CN105861419A - Bioartificial liver based on three-dimensional cell printing, and preparation method and application of bioartificial liver - Google Patents

Bioartificial liver based on three-dimensional cell printing, and preparation method and application of bioartificial liver Download PDF

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CN105861419A
CN105861419A CN201610258273.5A CN201610258273A CN105861419A CN 105861419 A CN105861419 A CN 105861419A CN 201610258273 A CN201610258273 A CN 201610258273A CN 105861419 A CN105861419 A CN 105861419A
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liver
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毛雷
毛一雷
杨华瑜
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Abstract

The invention provides a bioartificial liver. The bioartificial liver is a biological structure body which has a fixed steric configuration and is obtained by carrying out three-dimensional cell printing on cell printing ink containing primary hepatocyte or terminal differentiation hepatocyte (Hepa RG) together with a cytoskeleton material, cultivating and carrying out induced differentiation. The bioartificial liver has excellent biosecurity and structural stability, and has comprehensive and effective liver functions. The invention also provides a method for preparing the bioartificial liver, and application of the bioartificial liver in drug metabolism research.

Description

Based on Biotype artificial liver that cell three-dimensional prints and its preparation method and application
Technical field
The invention belongs to biomedical sector, relate to a kind of artificial tissue or organ, be specifically related to a kind of Biotype artificial liver, And its preparation method and application.
Background technology
Liver transplantation is the currently the only method that can effectively treat liver failure, but organ shortage has become as a global difficulty Topic.For a long time, medical industry has put into the difficult problem that substantial amounts of resource carries out studying to solving transplant organ deficiency.Due to me The hepatic failure patients that state's difference reason causes is the most, and at present for liver shortage, liver transplantation quantity is difficult to situation about increasing in a short time Under, artificial liver has obtained quick development and has been widely applied.
Artificial liver refers to by external machinery, chemical or biological device, temporarily and partly substitutes liver function, thus assists to control Treat hepatic insufficiency, liver failure or the method for relevant disease.At present, artificial liver of the prior art divides according to its composition and character It is three classes: physical, biotype and mixed type.These artificial livers can be all that liver regeneration is created by harmful substance in filtering blood Make the time.But existing artificial liver is also with some bio-safety problems, also limit its application.
In recent years, 3D prints becomes the emerging technology attracted attention in the whole world.Have been widely used for manufacturing industry, aerospace industry at present In field.3D is printed upon how the application of biomedical sector with manufacture anatomical model, surgical instruments, implant and prosthese is at present Main, specifically the application in surgery of liver is also mainly used for para-operative inanimate hepatic model to manufacture.
3D biometric print technology develop into biological tissue print and Organ printing bring hope and dawn.3D prints and is described as The representative art of " industrial revolution for the third time ", and 3D biometric print is 3D printing technique forefront and the richest vitality Research field.3D biometric print may be defined as with special biometric print machine as Main Means, with process active material include cell, Somatomedin, biomaterial etc. are main contents, new with rebuild tissue and the organ interdiscipline as main target and field Type regenerative medicine engineering.3D biometric print technology has incomparable compared to other tissue engineering bracket rapid shaping technique Advantage: the precision 1. building complex organization or organ is high, can really realize assembling and the structure of cell aspect.As constituting people The elementary cell of body, cell size in the range of several microns to tens microns, regulating cell distribution resolution need to 10 μm with Under, and use tradition tissue engineering technique to be difficult to the least resolution.2. cell and material organic assembling can be realized.3D beats Different cells in histoorgan and component can be carried out integrated molding by print.3. can be according to defective tissue or the actual feelings of organ Condition carries out instant, quick, controlled electronic 3-D model and reproduces, and carries out three-dimensional reconstruction by clinical image collection data. Re-use 3D printer and produce the product complying fully with patient's needs.4. individual character manufacturing complicated tissue organ, cost is controlled. For conventionally manufactured, product structure is the most complicated, and cost is the highest, and production quantity is the fewest, and manufacturing cost is the highest.And print at 3D In there is not this problem, completely personalized manufacture can on-demand produce, and printing labyrinth does not increase extra cost.
