CN102643778A - Method for separating, culturing and identifying primary hepatic cells of livestock and fowl - Google Patents

Method for separating, culturing and identifying primary hepatic cells of livestock and fowl Download PDF

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Publication number
CN102643778A
CN102643778A CN 201210145959 CN201210145959A CN102643778A CN 102643778 A CN102643778 A CN 102643778A CN 201210145959 CN201210145959 CN 201210145959 CN 201210145959 A CN201210145959 A CN 201210145959A CN 102643778 A CN102643778 A CN 102643778A
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livestock
cell
liver
liquid
poultry
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唐胜球
江青艳
朱晓彤
董小英
罗增福
宾艳芳
周桂炫
方心灵
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Shaoguan University
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Shaoguan University
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Abstract

The invention relates to a method for separating, culturing and identifying primary hepatic cells of livestock and fowl, which integrates a chelation process and an in-situ two-step perfusion process. Ca<2+> and Mg<2+> in hepatic tissues of livestock and fowl are previously removed so that the hepatic tissues maximally release the primary hepatic cells; and the formula of additives of the culture fluid is adjusted to perfect the serum-free culture system and identify the glycogen of the obtained primary hepatic cells. The invention can effectively enhance the quantity, motility and purity of the separated primary hepatic cells of livestock and fowl, and is applicable to various animals; and the obtained primary hepatic cells have the advantages of fewer traumas, high wall attaching tendency and high activity, can be widely used for physiological function adjustment, metabolic diseases, and pharmacological and toxicological mechanisms of hepatic cells of livestock and fowl, can establish a virus infected cell model, and can be used for research in the fields of physics and chemistry, toxicant factor influence and the like.

