CN204714806U - A kind of Vitro hepatic perfusion device - Google Patents
A kind of Vitro hepatic perfusion device Download PDFInfo
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- CN204714806U CN204714806U CN201520356633.6U CN201520356633U CN204714806U CN 204714806 U CN204714806 U CN 204714806U CN 201520356633 U CN201520356633 U CN 201520356633U CN 204714806 U CN204714806 U CN 204714806U
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Abstract
The utility model discloses a kind of Vitro hepatic perfusion device, it comprises the perfusate storage vessel group, four-way pipe, infusion pump, heat exchange system, oxygenator, de-soak device, perfusion catheter, Y-tube and the reflux pump that are made up of 3 perfusate storage vessels.The utility model can carry out three step liver perfusions in vitro liver, carry out effective perfusion of front perfusate and Digestive system to greatest extent rapidly, continuously, farthest Quality Control digestion hepatic tissue process, cost-saving, steady temperature, a large amount of liver cells that decreasing pollution, thus the liver cell that the acquisition Experiment and clinic presentations of guaranteeing both quality and quantity is used, especially hepatocyte transplantation, bioartificial liver etc. are required.
Description
Technical field
The utility model relates to a kind of medical experiment device, particularly relates to a kind of Vitro hepatic perfusion device.
Background technology
Primary porcine hepatocyte is most suitable xenogenesis liver cell source in BAL treatment, it is enzyme digestion that the liver cell of existing maturation is separated the method adopted, the method is that collagenase etc. is injected liver vessel, act on hepatic tissue, fiber complicated between liver cell is connected dissolve, thus obtain complete individual hepatocyte.For large animal, as pig, the liver etc. of dog and people, because volume is large, perfusate consumption is large, and the time is long, and original position or In vitro perfusion all cannot adopt the pouring-in perfusion of traditional manual syringe, can only select infusion pump.And when using infusion pump perfusion, owing to lacking special suite of equipment, cause in perfusion sepn process and very easily cause the pollutions such as bacterium.Because collagenase is very high to temperature requirement, 37 DEG C is its best use of temperature, special perifusion apparatus is not had to be difficult to control perfusate temperature, therefore, often because temperature controls bad, and have impact on final cell dissociation effect, required cell quantity and specification of quality can not be reached, and cause the waste of expensive collagenase, increase the hepatocellular cost of separation and Culture.
Therefore, need one to be suitable for digestion at present and be separated the hepatocellular Vitro hepatic perfusion device of large animal liver.
Utility model content
For solving the problem, the utility model provides a kind of Vitro hepatic perfusion device, plant Vitro hepatic perfusion device, it is characterized in that: comprise perfusate storage vessel group, four-way pipe, infusion pump, heat exchange system, oxygenator, de-soak device, perfusion catheter, Y-tube and reflux pump; Described perfusate storage vessel group comprises container A, container B and container C, and described container A, container B, container C are equipped with fluid inlet and the liquid outlet with stopping valve, and described container C is also provided with refluxing opening; Described infusion pump, heat exchanger, oxygenator, de-soak device, perfusion catheter and reflux pump are equipped with fluid inlet and liquid outlet; Four mouths of pipe of described four-way pipe are connected by flexible pipe with the fluid inlet of infusion pump with the liquid outlet of container A, the liquid outlet of container B, the liquid outlet of container C respectively; The liquid outlet of infusion pump is all connected by flexible pipe with the refluxing opening of container C with the fluid inlet of perfusion catheter, the liquid outlet of reflux pump with the fluid inlet of de-soak device, the fluid inlet of de-soak device with the fluid inlet of oxygenator, the liquid outlet of oxygenator with the liquid outlet of the fluid inlet of heat exchange system, heat exchange system; The liquid outlet of described perfusion catheter stretches in dress liver container, and the bottom of described dress liver container is also provided with liquid outlet; Described Y-tube is positioned at the below of dress liver container, and wherein two mouths of pipe of Y-tube are respectively by the hose connection liquid outlet of dress liver container, the fluid inlet of reflux pump, and the 3rd mouth of pipe of Y-tube connects sewer pipe, and described sewer pipe is provided with stopping valve.
