CN104845878B - In-vitro liver perfusion device and application thereof - Google Patents
In-vitro liver perfusion device and application thereof Download PDFInfo
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Abstract
The invention discloses an in-vitro liver perfusion device which comprises a perfusion liquid storage container set composed of 3 perfusion liquid storage containers, a four-way pipe, a perfusion pump, a heat exchange system, an oxygenator, a defoaming device, a perfusion catheter, a three-way pipe and a reflux pump. The in-vitro liver perfusion device can perform three-step perfusion on the in-vitro liver, maximally perform effective perfusion on the pre-perfusion liquid and digestive juice quickly and continuously, and maximally control the hepatic tissue digestion process, thereby saving the cost, keeping the temperature constant, reducing the pollution, and further obtaining sufficient experimental and clinical liver cells with high quality (especially abundant liver cells required by liver cell transplantation, biological artificial livers and the like).
Description
Technical field
The present invention its be related to a kind of Vitro hepatic perfusion device and application thereof.
Background technology
Primary porcine hepatocyte is most suitable xenogenesis hepatocyte source in bal treatment, and existing ripe hepatocyte separates to be adopted
Method is enzyme digestion, and the method is that collagenase etc. is injected liver vessel, acts on hepatic tissue, makes complicated between hepatocyte
Fiber connect dissolving, thus obtaining complete individual hepatocyte.For big animal, such as pig, liver of Canis familiaris L. and people etc., due to body
Long-pending big, perfusate consumption is big, and the time is long, and original position or In vitro perfusion all cannot be pouring-in using traditional manual syringe
Perfusion, can only select infusion pump.And when using infusion pump perfusion, due to lacking special complete set of equipments, cause perfusion to separate
Antibacterial etc. is easily caused to pollute in journey.Because collagenase is very high to temperature requirement, 37 DEG C is its best use of temperature, unspecial
Perifusion apparatus be difficult to control perfusion liquid temp, therefore, often due to temperature control is bad, and have impact on final cell dissociation
Effect is it is impossible to reach required cell quantity and prescription, and causes the waste of expensive collagenase, increases separation and Culture liver thin
The cost of born of the same parents.
Therefore, need one kind to be suitable to digestion at present and separate big animal liverss hepatocellular Vitro hepatic perfusion device.
Content of the invention
For solving the above problems, the invention provides a kind of Vitro hepatic perfusion device, plant Vitro hepatic perfusion device, its feature
Be: include perfusate storage container group, four-way pipe, infusion pump, heat-exchange system, oxygenator, debubbler, perfusion catheter, three
Siphunculus and reflux pump；Described perfusate storage container group includes container a, container b and container c, described container a, container b, container c
It is equipped with inlet and the liquid outlet with stop valve, described container c is additionally provided with refluxing opening；Described infusion pump, heat exchanger, oxygen
Clutch, debubbler, perfusion catheter and reflux pump are equipped with inlet and liquid outlet；Four mouths of pipe of described four-way pipe respectively with appearance
The liquid outlet of device a, the liquid outlet of container b, the liquid outlet of container c are connected by flexible pipe with the inlet of infusion pump；Infusion pump
The inlet of inlet, the liquid outlet of heat-exchange system and oxygenator of liquid outlet and heat-exchange system, the liquid outlet of oxygenator
Backflow with the inlet of the inlet of debubbler, the inlet of debubbler and perfusion catheter, the liquid outlet of reflux pump and container c
Mouth is all connected by flexible pipe；The liquid outlet of described perfusion catheter stretches in dress liver container, and the bottom of described dress liver container is also
It is provided with liquid outlet；Described tee T is located at the lower section of dress liver container, and the two of which mouth of pipe of tee T is respectively by flexible pipe even
The liquid outlet of tipping liver container, the inlet of reflux pump, the 3rd mouth of pipe of tee T connects sewer pipe, on described sewer pipe
It is provided with stop valve.
It is further preferred that the inlet of the inlet, the inlet of container b and container c of described container a is carrying liqs
The feed liquor funnel of sterilizing filter membrane, is located at the top of corresponding container respectively；The liquid outlet of described container a, the liquid outlet of container b and appearance
The liquid outlet of device c is respectively positioned on bottom；The top of described container a, container b and container c is equipped with the air-vent with air filter film；Institute
State container a, container b and container c and be equipped with liquidometer.
It is further preferred that described heat-exchange system is made up of heat exchanger, hot recycle pump and thermostat water bath.
It is further preferred that the volume of described container a is 4-10l, the volume of container b is 1-2l, and the volume of container c is 2-
4l.
It is further preferred that described dress liver container carries cap.
It is further preferred that the internal diameter of described flexible pipe is 6mm-10mm.
The present invention utilizes hot recycle pump to note 37-42 DEG C in water-bath of heat recirculated water with the speed of 100-200ml/min
Enter heating perfusate in heat exchanger, make perfusate keep 37 DEG C of constant temperature in whole perfusing course.
Carry out oxygenated treatment using oxygenator in perfusate, make dissolving partial pressure of oxygen in perfusate be maintained at 180-
260mmhg, reduces hepatocellular hypoxic exposure, reduces cavity in hepatocyte, improves hepatocellular vigor.
Remove size bubble in perfusate using debubbler before perfusate enters liver, it is to avoid bubble is in liver vessel
Form gas embolus, make liver perfusion more full and uniform.
Due to employing technique scheme, assembly of the invention has the advantage that
1. easy to use, reliable, perfusing course is carried out continuously, and single need to just can complete to grasp in one hour
Make, save human and material resources and time.
2. pass through heat exchanger, perfusate remains 37 DEG C of constant temperature in whole perfusing course, meets hepatocyte and divide
From and collagenase needed for constant temperature fixed it is ensured that the activity of digestion and isolating hepatocytes.
3. oxygenated treatment has been carried out to perfusate by oxygenator, reduced hepatocellular hypoxic exposure, reduced hepatocyte
Interior cavity, improves hepatocellular vigor.
4. debubbler avoid gas embolus in liver vessel formation it is ensured that in liver all cells can obtain uniformly
Perfusion.
