour after planting, so that the repolarizing speed of an in vitro hepatocyte population in a three-dimensional environment is improved, and primary mature hepatocytes with an approximate physiological function phenotype are provided for liver tissue engineering.
Description
Technical field
The present invention relates to liver tissue engineering to learn a skill field, particularly relate to the method for the external multipole of a kind of primary hepatocyte.
Background technology
Liver tissue engineering has important fundamental research and clinical value in drug screening, external supportive treatment and the field such as hepatic tissue physiology and disease simulation model.The primary mature hepatocytes with approximate physiological function phenotype is the important cells composition of liver tissue engineering.How to obtain one of core objective and successful gordian technique that the through engineering approaches hepatic tissue with function in closest body is this area research.
Mature hepatocytes has the complicated polar character different from other epithelial cells on function and structure, and this polarity is the basis that liver cell plays physiological function.Leave after in-vivo tissue environment through enzymolysis, mature hepatocytes loses polarity, and original function phenotype occurs to go down rapidly.Under certain conditions in vitro, liver cell recovers and maintains its polarity, and keep higher functional level, and maintain the long period, this phenomenon is called liver cell multipole.Liver cell multipole comprises following characteristics: reappear the intermediate polarity ultrastructure of typical liver cell and functional bile canaliculus excretion.It is generally acknowledged, the new primary hepatocyte be separated only has recovery cell polarity rear to possess normal cell function.Therefore, by various techniques and methods process to recover cell polarity, be significant observation index and the target of liver tissue engineering.
, under the ins and outs condition be applicable to, all likely there is multiple polarity in the primary mature hepatocytes that two and three dimensions is cultivated.Due to closer to physiological characteristic, promote that three-dimensional liver cell multipole is the impetus of current liver tissue engineering technical study.As liver cell microsphere, as a kind of three-dimensional hepatic tissue body obtaining more research, its formation embodies typical hepatocyte process of self-organization: the primary hepatocyte of fresh separated has the trend of self-assemble, and their can on the basis of aggregate, compact gradually and re-establish polar structure.Research, by optimization culture based component, improves oxygen supply mostly at present, and the chemistry of improved culture medium matter or the means such as physical structure and improvement planting patterns, make hepatocyte multipole.
RyoSudo etc. apply Collagen type-I sandwiched configuration and cultivate Rat Primary Hepatocytes, cell culture fluid by minimum medium (DMEM), 20mM4-hydroxyethyl piperazine ethanesulfonic acid, 25mM sodium bicarbonate, 30mg/LL-proline(Pro), 10
-7m dexamethasone, 10mM niacinamide, xitix-2-the phosphoric acid of 1mM, 10ng/mL epithelical cell growth factor and the dual anti-composition of penicillin streptomycin, after liver cell plantation, 72h first time detects the excretion function (SudoR. of functional bile canaliculus to fluorescein oxalic acid (FDA), MitakaT., IkedaM., andTanishitaK. (2005) Reconstructionof3Dstacked-upstructuresbyratsmallhepatocy tesonmicroporousmembranes.TheFASEBJournal19 (12): 1695-7.).The same method such as MarkA.Talamini cultivates Rat Primary Hepatocytes, after liver cell plantation, 48h detects the asymmetry polar contribution (Talamini of membrane antigen albumen, M.A., KappusB., this studies have reported that the time (48h-72h) the earliest appears in the formation of middle liver cell (structure and function) multipole andHubbardA. (1997) Repolarizationofhepatocytesinculture.Hepatology25 (1): 167-72.).
There is following main drawback in above-mentioned prior art:
1., although make primary hepatocyte there occurs the behavior of multipole under in vitro conditions, not that physiological is recovered.Such as, two-dimension single layer is cultivated primary hepatocyte and is not substantially formed liver plate spline structure, disappears after the vesica sample bile canaliculus fragment inoculation 48h of formation, and liver plasma membrane region protein distribution of specific is basic after 1 week after incubation to disappear.
2. the primary hepatocyte that sandwiched configuration is cultivated improves the ability of physiological primary hepatocyte multipole, and liver cell forms liver plate spline structure, and companion's bile canaliculus network is formed, and liver plasma membrane region protein is distribution of specific, but it is not remarkable to improve degree.Such as, primary hepatocyte albumin resultant quantity when 72h cultivated by sandwich is 30 μ g/10
6cell/sky, urea synthesis amount is 120 μ g/10
6cell/sky.