Not yet seeing the concrete report of 3D biometric print artificial liver tissue at present in public publication, the present invention is based on above-mentioned background A kind of new Biotype artificial liver is proposed.
Summary of the invention
The primary and foremost purpose of the present invention is: provide a kind of Biotype artificial liver approximating liver alive, has the biological safety of excellence And structural stability, and possesses liver function comprehensively and effectively.
Another object of the present invention is: the preparation method of the Biotype artificial liver described in offer.
The above-mentioned purpose of the present invention is achieved through the following technical solutions:
First, it is provided that a kind of Biotype artificial liver, it is by containing primary hepatocyte or terminal differentiation hepatocyte (HepaRG) Cell printing ink is after three-dimensional cell prints together with cell scaffold material, through cultivating and induction differentiation obtains has fixing sky Between the biological structure body of configuration.
In preferred embodiments of the present invention, in described biological structure body, there is liver cell group.
In the scheme that the present invention is further preferred, in described biological structure body, also there is gallbladder tube structure.
In preferred embodiments of the present invention, described cell printing ink contains collagen further；Described collagen accounts for cell printing ink The 85～95% of water, further preferred 90%.
In preferred embodiments of the present invention, in described cell printing ink, cell concentration is 0.8 × 106/ ml～1.2 × 106/ml。
In preferred embodiments of the present invention, described cell scaffold material contains gelatin and/or gelatine derivative.
In preferred embodiments of the present invention, described induction differentiation uses the DMSO solution of not higher than 2% concentration.
On this basis, the present invention also provides for a kind of method preparing described Biotype artificial liver, comprises the following steps:
1) preparation cell printing ink and cell scaffold material:
1.1) by the most cultured primary hepatocyte or terminal differentiation hepatocyte (HepaRG cell) and collagen one Rising and be hybridly prepared into suspension, regulation cell concentration is 0.8 × 106/ ml～1.2 × 106/ ml, standby；
1.2) preparation is containing gelatin and/or the cell scaffold material of gelatine derivative according to a conventional method, standby；
2) three-dimensional cell prints:
2.1) first by step 1.2) cell scaffold material prepared through 3D printer according to predetermined pattern file printout to predeterminated position Form described structure basal layer；
2.2) by step 1.1) the cell printing ink prepared and step 1.2) cell scaffold material prepared is mixed to get mixing material Material, in mixing material, cell concentration controls 0.8 × 105/ ml～1.2 × 105/ml；Described mixing materials'use 3D printer is pressed According to predetermined pattern file in step 2.1) successively print on the basal layer that obtains, until completing whole print procedure to obtain predetermined structure, The jet diameters of described 3D printer controls in 100～1000 μm；
2.3) by step 2.2) structure that printed uses the valeral solution soaking 2 of 0.5%～1% concentration～completes to fix for 5 minutes；
3) cultivate and induce differentiation
3.1) by step 2.3) fixing after structure cultivate in culture fluid at least 7 days, cultivation temperature is 35～37 DEG C, cultivates ring Border CO2Content is 4～6%, containing 8～the penicillin of the hyclone of 15%, 0.2～0.8%, 0.2～0.8% in described culture fluid Streptomycin and the DMEM cell culture medium of surplus；
3.2) in step 3.1) the DMSO solution that adds concentration not higher than 2% in cultured structure carries out induction differentiation, induction Differentiation was less than 7 days；Complete induction differentiation and i.e. obtain Biotype artificial liver structure body of the present invention.
In the method for the Biotype artificial liver described in currently preferred preparation, step 1.1) described in suspension in, collagen accounts for 85～95%, more preferably 90%；Control cell concentration 1 × 106/ml。
In the method for the Biotype artificial liver described in currently preferred preparation, step 2) described in printing, ambient temperature control System is at 18 22 DEG C, and printing precision is accurate to 50 μm.
In the method for the Biotype artificial liver described in currently preferred preparation, step 2.3) described in fixing, be use 0.5% The valeral solution soaking of concentration 3 minutes.