5. adopt jumbo perfusate storage container in device, and the infusion pump 4 of high flow rate at the uniform velocity irrigate it is ensured that greatly
The pressure of animal liverss perfusion and perfusate volume needs.
6., in whole filling process, all in the dress liver container having lid, perfusate is also at aseptic pipeline to liver
In container, it is to avoid antibacterial, the pollution of funguses.
In sum, the invention provides a kind of set of, easy to operate is thin for the digestion big animal liverss liver of separation
The Vitro hepatic perfusion device of born of the same parents, it can carry out three step liver perfusions in vitro liver, rapidly, continuously carries out front filling to greatest extent
Flow liquid and effective perfusion of Digestive system, farthest Quality Control digests hepatic tissue process, cost-effective, steady temperature, reduces dirty
Dye, thus that guarantees both quality and quantity obtains experiment and the hepatocyte used by clinic, especially hepatocyte transplantation, bioartificial liver etc. is required
A large amount of hepatocyte.
Present invention also offers a kind of side of the separation preparation carrying out primary hepatocyte using above-mentioned Vitro hepatic perfusion device
Method:
(1) perfusate is loaded in container a, perfusate loads in container b, and perfusate loads in container c；
(2) use perfusate pre-filled flexible pipe to export to debubbler, make to be full of perfusate between container a and debubbler outlet
ⅰ；
(3) take in vitro animal liverss to be placed in dress liver container, first use perfusate perfusion, perfusion rate is 300-
1000ml/min, Hemoperfusion time is 10-12 minute；Use perfusate perfusion again, perfusion rate is 300-1000ml/min, perfusion
Time is 2-3 minute；Finally use perfusate perfusion, perfusion rate is 300-1200ml/min, Hemoperfusion time divides for 25-40
Clock；
Wherein, every liter of described perfusate i contains following composition: 8.1-8.5 gram of sodium chloride, 0.5 gram of potassium chloride, 2.2-2.6
Gram hepes, 3.5-4.5 gram of nac, 0.9-1.1 gram of egta, remaining is deionized water；Every liter of described perfusate ii contain following become
Point: 8.1-8.5 gram of sodium chloride, 0.5 gram of potassium chloride, 2.2-2.6 gram of hepes, remaining is deionized water；Described perfusate iii is every
Rise and contain following composition: 3.8-4 gram of sodium chloride, 0.5 gram of potassium chloride, 23-25 gram of hepes, 0.07 gram of calcium chloride dihydrate, 3.5-
4.5 grams of albumin, 162-260w ü nsch unit collagenase, remaining is deionized water；
(4) isolating hepatocytes: the liver after washing step (3) process, collect hepatocyte suspension, filter, be centrifuged, collect and sink
Form sediment.
Hepes:4- hydroxyethyl piperazine ethanesulfonic acid.
Nac:n-acetylcysteine, n- acetyl group-l- cysteine.
Egta: ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA).
W ü nsch unit: be the special active unit of collagenase.
Further, in step (3), every liter of described perfusate i contains following composition: 8.3 grams of sodium chloride, 0.5 gram of chlorination
Potassium, 2.4 grams of hepes, 3.5-4.5 gram of nac, 0.9-1.1 gram of egta, remaining is deionized water；
Every liter of perfusate ii contains following composition: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes, and remaining is to go
Ionized water；
Every liter of perfusate iii is containing following composition: 3.9 grams of sodium chloride, 0.5 gram of potassium chloride, 24 grams of hepes, 0.07 gram two
Water calcium chloride, 4 grams of albumin, 162-260w ü nsch unit collagenase, remaining is deionized water.
Further, every liter of described perfusate i contains following composition: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams
Hepes, 4 grams of nac, 0.95 gram of egta, remaining is deionized water；
Every liter of perfusate ii contains following composition: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes, and remaining is to go
Ionized water；
Every liter of perfusate iii is containing following composition: 3.9 grams of sodium chloride, 0.5 gram of potassium chloride, 24 grams of hepes, 0.07 gram two
Water calcium chloride, 4 grams of albumin, 216w ü nsch unit collagenase, remaining is deionized water.
Wherein, in described perfusate iii, collagenase is collagenase serva nb8 or collagenase roche
liberasetm.
Wherein, the perfusion rate of described perfusate i is 400ml/min, and Hemoperfusion time is 10 minutes；
The perfusion rate of perfusate ii is 400ml/min, and Hemoperfusion time is 2 minutes；
The perfusion rate of perfusate iii is 500ml/min, and Hemoperfusion time is 30 minutes.
Wherein, in step (3), perfusate i, perfusate ii, the perfusion temperature of perfusate iii are 37 c.
Wherein, in step (3), the perfusion of perfusate iii is circulation perfusion mode.
Wherein, in step (3), the dissolving partial pressure of oxygen of perfusate i, perfusate ii and/or perfusate iii is 180-
260mmhg.
Wherein, in step (4), described filtration is to be filtered with four layers of absorbent carbasus.
Wherein, in step (4), described centrifugation is multiple centrifugation, comprises the concrete steps that:
A, centrifugation, abandoning supernatant.
B, resuspended precipitation, centrifugation；
C, process b is repeated once.
Wherein, the condition of centrifugation is: centrifuging temperature is 4 DEG C, and centrifugal force is 50-55 × g, and the time of centrifugation is 10 minutes.
Wherein, in step b, resuspended precipitation adopts hepatocyte cleaning mixture.
Wherein, in step b, washing is using the washing of hepatocyte cleaning mixture.
Wherein, contain following composition: 9.3-10.8 gram of williams'medium e powder in every liter of described hepatocyte cleaning mixture
End, 2.2 grams of sodium bicarbonate, 2.4-2.8 gram of hepes, 0.1 gram of streptomycin, 100000 units of Penicillin, remaining is deionized water.
Further, contain following composition: 9.3-10.8 gram of williams'medium in every liter of described hepatocyte cleaning mixture
E powder, 2.2 grams of sodium bicarbonate, 2.6 grams of hepes, 0.1 gram of streptomycin, 100000 units of Penicillin, remaining is deionized water.