3. evening time of multipole appearance.
Summary of the invention
In view of this, be necessary, for above-mentioned problem, to provide the method for the external multipole of a kind of primary hepatocyte.
To achieve these goals, the present invention adopts following technical scheme:
The method of the external multipole of a kind of primary hepatocyte, comprise the following steps: primary hepatocyte suspension is planted in cell culture insert by the mode of perfusion plantation, compact power is applied to primary hepatocyte simultaneously, make unit volume cell density reach more than 90%, carry out perfusion culture with hepatocytic phenotype serum-free medium afterwards.
Preferably, the method for perfusion plantation is that primary hepatocyte suspension flows through and is stranded in cell culture insert.
Preferably, during perfusion plantation, the flow velocity of primary hepatocyte suspension is 0.02mL/h.
Preferably, during perfusion culture, the flow velocity of hepatocytic phenotype serum-free medium is 0.02 ~ 0.06mL/h.
More preferably, after the compact plantation of liver cell, with the flow velocity of 0.02mL/h, perfusion culture is carried out to liver cell in 1h, with the flow velocity of 0.02 ~ 0.03mL/h, perfusion culture is carried out to liver cell in 2 to 4h, after 4h, with the flow velocity of 0.04 ~ 0.06mL/h, perfusion culture is carried out to liver cell.
Preferably, described perfusion plantation is circumfusion plantation.The plantation of described circumfusion adopts a certain amount of primary hepatocyte suspension to carry out perfusion to plant, the mode making the primary hepatocyte suspension flowed out in cell culture insert flow into cell culture insert again to carry out planting.
Preferably, to the concrete grammar that primary hepatocyte applies compact power be: being wriggled by ophthalmic tweezers under common inverted light microscope applies compact power to primary hepatocyte.
Preferably, hepatocellular incubation time is more than 12h.
More preferably, hepatocellular incubation time is 12h to 120h.
Still more preferably, hepatocellular incubation time is 12h to 48h.
Still more preferably, hepatocellular incubation time is 12h to 24h.
Preferably, the density of primary hepatocyte suspension is 4-6 × 10
6individual/mL.
Preferably, hepatocytic phenotype serum-free medium comprises basic medium HepatoZYMESFM (Invitrogen, LifeTechnologies, USA), 2mML-glutamine, 60 μMs of HEPES (4-hydroxyethyl piperazine ethanesulfonic acid), 50 μMs of dexamethasone and 1 × penicillin/streptomycin are dual anti-.
Contriver, by analysis to prior art, thinks that the reason why prior art exists multiple shortcoming may have: liver cell can not form high-density culture fast; Arrange loose between liver cell, do not form close contact.Therefore, contriver, to this has been improvement, defines the present invention.The cultural method of the external multipole of primary hepatocyte of the present invention carries out circumfusion cultivation after utilizing mechanics compact planting patterns plantation primary hepatocyte.The compact plantation of mechanics refers to and utilizes mechanical force repopulating cell, and cell tight is arranged, and realizes the planting patterns of the high-density planting of cell.The mechanical force applied can make cell compact, and therefore this mechanical force is called compact power.The present invention adopts the compact plantation primary hepatocyte of circumfusion mode, carries out circumfusion cultivation afterwards, is called micro-fluidic perfusion culture method.
Compared with prior art, the present invention's micro-fluidic perfusion culture method has following beneficial effect:
(1) utilize the compact plantation primary hepatocyte of mechanics in the present invention, can make to arrange between liver cell compact, form close contact, liver cell can form high-density culture fast.
(2) utilize the compact plantation primary hepatocyte of mechanics in the present invention, significantly improve the cell in vitro function of liver cell population, primary hepatocyte is at cultivation 72h, and albumin resultant quantity is 150 μ g/10
6cell/sky, urea synthesis amount is 750 μ g/10
6cell/sky, cultivates the synthesis level of primary hepatocyte far away higher than prior art.
(3) the present invention plants and dimensional culture the mechanics compact way that primary hepatocyte carries out hepatic tissue mechanics feature in analogue body, and after in the present invention, the multipole of liver cell in structure and function is all advanced to plantation, 12h is formed.
