CN106916781A - A kind of construction method of external 3 D human body hepatic tissue and its application - Google Patents
A kind of construction method of external 3 D human body hepatic tissue and its application Download PDFInfo
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Abstract
The present invention relates to a kind of construction method of external 3 D human body hepatic tissue, comprise the following steps：(1) solution of seed cell and bio-ink solution are uniformly mixed to get cell printing solution；(2) under the control of the computer, the cell printing solution is uniformly extruded according to the path and speed of design using cell printing machine, and the rapid condensation of cell printing solution is formed gel microfilament at a certain temperature；Operated more than repeating, the structure of multi-layer cellular structure is completed in the way of scanning layer by layer；(3) secondary cross-linking treatment is carried out to the multi-layer cellular structure, and it is cultivated using cell culture fluid, obtain constitutionally stable multi-layer cellular structure；(4) using constitutionally stable multi-layer cellular structure described in inducing culture culture, external 3 D human body hepatic tissue is obtained.The good external three-dimensional liver organization of method constructing function of the present invention, can be widely applied to the fields such as liver disease, drug development, drug screening.
Description
Technical field
The present invention relates to biological technical field, in particular it relates to a kind of three-dimensional bionic human liver group
The vitro construction method knitted.
Background technology
Liver is the maximum internal organs of human body, is responsible for removing toxic substances, metabolism, secretion of bile and exempts from
Various critical functions such as epidemic disease defence.China is hepatopathy big country, 300,000 people is there are about every year and dies from liver
Disease, the population that there are about 1/10th is hepatitis carrier, for the expense of liver disease
More than 30,000,000,000.For serious diseases such as liver cancer and acute hepatic failures, the optimal path for the treatment of is
Liver transfer operation, but the limitation in organ donor source is still inevitable problem.On the other hand, put down
A kind of R＆D costs of new drug are approximately 5,000,000,000 dollars, last the absorbed input of 12 years, greatly
Part funds and time all spend in screening compound and drug test stage.However, on medicine
Human toxicity and Adverse events are still likely to occur behind city, this be also medicine recall it is main
Reason, and wherein there are about the 50% hepatotoxicity problem for coming from medicine.
Conventional drug test model is the cell of animal model and two-dimension single layer culture at this stage.
But the species specificity and muroid due to multiple pathogens (such as hepatitis C) are with the mankind's
The huge difference of Telomerase regulation mechanism, causes the liver metabolism rule of animal body in many aspects
All have this qualitative difference with human body, therefore, animal model often cannot effective detection go out medicine
The side effects such as the toxicity for human liver.
The human liver cell of plane culture is drug toxicity correlative study " golden standard ", application
In including drug metabolism, drug toxicity, hepatites virus infections, antiviral drugs exploitation and liver
The research fields such as the new treatment of disease.But the human hepatocyte source of health is extremely limited, and puts down
Face monolayer culture conditions again limit distribution and the cybotactic state of cell surface receptor, disturb just
Often tissue in cell cortex protein signal transduction, therefore two-dimension single layer culture liver cell very
Its phenotype and functional character (less than 7 days) are lost soon, strongly limit the extensive use of the technology.
Therefore, the stem cell originated based on human primary's liver cell or human body, is lured in a three-dimensional structure
Lead and be differentiated to form functional liver cell structure, be that the emphasis studied at this stage and difficult point are asked
Topic.
Philosophy and technique based on organizational project, domestic and foreign scholars use biomaterial thin as liver
The timbering material of born of the same parents' culture, target is reconstituted cell growth polarity and function, and then forms function
The three-dimensional hepatic tissue of property.
The differentiation and maturation of human hepatocyte interacts for cell-ECM, cell-epimatrix phase
Interaction and oxygen content have demand higher, and above-described traditional tissue engineering technique is very
It is difficult that various kinds of cell composition is implanted into solid support, particularly it is difficult to be controlled in three dimensional scaffold structure
The spatial distribution of different types of cell and extracellular matrix is made, therefore, external structure large volume
, long-term tool functional human body hepatic tissue have very highly difficult, relevant report is had not seen at present.