Further, contain following composition: 9.5 grams of williams'medium e in every liter of described hepatocyte cleaning mixture
Powder, 2.2 grams of sodium bicarbonate, 2.6 grams of hepes, 0.1 gram of streptomycin, 100000 units of Penicillin, remaining is deionized water.
The method of the present invention can be not only used for pig liver, and goes for various and larger animal liver, such as
People, pig, Canis familiaris L., the liver of cattle, have the popularity of application.Using assembly of the invention, primary hepatocyte can be efficiently separated, point
From the hepatocellular quantity obtaining is many, motility rate and purity are high, it is hepatocyte transplantation, biological artificial liver support system treatment acute and chronic
Hepatopathy and hepatocyte external application model provide potential hepatocyte resource, and potential applicability in clinical practice is excellent.
Obviously, the above according to the present invention, according to ordinary technical knowledge and the customary means of this area, without departing from
Under the premise of the present invention above-mentioned basic fundamental thought, modification, replacement or the change of other various ways can also be made.
The specific embodiment of form by the following examples, remakes further specifically to the above of the present invention
Bright.But this scope being interpreted as the above-mentioned theme of the present invention should not be only limitted to Examples below.All based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description
Fig. 1 is the structural representation of the present invention.In figure: 1. perfusate storage container, volume 10l；2. perfusate storage
Container, volume 2l；3. perfusate storage container, volume 4l；4. infusion pump；5. heat exchanger；6. oxygenator；7. debubbler；
8. lidded container；9. reflux pump；10. waste fluid container；11. feed liquor buccal funnels；12. feed liquor buccal funnels；13. feed liquor buccal funnels；14.
Sterilization of liquids filter membrane；15. sterilization of liquids filter membranes；16. sterilization of liquids filter membranes；17. passages；18. air filter films；19. passages；
20. air filter films；21. passages；22. air filter films；23. liquidometers；24. liquidometers；25. liquidometers；26. liquid flow out
Mouthful；27. liquid flowing outlets；28. liquid flowing outlets；29. stop valves；30. stop valves；31. stop valves；32. liquid refluxing openings；
33. liquid flowing outlets；34. stop valves；35. thermostat water baths；36. hot recycle pumps；37. perfusion catheters；38. four-way pipes；39. 3
Siphunculus.
Specific embodiment
The Vitro hepatic perfusion device of embodiment 1 present invention
Vitro hepatic perfusion device as shown in Figure 1, comprises a perfusate storage container 1, volume 10l, storage container 1
There is feed liquor buccal funnel 11 upper end, and funnel 11 bottom is mounted with a sterilization of liquids filter membrane 14, and there is a ventilation storage container 1 upper end
Hole 17, is connected to an air filter film 18 thereon, has a liquidometer 23 on storage container 1 side wall, and storage container 1 lower end has one
Individual liquid flowing outlet 26, there is a stop valve 29 in exit.This device also comprises a perfusate storage container 2, volume 2l,
There is feed liquor buccal funnel 12 storage container 2 upper end, and funnel 12 bottom is mounted with a sterilization of liquids filter membrane 15, and bin 2 upper end has
One passage 19, is connected to an air filter film 20 thereon, has a liquidometer 2 on storage container 2 side wall), storage container 2
There is a liquid flowing outlet 27 lower end, and there is a stop valve 30 in exit.This device also comprises a perfusate storage container
3, volume 4l, there is feed liquor buccal funnel 13 storage container 3 upper end, and funnel 13 bottom is mounted with a sterilization of liquids filter membrane 16, storage
There is a passage 21 container 3 upper end, is connected to an air filter film 22 thereon, has a liquid in storage container 3 side wall upper part
Refluxing opening 32, opposite side side wall has a liquidometer 25, and there is a liquid flowing outlet 28 storage container 3 lower end, and exit has
One stop valve 31.This device also includes infusion pump 4；Heat exchanger 5, with heat exchanger 5 use cooperatively thermostat water bath 35 and
Hot recycle pump 36；Oxygenator 6, can be passed through oxygen；Debubbler 7；Dress liver container 8 with cover, covers thereon and can dispose perfusion catheter
37, liquid flowing outlet 33 is arranged at bottom, and flow export is connected to tee T 39, connects stop valve 34 below tee T；Reflux pump 9 and waste liquid hold
Device 10.
Different according to perfusion rate, the flexible pipe being 6-10mm with internal diameter is first respectively by perfusate bin 1,2,3 and four
Siphunculus 38 connects, and is then connected remaining for four-way pipe 38 one end with infusion pump 4, is then sequentially connected heat exchanger 5, oxygenator
6 and debubbler 7, finally connect perfusion catheter 37, be connected to dress liver container 8 with cover.Afterwards, below dress liver container 8 with cover
Tee T 33, the hose connection waste fluid container 10 being 6-10mm with stop valve 34 end internal diameter, another section of tee T 33 connects back to
Stream pump 9, finally connects liquid refluxing opening 32 in perfusate storage container 3.The hose connection heat exchanger 5 being 6mm with internal diameter
Upper heat recirculated water gateway and thermostat water bath 35 and hot recycle pump 36.
The using method of apparatus of the present invention is such that
The complete liver of normal bar horse pig is obtained in surgery mode, it is thin that full liver separately prepares primary Hepar Sus domestica as a example weighing 300 grams
As a example the perfusing course of born of the same parents, this device using method is described.
Sterilize after first a complete set of equipment being decoupled, carry out aseptic assembling afterwards.In three perfusate storage containers respectively
Add filtration sterilization perfusate 4-10l, perfusate 1-2l and perfusate 2-4l.After pipeline is connected, pre- with perfusate
Priming line exports to debubbler 7, makes to be full of perfusate between perfusate storage container 1 and debubbler 7 outlet.Then surgery
Win complete big animal liverss under aseptic condition or people supplies hepatic tissue, be placed in lid dress liver container 8, by perfusion catheter 37
With liver portal system UNICOM, the perfusion catheter other end and perfusion device UNICOM afterwards.Then, open infusion pump 4 with 400-
The perfusate of 37 DEG C of oxygenations is poured into liver by the speed of 1000ml/min, and the perfusate pouring into liver is at liver superior and inferior vena cava
Overflow, finally flow in waste fluid container 10, discard, Hemoperfusion time is 10-12 minute.This is first step perfusion, by perfusate
Rinse blood in liver, be that residual hepatocyte is rinsed well as far as possible, the connection simultaneously tentatively loosened between hepatocyte.
