CN107201336A - The method for preparing mouse cellularised liver again - Google Patents
The method for preparing mouse cellularised liver again Download PDFInfo
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- CN107201336A CN107201336A CN201710562199.0A CN201710562199A CN107201336A CN 107201336 A CN107201336 A CN 107201336A CN 201710562199 A CN201710562199 A CN 201710562199A CN 107201336 A CN107201336 A CN 107201336A
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Abstract
The invention discloses a kind of method for preparing mouse cellularised liver again.The present invention comprises the following steps：(1) perfusion liquid is added in perfusion system, perfusion liquid is included：DMEM culture mediums, and addition has 10% hyclone, 100U/mL penicillin, 100 μ g/mL streptomysins in the medium；(2) by perfusion liquid with 5mL/min velocity perfusion into liver biological support, infusion time 30min, wherein, perfusion liquid trans-portal vein injection liver biological support, from infrahepatic vena cava outflow；(3) stop perfusion, 5 × 10 are slowly injected into perfusion liquid6Individual cell is dissolved in 3mL perfusion liquids, injects the perfusion liquid with cell in liver biological support by 1mL/min speed；(4) by perfusion liquid with 10mL/min velocity perfusion liver biological support 10min；(5) repeat step (3) and (4) 4 times, produce again cellularised liver.The inventive method is simple, saves the time, reduces cost.
Description
Technical field
The present invention relates to liver tissue engineering field, and in particular to a kind of method for preparing mouse cellularised liver again.
Background technology
At present, organ transplant is the only effective method for the treatment of organs exhaustion, for the active demand of suitable organ transplant
Considerably beyond available organ number.Overlong time of the patient of a large amount of organ failures because waiting donor, in the process of wait
In finish life.Therefore, donor organ lacks the serious clinical treatment for constraining organ failure patient, reduces the total of patient
Body life cycle.In order to seek other replacement therapy methods, the research based on organizational project and regenerative medicine seems particularly heavy
Will so that provided for many patients be available for the parenchymatous organ of transplanting to be possibly realized in the future.
In the treatment of End-stage liver disease, the serious development for constraining liver transplant that liver donor source is not enough.In liver
In the research of replacement therapy, then the research field of liver replacement therapy has been widened in the preparation and application of cellularised liver.With liver
Biological support is that carrier prepares cellularised liver again, normal liver is successfully simulated in terms of morphology and function assessment two, not
The replacement therapy for being applied to operation transplantation takes precautions against significant.
A series of decellularization processing are carried out using normal liver, so that it may obtain complete liver biological support, this side
Method can not only remove substantial amounts of cell and nuclearity material, also retains support extracellular matrix and ultra micro vascular structure.Carefully
Extracellular matrix components are generally synthesized with discharging by the intrinsic cell of histoorgan, and keep dynamic equilibrium with ambient, external environment.
Micro-vascular structures provide growing environment in support, simulation liver for seed cell in liver, maintain iuntercellular reciprocation.Therefore, liver
Dirty biological support is a kind of preferable cell carrier.Cellularised liver is synthesized the mouse prepared on this basis compared with other biological again
Liver cellularised again prepared by material compares, with more excellent biological effect.But during preparation, because of it
The cell quantity of implantation, reperfusion mode, the different efficiency for determining that again cellularised liver is played such as infusion time are different.
By experiment grope we have found that：(1) in terms of perfusion cell quantity.The excessive cell of perfusion enters liver, can make
Cytotrophy supply is not enough, final apoptosis；Hypocellularity is irrigated, cell radix is not only reduced, can not set up effective thin
Intercellular communication.(2) in terms of perfusion channel.Trans-portal vein, arteria hepatica, vena hepatica perfusion can be divided into by classification to irrigate.Checking
It was found that easily perfusion liquid leakage occurs because of high pressure for trans-hepatic artery perfusion, vena hepatica retroperfusion cell is difficult disperse and enters vascular system
System.(3) in terms of perfusion number of times.Disposable perfusion is compared with gradation perfusion, on the basis of injection same number liver cell, point
Liver prepared by secondary perfusion plays more preferable liver function.Therefore, how cell again is improved by improving above-mentioned experimental method
Liver biological efficiency is current structure one of cellularised liver urgent problem to be solved again.