Further preferably, the fluid inlet of the fluid inlet of described container A, the fluid inlet of container B and container C is the feed liquor funnel of carry liquid sterilizing filter membrane, lays respectively at the top of corresponding container; The liquid outlet of the liquid outlet of described container A, the liquid outlet of container B and container C is all positioned at bottom; The top of described container A, container B and container C is equipped with the breather hole of band air filter film; Described container A, container B and container C are equipped with liquidometer.
Further preferably, described heat exchange system is made up of heat exchanger, hot recycle pump and thermostat water bath.
Further preferably, the volume of described container A is 4-10L, and the volume of container B is 1-2L, and the volume of container C is 2-4L.
Further preferably, described dress liver container is with cap.
Further preferably, the internal diameter of described flexible pipe is 6mm-10mm.
The utility model utilizes hot recycle pump that the heat recirculated water of 37-42 in water-bath DEG C is heated perfusate with the speed of 100-200mL/min injection heat exchanger, makes perfusate keep 37 DEG C of constant temperature in whole perfusing course.
Utilize oxygenator to carry out oxygenated treatment in perfusate, make dissolved oxygen dividing potential drop in perfusate remain on 180-260mmHg, reduce hepatocellular hypoxic exposure, reduce cavity in liver cell, improve hepatocellular vigor.
Utilize de-soak device before perfusate enters liver, remove size bubble in perfusate, avoid bubble in liver vessel, form gas embolus, make liver perfusion more full and uniform.
Owing to have employed technique scheme, device of the present utility model has following advantage:
1. easy to use, reliable, perfusing course carries out continuously, and single need just can complete operation in one hour, saves human and material resources and time.
2., by heat exchanger, perfusate remains 37 DEG C of constant temperature in whole perfusing course, meet liver cell be separated and constant temperature needed for collagenase fixed, ensure that the activity of digestion and isolating hepatocytes.
3. by oxygenator, oxygenated treatment has been carried out to perfusate, reduced hepatocellular hypoxic exposure, reduced cavity in liver cell, improve hepatocellular vigor.
4. de-soak device avoids the formation of gas embolus in liver vessel, ensure that in liver, all cells can obtain uniform perfusion.
5. adopt jumbo perfusate storage vessel in device, and the peristaltic pump of high flow rate at the uniform velocity pours into, ensure that pressure and the perfusate volume needs of large animal liver perfusion.
6. in whole filling process, liver is all in the sterile chamber of lid, and perfusate is also in aseptic pipeline and container, avoids the pollution of bacterium, fungi.
In sum, the utility model provides a kind of complete, easy to operate is separated the hepatocellular Vitro hepatic perfusion device of large animal liver for digesting, it can carry out three step liver perfusions in vitro liver, carry out effective perfusion of front perfusate and Digestive system to greatest extent rapidly, continuously, farthest Quality Control digestion hepatic tissue process, cost-saving, steady temperature, decreasing pollution, thus the liver cell that the acquisition Experiment and clinic presentations of guaranteeing both quality and quantity is used, a large amount of liver cells that especially hepatocyte transplantation, bioartificial liver etc. are required.
According to foregoing of the present utility model, according to ordinary technical knowledge and the customary means of this area, not departing under the utility model above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present utility model again.But this should be interpreted as that the scope of the above-mentioned theme of the utility model is only limitted to following example.All technology realized based on the utility model foregoing all belong to scope of the present utility model.
Accompanying drawing explanation
Fig. 1 is structural representation of the present utility model.In figure: 1. perfusate I storage vessel, volume 10L; 2. perfusate II storage vessel, volume 2L; 3. perfusate III storage vessel, volume 4L; 4. infusion pump; 5. heat exchanger; 6. oxygenator; 7. de-soak device; 8. lidded container; 9. reflux pump; 10. waste fluid container; 11. fluid inlet funnels; 12. fluid inlet funnels; 13. fluid inlet funnels; 14. sterilization of liquids filter membranes; 15. sterilization of liquids filter membranes; 16. sterilization of liquids filter membranes; 17. ventilating pits; 18. air filter films; 19. ventilating pits; 20. air filter films; 21. ventilating pits; 22. air filter films; 23. liquidometers; 24. liquidometers; 25. liquidometers; 26. liquid flowing outlets; 27. liquid flowing outlets; 28. liquid flowing outlets; 29. stopping valve; 30. stopping valve; 31. stopping valve; 32. liquid return mouths; 33. liquid flowing outlets; 34. stopping valve; 35. thermostat water baths; 36. hot recycle pumps; 37. perfusion catheters; 38. four-way pipes; 39. Y-tubes.