Secondly, the perfusate of 37 DEG C of oxygenations is poured into by liver with the speed of 400-1000ml/min, pours into the filling of liver
Flow liquid overflows at liver superior and inferior vena cava, finally flows in waste fluid container 10, discards, and Hemoperfusion time is 1-2 minute.This is the
Two step perfusions, are rinsed by perfusate and remain perfusate in liver, remove the inhibitor egta of collagenase activity in perfusate
Deng composition, it is that enzymic digestion is prepared.
Finally, the perfusate containing collagenase for 37 DEG C of oxygenations is poured into by liver with the speed of 500-1100ml/min,
The perfusate pouring into liver overflows at liver superior and inferior vena cava, is saved in equipped with the dress liver container 8 of liver, opens backflow
Pump 9, to be drawn back perfusate in perfusate storage container 3 with infusion pump 4 same speed, is formed and at the uniform velocity circulates perfusion liver
Dirty, after 25-40 minute, until Glisson's capsule is separated with hepatocyte, terminate perfusion.Perfusate flows in waste fluid container 10, discards.
This is the 3rd step liver perfusion, and main by irrigating collagenase to intrahepatic circulatory, the fiber between digest and decompose hepatocyte connects, point
From hepatocyte.
The dress liver container 8 that will be equipped with liver afterwards takes out from perfusion device, moves in superclean bench, enters follow-up
Hepatocyte suspension collect and washing process.The process of described hepatocyte washing is: first, is cooled to 4 DEG C of williams' in advance
Medium e washs culture medium, and 300-1000ml adds in aseptic basin, scratches Glisson's capsule with operating scissorss, separates indigested liver group
Knit and connective tissue, collect hepatocyte suspension.After above-mentioned hepatocyte suspension filters through four layers of absorbent carbasus, under the conditions of 4 DEG C, centrifugation
Power 50-55 × g is centrifuged 10 minutes.Afterwards, discard centrifuged supernatant, wash culture basic weight with 4 DEG C of williams'medium e
The hepatocyte of outstanding precipitation, under the conditions of 4 DEG C, centrifugal force 50-55 × g is centrifuged 10 minutes.Said process is repeated once.Finally, abandon
Go centrifuged supernatant, precipitation hepatocyte is resuspended with serum free hepatocyte medium, adjusting cell concentration is the every milli in 2-5 × 107
Rise, put standby in ice bath.
Every liter of above-described perfusate contains: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes, 3-6g
Nac, 0.9-1.1g egta, balance of deionized water；
Every liter of perfusate contains: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes, balance of deionized water；
Every liter of perfusate contains: 3.9 grams of sodium chloride, 0.5 gram of potassium chloride, 24 grams of hepes, 0.07 gram of calcium chloride dihydrate
(cacl22h2o), 4 grams of albumin, 68-130pz active unit collagenase, balance of deionized water.
Contain in williams'medium e every liter of culture medium of washing: 9.5-10.8 gram of williams'medium e powder
End, 2.2 grams of sodium bicarbonate, 2.6 grams of hepes, 0.1g streptomycin, 100000 units of Penicillin, balance of deionized water.
Embodiment 2 carries out, using Vitro hepatic perfusion device of the present invention, the method that primary hepatocyte separates preparation
First, test reagent
Perfusate i, perfusate ii, perfusate iii, hepatocyte cleaning mixture；
Respective preparation method is as follows:
1. perfusate: taking prepare 1 liter of reagent as a example:
(1) take following composition: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes, 4 grams of nac, egta 0.95
Gram；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.During use, it is preheated to 37 DEG C.
2. perfusate: taking prepare 1 liter of reagent as a example:
(1) take following composition: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.During use, load perfusate storage container
In 2, it is preheated to 37 DEG C.
3. perfusate: taking prepare 1 liter of reagent as a example:
(1) take following composition: 3.9 grams of sodium chloride, 0.5 gram of potassium chloride, 24 grams of hepes, calcium chloride dihydrate (cacl2·
2h2O) 0.07 gram, 4 grams of albumin, 200 milligrams of collagenase serva nb8 (being 1.08 w ü nsch units inside 1mg enzyme) (i.e.
216w ü nsch unit)；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.6, with 0.2 micron membrane filter filtration sterilization, is configured using first 15 minutes, during use, loads perfusate storage
In container 3, it is preheated to 37 DEG C.
4. hepatocyte cleaning mixture: taking prepare 1 liter of reagent as a example:
(1) take following composition: 9.5 grams of williams'medium e powder, 2.2 grams of sodium bicarbonate, 2.6 grams of hepes, chain
0.1 gram of mycin, penicillin 100000 unit；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.3, and with 0.2 micron membrane filter filtration sterilization, 4 DEG C preserve and use.
2nd, test method
Obtain the complete liver of normal bar horse pig, so that full liver weighs 300 grams as a example separate and prepare primary porcine hepatocyte.Respectively will
Perfusate loads in perfusate storage container 1, perfusate is loaded in perfusate storage container 2, perfusate is filled
Enter in perfusate storage container 3.Described preheating is all preheated by heat exchanger 5.
A, the In vitro perfusion of liver
By the complete liver won, it is placed in dress liver container 8, using internal diameter 6mm flexible pipe trans-portal vein intubation, will filter
Sterilizing, it is preheated to 37 DEG C of 4000ml perfusates, through infusion pump 4 with 400ml/min speed perfusion 10 minutes, discard perfusate；Connect
With filtration sterilization, be preheated to 37 DEG C of 800ml perfusates, with 400ml/min speed perfusion 2 minutes, discard perfusate；Afterwards
By filtration sterilization, it is preheated to 37 DEG C of 2000ml perfusate, with 500ml/min speed loop perfusion 30 minutes,.Above perfusion
During, all perfusates are all through the oxygenation process of oxygenator 6 it is ensured that dissolving partial pressure of oxygen in perfusate is 200mmhg.