Biotechnology based on 3D printing has in the manufacture of personalized external biological structural model
Unique advantage, the application of this technology is increasingly becoming an emerging field for multi-crossed disciplines-thin
Born of the same parents' 3D printing.The technology can quickly be formed the internal three-dimensional structure containing cell without
Cell seeding process；Can the spatially different types of cell of accurate deposition；Can be in structure
Straight through tube is designed to improve the ability of mass transfer, is carried to build large volume of active mass
Supply possible, the external structure research and drug test of Various Tissues have been had been applied at present
In, including adipose tissue, energy metabolism system, cancerous tissue etc..In January, 2014, the U.S.
Biotech company Organovo utilizes cell inkjet technology (inkjet cell printing
Technology the miniaturization containing liver cell, astrocyte and vascular endothelial cell) is constructed
" mini people's liver ", receives social extensive concern, it is shown that cell 3D printing technique it is huge
Prospect, but due to changing the limitation of technology print speed, the full-size of the mini liver is 2mm,
And the report of formal document is not yet seen at present, price is very expensive.In addition, at present still
Have not seen that three-dimensional printing technology builds other reports of human hepatocyte.
The content of the invention
(1) technical problem to be solved
Bad, the size that there is functional character for the external human body hepatic tissue for building in the prior art
The uncontrollable defect of size, present invention firstly provides a kind of volume structure it is adjustable, have for a long time
The construction method of the external human body hepatic tissue of function.
(2) technical scheme
Technical solutions according to the invention are comprised the following steps：
(1) solution of seed cell and bio-ink solution are uniformly mixed to get cell printing
Solution；
(2) under the control of the computer, the cell printing solution is pressed using cell printing machine
Path and speed according to design are uniformly extruded, and make cell printing solution at a certain temperature
Rapid condensation forms gel microfilament；Operated more than repeating, complete many in the way of scanning layer by layer
The structure of confluent monolayer cells structure；
(3) secondary cross-linking treatment is carried out to the multi-layer cellular structure, and is trained using cell
Nutrient solution is cultivated it, obtains constitutionally stable multi-layer cellular structure；
(4) using constitutionally stable multi-layer cellular structure described in inducing culture culture, obtain
To external 3 D human body hepatic tissue.
In the present invention, the bio-ink is selected from comprising the molten of one or more following material
Liquid：Gelatin, gelatine derivative, alginate, alginate derivative, agar, matrix
Glue, collagen, proteoglycan, glycoprotein, hyaluronic acid, shitosan, layer connection egg
In vain, fine connection albumen, fibrin, vitronectin, osteopontin, peptide fragment hydrogel.
In the present invention, the bio-ink is preferably the mixed liquor of gelatin and alginate, bright
The mixed liquor or sodium alginate of glue, sodium alginate and matrigel, collagen and fibrin
Former mixed liquor；Preferably, wherein in the bio-ink, the concentration of alginate is
0.1%~5%.Above material is natural biologic material, and cell compatibility is good.Alginate
Can physiological concentration calcium ion effect under Quick cross-linking, and maintain for a long time it is cyto-architectural
Stability under culture environment；Gelatin has temperature-sensing property, can be protected by adjusting molding condition
Low shear conditions are held so as to ensure cell survival；Matrigel, fibrinogen, gelatin etc.
Material has fabulous cell induction and a tissue inductivity, differentiation beneficial to three-dimensional liver cell,
Ripe and hepatic tissue is formed.
In the present invention, the seed cell include people's liver stem cells, people's liver parenchymal cell,
Liver cell, people's inductivity that people's liver cell system, human embryo stem cell induction are differentiated to form
Liver cell, human mesenchymal stem cell induction differentiation shape that myeloid-lymphoid stem cell induction is differentiated to form
Into liver cell, the liver cell that is differentiated to form of people's tissue stem cell induction, people's endothelium it is thin
In born of the same parents, HF, people's bile duct epithelial cell, people's Kupffer cell, Human astrocyte
One or more mixture.