Embodiment
Embodiment 1
Vitro hepatic perfusion device as shown in Figure 1, comprise perfusate I storage vessel 1, volume 10L, there is fluid inlet funnel 11 storage vessel 1 upper end, and funnel 11 bottom is mounted with a sterilization of liquids filter membrane 14, and there is a ventilating pit 17 storage vessel 1 upper end, it is connected to an air filter film 18, storage vessel 1 sidewall has a liquidometer 23, and there is a liquid flowing outlet 26 storage vessel 1 lower end, and there is a stopping valve 29 in exit.This device also comprises perfusate II storage vessel 2, volume 2L, there is fluid inlet funnel 12 storage vessel 2 upper end, funnel 12 bottom is mounted with a sterilization of liquids filter membrane 15, there is a ventilating pit 19 holder 2 upper end, it is connected to an air filter film 20, and storage vessel 2 sidewall has a liquidometer 2), there is a liquid flowing outlet 27 storage vessel 2 lower end, and there is a stopping valve 30 in exit.This device also comprises perfusate III storage vessel 3, volume 4L, there is fluid inlet funnel 13 storage vessel 3 upper end, funnel 13 bottom is mounted with a sterilization of liquids filter membrane 16, and there is a ventilating pit 21 storage vessel 3 upper end, it is connected to an air filter film 22, a liquid return mouth 32 is had in storage vessel 3 side wall upper part, opposite side sidewall has a liquidometer 25, there is a liquid flowing outlet 28 storage vessel 3 lower end, and there is a stopping valve 31 in exit.This device also comprises infusion pump 4; Heat exchanger 5, with heat exchanger 5 with the use of thermostat water bath 35 and hot recycle pump 36; Oxygenator 6, can pass into oxygen; De-soak device 7; Band mounts cover liver container 8, and it covers and can settle perfusion catheter 37, liquid flowing outlet 33 is arranged at bottom, and spout is connected to Y-tube 39, connects stopping valve 34 below Y-tube; Reflux pump 9 and waste fluid container 10.
Different according to perfusion rate, be that the flexible pipe of 6-10mm is first respectively by perfusate holder 1 with internal diameter, 2,3 are connected with four-way pipe 38, then remaining for four-way pipe 38 one end is connected with infusion pump 4, then connects heat exchanger 5 successively, oxygenator 6 and de-soak device 7, finally connect perfusion catheter 37, be connected to band and mount cover liver container 8.Afterwards, band mounts cover Y-tube 33 below liver container 8, and band stopping valve 34 end internal diameter is the hose connection waste fluid container 10 of 6-10mm, and another section of Y-tube 33 connects reflux pump 9, finally connects liquid return mouth 32 on perfusate III storage vessel 3.With internal diameter be 6mm hose connection heat exchanger 5 on heat recirculated water gangway and thermostat water bath 35 and hot recycle pump 36.
The using method of the utility model device is such:
Obtain the complete liver of normal bar horse pig in surgery mode, it is example that full liver weighs 300 grams for example is separated the perfusing course preparing primary porcine hepatocyte, and this device using method is described.
