CN105695392A - Culturing method for improving in-vitro differentiation phenotype and function of hepatic cells - Google Patents
Culturing method for improving in-vitro differentiation phenotype and function of hepatic cells Download PDFInfo
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- CN105695392A CN105695392A CN201510937399.0A CN201510937399A CN105695392A CN 105695392 A CN105695392 A CN 105695392A CN 201510937399 A CN201510937399 A CN 201510937399A CN 105695392 A CN105695392 A CN 105695392A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Abstract
The invention discloses a culturing method for improving the in-vitro differentiation phenotype and function of hepatic cells. The culturing method is characterized by comprising the following steps that the hepatic cells are adopted as seed cells, a porous silk protein scaffold is taken as an in-vitro culturing carrier of the hepatic cells, the hepatic cells, hepatogenic interstitial cells and an extracellular matrix are inoculated to the porous silk protein scaffold according to a certain proportion, and the porous silk protein scaffold is put in a simulated hepatic cell in-vitro culturing environment to be cultured. A three-dimensional hepatic cell co-culturing system built through the method not only can be applied to tissue-engineered hepatic cell function units, but also can be applied to in-vitro evaluation of an influence of drugs on a hepatic metabolism enzyme and hepatic toxicity detection, and therefore a novel screening and evaluating tool is supplied to new drug research and development.
Description
Technical field
The present invention relates to a kind of hepatocellular extracorporeal culturing method, particularly a kind of cultural method improving hepatocyte vitro differentiation phenotype and function。
Background technology
Primary hepatocyte does not still build the desirable seed cell source of engineered liver, and is the important models that vitro Drug ADME/T character carries out accurate evaluation。But hepatocyte very easily " dedifferenting " occurs in traditional external plane incubation, the reduction of activity namely occur, the reduction of the change of phenotype and functional activity (including synthesis and metabolic activity) even completely loses。Research confirms: the human liver cell of fresh separated is under condition of suspension culture, and its P450 enzymatic activity only maintains 4h；In the coated culture plate of collagen, the P450 expression of enzymes amount of attached cell and enzymatic activity will significantly reduce after 24-48h。And due to the reduction of enzyme functional activity, the sensitivity in hepatotoxic medication screens of the hepatocyte monolayer method only has 50%。Therefore overcome in how cultivating in vitro or delay hepatocellular " dedifferenting " phenomenon, thus effectively maintaining hepatocellular desirable differentiation state, namely maintain its phenotype and correlation function " bottleneck " problem that to be always up in liver cell culture and applied research urgently to be resolved hurrily。
Summary of the invention
The present invention is to solve the above-mentioned deficiency existing for prior art, it is proposed to one desirable can maintain hepatic cell growth activity, and significantly improves the new cultural method of Cultured Hepatocytes in vitro phenotype and functional activity。
The technical solution of the present invention is: a kind of cultural method improving hepatocyte vitro differentiation phenotype and function, it is characterized in that: described method carries out according to following steps: employing hepatocyte is seed cell, using porous silk albumen support as Hepatocytes culture in vetro carrier, hepatocyte, liver source property Interstitial cell and extracellular matrix are inoculated on porous silk albumen support by a certain percentage, and prop up porous silk albumen to be placed in the hepatocellular In vitro culture microenvironment of simulation and cultivate
Described by hepatocyte, the concrete operation step that liver source property Interstitial cell and extracellular matrix are inoculated on porous silk albumen support by a certain percentage is as follows: mixed according to the ratio of cell number 3:1 by the hepatocyte regulating liver-QI star Interstitial cell of fresh separated, and the cell mixing obtained is mixed with extracellular matrix, obtain cell-matrix mixture, described extracellular matrix is mixed according to the ratio that volume ratio is 1:1 by collagen and matrigel, then cell-matrix mixture multiple spot is seeded to multiple porous silk albumen support, and ensure that hepatocyte quantity is 600, 000cells/ support, the volume of matrigel is 30 μ L/ supports,