In the method for the Biotype artificial liver described in currently preferred preparation, step 3) preferably by step 2.3) fixing after Structure is according to described CMC model 7 days and induces differentiation 7 days.
In the method for the Biotype artificial liver described in the preparation that the present invention is further preferred, step 3.2) preferably in step 3.1) In cultured structure add concentration 0.5%～2% DMSO solution carry out induction differentiation.
Compared with artificial liver device of the prior art, the artificial liver of the present invention is the hepatic tissue knot with real biological significance Structure body, whether structure or function are all more nearly hepatic tissue alive.
In prior art, be theoretically utilized in 3D print hepatic tissue cell have a variety of, but its print after breed and induce divide The effect significant difference changed.The present inventor, through repeatedly verifying and screening, finds that HepaRG cell has the most excellent cell Dryness, and primary hepatocyte and bile duct epithelial cell can be induced under specific induction differentiation condition.This cell exists The present invention cultivates and can be divided into primary hepatocyte in the 3D print structure body under inductive condition, is attended by the knot of biliary ductal tree simultaneously Structure so that the structure after differentiation is more nearly hepatic tissue architectural feature alive.
On this basis, the present invention optimizes the condition of culture after printing and induction differentiation condition further, have selected DMSO molten Liquid is as induction agent, and determines the concentration of DMSO solution through substantial amounts of experiment sieving with checking, and the present invention is lured by this Lead atomization and serve pivotal role.DMSO solution is organic solvent conventional in a kind of biomedical research, has certain Cytotoxicity.But the DMSO of low concentration can have the function of differentiation of stem cells.Inventor sends out through lot of experiments Existing, under the culture environment that 3D of the present invention prints, the concentration DMSO solution less than 2% has for inducement structure body Good result.Wherein, after the concentration DMSO solution close to 1% is induced, structure liver metabolism function is preferable, to drug metabolism ratio More sensitive；After the concentration DMSO solution close to 2% is induced, structure physical ability produces more bile duct tree.And concentration exceedes The DMSO solution of 2% then has certain cytotoxicity, it is impossible to the effectively differentiation of inducement structure cells in vivo.
Additionally, other conditions that the present invention cultivates and induction differentiation is used, including cultivating and the induction control of divergaence time, training The control of the condition of supporting, and the composition of culture fluid etc., it is all the result of the optimization that inventor screens through a large amount of repetition tests.
The most all many selections and optimization, finally make hepatocellular survival in the Biotype artificial liver structure body of the present invention Rate is up to more than 95%, occurs obvious hepatic lineage, and the agglomerating growth of hepatocyte after differentiation；The various hepatocyte functions of representing Albumen is all expressed substantially in structure；Generally structure good stability, favorable repeatability, have liver function.Experiments verify that, Can continue under the artificial liver construction conventional organization condition of culture of the present invention at least 8 week structures dissipate, cell the most dead.
At present, regular growth cultivation is the main method of drug metabolism study, but is inaccurate.And the present invention through 3D cell Printing and 3D obtain after cultivating, and have fixed space configuration, have the organizational structure similar with the liver that lives and the structure of function Body, it has been investigated that, it can inspire in the processing procedure of medicine cultivates higher compensatory capacity compared with conventional cell, therefore For drug metabolism study, there is higher using value.
Therefore, the present invention also provides for described Biotype artificial liver in drug metabolism study as the application of simulated liver.
Accompanying drawing explanation
Fig. 1 embodies the apparent state of the Biotype artificial liver of Example 1 and Example 2 of the present invention.
Fig. 2 embodies the cell differentiated result of the Biotype artificial liver of Example 1 and Example 2 of the present invention.Wherein, A is The DMSO adding 0.5% concentration described in embodiment 2 induces the Microscopic of structure after 7 days；B is the partial enlarged drawing of A； C is that the DMSO adding 2% concentration described in embodiment 1 induces the Microscopic of structure after 7 days；D is the partial enlarged drawing of C.