The content of the invention
It is an object of the invention to provide a kind of method for preparing mouse cellularised liver again.
The present invention is achieved through the following technical solutions：
The method for preparing mouse cellularised liver again, comprises the following steps：
(1) perfusion liquid is added in perfusion system, perfusion liquid is included：DMEM culture mediums, and addition has in the medium
10% hyclone, 100U/mL penicillin, 100 μ g/mL streptomysins；
(2) by perfusion liquid with 5mL/min velocity perfusion into liver biological support, infusion time 30min, wherein, fill
Fluid injection trans-portal vein injects liver biological support, from infrahepatic vena cava outflow；
(3) stop perfusion, 5 × 10 are slowly injected into perfusion liquid6Individual cell (being dissolved in 3mL perfusion liquids), by 1mL/min's
Speed injects the perfusion liquid with cell in liver biological support；
(4) by perfusion liquid with 10mL/min velocity perfusion liver biological support 10min；
(5) repeat step (3) and (4) 4 times, produce again cellularised liver.
Further, the preparation method of the liver biological support is as follows：
A, portal vein perfusion heparin containing 12.5U/ml through in vitro liver phosphate buffered saline solution, wherein, with 5ml/min
Velocity perfusion 10min；
B, in vitro liver placed into 24h under conditions of -80 DEG C；
C, frozen liver thawed at ambient temperature, then by phosphate buffered saline solution with 1ml/min speed through door
Venous perfusion 1h；
D, 1% sodium dodecyl sulfate solution with 5ml/min speed trans-portal vein irrigated into 1h；
E, by 1% Triton X-100 solution with 0.1% trypsase it is quiet through door with 5ml/min speed
Arteries and veins irrigates 2h；
F, 1 × phosphate buffered saline solution with 5ml/min speed trans-portal vein irrigated into 2h；
G, the phosphate buffered saline solution containing mycillin with 5ml/min speed trans-portal vein irrigated into 20min, produced
Liver biological support.
Yet further, step G also includes rinsing liver surface with the phosphate buffered saline solution containing mycillin.
Further, perfusion is the outlet using infrahepatic vena cava as liquid every time.
In addition, perfusion is irrigated by way of putting pipe in vitro liver portal vein every time.
In addition, perfusion is completed in operation ware every time.
The present invention has advantages below and beneficial effect：
(1) the overall liver cell quantity that method of the invention is irrigated can reach 20 × 106, it is inventor after research
Result, the growth and the expression of liver function of the optimum mouse middle liver cell of cellularised liver again.
(2) liver cell perfusion is by portal vein perfusion in method of the invention, and point 4 minor ticks perfusion, cell spills rate
Minimum, liver function expression is optimal.
The mouse of the invention prepared with current conventional method compared with cellularised liver, can obtain faster liver cell and increase again
Speed is grown, more preferable liver function is given expression to, is embodied in and secrets out of more albumin and urea components.
Brief description of the drawings
Fig. 1 is the schematic diagram after normal liver cell is dyed with liver cellularised again of the invention through H＆E.
Fig. 2 is the schematic diagram after normal liver cell is dyed with liver cellularised again of the invention through Masson.
Fig. 3 is cellularised liver inner cell fluorogram again.
Fig. 4 increases statistical chart for cellularised liver inner cell again.
Fig. 5 is cellularised liver words spoken by an actor from offstage protein secretion situation map again.
Fig. 6 is urea secretion situation map in cellularised liver again.
Embodiment
With reference to embodiment, the present invention is further illustrated, but embodiments of the present invention are not limited to this.
Embodiment 1
The method for preparing mouse cellularised liver again, comprises the following steps：
(1) perfusion liquid is added in perfusion system, perfusion liquid is included：DMEM culture mediums, and addition has in the medium
10% hyclone, 100U/mL penicillin, 100 μ g/mL streptomysins；
(2) by perfusion liquid with 5mL/min velocity perfusion into liver biological support, infusion time 30min, wherein, fill
Fluid injection trans-portal vein injects liver biological support, from infrahepatic vena cava outflow；
(3) stop perfusion, 5 × 10 are slowly injected into perfusion liquid6Individual cell (being dissolved in 3mL perfusion liquids), by 1mL/min's
Speed injects the perfusion liquid with cell in liver biological support；
(4) by perfusion liquid with 10mL/min velocity perfusion liver biological support 10min；
(5) repeat step (3) and (4) 4 times, produce again cellularised liver.