Accompanying drawing explanation
Fig. 1 is simple microscope observation figure and laser co-focusing fluorescence microscopy figure in effect example 1.Figure 1A (i) and Figure 1B (i) is respectively the simple microscope observation figure of control group and treatment group, and Figure 1A (ii) and Figure 1B (ii) is respectively the laser co-focusing fluorescence microscopy figure after the fluorescent mark of control group and compact power group.Liver cell liver cell Fig. 2 is liver cell planting density histogram in effect example 1.
Fig. 3 is liver cell shape figure in effect example 2.Fig. 3 A is cellular control unit shape figure, Fig. 3 B, and to be treatment group cell shape figure, Fig. 3 C be is planted in the liver cell shape figure had in the sinusoidal liver cell section of typical liver cell.
Fig. 4 is liver cell contact distance map in effect example 2.Fig. 4 A is control group contact distance map, and Fig. 4 B is treatment group contact distance map.
Fig. 5 is liver cell contact area histogram in effect example 2.
Fig. 6 is bile canaliculus excretion FDA situation map in effect example 3.
Fig. 7 is the ultrastructure figure of liver cell under scanning electron microscope in effect example 4.
Fig. 8 is albumin resultant quantity histogram in effect example 5.
Fig. 9 is urea synthesis amount histogram in effect example 5.
Figure 10 is the polarity layout viewing of Hepatocyte in effect example 6.
Figure 11 is liver cell MRP2/F-actin fluorescent mark bile canaliculus cavity area and hepatic tissue area ratio in effect example 6.
Embodiment
For a better understanding of the present invention, be described further below in conjunction with the drawings and specific embodiments.In the present invention, agents useful for same or instrument all can be buied by market, and the detection method of use is all known in the art, does not repeat them here.
Embodiment 1, micro-fluidic perfusion culture method
The present embodiment provides the method for the external multipole of a kind of primary hepatocyte, comprises the following steps: be 5 × 10 by 50 μ L density
6the primary hepatocyte suspension of individual/mL to be flowed through with 0.02mL/h flow velocity by the mode of circumfusion and is naturally stranded in cell culture insert.Wriggled gently by ophthalmic tweezers under common inverted light microscope simultaneously and simple compact power is applied to primary hepatocyte group, make unit volume cell density reach more than 90%.Cell uses hepatocytic phenotype serum-free medium at 37 DEG C, 5%CO after planting
2perfusion culture is carried out under condition.Use hepatocytic phenotype serum-free medium with 0.02mL/h flow velocity perfusion culture in 1h, with the low flow velocity (0.02 ~ 0.03mL/h) of close inoculation, perfusion culture is carried out to cell with hepatocytic phenotype serum-free medium in 2 to 4h, after 4h, perfusion flow velocity is increased to 0.04 ~ 0.06mL/h, for Growth of Cells continues to supply fresh nutrient solution.When perfusion culture, bring into use low flow velocity to carry out perfusion culture, namely can ensure the demand of cell to nutrition, be convenient to again liver cell adherent; At the adherent rear increasing perfusion flow velocity of liver cell, be the well-off nutrition of Growth of Cells, be convenient to cell and grow multipole fast.
Described hepatocytic phenotype serum-free medium comprises basic medium HepatoZYMESFM (Invitrogen, LifeTechnologies, USA), 2mML-glutamine, 60 μMs of HEPES, 50 μMs of dexamethasone and 1 × penicillin/streptomycin dual anti-(penicillin 100U/mL, Streptomycin sulphate 100 μ g/mL).L-glutaminate provides energy for liver cell culture, participates in hepatocellular albumen and nucleic acid synthesis.HEPES is the damping fluid of cell cultures, regulates the pH value of cell culture fluid about about 7.4.Dexamethasone regulates hepatocellular secreting function etc., and dual anti-bacteria growing inhibiting, avoids cell contamination.
Primaryly in the present embodiment be hepatocellularly separated acquisition in the following manner: with reference to Selgen two step perfusion method, be separated Wistar rat primary mature hepatocytes.Lysed cells suspension is through two times centrifugal, and the centrifugal 10min of each 50g, leaves and takes eccentric cell precipitation, be prepared into primary hepatocyte suspension with hepatocytic phenotype serum-free medium.Cell viability (platform the expects blue staining) separating sample more than 90% can be used for inoculation and carries out external multiple Polarity experiment.