Described being propped up by porous silk albumen is placed on that to simulate the concrete operation step carrying out cultivating in hepatocellular In vitro culture microenvironment as follows: be placed in porous cell culture plate by the porous silk albumen support inoculated according to the density in 1 support/hole, and porous cell culture plate is placed in cell culture incubator, at 37 DEG C, 5%CO2When gelation 2h, then add in each hole of porous cell culture plate and co-culture base, the described base that co-cultures is hepatocyte culture medium and star Interstitial cell culture medium mixes according to the ratio that volume ratio is 1:1。
The present invention compared with the existing technology, has the advantage that
Hepatocellular extracorporeal culturing method disclosed in this invention, this cultural method utilizes between cell and cell and the interaction of complexity between cell and extracellular matrix, form the microenvironment similar with internal hepatocyte, make hepatocellular growthform closer to internal three dimensional growth mode；It adopts porous fibroin albumen as cultivating support, add collagen and matrigel as culture matrix simultaneously, construct a compound criteria system, this compound criteria system is not only able to provide three dimensional growth space into hepatocyte, and it has biocompatibility, material plasticity and supports hepatic cell growth and organized feature, additionally, its porous character is also greatly improved the material transmission in cultivating system, thus being conducive to hepatocyte incubation in vitro maintains its activity；Biocompatibility, mechanical strength feature and biodegradation character specific to porous silk albumen support, this cultivating system is made to be easily achieved Hepatocyte transplant and recovery, this not only can advance hepatocyte replacement therapy, but also be likely and inquire into the correlational study desirable study model system of offer about hepatocyte transplantation；In the model that this cultural method is set up, iuntercellular sets up intercellular communication each other, and bile capillary structure can be differentiated to form, illustrate that constructed liver cell culture system is more for systematism feature, thus being conducive to hepatocyte incubation in vitro maintains its phenotype and function；In the cultivating system that the method is set up, hepatocellular albumin secretion and urea synthesis function are all remarkably higher than plane cultivation group and " sandwich " cultivation group, closer at the hepatocellular functional activity of body, in this cultivating system, hepatocellular metabolic enzyme activity is significantly higher than classical culture protocols (plane cultivation group and " sandwich " cultivation group) simultaneously, it is more conducive to drug metabolism activity and liver toxicity are carried out in-vitro evaluation accurately, consequently, it is possible to provide new appraisal tool for new drug development。
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph (A: prepared by salting out method of porous silk albumen support；B: prepared by organic solvent method)。
In order to utilize live cell fluorescent labeling method display Rat Primary Hepatocytes (redness) and the star Interstitial cell (blue-fluorescence) growthform on porous silk albumen support, (A:3D co-cultures group to Fig. 2；B: matched group) and cytoactive (C)。
Fig. 3 is the morphosis (A1-2: cultivation group of the present invention of the rat hepatocytes that HE dyeing display utilizes the method for the invention to cultivate；B: matched group)。
Fig. 4 is scanning electron microscope (SEM) detection display Rat Primary Hepatocytes growth morphology on porous silk albumen support and feature (A1-2)；Transmission electron microscope (TEM) detection display Rat Primary Hepatocytes forms cell and connects (B)。
Fig. 5 is albumin secretion (A) and the urea synthesis function (B) of the rat hepatocytes that the method for the invention is cultivated。
Fig. 6 utilizes qRT-PCR method to detect the method for the invention Rat Primary Hepatocytes metabolic enzyme gene expression is affected one of legend。
Fig. 7 utilizes qRT-PCR method to detect the method for the invention Rat Primary Hepatocytes metabolic enzyme gene expression is affected the two of legend。