In the present invention, the density of seed cell is 10 in the cell printing solution5~107It is individual
/mL。
In the present invention, the secondary cross-linking solution includes being selected from one or more following material
Solution：Calcium chloride, Geniposide, glutaraldehyde, thrombin solution；Preferably, described two
Secondary crosslinker solution is that the calcium chloride or concentration of concentration 100mM~500mM are
The fibrin ferment of 10ng/ml~1mg/ml.
In the present invention, the inducing culture is to add concentration in liver cell amplification cultivation liquid
It is 0.5%~2% DMSO.
The liver cell amplification culture medium that the present invention is used is addition in DMEM nutrient solutions
10%FBS serum, 0.05% insulin, 5 × 10-5M hydrocortisone succinates, 1% is blue or green
Streptomysin, 1%GlutaMAXTMSupplement and 1%MEM Non-Essential
Amino Acids Solution.The concentration addition of DMSO is different, the hepatic tissue of induced synthesis
Structure it is different, when it is 0.5%DMSO to add concentration, can both form liver cell, also may be used
Form bile duct network structure；When it is 2% DMSO to add concentration, can only liver be formed thin
Born of the same parents, when it is 1% to add concentration, early stage can form bile duct network structure, and the later stage disappears.
Another object of the present invention is the external three-dimensional that the Sustainable use method of the present invention builds
Human body hepatic tissue.
Final object of the present invention is that protection external 3 D human body hepatic tissue of the invention exists
1) material of in-vivo tissue reparation or regeneration is prepared；2) vitro detection use in medicament-induced hepatotoxicity is prepared
Material；3) liver disease；4) drug development, drug screening, drug test 5) build
Application in terms of pharmacological model, pathological model, tissue/organ model.
(3) beneficial effect
Method of the present invention, with following beneficial effect
1) volume and size are controllable
Hepatic tissue in the present invention is prepared from by extruded type cell three-dimensional printing technique, phase
For ink jet type cell printing technology, print speed is fast, efficiency high, inside configuration can
Conveniently realize that through channel is transmitted so as to nutriment, can both form the micro- of grade yardstick
Type hepatic tissue, can also form centimetre, the large-scale hepatic tissue of even decimeter grade yardstick.Both
All can as liver disease research platform, pathology/pharmacological model and histoorgan model,
The former is particularly suited for high-throughput drug detection research, and can coordinate the biochip device to make
With the latter is more suitable for internal disease damage tissue renovation material.
2) hepatic tissue structure-controllable
In the present invention, stem cell can break up to multiple directions simultaneously in a three-dimensional structure, shape
Into the various kinds of cell in hepatic tissue, and, can be formed by adjusting the concentration of inducing solution
With or without the multiple-limb bile duct network of micron-sized insertion.
3) hepatic tissue better function
Stem cell remains right to the maturity and long-term function of liver cell induction differentiation
It is all higher in the requirement of carrier material, surrounding three aspects of other cells and oxygen concentration,
In existing report or stem cell differentiation efficiency is low, maturity is poor, or liver cell
Function is held time shorter.Containing various thin to liver in cell three-dimensional structure of the invention
Born of the same parents' function has the carrier material of facilitation, such as collagen, matrigel.Additionally, being based on
Three-dimensional printing technology, it is ensured that between the Breadth Maximum and microfilament per root timber material microfilament
Away from, cell culture fluid is filled between microfilament, with meet liver cell and stem cell to compared with
The requirement of high oxygen concentration.With the same cell under identical inductive condition of two dimension culture
Compare, the albumin secretion level of three-dimensional cell structure of the invention is 711 times, urinate
Plain secretion level is 16 times, as shown in Figure 3.The table of liver cell key gene albumin
It is 18 times up to level, the expression of bile duct cell key gene CK19 is 18.6 times,
The expression of hepatocytes secrete function key gene MRP2 is 10 times, liver cell medicine
The expression of thing metabolic function key gene CYP3A4 genes is 189 times, such as Fig. 4
It is shown.