Comparative example 1, do not apply the method for the external multipole of primary hepatocyte of centrifugal action
The present embodiment provides the method for the external multipole of a kind of primary hepatocyte, does not apply compact power, and other steps are identical with embodiment 1 with condition, specifically comprise the following steps:
Be 5 × 10 by 50 μ L density
6the primary hepatocyte suspension of individual/mL to be flowed through with 0.02mL/h flow velocity by the mode of circumfusion and is naturally stranded in cell culture insert.Cell uses hepatocytic phenotype serum-free medium at 37 DEG C, 5%CO after planting
2perfusion culture is carried out under condition.Use hepatocytic phenotype serum-free medium with 0.02mL/h flow velocity perfusion culture in 1h, with the low flow velocity (0.02 ~ 0.03mL/h) of close inoculation, perfusion culture is carried out to cell with hepatocytic phenotype serum-free medium in 2 to 4h, after 4h, perfusion flow velocity is increased to 0.04 ~ 0.06mL/h, for Growth of Cells continues to supply fresh nutrient solution.
Effect example 1, liver cell planting density
Detect hepatocellular planting density (treatment group) in embodiment 1, with liver cell in comparative example 1 in contrast.Concrete measuring method is:
Wash liver cell 3 times with phosphate buffered saline buffer (PBS), after each washing 5min, fix 30min by 3.7% (w/v) paraformaldehyde (PFA) room temperature, PBS vibration washing 3 times, washs 5min at every turn; 250nmol/mLSytoxGreen fluorescence dye (Invitrogen, LifeTechnologies, USA) and 200 μ g/mLRNAse (Sigma, USA) labeled cell core, room temperature dyeing 30min, PBS vibration washing 3 times, washs 5min at every turn.Observe with simple microscope and laser confocal fluorescence microscope and take pictures and obtain 3-D view.
Nucleus in laser co-focusing image uses the blob detection module qualification in Imarissoftware (version7.1.0, Bitplane, Switzerland).Total cellular score is the nucleus sum after correcting multinuclear liver cell, and the multiple nucleus being less than 20 microns as interval are considered as a nucleus.Calculate cell number in each confocal amount, obtain unit volume inner cell quantity, i.e. cell seeding density.By detecting hepatocellular mean diameter, estimating the cell volume in each culturing room further, obtaining unit space liver cell occupation proportion.Result as shown in Figure 1-2.
From Fig. 1-2, in control group, cell seeding density is 7.8 × 10
7± 4.5 × 10
6individual/cm
3, higher than common liver cell planting density 1 × 10
6individual/cm
3, in unit space, cell occupation proportion is 69.9% ± 4%, is 63.4% slightly larger than rigid spheres occupation proportion in Theoretical Calculation unit space; And in compact power treatment group, cell seeding density is 1 × 10
8± 3.9 × 10
6individual/cm
3, unit space liver cell occupation proportion is 93.1% ± 3.1%, carries ratio much larger than control group and theoretical maximum rigidity ball-joint.Above result shows, applies the compact power of simple mechanics, achieve liver cell high-density culture in vitro to plantation liver cell population.
Effect example 2, liver cell shape and contact area
The primary hepatocyte of fresh separated is with CellMaskOrange (Invitrogen, LifeTechnologies, the USA) dye marker of 5 μ g/mL, and after room temperature effect 15min, substratum washing once.According to the method multipole described in embodiment 1 as treatment group, according to the method multipole described in comparative example 1 as a control group.Observe and measure shape and the contact area of the liver cell population obtained after cultivating 48h.
Sample treatment is as follows: discard cell culture fluid, and PBS vibration washing 3 times, washs 5min at every turn; Fix 30min by 3.7% (w/v) PFA room temperature, PBS vibration washing 3 times, washs 5min at every turn.Liver cell after laser confocal fluorescence microscope observes above-mentioned process is also taken pictures and obtains 3-D view.In order to the exposure level between observation of cell and cell, three-dimensional Confocal Images utilizes ImageJ1.43 to rebuild.By measuring the distance of 20 pairs of exposing cells in each culturing room, estimate the contact surface area between cell and cell.The distance (three-dimensional stacked height) of exposing cell is determined by image come into contact part.Suppose the isometric compression of cell, intercellular contact surface area calculates with the square value of contact height.Result as in Figure 3-5.
As shown in Figure 3-4, in treatment group, mechanics is compact transmits between liver cell, and cell individual is subject to squeezing from the three-dimensional of peripheral cell touching, and shape changes.Although technique means directly can't measure the accurate size of the inner compact power of cell mass at present, carry out microscopic examination to fluorescently-labeled cytolemma, compared with control group, the most cell for the treatment of group occurs by spherical to square change.In body, liver cell is polygon, and relative to the spherule cell of control group, the square cell for the treatment of group is compared with form hepatocellular in close proximity to body.