Fig. 8 utilizes qRT-PCR method to detect the method for the invention Rat Primary Hepatocytes metabolic enzyme gene expression is affected the three of legend。
Fig. 9 is the method for the invention impact on Rat Primary Hepatocytes metabolic enzyme CYP3A1 activity。
Detailed description of the invention
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described。As shown in Figures 1 to 7:
The preparation of porous silk albumen support:
First the silk fibroin protein solution that concentration is 6-7% is prepared, employing saltouts-and seasoning prepares porous fibroin albumen support, control support aperture by salt particle size and be 500-600 μm, fully it is infiltrated in deionized water after drying, then the three-dimensional rack (i.e. porous silk albumen support) that standard hole device prepares diameter 5mm, thickness 3mm is adopted。Porous silk albumen support needs to process through autoclave sterilization before inoculating cell, and is immersed in culture medium and carries out pre-equilibration 12h。
The separation and Culture of Rat Primary Hepatocytes:
Rat through anesthesia after adopt collagenase two step perfusion method (0.5mg/ml,Collagenase Type) separate acquisition rat (growing up, male Sprague Dawley Rats) primary hepatocyte, it is purified after gradient percoll liquid separates, is enriched with。Generally can obtain 2 × 108Cell total amount, and competent cell > 90%. fresh separated liver cell culture in William ' sE culture medium add 0.5U/mL insulin (Invitrogen), 7ng/mL glucagon (Sigma), 7.5 μ g/mL cetacort (Sigma) and 1% antibiotic (Invitrogen)
Rat primary liver star Interstitial cell purchases U.S. ScienCell, is incubated at Stellate culture medium, carries out 1:3 routine passage cultivation when Growth of Cells is to nearly 90% degrees of fusion,
All cells is all incubated at 37 DEG C, in 5%CO2 cell culture incubator。
Carry out the In vitro culture of rat hepatocytes:
The Rat Primary Hepatocytes obtained in above-mentioned steps is seed cell, using porous silk albumen support as Hepatocytes culture in vetro carrier, hepatocyte, liver source property Interstitial cell and extracellular matrix are inoculated on porous silk albumen support by a certain percentage, and prop up porous silk albumen to be placed in the hepatocellular In vitro culture microenvironment of simulation and cultivate
The hepatocyte regulating liver-QI star Interstitial cell of fresh separated is mixed according to the ratio of cell number 3:1, and the cell mixing obtained is mixed with extracellular matrix, obtain cell-matrix mixture, described extracellular matrix is mixed (product that described collagen and matrigel all select BDBiosciences) by collagen and matrigel according to the ratio that volume ratio is 1:1, then cell-matrix mixture multiple spot is seeded to multiple porous silk albumen support, and ensure that hepatocyte quantity is 600,000cells/ support, the volume of matrigel is 30 μ L/ supports
The porous silk albumen support inoculated is placed in porous cell culture plate according to the density in 1 support/hole, and porous cell culture plate is placed in cell culture incubator, at 37 DEG C, 5%CO2When abundant gelation 2h, in each hole of porous cell culture plate, then be slowly added to co-culture base, the described base that co-cultures is hepatocyte culture medium and star Interstitial cell culture medium mixes according to the ratio that volume ratio is 1:1,
By above-mentioned steps, construct hepatocellular three-dimensional co-culture model。
The Rat Primary Hepatocytes of batch fresh separated distinguishes direct inoculation in through collagen coated 24 orifice plates 1(plane cultivation group as a control group with equal densities together)；Concurrently setting tradition sandwich cultivation group is matched group 2。
Set up the morphologic detection platform evaluating hepatocyte three-dimensional co-culture model:
Hepatic cell growth form and Activity determination: in order to better observe different cells in three-dimensional co-culture system, initially with viable cell labelling dyestuff DiI and DiD(Invitrogen) distinguish labelling primary hepatocyte and star Interstitial cell, directly observe under laser confocal microscope after cultivating 5 days。Cytoactive dyeing adopts calcein-AM/EthD-1 staining kit (Invitrogen), hatches 2h for 37 DEG C, and all the other operation by specifications carry out。Observe under laser confocal microscope after dyeing, take pictures。