B, isolating hepatocytes
Discard perfusate, by hepatic metastasis to new aseptic basin, pour the hepatocyte cleaning mixture that 600ml is cooled to 4 DEG C in advance into,
Scratch Glisson's capsule, remove indigested hepatic tissue and connective tissue, collect hepatocyte suspension.Above-mentioned hepatocyte suspension is de- through four layers
After fat filtered through gauze, be averagely sub-packed in two volumes be 750ml sterile centrifuge bottles in, under the conditions of 4 DEG C, centrifugal force 50 × g from
The heart 10 minutes.Discard centrifuged supernatant, repeat and add 4 DEG C of hepatocyte cleaning mixture 500ml resuspended heavy in each centrifuge bottle
The hepatocyte formed sediment, under the conditions of 4 DEG C, centrifugal force 50 × g is centrifuged 10 minutes.Discard centrifuged supernatant, repeat to each centrifuge bottle
The hepatocyte of the middle resuspended precipitation of hepatocyte cleaning mixture 500ml adding 4 DEG C, under the conditions of 4 DEG C, centrifugal force 50 × g is centrifuged 10 minutes.
Abandoning supernatant, collects precipitation, the hepatocyte as separately obtaining.
3rd, detection method
After precipitation hepatocyte is weighed, the serum-free liver cell culture liquid being cooled to 4 DEG C with 500ml in advance is resuspended, calculates for the first time
Extension rate, then takes out 10ml hepatocyte suspension, is diluted to 400 times with serum-free liver cell culture liquid further, to dilution
To 400 times of hepatocyte carrying out Trypan Blue, with blood cell counting plate, counted under microscope, nucleus indigo plant contaminates for extremely thin
Born of the same parents, calculate Cell viability, calculate cell purity according to cellular morphology under microscope.Pass through grain count instrument simultaneously
(multisizer 3 beckman coulter counter) measures total cellular score and cell concentration.Remaining hepatocyte suspension is put
Standby in ice bath, and cultivated further finally according to the purposes regulation cell concentration of required cell.
4th, result of the test
The hepatocyte result of present invention preparation is as shown in the table:
| Fresh separated primary porcine hepatocyte index | The inventive method |
| Hepatocyte motility rate (%) | 99-99.5 |
| Obtained hepatocyte sum (individual/gram hepatic tissue) | 6.93-8.39×107 |
| Hepatocyte purity (%) | 95-98 |
Experimental result illustrates, it is equal in terms of motility rate, quantity and purity that the inventive method separates the primary porcine hepatocyte obtaining
Very excellent.
Embodiment 3 carries out, using Vitro hepatic perfusion device of the present invention, the method that primary hepatocyte separates preparation
First, test reagent
1. perfusate: taking prepare 1 liter of reagent as a example:
(1) take following composition: 8.1 grams of sodium chloride, 0.5 gram of potassium chloride, 2.6 grams of hepes, 4.5 grams of nac, egta 1.1
Gram；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.During use, it is preheated to 37 DEG C.
2. perfusate: taking prepare 1 liter of reagent as a example:
(1) take following composition: 8.1 grams of sodium chloride, 0.5 gram of potassium chloride, 2.6 grams of hepes；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.During use, it is preheated to 37 DEG C.
3. perfusate: taking prepare 1 liter of reagent as a example:
(1) take following composition: 3.8 grams of sodium chloride, 0.5 gram of potassium chloride, 25 grams of hepes, calcium chloride dihydrate (cacl2·
2h2O) 0.07 gram, 4.5 grams of albumin, collagenase roche liberasetm(being 5.2 w ü nsch units inside 1mg enzyme) 50 milli
Gram (i.e. 260w ü nsch unit)；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.6, with 0.2 micron membrane filter filtration sterilization, was configured using first 15 minutes, during use, is preheated to 37 DEG C.
4. hepatocyte cleaning mixture: taking prepare 1 liter of reagent as a example:
(1) take following composition: 10.8 grams of williams'medium e powder, 2.2 grams of sodium bicarbonate, 2.8 grams of hepes,
0.1 gram of streptomycin, penicillin 100000 unit；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.3, and with 0.2 micron membrane filter filtration sterilization, 4 DEG C preserve and use.
2nd, test method
Obtain the complete liver of normal bar horse pig, so that full liver weighs 800 grams as a example separate and prepare primary porcine hepatocyte.Respectively will
Perfusate loads in perfusate storage container 1, perfusate is loaded in perfusate storage container 2, perfusate is filled
Enter in perfusate storage container 3.Described preheating is all preheated by heat exchanger 5.
A, the In vitro perfusion of liver
It is placed in the complete liver won in dress liver container 8, using internal diameter 10mm flexible pipe trans-portal vein intubation, incited somebody to action
Filter sterilization, it is preheated to 37 DEG C of perfusate 12000ml, through infusion pump 4 with 1000ml/min speed perfusion 12 minutes, discard filling
Flow liquid；Then with filtration sterilization, it is preheated to 37 DEG C of perfusate 3000ml, with 1000ml/min speed perfusion 3 minutes, discard
Perfusate；Afterwards by filtration sterilization, it is preheated to 37 DEG C of perfusate 4000ml, with 40 points of 1200ml/min speed loop perfusion
Clock.In above perfusing course, all perfusates all through the oxygenation process of oxygenator 6 it is ensured that in perfusate dissolving partial pressure of oxygen be
200mmhg.
B, isolating hepatocytes
After above-mentioned hepatocyte suspension filters through four layers of absorbent carbasus, be averagely sub-packed in four volumes be 750ml aseptic from
In heart bottle, under the conditions of 4 DEG C, centrifugal force 50 × g is centrifuged 10 minutes.Discard centrifuged supernatant, then add 4 DEG C in each centrifuge bottle
The resuspended precipitation of hepatocyte cleaning mixture 500ml, under the conditions of 4 DEG C, centrifugal force 50 × g be centrifuged 10 minutes.Discard centrifuged supernatant, then
4 DEG C of the resuspended precipitation of hepatocyte cleaning mixture 500ml is added in each centrifuge bottle, under the conditions of 4 DEG C, centrifugal force 50 × g centrifugation 10
Minute.Abandoning supernatant, collects precipitation, as primary hepatocyte.