Fig. 3 embodies the Biotype artificial liver liver function correlative protein expression described in the embodiment of the present invention 1.Wherein, a embodies The expression of albumin A LB；What b embodied is the expression of recombined human Cytokeratin 18；What c embodied is liver plasma membrane transhipment The expression of albumen MRP2；What d embodied is the expression of cytochrome enzyme CYP3A4；That e embodies is tight junction protein ZO 1 Express.
Fig. 4 embodies the biliary ductal tree structure in the Biotype artificial liver described in the embodiment of the present invention 1.A therein passes through The expression of CK19/ZO 1, imply that the structure of bile duct wall；B embodies hepatocyte/bile duct cell essence by the expression of ALB/MRP2 Fine texture.
Fig. 5 embodies the contrast of the liver function index of the Biotype artificial liver described in 2D culture hepatocyte and embodiment 2.
Fig. 6 embodies and utilizes the Biotype artificial liver described in the embodiment of the present invention 2 acetaminophen to carry out liver metabolism removing toxic substances in fact The result tested.
Detailed description of the invention
Mode by the following examples elaborates the present invention, but the thought of the present invention is not limited to cited embodiment. In cited embodiment, the reagent of all uses, material and apparatus are existing product.
Embodiment 1
A kind of Biotype artificial liver, it be by the cell printing ink containing HepaRG cell together with cell scaffold material three After dimension cell printing, through the biological structure body with fixed space configuration cultivated and induction differentiation obtains；There is liver cell Group and gallbladder tube structure.
Above-mentioned Biotype artificial liver is prepared by following steps:
1) preparation cell printing ink and cell scaffold material:
1.1) HepaRG cell is purchased from Invitrogen company, and by specification requires to cultivate, and is suspended in culture fluid, centrifugal, and Being configured to suspension after collagen mixing, adjusting cell concentration is 1 × 106/ ml, standby；
1.2) buying cell scaffold material test kit from Tianjin Lutao company, by specification is configured to derive containing gelatin and/or gelatin The cell scaffold material of thing, standby；
2) three-dimensional cell prints:
2.1) first by step 1.2) cell scaffold material prepared through 3D printer according to predetermined pattern file printout to predeterminated position Form described structure basal layer；Ambient temperature controls at 18 DEG C
2.2) by step 1.1) the cell printing ink prepared and step 1.2) cell scaffold material prepared is mixed to get mixing material Material, in mixing material, cell concentration controls 1 × 105/ml；By described mixing materials'use 3D printer according to predetermined pattern File is in step 2.1) successively print, until completing whole print procedure to obtain predetermined structure, described 3D on the basal layer that obtains The jet diameters of printer controls in 500 μm；Ambient temperature controls at 18 DEG C, and printing precision is accurate to 50 μm.
2.3) by step 2.2) structure that printed uses the valeral solution soaking of 0.5% concentration to complete to fix for 3 minutes；
3) cultivate and induce differentiation
3.1) by step 2.3) fixing after structure cultivate in culture fluid 7 days, cultivation temperature is 37 DEG C, culture environment CO2 Content is 5%, in described culture fluid containing 10% hyclone, the penicillin of 0.5% and the streptomycin of 0.5% and surplus DMEM cell culture medium；
3.2) in step 3.1) cultured structure adds the DMSO solution that concentration is 2% carry out induction differentiation, induction differentiation 7 days；Complete induction differentiation and i.e. obtain Biotype artificial liver structure body of the present invention.
Biotype artificial liver prepared by the present embodiment apparent as it is shown in figure 1, by immunofluorescence staining observe albumin secretion, Hepatocyte function correlative protein expression and bile duct cell correlative protein expression, it is seen that the following architectural feature in this structure:
As shown in B, D in Fig. 2, hepatocyte presents the sample growth of uniting of allied organization's structure；And as it is shown on figure 3, generation The albumen of table hepatocyte function is expressed substantially in structure: the existence of ALB, illustrates that the cell of this cultivation has secretory function； CK18 is recombined human CK18, and it is structural protein main in liver cell；MRP2 is liver plasma membrane transport protein, There is explanation liver cell and there is film transport function in it；CYP3A4 is a kind of main drug metabolism enzyme of human body, and its existence illustrates this There is the ability of drug metabolism in class cell；ZO 1 is tight junction protein, is often used to observe various organizational structure compact siro spinning technology screen Barrier function and the index of penetrating function.