H&E dyes: fixes 24h under PBS flushing, 4% paraformaldehyde room temperature after sample collection, morphology center prepares paraffin section。Sequentially pass through dewaxing, H&E dyeing and mounting again, prepare H&E stained。Face optical microphotograph Microscopic observation, take pictures。
Scanning electron microscope and transmission electron microscope observing: collected sample, after PBS fully cleans, after fixing 4-6h, PBS flushing, fixes 2h after 2% osmic acid before 2.5% glutaraldehyde solution, and after PBS fully cleans again, sample segment is placed in vacuum drying oven inner drying overnight。Dried sample, after metal spraying 15min, is observed under scanning electron microscope and is taken pictures。Transmission electron microscope inspection center delivered to by all the other samples, prepares ultrathin section after resin embedding, observes, takes pictures under transmission electron microscope。
Set up the functional activity detection platform evaluating hepatocyte three-dimensional co-culture model:
Albumin content measures and urea synthesis measures: culture medium in each cultivation group of periodic collection in incubation, measure batch albumin and urea content after-80 DEG C of preservations。Wherein, albumin measuring adopts rat albumin ELISA kit, and the mensuration of urea synthesis adopts QuantiChrom carbamide detection kit。Concrete operations all complete according to description。
Quantitative RT-PCR measures metabolic enzyme gene and expresses: periodic collection sample, directly adopts TRIZOL cell lysis, RNAeasy test kit (Qiagen). after extracting total serum IgE, adopt PrimeScriptTMRT test kit and SYBRPremixExTaqTMII test kit (TaKaRa) carries out quantitative RT-PCR amplification。Quantitative analysis (ABIsmartI) is carried out, it is determined that the expression of each genes of interest according to the Ct value of each genes of interest and the Ct value of reference gene。The primer sequence of institute's amplification gene is as follows:
The primer sequence of quantitative RT-PCR detection
| Gene | Forward | Reverse |
| rGAPDH | CAAGGTCATCCATGACAACTTTG | GGGCCATCCACAGTCTTCTG |
| rCYP1A2 | CGGTGATTGGCAGAGATCGG | GTCCCTCGTTGTGCTGTGG |
| rCYP2B1 | CTCCAAAAACCTCCAGGAAATCCTC | GTGGATAACTGCATCAGTGTATGGC |
| rCYP3A1 | GAGGAGTAATTTGCTGACAGAACCTGC | CCAGGAAT CCCCTGTTTCTTGAA |
r:Rat-specificprimer
Hepatocyte metabolic enzyme activity measures: in cultivating the 6th day, changes serum-free medium, and adds 50 μMs of testosterones, 37 DEG C hatch 4h after collect culture medium, 4 DEG C centrifugal (12000rpm, 10min) collects supernatant afterwards and preserves extremely-80 DEG C of refrigerators。Carry sample is collected after terminating, HPLC method is adopted to carry out level (testing conditions: A (methanol): B (water)=52:48,0-15min, the B48-30 of detection CYP3A metabolite 6 β hydroxyl testosterone, 15-20min30-20, UV254nm)。
Result:
1. hepatic cell growth form and activity: as in figure 2 it is shown, primary hepatocyte growth conditions is good on porous silk albumen support, cytoactive dyeing display most cells is green fluorescence, illustrates that its activity is desirable。Only have a small amount of cell death, show red fluorescence。Cell marking picture prompting, hepatocyte presents agglomerate growth pattern at three-dimensional co-culture system, and is prone to polymerized together with star Interstitial cell agglomerating；And cellular control unit comparatively disperses, have no cell agglomerating phenomenon polymerized together。Analyze the hepatocyte agglomerating hepatocyte incubation in vitro of having reason of polymerization and better maintain its phenotype and function。
2.H&E dyes the formation and morphological characteristic that (Fig. 3) result proves that hepatocyte rolls into a ball further。Visible: the agglomerate growth on porous silk albumen support of major part hepatocyte and star Interstitial cell。Wherein, hepatocyte presents polygon, it is seen that monokaryon or double-core, and star Interstitial cell is distributed in hepatocyte group, and the two closely condenses together, and supports that two kinds of iuntercellulars would be likely to occur cell-ECM interphase interaction。