First by sterilizing after a complete set of equipment partition, aseptic assembling is carried out afterwards.Filtration sterilization perfusate I 4-10L is added respectively, perfusate II 1-2L and perfusate III 2-4L in three perfusate storage vessels.After pipeline is connected, export to de-soak device with the whole pipeline of perfusate I pre-filled.Then win complete large animal liver or people's confession hepatic tissue under surgical sterilized condition, be placed in lid sterile chamber, by perfusion catheter and liver portal system UNICOM, the perfusion catheter the other end and perfusion device UNICOM afterwards.Then, open infusion pump, with the speed of 400-1000mL/min, the perfusate I of 37 DEG C of oxygenations is poured into liver, the perfusate I pouring into liver overflows from liver superior and inferior vena cava, and in final inflow waste fluid container, discard, Hemoperfusion time is 10-12 minute.This is the first step perfusion, rinses liver inner blood by perfusate I, is that residual hepatocyte is rinsed well as far as possible, the connection of simultaneously tentatively loosening between liver cell.
Secondly, with the speed of 400-1000mL/min, the perfusate II of 37 DEG C of oxygenations is poured into liver, the perfusate II pouring into liver overflows from liver superior and inferior vena cava, and in final inflow waste fluid container, discard, Hemoperfusion time is 1-2 minute.This is second step perfusion, rinses residual perfusate I in liver, remove the compositions such as the inhibitor EGTA of collagenase activities in perfusate I, for enzymic digestion is prepared by perfusate II.
Finally, with the speed of 500-1100mL/min, the perfusate III containing collagenase of 37 DEG C of oxygenations is poured into liver, the perfusate III pouring into liver overflows from liver superior and inferior vena cava, be kept at and be equipped with in the sterile chamber of liver, open reflux pump with the speed identical with infusion pump, perfusate III to be drawn back in its original storage vessel, form at the uniform velocity circulation perfusion liver, after 25-40 minute, until Glisson's capsule is separated with liver cell, terminate perfusion.Perfusate III flows in waste fluid container, discards.This is the 3rd step liver perfusion, and mainly through to liver internal recycle potting compound protoenzyme, the fiber between digest and decompose liver cell connects, isolating hepatocytes.
Afterwards the lidded container that liver is housed is taken out from perfusion device, move in Bechtop, enter follow-up hepatocyte suspension and collect and washing process.The process of described liver cell washing is: first, and the Williams'Medium E being chilled to 4 DEG C is in advance washed substratum, and 300-1000mL adds in aseptic basin, scratches Glisson's capsule with operating scissors, is separated indigested hepatic tissue and reticular tissue, collects hepatocyte suspension.Above-mentioned hepatocyte suspension after four pull-up fat filtered through gauze, under 4 DEG C of conditions, centrifugal 10 minutes of centrifugal force 50-55 × g.Afterwards, discard centrifuged supernatant, wash the liver cell of the resuspended precipitation of substratum with the Williams'Medium E of 4 DEG C, under 4 DEG C of conditions, centrifugal 10 minutes of centrifugal force 50-55 × g.Said process is repeated once.Finally, discard centrifuged supernatant, precipitation liver cell is resuspended with serum free hepatocyte medium, regulates cell concn to be every milliliter, 2-5 × 107, puts in ice bath for subsequent use.
Above-described perfusate I often liter contain: 8.3 grams of sodium-chlor, 0.5 gram of Repone K, 2.4 grams of HEPES, 3-6g NAC, 0.9-1.1g EGTA, surplus is deionized water;
Perfusate II often liter contain: 8.3 grams of sodium-chlor, 0.5 gram of Repone K, 2.4 grams of HEPES, surplus is deionized water;
Perfusate III often liter contain: 3.9 grams of sodium-chlor, 0.5 gram of Repone K, 24 grams of HEPES, 0.07 gram of Calcium dichloride dihydrate (CaCl
22H2O), 4 grams of albumin, 68-130PZ activity unit collagenase, surplus is deionized water.
Williams'Medium E washs during substratum often rises and contains: 9.5-10.8 gram of Williams'MediumE powder, 2.2 grams of sodium bicarbonates, 2.6 grams of HEPES, 0.1g Streptomycin sulphates, 100000 units of Penicillin, surplus is deionized water.