Additionally, as shown in Figure 4, the expression of some albumen may imply that cell can break up in structure and present bile duct Structure: what Fig. 4 a embodied is the expression of CK19/ZO 1, and CK19 is recombined human Cyfra21-1, expresses at tubular epithelium more In cell；ZO 1 is tight junction protein, is often used to observe various organizational structure tight junction barrier functions and penetrating function Index；The expression of both albumen means that tree-shaped bile duct network is formed, and may imply that the existence of gallbladder tube structure；Fig. 4 b embodies Be the expression of ALB/MRP2, it can be seen that hepatocyte/bile duct cell fine structure.
Embodiment 2
A kind of Biotype artificial liver, it be by the cell printing ink containing human primary hepatocyte together with cell scaffold material three After dimension cell printing, through the biological structure body with fixed space configuration cultivated and induction differentiation obtains；There is liver cell Group and gallbladder tube structure.
Above-mentioned Biotype artificial liver is prepared by following steps:
2) preparation cell printing ink and cell scaffold material:
1.1) the discarded liver organization produced when utilizing hepatectomy, after using collagenase perfusion, it is thin that digestion obtains the primary liver of people Born of the same parents, cultivate 1 day in culture dish, centrifugal, and are configured to suspension after collagen mixing, and adjusting cell concentration is 0.8 × 106/ ml, Standby；
1.2) buying cell scaffold material test kit from Tianjin Lutao company, by specification is configured to derive containing gelatin and/or gelatin The cell scaffold material of thing, standby；
2) three-dimensional cell prints:
2.1) first by step 1.2) cell scaffold material prepared through 3D printer according to predetermined pattern file printout to predeterminated position Form described structure basal layer；Ambient temperature controls at 20 DEG C
2.2) by step 1.1) the cell printing ink prepared and step 1.2) cell scaffold material prepared is mixed to get mixing material Material, in mixing material, cell concentration controls 0.8 × 105/ml；By described mixing materials'use 3D printer according to predetermined pattern File is in step 2.1) successively print, until completing whole print procedure to obtain predetermined structure, described 3D on the basal layer that obtains The jet diameters of printer controls in 800 μm；Ambient temperature controls at 20 DEG C, and printing precision is accurate to 50 μm.
2.3) by step 2.2) structure that printed uses the valeral solution soaking of 0.5% concentration to complete to fix for 5 minutes；
3) cultivate and induce differentiation
3.1) by step 2.3) fixing after structure cultivate in culture fluid 8 days, cultivation temperature is 37 DEG C, culture environment CO2 Content is 5%, in described culture fluid containing 12% hyclone, the penicillin of 0.2% and the streptomycin of 0.8% and surplus DMEM cell culture medium；
3.2) in step 3.1) cultured structure adds the DMSO solution that concentration is 0.5% carry out induction differentiation, induction point Change 7 days；Complete induction differentiation and i.e. obtain Biotype artificial liver structure body of the present invention.
Biotype artificial liver prepared by the present embodiment is apparent and microstructure characteristic is similar to Example 1.Observe its microstructure, As shown in A, C in Fig. 2, hepatocyte presents the sample growth of uniting of allied organization's structure.