Worth explanation is that, when preparing cellularised liver again, above-mentioned perfusion is circumfusion, and perfusion liquid is entered from portal vein
Enter, from infrahepatic vena cava outflow, trans-portal vein enters liver biological support, such circumfusion to the perfusion liquid of outflow again.Such as
Shown in Fig. 3~6, it can be seen that liver inner cell growth pattern cellularised again of the invention, and albumin and urea secretion feelings
Condition.
Above-mentioned liver biological support need to can only be carried out thin again directly using product on the market according to the above method of the present invention
Born of the same parentsization, but present invention employs above-mentioned culture medium, and special method for filling, hepatocyte growth speed can be accelerated,
More preferable liver function is given expression to, is embodied in and secrets out of more albumin and urea components.
Embodiment 2
The present embodiment is that the liver biological support of the present embodiment is prepared using following methods with the difference of embodiment 1
And obtain：
A, portal vein perfusion heparin containing 12.5U/ml through in vitro liver phosphate buffered saline solution, wherein, with 5ml/min
Velocity perfusion 10min；
B, in vitro liver placed into 24h under conditions of -80 DEG C；
C, frozen liver thawed at ambient temperature, then by phosphate buffered saline solution with 1ml/min speed through door
Venous perfusion 1h；
D, 1% sodium dodecyl sulfate solution with 5ml/min speed trans-portal vein irrigated into 1h；
E, by 1% Triton X-100 solution with 0.1% trypsase it is quiet through door with 5ml/min speed
Arteries and veins irrigates 2h；
F, phosphate buffered saline solution with 5ml/min speed trans-portal vein irrigated into 2h；
G, the phosphate buffered saline solution containing mycillin with 5ml/min speed trans-portal vein irrigated into 20min, be used in combination
Phosphate buffered saline solution containing mycillin rinses liver surface, produces liver biological support.
Wherein, in prepared product support, perfusion every time is the outlet using infrahepatic vena cava as liquid；Perfusion every time
It is irrigated by way of putting pipe in vitro liver portal vein.In addition, perfusion is completed in operation ware every time.
What deserves to be explained is, the in vitro liver of the present embodiment is the liver taken out using conventional method out of rat body.
By the way that cellularised liver and normal liver cell are carried out to the institute of such as Fig. 1~2 again made from the method for the present invention
Show, as shown in Figure 1, find that cytotostatic is attached in support after H＆E dyeing；As shown in Figure 2, found again after Masson dyeing thin
Born of the same parentsization liver remains extracellular matrix (fibrin, elastin laminin etc.) composition.
The method that the present embodiment prepares liver biological support has the characteristics that：
(1) osmotic pressure inside and outside in vitro liver biological support can be kept using the phosphate buffered saline solution of the heparin containing 12.5U/mL
Uniformity.The anticoagulation of the heparin of suitable concn can avoid residual blood solidification obstruction micro-vascular structures, and the influence later stage fills
Note.
(2) liver branch is placed on 24h under -80 DEG C of cryogenic conditions, promotes cell component to dissociate by frozen-thaw process, accelerated
Later stage removes cell processes.
(3) lipid and albumen on cell membrane, and then dissolving film egg are can dissolve using 1% sodium dodecyl sulfate solution
It is white and destroy cell membrane, into cell after nucleoprotein in depolymerization cell.
(4) using 1% Triton X-100 solution and 0.1% trypsase can effectively detachment DNA and
Contact between protein.
(5) using the phosphate buffered saline solution containing mycillin can effectively suppress in filling process to occur it is micro-
Biological infection.
(6) cell can be removed to greatest extent using 5mL/min rate of flooding and reduced to microvascular and extracellular base
The destruction of matter, is the result after research repeatedly.
In summary, the physical condition and chemical reagent employed in above-mentioned liver biological support preparation can be eluted completely
Liver inner cell and nuclearity composition, and reagent rate of flooding and perfusion concentration maximizing reduce itself and liver biological support
Action time, reduce destruction to biological support structural proteins and extracellular matrix components, most effectively remain its biological
Integrality.Therefore, the liver biological support that prepared by the present invention, while cell is removed completely, can be sufficiently reserved the thin of liver
Extracellular matrix.The liver biological support of characteristic is learned with outstanding tissue biological by preparing, is follow-up liver cellularised again
Dirty preparation provides a splendid carrier.