3rd, detection method
With embodiment 2.
4th, result of the test
The hepatocyte result of present invention preparation is as shown in the table:
| Fresh separated primary porcine hepatocyte index | The inventive method |
| Hepatocyte motility rate (%) | 96-99 |
| Obtained hepatocyte sum (individual/gram hepatic tissue) | 6.55-7.28×107 |
| Hepatocyte purity (%) | 95-98 |
Experimental result illustrates, it is equal in terms of motility rate, quantity and purity that the inventive method separates the primary porcine hepatocyte obtaining
Very excellent.
Embodiment 4 carries out, using Vitro hepatic perfusion device of the present invention, the method that primary hepatocyte separates preparation
First, test reagent
1. perfusate: taking prepare 1 liter of reagent as a example:
(1) take following composition: 8.5 grams of sodium chloride, 0.5 gram of potassium chloride, 2.2 grams of hepes, 3.5 grams of nac, egta 0.9
Gram；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.During use, it is preheated to 37 DEG C.
2. perfusate: taking prepare 1 liter of reagent as a example:
(1) take following composition: 8.5 grams of sodium chloride, 0.5 gram of potassium chloride, 2.2 grams of hepes；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.During use, it is preheated to 37 DEG C.
3. perfusate: taking prepare 1 liter of reagent as a example:
(1) take following composition: 4 grams of sodium chloride, 0.5 gram of potassium chloride, 23 grams of hepes, calcium chloride dihydrate (cacl2·2h2o)
0.07 gram, 3.5 grams of albumin, 150 milligrams of (i.e. 162w of collagenase serva nb8 (being 1.08 w ü nsch units inside 1mg enzyme)
ü nsch unit)；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.6, with 0.2 micron membrane filter filtration sterilization, was configured using first 15 minutes, during use, is preheated to 37 DEG C.
4. hepatocyte cleaning mixture: taking prepare 1 liter of reagent as a example:
(1) take following composition: 9.3 grams of williams'medium e powder, 2.2 grams of sodium bicarbonate, 2.4 grams of hepes, chain
0.1 gram of mycin, penicillin 100000 unit；
(2) mentioned component is mixed, be dissolved in deionized water, stirring 10 minutes under room temperature, so as to abundant dissolve, is settled to
1l, adjusts ph to 7.3, and with 0.2 micron membrane filter filtration sterilization, 4 DEG C preserve and use.
2nd, test method
Obtain the complete liver of normal bar horse pig, so that full liver weighs 200 grams as a example separate and prepare primary porcine hepatocyte.Respectively will
Perfusate loads in perfusate storage container 1, perfusate is loaded in perfusate storage container 2, perfusate is filled
Enter in perfusate storage container 3.Described preheating is all preheated by heat exchanger 5.
A, the In vitro perfusion of liver
By the complete liver won, it is placed in dress liver container 8, using internal diameter 4mm flexible pipe trans-portal vein intubation, will filter
Sterilizing, it is preheated to 37 DEG C of perfusate 3300ml, through infusion pump 4 with 300ml/min speed perfusion 11 minutes, discard perfusate；
Then with filtration sterilization, it is preheated to 37 DEG C of perfusate 750ml, with 300ml/min speed perfusion 2.5 minutes, discard perfusion
Liquid；Afterwards by filtration sterilization, it is preheated to 37 DEG C of perfusate 1500ml, with 300ml/min speed loop perfusion 25 minutes.With
In upper perfusing course, all perfusates all through the oxygenation process of oxygenator 6 it is ensured that in perfusate dissolving partial pressure of oxygen be
200mmhg.
B, isolating hepatocytes
After above-mentioned hepatocyte suspension filters through four layers of absorbent carbasus, be averagely sub-packed in two volumes be 750ml aseptic from
In heart bottle, under the conditions of 4 DEG C, centrifugal force 50 × g is centrifuged 10 minutes.Discard centrifuged supernatant, then add 4 DEG C in each centrifuge bottle
The resuspended precipitation of hepatocyte cleaning mixture 500ml, under the conditions of 4 DEG C, centrifugal force 50 × g be centrifuged 10 minutes.Discard centrifuged supernatant, then
4 DEG C of the resuspended precipitation of hepatocyte cleaning mixture 500ml is added in each centrifuge bottle, under the conditions of 4 DEG C, centrifugal force 50 × g centrifugation 10
Minute.Abandoning supernatant, collects precipitation, as primary hepatocyte.
3rd, detection method
With embodiment 2.
4th, result of the test
The hepatocyte result of present invention preparation is as shown in the table:
| Fresh separated primary porcine hepatocyte index | The inventive method |
| Hepatocyte motility rate (%) | 99-99.5 |
| Obtained hepatocyte sum (individual/gram hepatic tissue) | 7.33-8.55×107 |
| Hepatocyte purity (%) | 95-98 |
Experimental result illustrates, it is equal in terms of motility rate, quantity and purity that the inventive method separates the primary porcine hepatocyte obtaining
Very excellent.
Illustrate beneficial effects of the present invention below by way of test example.
The primary hepatocyte method for separating and preparing of test example 1 present invention is compared with other methods
First, existing method prepares hepatocyte
Four step perfusion methods preparation hepatocyte disclosed in existing 1:cn1632107a, the method obtained hepatocyte sum 6.25 ×
107Individual/gram hepatic tissue, Cell viability are 90-99%, cell purity is 90-95%.
Two step perfusion methods preparation hepatocyte disclosed in existing 2:cn1632107a, the method obtained hepatocyte sum 1.25 ×
107Individual/gram hepatic tissue, Cell viability are 80-90%, cell purity is 80-90%.
Existing 3:zhang zg etal., an updated method for the isolation and culture
Of primary calf hepatocytes, vet j, method preparation hepatocyte, the method disclosed in 2012,191:323-326
Obtained hepatocyte sum 1.12 × 107Individual/gram hepatic tissue, Cell viability are 85.7%.