Biotype artificial liver described in the present embodiment is carried out following experiment in vitro detection:
1. Liver function grade:
After structure embodiment 2 prepared cultivates the different time according to conventional organization cultural method, detect its liver merit respectively Can, including:
1.1 observe albumin secretion, hepatocyte function correlative protein expression, bile duct cell associated protein by immunofluorescence staining (observed result is close with embodiment 1)；
1.2 hepatocyte cultivated with conventional 2D, as compared with control cells, use ELISA method, the hepatocyte (figure that detection 2D cultivates respectively In with * labelling) print through 3D with embodiment 2 and induce the structure (without * labelling in figure) that obtains of differentiation at identical culture fluid After the middle cultivation identical time, ALB in culture fluid supernatant, the content of AST, Fe；Use the test kit of promega company simultaneously The enzymatic activity of multiple CYP isozyme in detection supernatant；Result is as follows:
As it is shown in figure 5, abscissa is the incubation time after having induced in each figure, along with the prolongation of incubation time, cell exists Embodiment 2 structure can maintain certain ALB secretory function and CYP3A4 enzymatic activity, and cell injury is less than compared with control cells (because AST and Fe is relatively low, illustrate that cell damage is few).Induce latter 1 day, compared with control cells and the training of embodiment 2 structure In nutrient solution supernatant, the level of AST, Fe, ALB and CYP3A4 activity does not has significant difference.After having induced 7 days, embodiment 2 structure culture fluid AST concentration 23 ± 7U/L, less than 27 ± 6U/L of compared with control cells, illustrate embodiment 2 structure hepatocyte Damage less, and after induction completes 14 days, this difference is more significantly, embodiment 2 structure culture fluid AST concentration 24 ± 6U/L, less than 38 ± 11U/L of compared with control cells.Fe content in compared with control cells and embodiment 2 structure culture fluid supernatant is also Presenting this trend, after having induced 7 days, embodiment 2 structure culture fluid Fe concentration 8 ± 3U/L, less than compared with control cells 16±6U/L；After having induced 14 days, embodiment 2 structure culture fluid Fe concentration 6 ± 2U/L, less than the 17 of compared with control cells ± 8U/L, and have significant difference.The ability of the structure secretion ALB of embodiment 2, far above compared with control cells, completes in induction After 7 days, in the supernatant of embodiment 2 structure, ALB concentration is 43 ± 15g/L, is ALB concentration in compared with control cells supernatant In twice；And after induction completes 14 days, in embodiment 2 structure culture fluid supernatant, ALB concentration reaches compared with control cells cultivation 3.7 times (44 ± 21g/L vs 12 ± 4g/L) in liquid supernatant.After induction completes 7 days, the supernatant of embodiment 2 structure Middle CYP3A4 activity is 3043 ± 343, and compared with control cells is 1651 ± 273.After having induced 14 days, embodiment 2 structure In the culture fluid supernatant of body, CYP3A4 activity is 1951 ± 287, is 3 times of compared with control cells.
2. drug metabolism study:
Take the Biotype artificial liver structure body that the embodiment of the present invention 2 method obtains, in culture fluid, add normal saline respectively (empty White comparison Blank control) and the acetaminophen (APAP) of 600 μm ol/L, detect each group of viable count after acting on the identical time Amount, cell proliferative conditions, multiple CYP enzymatic activity and albumen and mrna expression amount, result such as Fig. 6 shows, the present invention implements After the artificial liver structure culture fluid of example 2 preparation adds acetaminophen, the activity (2136 ± 102vs of CYP3A4 enzyme 2956 ± 387) and protein expression (0.890 ± 0.69vs 10.9 ± 7.70) substantially increases, the artificial liver construction of the present invention is described Drug metabolism can inspire the strongest compensatory capacity.

Claims (10)

1. a Biotype artificial liver, it is characterised in that: it is by containing primary hepatocyte or terminal differentiation hepatocyte (HepaRG) Cell printing ink together with cell scaffold material three-dimensional cell print after, through cultivation and induction differentiation obtain have fixing The biological structure body of steric configuration.
2. the Biotype artificial liver described in claim 1, it is characterised in that: in described biological structure body, there is liver cell Group.
3. any one Biotype artificial liver described in claim 1 or 2, it is characterised in that: in described biological structure body There is gallbladder tube structure.
4. the Biotype artificial liver described in claim 1, it is characterised in that: described cell printing ink contains glue further Former；Described collagen accounts for the 85～95% of cell printing ink, and preferably 90%.
5. the Biotype artificial liver described in claim 1, it is characterised in that: in described cell printing ink, cell concentration is 0.8×106/ ml～1.2 × 106/ml。
6. the Biotype artificial liver described in claim 1, it is characterised in that: described induction differentiation use not higher than 2% is dense The DMSO solution of degree.