According to above-described embodiment, the present invention just can be realized well.What deserves to be explained is, the premise based on above-mentioned design
Under, to solve same technical problem, some made in the present invention are used without substantial change or polishing
The essence of technical scheme is still as the present invention, therefore it should also be as within the scope of the present invention.
Claims (5)
1. a kind of method for preparing mouse cellularised liver again, it is characterised in that comprise the following steps：
(1) perfusion liquid is added in perfusion system, perfusion liquid is included：DMEM culture mediums, and addition has 10% tire in the medium
Cow's serum, 100U/mL penicillin, 100 μ g/mL streptomysins；
(2) by perfusion liquid with 5mL/min velocity perfusion into liver biological support, infusion time 30min, wherein, perfusion liquid
Trans-portal vein injects liver biological support, from infrahepatic vena cava outflow；
(3) stop perfusion, 5 × 10 are slowly injected into 3mL perfusion liquids6Individual cell, by 1mL/min speed by with cell
In perfusion liquid injection liver biological support；
(4) by perfusion liquid with 10mL/min velocity perfusion liver biological support 10min；
(5) repeat step (3) and (4) 4 times, produce again cellularised liver.
2. the method according to claim 1 for preparing mouse cellularised liver again, it is characterised in that the liver biology branch
The preparation method of frame is as follows：
A, portal vein perfusion heparin containing 12.5U/ml through in vitro liver phosphate buffered saline solution liquid, wherein, with 5ml/min
Velocity perfusion 10min；
B, in vitro liver placed into 24h under conditions of -80 DEG C；
C, frozen liver thawed at ambient temperature, then by phosphate buffered saline solution with 1ml/min speed trans-portal vein
Irrigate 1h；
D, 1% sodium dodecyl sulfate solution with 5ml/min speed trans-portal vein irrigated into 1h；
E, 1% Triton X-100 solution and 0.1% trypsase filled with 5ml/min speed trans-portal vein
Note 2h；
F, phosphate buffered saline solution with 5ml/min speed trans-portal vein irrigated into 2h；
G, the phosphate buffered saline solution containing mycillin with 5ml/min speed trans-portal vein irrigated into 20min, produce liver
Biological support.
3. the method according to claim 2 for preparing mouse cellularised liver again, it is characterised in that step G also includes using
Phosphate buffered saline solution containing mycillin rinses liver surface.
4. the method according to claim 2 for preparing mouse cellularised liver again, it is characterised in that every time perfusion be with
Infrahepatic vena cava as liquid outlet.
5. the method according to claim 2 for preparing mouse cellularised liver again, it is characterised in that perfusion passes through every time
The mode that pipe is put in vitro liver portal vein is irrigated.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710562199.0A CN107201336A (en) | 2017-07-11 | 2017-07-11 | The method for preparing mouse cellularised liver again |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753686A (en) * | 2018-06-22 | 2018-11-06 | 北京达博威迎医药技术有限公司 | Organizational project hepatic model, its construction method and its application |
| CN111480644A (en) * | 2019-01-27 | 2020-08-04 | 宋红丽 | Normal-temperature mechanical perfusion combined with gene modification for repairing small animal organ system by stem cells |
-
2017
- 2017-07-11 CN CN201710562199.0A patent/CN107201336A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753686A (en) * | 2018-06-22 | 2018-11-06 | 北京达博威迎医药技术有限公司 | Organizational project hepatic model, its construction method and its application |
| CN108753686B (en) * | 2018-06-22 | 2021-06-22 | 中国人民解放军军事科学院军事医学研究院 | Tissue engineering liver model, construction method and application thereof |
| CN111480644A (en) * | 2019-01-27 | 2020-08-04 | 宋红丽 | Normal-temperature mechanical perfusion combined with gene modification for repairing small animal organ system by stem cells |
| CN111480644B (en) * | 2019-01-27 | 2024-02-27 | 宋红丽 | Normal temperature mechanical perfusion and gene modification combined stem cell repair small animal organ system |
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