2nd, the inventive method preparation hepatocyte
The inventive method prepares hepatocyte: prepares hepatocyte, the method obtained hepatocyte sum 6.93- according to embodiment 2
8.39×107Individual/gram hepatic tissue, Cell viability are 99-99.5%, cell purity is 95-98%.
3rd, control methods preparation hepatocyte
The step except not containing the perfusate perfusion shown in the embodiment of the present invention 2 for the separation method, remaining same present invention
Embodiment 2, result obtained hepatocyte sum 2.62-4.19 × 107Individual/gram hepatic tissue, Cell viability are 90-95%, cell is pure
Spend for 86-92%.
The effects of five kinds of connection agents are summarized as follows table:
As can be seen from the above table:
1st, compared with existing three step perfusion methods and two step perfusion methods, it is thin that the present invention three step perfusion method is used for the primary liver of separation
During born of the same parents, the hepatocyte sum of acquisition, Cell viability and cell purity are all significantly increased.
2nd, compared with existing four step perfusion methods, when the present invention three step perfusion method is used for separating primary hepatocyte, obtain
Slightly excellent on hepatocyte sum, Cell viability, hepatocyte purity significantly improves.
3 and control methods, that is, the two step perfusion methods lacking perfusate perfusion are compared, and the present invention three step perfusion method is used for
When separating primary hepatocyte, the hepatocyte sum of acquisition, Cell viability and cell purity all have significantly more excellent.
Comparative result illustrates, the inventive method, by the combination of specific perfusate and perfusion step, can be divided effectively
From primary hepatocyte, separating effect is superior to existing perfusion, and only three kinds of perfusate, cost is not high.
To sum up, primary liver can be efficiently separated using the method that apparatus of the present invention carry out primary hepatocyte separation preparation thin
Born of the same parents, the hepatocellular quantity that separation obtains is many, motility rate and purity are high, is hepatocyte transplantation, biological artificial liver support system treatment
Acute and chronic hepatitis and hepatocyte external application model provide potential hepatocyte resource, and potential applicability in clinical practice is excellent.
Claims (22)
1. a kind of Vitro hepatic perfusion device it is characterised in that: include perfusate storage container group, four-way pipe, infusion pump, heat exchange
System, oxygenator, debubbler, perfusion catheter, tee T and reflux pump；
Described perfusate storage container group includes container a, container b and container c, described container a, container b, container c be equipped with into
Liquid mouth and the liquid outlet with stop valve, described container c is additionally provided with refluxing opening；
Described infusion pump, heat-exchange system, oxygenator, debubbler, perfusion catheter and reflux pump are equipped with inlet and liquid outlet；
Four mouths of pipe of described four-way pipe respectively with the liquid outlet of container a, the liquid outlet of container b, the liquid outlet of container c and perfusion
The inlet of pump passes through flexible pipe and is connected；The liquid outlet of infusion pump and the inlet of heat-exchange system, the liquid outlet of heat-exchange system
With entering of the inlet of the inlet of oxygenator, the liquid outlet of oxygenator and debubbler, the liquid outlet of debubbler and perfusion catheter
Liquid mouth, the liquid outlet of reflux pump are all connected by flexible pipe with the refluxing opening of container c；
The liquid outlet of described perfusion catheter stretches in dress liver container, and the bottom of described dress liver container is additionally provided with liquid outlet；
Described tee T is located at the lower section of dress liver container, and the two of which mouth of pipe of tee T passes through hose connection dress liver respectively
The liquid outlet of container, the inlet of reflux pump, the 3rd mouth of pipe of tee T connects sewer pipe, and described sewer pipe is provided with cut-off
Valve.
2. Vitro hepatic perfusion device according to claim 1 it is characterised in that:
The inlet of the inlet, the inlet of container b and container c of described container a is the feed liquor leakage of carrying liqs sterilizing filter membrane
Bucket, is located at the top of corresponding container respectively；
The liquid outlet of the liquid outlet, the liquid outlet of container b and container c of described container a is respectively positioned on bottom；
The top of described container a, container b and container c is equipped with the air-vent with air filter film；
Described container a, container b and container c are equipped with liquidometer.
3. Vitro hepatic perfusion device according to claim 1 it is characterised in that: described heat-exchange system by heat exchanger,
Hot recycle pump and thermostat water bath composition.
4. Vitro hepatic perfusion device according to claim 1 it is characterised in that: the volume of described container a be 4-10l, hold
The volume of device b is 1-2l, and the volume of container c is 2-4l.
5. Vitro hepatic perfusion device according to claim 1 it is characterised in that: described dress liver container carry cap.
6. the Vitro hepatic perfusion device according to any one of claim 1-5 it is characterised in that: the internal diameter of described flexible pipe is
6mm-10mm.
7. a kind of usage right requires Vitro hepatic perfusion device described in any one of 1-6 to carry out the side that primary hepatocyte separates preparation
Method it is characterised in that:
(1) perfusate is loaded in container a, perfusate loads in container b, and perfusate loads in container c；
(2) use perfusate pre-filled flexible pipe to export to debubbler, make to be full of perfusate between container a and debubbler outlet；
(3) take in vitro animal liverss to be placed in dress liver container, first use perfusate perfusion, perfusion rate is 300-1000ml/
Min, Hemoperfusion time is 10-12 minute；Use perfusate perfusion again, perfusion rate is 300-1000ml/min, Hemoperfusion time is
2-3 minute；Finally use perfusate perfusion, perfusion rate is 300-1200ml/min, Hemoperfusion time is 25-40 minute；
Wherein, every liter of described perfusate i contains following composition: 8.1-8.5 gram of sodium chloride, 0.5 gram of potassium chloride, 2.2-2.6 gram
Hepes, 3.5-4.5 gram of nac, 0.9-1.1 gram of egta, remaining is deionized water；
Every liter of described perfusate ii contains following composition: 8.1-8.5 gram of sodium chloride, 0.5 gram of potassium chloride, 2.2-2.6 gram of hepes,
Remaining is deionized water；
Every liter of described perfusate iii contains following composition: 3.8-4 gram of sodium chloride, 0.5 gram of potassium chloride, 23-25 gram of hepes, and 0.07
Gram calcium chloride dihydrate, 3.5-4.5 gram of albumin, 162-260w ü nsch unit collagenase, remaining is deionized water；
(4) isolating hepatocytes: the liver after washing step (3) process, collect hepatocyte suspension, filter, be centrifuged, collecting precipitation is
Can.