As illustrated in figures 4-5, mechanics compact treatment group iuntercellular distance is 13.9 ± 0.3 μm, and cellular control unit spacing is 15.0 ± 0.2 μm; Mechanics compact treatment group cell contact area is 200 ± 0.6 μm
2, cellular control unit Contact area is 100 ± 0.5 μm
2, mechanics compact treatment group cell contact area is approximately the twice of control group, and difference has statistical significance (P < 0.05).Therefore, it is compacter that compact power makes liver cell population arrange, and cell contact more directly closely.
Effect example 3, bile canaliculus excretion FDA
Detect the situation (treatment group) that embodiment 1 cultivates the liver cell population bile canaliculus excretion FDA that 12h, 24h and 48h obtain.With comparative example 1 for control group, detect the situation that comparative example 1 cultivates the bile canaliculus excretion FDA of the liver cell population that 12h, 24h and 48h obtain.
Treatment group and the process of control group liver cell population each sample as follows: be divided into two parts respectively, a liver cell population PBS washs 3 times, each 5min, and rear use, containing substratum 37 DEG C of culturing cell 40min of 20 μ g/mLFDA, to be vibrated washed cell 3 times with PBS, each 5min.Another part of liver cell population does not dye and carries out laser confocal fluorescence microscope observation and take pictures, and gets rid of the impact of liver cell autofluorescence.Observe with laser confocal fluorescence microscope and take pictures.The timid tube chamber size that FDA location obtains uses the threshold value of ImageJ software and conjury stick function to carry out identification with quantitative.Measure the bile canaliculus size of each condition more than 5 random field.
The present embodiment repeats experimental verification through more than 5 times, result as shown in Figure 6, be no more than 12h after the inoculation for the treatment of group liver cell population and just generally occur diacetic acid fluorescein (FDA) excretion phenomenon, and control group liver cell population needs 48h just to occur FDA excretion phenomenon, compared with just there is FDA excretion phenomenon with 72h in the sandwich dimensional culture model reported in the past, all have and significantly shift to an earlier date.This may be because adopt micro-fluidic perfusion culture method culture hepatocyte in the present embodiment, and the sandwich that cell seeding density ratio is conventional is cultivated high.
The Ultrastructural observation of effect example 4, liver cell population
The present embodiment has carried out Ultrastructural observation to the liver cell population of 12h and 48h after plantation further.Liver cell population 2.5% (w/v) glutaraldehyde fixes 30min, under microscopic visualization, utilizes microcosmic to clamp and cell mass is shifted out from micropore.Cell mass 1% (w/v) perosmic anhydride is fixed, working concentration gradient (25%, 50%, 75%, 95%, 100%) ethanol dehydration, each mass action 15min, subsequently with 100% acetone dehydration twice, each 15min.The sample acetone of volume ratio 1:1 and epoxy resin mixed solution incubated at room 4h, then use acetone and the epoxy resin overnight incubation of volume ratio 1:6.Sample is embedded in 100% epoxy resin, 60 DEG C of oven for curing 24h.Utilize Lycra EMUC6 ultramicrotome to cut into slices, sample collection is in the copper sieve of 200 order meshes.Sample utilizes transmission electron microscope observation and takes pictures after using the two dyeing of 3% acetic acid uranium-lead citrate.
As shown in Figure 7, the treatment group liver cell of compact power effect after planting 12h just forms functional bile canaliculus, and along with microvillus and close-connected formation, this ultrastructure is retained to more than 48h, further determined that the existence of rebuilding bile canaliculus (BC); On the contrary, after plantation 12h control group primary hepatocyte in not there is the functional bile canaliculus structure that formed along with microvillus (MV) and compact siro spinning technology (TJ), only there is the little tubular structure being dispersed in distribution in 48h after planting.Above result clearly illustrates that, the structure and function characteristic phenotypic that micro-fluidic perfusion culture method significantly accelerates liver cell population is formed.