4) it is transparent
Eucaryotic cell structure body transparency of the invention is high, can monitor it by light microscope
The growing state of the cell for being loaded and be conducive to real-time monitoring three-dimensional hepatic tissue life
Thing performance.
Brief description of the drawings
Fig. 1 is hepatic tissue pattern of the present invention.
(A) hepatic tissue microstructure, spherical is liver cell cluster, as shown in blue arrow,
Fusiformis is bile duct cell, as illustrated with black arrow；
(B) the hepatic tissue microstructure of liver cell is comprised only, spherical is liver cell cluster,
As shown in blue arrow；
Fig. 2 represents the key point protein expression situation of various kinds of cell in hepatic tissue；
Fig. 3 represents hepatic tissue albumin and urea secretion level, the bar of the same race with two dimension culture
Part compares, and * represents that data have significant difference；
Fig. 4 represents the key point expression conditions of hepatic tissue, same with two dimension culture
The condition of kind compares, and * represents that data have significant difference.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Term " 3 D-printing " used herein refers to：Via with it is automatic or automanual,
The method that computer assisted three-dimensional modeling apparatus (such as three-dimensional printer) match, utilizes
The three-dimensional accurate deposition that the compatible raw material of 3 D-printing is carried out.
In some embodiments, three-dimensional structure of the invention is engineering.Term " work
Journey " refer to：It is (such as three-dimensional by computer assisted device according to computer script
Printer), by raw material (such as bio-ink raw material and/or cell printing solution) and they
Layer is placed to form three-dimensional structure.In further embodiment, computer script, for example
It is one or more computer programs, computer application or computer module.
In some embodiments, 3 D-printing method is continuous and/or substantially continuous.
Continuously the non-limiting examples of 3 D-printing method are：Via being connected to marking ink holder
Distribution end (for example, syringe, capillary etc.) distributes marking ink (example from three-dimensional printer
Such as bio-ink raw material and/or cell printing solution).In further non-limiting embodiments
In, continuous 3 D-printing method is to distribute printing by the repeat patterns (pattern) of functional unit
Ink.
In each embodiment, repeat function unit has any suitable geometry, bag
Include for example：Circle, square, rectangle, triangle, polygon and irregular geometry,
Obtained by the spatial patterned of unique marking ink and/or clearance space so as to produce to have
Plane geometric shape one or more organized layers.It is three-dimensional in further embodiment
One repeat patterns of the functional unit of printing include a layer, are adjacent to 3 D-printing (for example
Stacking) multiple layers to be forming the engineered constructs body with stratiform geometry.In each implementation
Scheme, be adjacent to 3 D-printing (such as stack) 2,3,4,5,6,7,8,9,10,
11st, 12,13,14,15 or more layers, to form engineered constructs body.Entering one
In the embodiment of step, one or more layers of the tissue with stratiform geometry also have puts down
Face geometry.
In some embodiments, the method for continuous 3 D-printing includes：Independently or relative to
Optimize and/or balance the parameter of such as printing height, pump speed, robot speed or its combination each other.
In an example, for deposit 3 D-printing head speed be 3mm/s, ground floor point
It is 0.5mm with height, and the distribution of each layer highly increases 0.4mm below.In some implementations
In scheme, distribution height is of substantially equal with the diameter of three-dimensional printer distribution end.It is unrestricted
System ground, suitable and/or optimal distribution distance will not cause material to flatten or be attached to distribution
On pin.In each embodiment, three-dimensional printer distribution end have about 20,50,100,
150、200、250、300、350、400、450、500、550、600、650、700、
750th, 800,850,900,950,1000 μm or bigger internal diameter, including wherein any model
Enclose.In each embodiment, the marking ink holder of three-dimensional printer has about 0.05,
0.1、0.5、1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、
40、45、50、55、60、65、70、75、80、85、90、95、100cm3Or it is bigger
Volume, including wherein any range.In some cases, when the residual pressure in system is accumulated
When tired relatively low, the pump speed is suitable or optimal.In some cases, favourable pump speed takes
The certainly ratio between the cross-sectional area and distribution pin of holder, the ratio is bigger, then need to get over
Low pump speed.In some embodiments, suitable and/or optimal print speed makes it possible to
The line of enough depositing homogeneous, the mechanical integrity without influenceing material.