8. method according to claim 7 it is characterised in that: in step (3),
Every liter of described perfusate i contains following composition: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes, 3.5-4.5 gram
Nac, 0.9-1.1 gram of egta, remaining is deionized water；
Every liter of perfusate ii contains following composition: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes, and remaining is deionization
Water；
Every liter of perfusate iii contains following composition: 3.9 grams of sodium chloride, 0.5 gram of potassium chloride, 24 grams of hepes, 0.07 gram of two water chlorine
Change calcium, 4 grams of albumin, 162-260w ü nsch unit collagenase, remaining is deionized water.
9. method according to claim 8 it is characterised in that:
Every liter of described perfusate i contains following composition: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes, 4 grams of nac,
0.95 gram of egta, remaining is deionized water；
Every liter of perfusate ii contains following composition: 8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of hepes, and remaining is deionization
Water；
Every liter of perfusate iii contains following composition: 3.9 grams of sodium chloride, 0.5 gram of potassium chloride, 24 grams of hepes, 0.07 gram of two water chlorine
Change calcium, 4 grams of albumin, 216w ü nsch unit collagenase, remaining is deionized water.
10. method according to claim 9 it is characterised in that: in described perfusate iii, collagenase be collagenase serva
Nb8 or collagenase roche liberasetm.
11. methods according to any one of claim 7-10 it is characterised in that:
The perfusion rate of described perfusate i is 400ml/min, and Hemoperfusion time is 10 minutes；
The perfusion rate of perfusate ii is 400ml/min, and Hemoperfusion time is 2 minutes；
The perfusion rate of perfusate iii is 500ml/min, and Hemoperfusion time is 30 minutes.
12. methods according to any one of claim 7-10 it is characterised in that: in step (3), perfusate i, perfusate
Ii, the perfusion temperature of perfusate iii are 37 DEG C.
13. methods according to any one of claim 7-10 it is characterised in that: in step (3), the perfusion of perfusate iii
Mode is circulation perfusion.
14. methods according to any one of claim 7-10 it is characterised in that: in step (3), perfusate i, perfusate ii
And/or the dissolving partial pressure of oxygen of perfusate iii is 180-260mmhg.
15. methods according to any one of claim 7-10 it is characterised in that: in step (4), described filtration is with four layers
Absorbent carbasuss filter.
16. methods according to any one of claim 7-10 it is characterised in that: in step (4), described centrifugation be repeatedly from
The heart, comprises the concrete steps that:
A, centrifugation, abandoning supernatant；
B, resuspended precipitation, centrifugation；
C, process b is repeated once.
17. methods according to claim 7 or 10 it is characterised in that: the condition of centrifugation is: centrifuging temperature is 4 DEG C, centrifugation
Power is 50-55 × g, and the time of centrifugation is 10 minutes.
18. methods according to claim 16 it is characterised in that: in step b, resuspended precipitation adopt hepatocyte cleaning mixture.
19. methods according to claim 7 it is characterised in that: in step (4), washing using hepatocyte cleaning mixture washing.
20. methods according to claim 18 or 19 it is characterised in that: containing as follows in every liter of described hepatocyte cleaning mixture
Composition: 9.3-10.8 gram of williams'medium e powder, 2.2 grams of sodium bicarbonate, 2.4-2.8 gram of hepes, 0.1 gram of strepto-
Element, 100000 units of Penicillin, remaining is deionized water.
21. methods according to claim 20 it is characterised in that: in every liter of described hepatocyte cleaning mixture containing following become
Point: 9.3-10.8 gram of williams'medium e powder, 2.2 grams of sodium bicarbonate, 2.6 grams of hepes, 0.1 gram of streptomycin,
100000 units of Penicillin, remaining is deionized water.
22. methods according to claim 21 it is characterised in that: in every liter of described hepatocyte cleaning mixture containing following become
Point: 9.5 grams of williams'medium e powder, 2.2 grams of sodium bicarbonate, 2.6 grams of hepes, 0.1 gram of streptomycin, 100000 units
Penicillin, remaining is deionized water.
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| CN1565652A (en) * | 2003-06-24 | 2005-01-19 | 中国人民解放军第三军医大学 | Simple extraneous liver perfusion device for digesting and separating liver cells |
| WO2011002926A2 (en) * | 2009-07-01 | 2011-01-06 | The General Hospital Corporation | Isolated adult cells, artificial organs,rehabilitated organs, research rools, organ encasements, organ perfusion systems, and methods for preparing and utilizing the same |
| CN104312903A (en) * | 2014-10-29 | 2015-01-28 | 贵阳医学院 | Experimental apparatus for in-vitro circulating perfusion and digestion of liver tissues |
| CN104611225A (en) * | 2015-02-16 | 2015-05-13 | 昆明市第一人民医院 | Bioreactor for constructing tissue engineering liver by using in-vitro circulatory perfusion |
| CN204714806U (en) * | 2015-05-28 | 2015-10-21 | 四川大学华西医院 | A kind of Vitro hepatic perfusion device |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1565652A (en) * | 2003-06-24 | 2005-01-19 | 中国人民解放军第三军医大学 | Simple extraneous liver perfusion device for digesting and separating liver cells |
| WO2011002926A2 (en) * | 2009-07-01 | 2011-01-06 | The General Hospital Corporation | Isolated adult cells, artificial organs,rehabilitated organs, research rools, organ encasements, organ perfusion systems, and methods for preparing and utilizing the same |
| CN104312903A (en) * | 2014-10-29 | 2015-01-28 | 贵阳医学院 | Experimental apparatus for in-vitro circulating perfusion and digestion of liver tissues |
| CN104611225A (en) * | 2015-02-16 | 2015-05-13 | 昆明市第一人民医院 | Bioreactor for constructing tissue engineering liver by using in-vitro circulatory perfusion |
| CN204714806U (en) * | 2015-05-28 | 2015-10-21 | 四川大学华西医院 | A kind of Vitro hepatic perfusion device |
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