Claims (6)
1. a Vitro hepatic perfusion device, is characterized in that: comprise perfusate storage vessel group, four-way pipe, infusion pump, heat exchange system, oxygenator, de-soak device, perfusion catheter, Y-tube and reflux pump;
Described perfusate storage vessel group comprises container A, container B and container C, and described container A, container B, container C are equipped with fluid inlet and the liquid outlet with stopping valve, and described container C is also provided with refluxing opening;
Described infusion pump, heat exchanger, oxygenator, de-soak device, perfusion catheter and reflux pump are equipped with fluid inlet and liquid outlet;
Four mouths of pipe of described four-way pipe are connected by flexible pipe with the fluid inlet of infusion pump with the liquid outlet of container A, the liquid outlet of container B, the liquid outlet of container C respectively; The liquid outlet of infusion pump is all connected by flexible pipe with the refluxing opening of container C with the fluid inlet of perfusion catheter, the liquid outlet of reflux pump with the fluid inlet of de-soak device, the fluid inlet of de-soak device with the fluid inlet of oxygenator, the liquid outlet of oxygenator with the liquid outlet of the fluid inlet of heat exchange system, heat exchange system;
The liquid outlet of described perfusion catheter stretches in dress liver container, and the bottom of described dress liver container is also provided with liquid outlet;
Described Y-tube is positioned at the below of dress liver container, and wherein two mouths of pipe of Y-tube are respectively by the hose connection liquid outlet of dress liver container, the fluid inlet of reflux pump, and the 3rd mouth of pipe of Y-tube connects sewer pipe, and described sewer pipe is provided with stopping valve.
2. Vitro hepatic perfusion device according to claim 1, is characterized in that:
The fluid inlet of the fluid inlet of described container A, the fluid inlet of container B and container C is the feed liquor funnel of carry liquid sterilizing filter membrane, lays respectively at the top of corresponding container;
The liquid outlet of the liquid outlet of described container A, the liquid outlet of container B and container C is all positioned at bottom;
The top of described container A, container B and container C is equipped with the breather hole of band air filter film;
Described container A, container B and container C are equipped with liquidometer.
3. Vitro hepatic perfusion device according to claim 1, is characterized in that: described heat exchange system is made up of heat exchanger, hot recycle pump and thermostat water bath.
4. Vitro hepatic perfusion device according to claim 1, is characterized in that: the volume of described container A is 4-10L, and the volume of container B is 1-2L, and the volume of container C is 2-4L.
5. Vitro hepatic perfusion device according to claim 1, is characterized in that: described dress liver container is with cap.
6. the Vitro hepatic perfusion device according to any one of claim 1-5, is characterized in that: the internal diameter of described flexible pipe is 6mm-10mm.
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| CN201520356633.6U CN204714806U (en) | 2015-05-28 | 2015-05-28 | A kind of Vitro hepatic perfusion device |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845878A (en) * | 2015-05-28 | 2015-08-19 | 四川大学华西医院 | In-vitro liver perfusion device and application thereof |
| CN114107045A (en) * | 2021-10-12 | 2022-03-01 | 江苏蒙彼利生物科技有限公司 | Perfusion device and perfusion method for culturing non-blood stem cells |
| CN114617119A (en) * | 2022-03-04 | 2022-06-14 | 杭州莱普晟医疗科技有限公司 | Systems and methods for pancreatic perfusion |
-
2015
- 2015-05-28 CN CN201520356633.6U patent/CN204714806U/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845878A (en) * | 2015-05-28 | 2015-08-19 | 四川大学华西医院 | In-vitro liver perfusion device and application thereof |
| CN104845878B (en) * | 2015-05-28 | 2017-01-25 | 四川大学华西医院 | In-vitro liver perfusion device and application thereof |
| CN114107045A (en) * | 2021-10-12 | 2022-03-01 | 江苏蒙彼利生物科技有限公司 | Perfusion device and perfusion method for culturing non-blood stem cells |
| CN114107045B (en) * | 2021-10-12 | 2023-08-11 | 江苏蒙彼利生物科技有限公司 | Serum-free stem cell culture perfusion device and perfusion method |
| CN114617119A (en) * | 2022-03-04 | 2022-06-14 | 杭州莱普晟医疗科技有限公司 | Systems and methods for pancreatic perfusion |
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