In some embodiments, the speed of technology disclosed herein and method and can amplification
For designing, building and operate for producing for being implanted into the three-dimensional structure that uses or for generating
The industry of the instrument (for researching and developing, such as analyzed in vitro) based on cell and/or business
Industry facility.In further embodiment, three-dimensional bionic human body hepatic tissue and its array are given birth to
Produce, store, distribute, list, publicize and sell, such as bioanalysis and high pass
Measure cellular array (such as microarray or chip), tissue array (such as micro- battle array of drug screening
Row or chip) and kit.In other embodiments, three-dimensional bionic human body hepatic tissue and
Its array is manufactured and is used for carrying out the bioanalysis and/or drug screening as service.
Embodiment 1
The present embodiment is related to a kind of construction method of external 3 D human body hepatic tissue, including following step
Suddenly：
Preparation process：
1) preparation of bio-ink original solution
Gelatin solution：It is according to mass ratio with deionized water by gelatin powder (Sigma, G1890)
15:100 ratio mixing, heating makes its uniform dissolution, Zhi Houfen in 3 hours under the conditions of 80 DEG C
Dress, in 4 DEG C or -20 DEG C of Cord bloods.10min is incubated in cell culture incubator using preceding every time
After it is melted as homogeneous solution.
Sodium alginate soln：By sodium alginate powder (Sigma, A0682) and deionized water according to
Mass ratio is 4:100 ratio mixing, heating makes its uniform dissolution in 3 hours under the conditions of 80 DEG C,
Dispense afterwards, in 4 DEG C or -20 DEG C of Cord bloods.Protected in cell culture incubator using preceding every time
It is set to melt as homogeneous solution after warm 10min.
2) culture of seed cell
HepaRG cells (sigma, HPRGC1) are trained in cell amplification cultivation liquid
Support, the composition of cell amplification cultivation liquid in DMEM (Gibco, 11960044) nutrient solution to add
Plus 10%FBS serum (Gibco, 16000), 0.05% insulin (Sigma, I9278), 5 × 10-5M
Hydrocortisone succinate (Sigma, H4881), 1% mycillin (Gibco, 15140122),
1%GlutaMAXTMSupplement (Gibco, 35050061) and 1%MEM
Non-Essential Amino Acids Solution(Gibco,11140050).When cell 90% converges
When according to 1:5 ratio passage, a nutrient solution was changed per 2-3 days.
Specific implementation step：
1) it is the μ L of previously prepared gelatin solution 600 is equal in room temperature with the μ l of sodium alginate soln 450
Even mixing, obtains bio-ink solution, and is deposited into being preheated in 37 DEG C of cell culture incubator
20min is standby；Seed cell is digested with the digestive juice of 0.25% trypsin/EDTA after collecting
With 6 × 106The density suspension of individual/ml obtains seed cell solution in cell amplification cultivation liquid；Take
100 μ L seed cells solution and bio-ink uniformly after mixing cell printing solution, and by its
It is loaded into 1mL disposable sterilized injectors；
2) asepsis injector is loaded into biological 3 D-printing equipment (specific method can be found in
Rui Yao,et al.,In Vitro Angiogenesis of 3D Tissue Engineered Adipose
Tissue,Journal of Bioactive and Compatible Polymer,2009；24：5), often
Under temperature, under computer software (Microsoft, AT640, Redmond, WA) control, with
Stepper motor speed 2mm/s, under the Parameter Conditions of sweep speed 10mm/s, in aseptic plane
3 D-printing on platform, forms the pregel three-dimensional structure that volume is 8mm × 8mm × 5mm
Body.
3) the pregel structure that will 2) obtain is crosslinked 3min with the calcium chloride solution of 100mM,
Then suck calcium chloride solution and add HepaRG cell amplification cultivation liquid, (37 under normal condition
DEG C, 5%CO2 incubators) cultured cells three-dimensional structure, changes liquid in every 2~3 days in incubation.