3. as shown in Figure 4, scanning electron microscope result display hepatocyte compact growth is in porous silk albumen rack surface and three-dimensional pore space, cellular morphology rule, and secretes substantial amounts of extracellular matrix。It is connected with each other to each other, forms the apparent form feature of class loading。Saturating color electronic microscope photos shows further can set up cell tight junction between hepatocyte and hepatocyte, there is exchange to each other, present " class loading " feature。These all support the excellent activity in hepatocyte incubation in vitro, systematism feature and desired phenotypes。
As it is shown in figure 5, hepatocyte albumin secretion and urea synthesis testing result show: compare with matched group, co-culture hepatocyte in group based on the three-dimensional of fibroin support and can secrete the albumin of higher level；In addition its urea synthesis level is also significantly increased (P < 0.05) than matched group。This result illustrates, primary hepatocyte is able to maintain that desirable complex functionality activity under this cultivating system。
The evaluation of hepatocyte metabolic enzyme expression and enzyme assay: the change of hepatocyte metabolic enzyme is the important indicator that hepatocyte metabolic function is evaluated。As shown in Fig. 6,7,8,9, after three-dimensional co-cultures, the various kinds of cell metabolism of pigment enzyme gene table level of Rat Primary Hepatocytes is significantly higher than matched group, and found by specific substrate metabolic analysis: compare with compareing cultivation group, in dimensional culture system, the metabolism of testosterone is improved by hepatocyte, prove that the metabolic activity of its CYP3A also is able to desirable maintenance in this cultivating system, thus having provided an experiment argument for hepatocellular ideal functionality。
Claims (1)
1. the cultural method that can improve hepatocyte vitro differentiation phenotype and function, it is characterized in that: described method carries out according to following steps: employing hepatocyte is seed cell, using porous silk albumen support as Hepatocytes culture in vetro carrier, hepatocyte, liver source property Interstitial cell and extracellular matrix are inoculated on porous silk albumen support by a certain percentage, and prop up porous silk albumen to be placed in the hepatocellular In vitro culture microenvironment of simulation and cultivate
Described by hepatocyte, the concrete operation step that liver source property Interstitial cell and extracellular matrix are inoculated on porous silk albumen support by a certain percentage is as follows: mixed according to the ratio of cell number 3:1 by the hepatocyte regulating liver-QI star Interstitial cell of fresh separated, and the cell mixing obtained is mixed with extracellular matrix, obtain cell-matrix mixture, described extracellular matrix is mixed according to the ratio that volume ratio is 1:1 by collagen and matrigel, then cell-matrix mixture multiple spot is seeded to multiple porous silk albumen support, and ensure that hepatocyte quantity is 600, 000cells/ support, the volume of matrigel is 30 μ L/ supports,
Described being propped up by porous silk albumen is placed on that to simulate the concrete operation step carrying out cultivating in hepatocellular In vitro culture microenvironment as follows: be placed in porous cell culture plate by the porous silk albumen support inoculated according to the density in 1 support/hole, and porous cell culture plate is placed in cell culture incubator, at 37 DEG C, 5%CO2When gelation 2h, then add in each hole of porous cell culture plate and co-culture base, the described base that co-cultures is hepatocyte culture medium and star Interstitial cell culture medium mixes according to the ratio that volume ratio is 1:1。
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Cited By (3)
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| CN107446885A (en) * | 2017-09-06 | 2017-12-08 | 大连医科大学 | A kind of timbering material of derived mesenchymal stem cells in vitro Osteoinductive differentiation and its application |
| CN110381968A (en) * | 2017-02-01 | 2019-10-25 | 菲尼克松生物制品公司 | The composition and method of coculture comprising liver cell |
| CN110384823A (en) * | 2019-07-19 | 2019-10-29 | 大连医科大学 | Bionical lobuli hepatis and construction method based on silk fibroin bracket |
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| CN110384823A (en) * | 2019-07-19 | 2019-10-29 | 大连医科大学 | Bionical lobuli hepatis and construction method based on silk fibroin bracket |
| CN110384823B (en) * | 2019-07-19 | 2021-08-03 | 大连医科大学 | Bionic liver lobule based on silk fibroin scaffold and construction method |
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