Effect example 5, albumin and urea secretion are tested
Utilize 5mL syringe collecting to adopt compact power process to cultivate the liver cell population substratum after 24h, 48h, 72h, 96h and 120h according to method in embodiment 1, measure the changes of function (treatment group) of liver cell population synthesis albumin and urea.Traditional two-dimentional collagen protein culture hepatocyte is as blank group.In substratum, albuminous concentration uses rat albumin enzyme linked immunosorbent assay (ELISA) test kit (BethylLaboratories, USA) carry out quantitatively, urea concentration then uses Urea Urea nitrogen test (BUN) test kit (StanbioLaboratory, USA) to carry out quantitatively.Utilize multi-functional microplate reader to measure albumin sample respectively at 450nm wavelength, urea sample is at the light intensity light absorption value of 520nm wavelength.
According to bibliographical information, it is 30 μ g/10 that sandwich cultivates primary hepatocyte albumin resultant quantity when 72h
6cell/sky, urea synthesis amount is 120 μ g/10
6cell/sky.The result of the present embodiment is as shown in table 1 and Fig. 8-9, and treatment group primary hepatocyte is when cultivating 72h, and albumin resultant quantity is 150 μ g/10
6cell/sky, urea synthesis amount is 750 μ g/10
6cell/sky, cultivates the synthesis level of primary hepatocyte far away higher than prior art.Three days that cultivate after inoculation, the albumin synthesis capability of compact power treatment group liver cell population was control group (two-dimension single layer cultivation) hepatocellular 2-10 times, then drops to the suitable level of monolayer culture liver cell after three days.Compact power process liver cell population urea synthesis amount, in cultivate after inoculation 5 days relatively stable (ANOVA, p=0.131), exceeds nearly 20 times than two-dimension single layer culture hepatocyte.Above result clearly shows, micro-fluidic perfusion culture method significantly improves external function and the vigor of liver cell population.
Table 1 albumin and urea synthesis scale
The immunofluorescence dyeing of effect example 6, liver cell population polar protein and the specific stain of Actin muscle F-actin
Method described in embodiment 1 is used to cultivate the liver cell population of 12h, 24h and 48h as treatment group, cultivate the liver cell population of corresponding time for control group with method described in comparative example 1, detect the immunofluorescence dyeing of liver cell population polar protein and the specific stain of Actin muscle F-actin.
Sample treatment is: liver cell population PBS washs 3 times, and after each 5min, fix 30min by 3.7% (w/v) PFA room temperature, PBS washs 3 times, washs 5min at every turn; The PBS room temperature of 0.1%TritonX-100 leaves standstill penetrating liver cell 30min; PBS washs 3 times, washs 5min at every turn.Under microscopic visualization, utilize microcosmic to clamp cell mass is shifted out from micropore.Use PBS4 DEG C of 2% bovine serum albumin (BSA) and 0.1%TritonX-100 to close to spend the night, PBS washs 3 times, washs 5min at every turn.All anti-antibodys cover sample surface, 4 DEG C of overnight incubation or room temperature effect 2h; PBS washs 3 times, washs 5min at every turn; Two all anti-antibodys cover sample surface, and 37 DEG C of lucifuges hatch 60min; PBS lucifuge washs 3 times, washs 5min at every turn.Multidrug resistant associated protein MRP2 primary antibodie (M8316, Sigma, USA) extent of dilution is 1:80, adhesion junction albumen E-cadherin primary antibodie (610182, BDBiosciences, USA) extent of dilution is 1:100, the extent of dilution of total adhesion junction albumen pan-cadherin primary antibodie (C1821, Sigma, USA) is 1:500, the extent of dilution of rhodamine mark goat-anti rabbit two anti-(Sigma, USA) is 1:200.Diluted fresh Hoechst33342 (H3570, Invitrogen, LifeTechnologies, USA, 1mg/mL) covers cell surface, and room temperature lucifuge leaves standstill 10min; PBS lucifuge vibration washing 3 times, washs 5min at every turn.Laser confocal imaging fluorescent microscope (ZeissLSM, Germany) carries out observing and taking pictures.ImageJ software is utilized to carry out quantitatively fluorescent mark bile canaliculus cavity area.
During the specific stain of Actin muscle F-actin, the previously described method of cell is fixed.At microchannel or the labeled in situ F-actin Actin muscle of random excision.On the microchannel of random excision, 100U/mLAlexafluor488phalloidin adds when two resist and hatch, and then resists the step after hatching to operate according to two.Original position F-actin dyeing is poured into 100U/mLAlexafluor594phalloidin and 1 μ g/mL nuclear staining agent Hoechst33342, and flow velocity is 0.5mL/h, effect 2h.After PBS washs 1h, laser confocal fluorescence microscope is used to carry out observing and taking pictures.