4) cell induction nutrient solution is replaced by after cultivating 7 days, continues to cultivate 7 days, the cell
Induction broth is 0.5%DMSO to add concentration in cell amplification cultivation liquid.
Embodiment 2
Compared with embodiment 1, difference in this case is that, step 4) in, the cell is lured
It is 2%DMSO that nutrient solution is led to add concentration in cell amplification cultivation liquid.
Embodiment 3
Compared with embodiment 1, difference in this case is that, step 4) in, the cell is lured
It is 1%DMSO that nutrient solution is led to add concentration in cell amplification cultivation liquid.
Experimental example
1st, the metamorphosis of cell and tissue
Observation of cell metamorphosis and taken a picture daily with light microscope (Olympus, CX40).
Gained three-dimensional bionic human body hepatic tissue microscopic appearance is as shown in figure 1, Fig. 1 (A) is embodiment 1
Obtaining existing liver cell (shown in grey arrow) also has bile duct network (shown in black arrow)
External hepatic tissue microstructure.Fig. 1 (B) obtains the liver for comprising only liver cell by embodiment 2
Tissue microstructure, as shown in grey arrow.
2nd, the related crucial egg of three-dimensional hepatic tissue in routine immunization fluorescence colour detection embodiment 1
White expression.Concrete operation step is：
Nutrient solution is sucked, the gained three-dimensional bionic of embodiment 1 is rinsed with phosphate buffer (Sigma)
Hepatic tissue 1 time；
With 2.5% glutaraldehyde (Sigma) fixing organization 4 hours at room temperature, phosphate buffer is used
(Sigma) tissue is rinsed 3 times, every time 5 minutes；
20min is processed with 0.25%Triton-X ruptures of membranes, is rinsed with phosphate buffer (Sigma)
Tissue 3 times, every time 5 minutes；
1h is closed with 5% fetal bovine serum albumin, is rinsed with phosphate buffer (Sigma) and organized
3 times, every time 5 minutes；
Add primary antibody solution, such as ZO-1 (ab59720, Abcam, 1 μ g/ml), MRP2
(ab3373, Abcam, 10 μ g/ml), ALB (ab83465, Abcam, 5 μ g/ml),
CYP3A4 (ab3572, Abcam, 5 μ g/ml), CK18 (AF7619, R＆D Systems,
1 μ g/ml), HNF-4 α (MAB4605, R＆D Systems, 15 μ g/ml), CK19
(MAB3506, R＆D Systems, 15 μ g/ml), AFP (MAB1368, R＆D
Systems, 15 μ g/ml), 4 DEG C are overnight.Tissue is rinsed with phosphate buffer (Sigma) 3 times,
5 minutes every time；
Add correspondence secondary antibody, such as Alexa594 (Abcam, 150080, dilution 1000
Again), Alexa488 (Abcam, 150113, dilute 1000 times), add corresponding two
It is anti-, after room temperature lucifuge is incubated 2h, rinse tissue 3 times with phosphate buffer (Sigma), every time
5 minutes；
The DAPI staining cell cores of 1 μ g/ml are added, room temperature lucifuge is incubated 15min；
Picture observation is carried out using laser confocal microscope (LSCM, Nikon, Z2).
The key protein of three-dimensional bionic hepatic tissue：ALB、AFP、CYP3A4、CK19、ZO-1
Staining conditions are shown in Fig. 2.
As shown in Figure 2, this several albumen detects expression high, wherein ALB and CYP3A4
It is the significant albumen of mature hepatocytes secreting function and drug-metabolizing function；AFP is that liver ancestral is thin
Born of the same parents' mark, CK19 is the mark of bile duct cell；ZO-1 is that liver cell forms sense of organization row
Occurs the significant albumen of polarity and timid tubular construction after row.As seen from the figure, liver cell is formed tightly
The Cluster Structures of solid matter row and expression albumin (ALB) high, drug metabolism albumen (CYP3A4)
With cell polarity albumen (ZO-1), bile duct cell is self-assembly of hollow tubular structure and table high
Up to CK19 albumen.