7. the method preparing Biotype artificial liver described in claim 1, comprises the following steps:
1) preparation cell printing ink and cell scaffold material:
1.1) by the most cultured primary hepatocyte or terminal differentiation hepatocyte (HepaRG cell) and collagen one Rising and be hybridly prepared into suspension, regulation cell concentration is 0.8 × 106/ ml～1.2 × 106/ ml, standby；
1.2) preparation is containing gelatin and/or the cell scaffold material of gelatine derivative according to a conventional method, standby；
2) three-dimensional cell prints:
2.1) first by step 1.2) cell scaffold material prepared through 3D printer according to predetermined pattern file printout to predeterminated position Form described structure basal layer；
2.2) by step 1.1) the cell printing ink prepared and step 1.2) cell scaffold material prepared is mixed to get mixing material Material, in mixing material, cell concentration controls 0.8 × 105/ ml～1.2 × 105/ml；Described mixing materials'use 3D printer is pressed According to predetermined pattern file in step 2.1) successively print on the basal layer that obtains, until completing whole print procedure to obtain predetermined structure, The jet diameters of described 3D printer controls in 100～1000 μm；
2.3) by step 2.2) structure that printed uses the valeral solution soaking 2 of 0.5%～1% concentration～completes to fix for 5 minutes； The valeral solution soaking 3 minute of 0.5% concentration is preferably used；
3) cultivate and induce differentiation
3.1) by step 2.3) fixing after structure cultivate in culture fluid at least 7 days, preferably 7 days, cultivation temperature was 35～37 DEG C, Culture environment CO2Content is 4～6%, in described culture fluid containing 8～the penicillin of the hyclone of 15%, 0.2～0.8% and 0.2～the DMEM cell culture medium of the streptomycin of 0.8% and surplus；
3.2) in step 3.1) cultured structure adds concentration it is not higher than the DMSO solution of 2%, preferred concentration is 0.5%～2% DMSO solution, carry out induction differentiation, induction differentiation less than 7 days, preferably 7 days；Complete induction differentiation and i.e. obtain described Biotype artificial liver structure body.
8. the method described in claim 7, it is characterised in that: step 1.1) described in suspension in, collagen accounts for 85～95%, Preferably 90%；Control cell concentration 1 × 106/ml。
9. the method described in claim 7, it is characterised in that: step 2) described in printing, ambient temperature controls at 18 22 DEG C, Printing precision is accurate to 50 μm.
10. the application in drug metabolism study of the Biotype artificial liver described in claim 1.
CN201610258273.5A 2016-04-22 2016-04-22 Bioartificial liver based on three-dimensional cell printing, and preparation method and application of bioartificial liver Withdrawn CN105861419A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109550093A (en) * 2017-09-23 2019-04-02 孙奕 Cell membrane artificial liver
CN110551679A (en) * 2019-08-02 2019-12-10 浙江大学 Accurate printing construction method of liver unit chip containing acinar three-vessel structure
CN111534489A (en) * 2020-04-29 2020-08-14 清华大学 T lymphocyte amplification method based on 3D printing
CN113667642A (en) * 2020-05-15 2021-11-19 北京大橡科技有限公司 3D human liver organ model construction method, 3D human liver organ model and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109550093A (en) * 2017-09-23 2019-04-02 孙奕 Cell membrane artificial liver
CN110551679A (en) * 2019-08-02 2019-12-10 浙江大学 Accurate printing construction method of liver unit chip containing acinar three-vessel structure
CN110551679B (en) * 2019-08-02 2021-10-15 浙江大学 Accurate printing construction method of liver unit chip containing acinar three-vessel structure
CN111534489A (en) * 2020-04-29 2020-08-14 清华大学 T lymphocyte amplification method based on 3D printing
CN113667642A (en) * 2020-05-15 2021-11-19 北京大橡科技有限公司 3D human liver organ model construction method, 3D human liver organ model and application thereof

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