From Figure 10 and Figure 11, under same culture conditions, control group multipole time of occurrence is 48h after plantation, and treatment group then significantly accelerates to the rear 12h of plantation.The time that this time has bibliographical information more so far significantly shortens.At 12h, we find that treatment group Actin muscle F-actin to strengthen being distributed within the scope of BC and prolongation in time in advance, strengthen the range extension of distribution.The characteristic distributions of being redyed by MRP2 can judge, F-actin and BC is formed, accelerate formation and maturation has inseparable relation.The ratio of MRP2/F-actin fluorescent mark bile canaliculus cavity area and hepatic tissue area, increases progressively along with incubation time and raises.12h is put in detection time, 24h and 48h, the MRP2/F-actin fluorescent mark bile canaliculus cavity area for the treatment of group and the ratio of hepatic tissue area are respectively 5.0% ± 0.2%, 12.5% ± 0.3%, 17.5% ± 0.4%, the MRP2/F-actin fluorescent mark bile canaliculus cavity area of control group and the ratio of hepatic tissue area are respectively 1.5% ± 0.1%, 4.5% ± 0.6%, 4.5% ± 0.3%.The MRP2/F-actin fluorescent mark bile canaliculus cavity area for the treatment of group and the ratio of hepatic tissue area are all about three times of control group.Above result shows, micro-fluidic perfusion culture method significantly accelerates the multipole speed of hepatocyte group under three-dimensional environment, and liver cell polarity is also fully maintained afterwards.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. the method for the external multipole of primary hepatocyte, it is characterized in that, comprise the following steps: primary hepatocyte suspension is planted in cell culture insert by the mode of perfusion plantation, compact power is applied to primary hepatocyte simultaneously, make unit volume cell density reach more than 90%, carry out perfusion culture with hepatocytic phenotype serum-free medium afterwards.
2. the method for the external multipole of primary hepatocyte according to claim 1, is characterized in that, the method for perfusion plantation is that primary hepatocyte suspension flows through and is stranded in cell culture insert.
3. the method for the external multipole of primary hepatocyte according to claim 1, is characterized in that, during perfusion plantation, the flow velocity of primary hepatocyte suspension is 0.02mL/h.
4. the method for the external multipole of primary hepatocyte according to claim 1, is characterized in that, during perfusion culture, the flow velocity of hepatocytic phenotype serum-free medium is 0.02 ~ 0.06mL/h.
5. the method for the external multipole of primary hepatocyte according to claim 4, it is characterized in that, after the compact plantation of liver cell, with the flow velocity of 0.02mL/h, perfusion culture is carried out to liver cell in 1h, with the flow velocity of 0.02 ~ 0.03mL/h, perfusion culture is carried out to liver cell in 2 to 4h, after 4h, with the flow velocity of 0.04 ~ 0.06mL/h, perfusion culture is carried out to liver cell.
6. the method for the external multipole of primary hepatocyte according to claim 1, is characterized in that, perfusion plantation is circumfusion plantation.
7. the method for the external multipole of primary hepatocyte according to claim 1, is characterized in that, concrete grammar primary hepatocyte being applied to compact power is: being wriggled by ophthalmic tweezers under common inverted light microscope applies compact power to primary hepatocyte.
8. the cultural method of the external multipole of primary hepatocyte according to claim 1, is characterized in that: hepatocellular incubation time is more than 12h.
9. the method for the external multipole of primary hepatocyte according to claim 1, is characterized in that, the density of primary hepatocyte suspension is 4-6 × 10
6individual/mL.
10. the method for the external multipole of primary hepatocyte according to claim 1, it is characterized in that, hepatocytic phenotype serum-free medium comprises basic medium HepatoZYMESFM, 2mML-glutamine, 60 μMs of HEPES, 50 μMs of dexamethasone and 1 × penicillin/streptomycin are dual anti-.
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| CN110923192A (en) * | 2019-11-27 | 2020-03-27 | 中国人民解放军第二军医大学 | Long-term in vitro culture method of mature hepatocytes |
| CN110923192B (en) * | 2019-11-27 | 2022-03-08 | 中国人民解放军第二军医大学 | Long-term in vitro culture method of mature hepatocytes |
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