3rd, the secreting function of the secretion of detection hepatic tissue albumin and urea
Detection kit (Bethyl, E80-129, E101, E115) is secreted using albumin
With urea secretion detection kit (BIO ASSAY SYSTEMS, DIUR-500) according to
The albumin secretion of the kit specification detection gained hepatic tissue of embodiment 1~3 and urea secretion
Function, as a result as shown in figure 3, wherein 2D-0.5 represents the DMSO under conditions of 2D printings
Concentration represent that the concentration of the DMSO under conditions of 3D printing is 0.5 for 0.5,2D-0.5,
Other expressions the like.Result show embodiment 2 compared with the cell of conventional plane culture,
In the case where cell culture fluid and induction broth composition, culture environment are just the same, three
The albumin secretion level for tieing up bionic liver tissue is 711 times of plane cultured cells, urea point
It is 16 times of plane cultured cells that bleeding is put down.
4th, hepatic tissue related gene expression is detected
Using standard quantitative DNA test (Quantitative real time polymerase
Chain reaction) detection the three-dimensional bionic hepatic tissue of embodiment 1~3 hepatic tissue related gene table
Reach, it is as follows that hepatic tissue RNA extracts operating procedure：
1) nutrient solution is sucked, gained in embodiment 3 is rinsed with phosphate buffer (Sigma)
Three-dimensional bionic hepatic tissue 3 times；
2) each hepatic tissue adds 1mlReagent (Gibco, 15596026), instead
Multiple piping and druming is mixed, and is then stored at room temperature 10min；
3) 0.2ml chloroforms (TaKaRa) is added, acutely vibration 30 seconds, room temperature places 5min；
4) at 4 DEG C, it is centrifuged 15 minutes using centrifuge (sigma3K15) 12000g；
5) Aspirate supernatant, adds isometric isopropanol, and room temperature places 10min；
6) 4 DEG C of 10000 × g are centrifuged 10 minutes；
7) supernatant is abandoned, with 75% ethanol wash RNA precipitate；
8) after air-drying, with DEPC (TaKaRa) water dissolves.
9) detected with spectrophotometer (Thermo Scientific) RNA concentration and
Purity.
RNA reverse transcription operating procedures：
Using PrimeScriptTMII 1st strand cDNA Synthesis Kit (TaKaRa,
6210), operated fully according to kit specification.Rna content is adjusted to 5ng.
Primer is：Oligo dT Primer.Reverse transcription PCR program is：42 DEG C of 50min, 95 DEG C
5min, 4 DEG C of insulations, PCR instrument (ABI, SimpliAmpTM thermal cycler) used.
Quantitative fluorescent PCR operating procedure：
Kit：Maxima SYBR Green qPCR Master Mix(Thermo
Scientific, K0251), operated fully according to kit specification.Response procedures are：95
DEG C 10min, 95 DEG C of 15s, 60 DEG C of 30s, 40 circulations, 72 DEG C of 30s, 72 DEG C of 10min.
Detecting instrument：Bio-Rad,CFX96.The primer sequence is as follows：
GAPDH-F TGCACCACCAACTGCTTAGC
GAPDH-R GGCATGGACTGTGGTCATGAG
ALB-F GCACAGAATCCTTGGTGAACAG
ALB-R ATGGAAGGTGAATGTTTCAGCA
MRP2-F TGAGCAAGTTTGAAACGCACAT
MRP2-R AGCTCTTCTCCTGCCGTCTCT
CYP3A4-F TAACAGTCTTTCCATTCCTC
CYP3A4-R GGACTCAGTTTCTTTTGAAT
CK19-F ATGGCCGAGCAGAACCGGAA
CK19-R CCATGAGCCGCTGGTACTTCC
Wherein, ALB and CYP3A4 are mature hepatocytes secreting function and drug-metabolizing function
Significant albumen；CK19 is the mark of bile duct cell；MRP2 is that liver cell forms the sense of organization
Occurs the significant albumen of polarity and timid tubular construction after arrangement.As shown in Figure 4：3 D-printing
The expression of the various genes of structure is above gene expression dose in two dimension printing, is equal
Under the conditions of 7 times of two-dimentional cultured cells, MRP2 gene expression doses are in 3 D-printing structure
15 times of two-dimentional cultured cells, CYP3a4 genes table in 3 D-printing structure under equal conditions
20 times of two-dimentional cultured cells, CK19 in 3 D-printing structure under being equal conditions up to level
Gene expression dose is 19 times of two-dimentional cultured cells under equal conditions.
Although general explanation, specific embodiment and experiment have above been used, to this hair
It is bright to have made detailed description, but on the basis of the present invention, it can be made some modifications or improvements,
This will be apparent to those skilled in the art.Therefore, without departing from spirit of the invention
On the basis of these modifications or improvements, belong to the scope of protection of present invention.
Claims (10)
1. a kind of construction method of external 3 D human body hepatic tissue, it is characterised in that including such as
Lower step：
(1) solution of seed cell and bio-ink solution are uniformly mixed to get cell printing
Solution；
(2) path using cell printing machine by the cell printing solution according to design and speed
The uniform extrusion of degree, and the rapid condensation of cell printing solution is formed gel microfilament；More than repeating
Operation, completes the structure of multi-layer cellular structure in the way of scanning layer by layer；
(3) secondary cross-linking treatment is carried out to the multi-layer cellular structure, and is trained using cell
Nutrient solution is cultivated it, obtains constitutionally stable multi-layer cellular structure；
(4) using constitutionally stable multi-layer cellular structure described in inducing culture culture, obtain
To external 3 D human body hepatic tissue.
2. method according to claim 1, it is characterised in that the bio-ink choosing
The solution of self-contained one or more following material：Gelatin, gelatine derivative, alginic acid
Salt, alginate derivative, agar, matrigel, collagen, proteoglycan, sugared egg
In vain, hyaluronic acid, shitosan, layer connection albumen, fine connection albumen, fibrin, glass
Connect albumen, osteopontin, peptide fragment hydrogel.
3. method according to claim 2, it is characterised in that the bio-ink is
Mixed liquor or the sea of the mixed liquor of gelatin and alginate, gelatin, alginate and matrigel
The mixed liquor of alginates, collagen and fibrinogen.
4. method according to claim 3, it is characterised in that in the bio-ink,
The concentration of alginate is 0.1%~5%.
5. the method according to claim 1 or 4, it is characterised in that the kind is careful
Born of the same parents include that people's liver stem cells, people's liver parenchymal cell, people's liver cell system, Human embryo are done
Liver cell that cell induction is differentiated to form, the induction of people's inductivity myeloid-lymphoid stem cell are differentiated to form
Liver cell, the liver cell that is differentiated to form of human mesenchymal stem cell induction, people's tissue it is dry
Liver cell, HEC, HF, people's courage that cell induction is differentiated to form
One or more mixture in pipe epithelial cell, people's Kupffer cell, Human astrocyte.
6. method according to claim 5, it is characterised in that the cell printing is molten
The density of seed cell is 10 in liquid5~107Individual/mL.
7. the method according to claim 1 or 6, it is characterised in that the secondary friendship
Connection solution includes the solution selected from one or more following material：Calcium chloride, Geniposide,
Glutaraldehyde, thrombin solution；Preferably, the secondary cross-linking solution is for concentration
The calcium chloride or concentration of 100mM~500mM are the fibrin ferment of 10ng/ml~1mg/ml.
8. the method according to claim 1 or 7, it is characterised in that the induction training
Foster base is that the DMSO that concentration is 0.5%~2% is added in liver cell amplification cultivation liquid.
9. the external 3 D human body liver group that a kind of any one of claim 1~8 methods described builds
Knit.
10. the external 3 D human body hepatic tissue described in claim 9 is repaiied preparing in-vivo tissue
Multiple or regeneration material；Prepare the material of vitro detection use in medicament-induced hepatotoxicity；Liver disease；Medicine
Thing exploitation, drug screening, drug test or drug test and structure pharmacological model, pathology mould
Application in terms of type, tissue/organ model.
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