Description

The method that a kind of livestock and poultry primary hepatocyte separates, cultivates and identify
Technical field
The invention belongs to biological technical field, relate to a kind of livestock and poultry primary hepatocyte separation of all kinds of animals of livestock and poultry, method of cultivating and identifying of being applicable to.
Background technology
Liver is a body performance metabolic function, and deoxidation stores glycogen, and the vitals of various biological functions such as synthesis secretion property protein and bio-transformation also are the main place of medicine performance toxic action.Therefore; It is biomaterial that many vitro studies are all selected the livestock and poultry primary hepatocyte for use, like the adjusting of liver cell physiological function, metabolic disease, and pharmacology, pathology and toxicology mechanism; The foundation of virus infected cell model, and the fields such as influence of physics and chemistry and poisonous substance factor.
It is thus clear that, the separation of primary hepatocyte, cultivate and identify it is the primary link of accomplishing above in vitro study, and liver cell external can the serum-free culture success, its separate and cultural method most important.In recent years, domestic and international many scholars have done a large amount of explorations on primary hepatocyte separation and culture technique, are obtaining many achievements aspect the practical application of liver cell separation, culture technique.The early stage main non-enzyme process isolating hepatocytes that adopts comprises mechanical phonograph recorder separation and chelating method.Mechanical phonograph recorder separation is to utilize the method that mechanical shearing, supercharging are sieved and physical means such as powerful piping and druming comes isolating hepatocytes, and big to hepatocellular injury, the cell motility rate is low.The chelating method is with combining Ca 2+, Mg 2+Sequestrant (like EDTA) remove Ca 2+The dependency adhesion factor make the fracture of iuntercellular desmosome and isolating hepatocytes, but the independent result of use of sequestrant is bad, often needs to be used in combination with enzyme process.Later stage changes the enzyme digestion isolating hepatocytes gradually into, comprises collagenase digestion and tryptic digestion method etc.Enzyme digestion is to utilize enzyme to destroy the bridging between the liver cell, makes the isolating a kind of method of liver cell.The common way of enzyme digestion is that the digestive ferment of an amount of concentration of adding after tissue is shredded is put into 37 ℃ of shaking baths digestion appropriate times.But,, adopt the common cytoactive that obtains of separating of tissue block digestion method lower, and a large amount of red corpuscle are difficult to removal in the hepatic tissue blocking for liver.When carrying out enzyme digestion, very strict to the concentration and requirement action time thereof of enzyme, especially trypsinase is a kind of strong protein digestion enzyme, and is serious to the liver plasma membrane damage, is prone to cause liver cell death.1972; Seglen adopts in-situ two-step collagenase perfusion method isolated from rat liver cell successfully to obtain high motility rate first; Because adopt collagenase digestion to separate primary hepatocyte, less to hepatocellular injury, and liver plasma membrane suffered infringement in digestive process can obtain repairing after cultivation.After this, numerous investigators have carried out certain improvement to this method, and are used for the hepatocellular separation of different plant species.Be used for the separation of adult chicken liver cell like improvement in-situ two-step perfusion methods such as Fraslin; Douaire etc. set up the chicken liver cell serum free culture system of growing up with Williams ' medium E as basic culture solution; One Chinese patent application (200410084625.7) has been reported the preparation method of a kind of bioartificial liver with pig and human liver cell; One Chinese patent application (200610050853.1) obtains fresh former generation porcine hepatocyte etc. of high motility rate with Dispase-collagenase perfusion method.But the limitation that above separation method is all certain mainly shows as: be that primary hepatocyte yield and purity are not high on the one hand, vigor is not strong, and process is more loaded down with trivial details; For the serum culture system is arranged, can't biologic material be provided on the other hand for the part Liver Function.
Summary of the invention
For overcoming the above-mentioned shortcoming of prior art; The present invention provides a kind of can effectively improve former generation livestock and poultry liver cell quantity, motility rate and the purity that is separated to, the primary hepatocyte that is obtained damage less, be prone to the method that adherent and active good livestock and poultry primary hepatocyte separates, cultivates and identifies.
The present invention solves the technological method that its technical problem adopts: a kind of livestock and poultry primary hepatocyte separates, cultivates and the method for identifying, chelating method and in-situ two-step perfusion method are integrated, and sloughs the interior Ca of livestock and poultry liver organization in advance 2+With Mg 2+, making it as often as possible to discharge primary hepatocyte, the additive prescription through the adjustment nutrient solution improves its serum free culture system, and carries out glycogen with regard to obtaining primary hepatocyte and identify.
Its operation steps is:
One, the separation of livestock and poultry primary hepatocyte
Fasting before the operation of
Figure 180671DEST_PATH_IMAGE001
livestock and poultry; Freely drink water and spend the night; The anti-freezing of neck injection 1400IU/kg dosage heparin sodium, abdominal injection vetanarcol (50mg/kg) anesthetized animal;
Figure 395621DEST_PATH_IMAGE002
with postanesthetic livestock and poultry dorsal position Baoding on the animalcule operating table; This operating table is placed on the big porcelain dish; With 75% alcohol wipe whole body, change in the Bechtop;
Open the abdominal cavity under
Figure 577203DEST_PATH_IMAGE003
aseptic condition; Pull to the body right side to enteron aisle gently; Expose mesenteric vein; Ligation postcava two ends, and import hepatic vein direction intubate therein, unclamp an end that flows to liver then;
Carry out perfusion and separate, at first digest preceding perfusion, be divided into two portions again: first part is with the perfusate of 37 ℃ of preheatings of speed perfusion of 50ml/min
Figure 558377DEST_PATH_IMAGE005
, this perfusate
Figure 62171DEST_PATH_IMAGE005
In contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O and 5mM EDTA treat that liver cuts off precava slightly during bulging, continue 20min, then with the perfusate of 37 ℃ of preheatings of same speed perfusion
Figure 285210DEST_PATH_IMAGE006
, this perfusate
Figure 911364DEST_PATH_IMAGE006
In contain 10mM HEPES, 137mM NaCl, 3mM KCl and 3mM Na 2HPO 4.12H 2O continues 5min;
After treating that effusive liquid becomes clearly, carry out second step digestion perfusion, promptly pour into the perfusate of 37 ℃ of preheatings with 20ml/min speed
Figure 28804DEST_PATH_IMAGE008
, this perfusate
Figure 184978DEST_PATH_IMAGE008
In contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O, 5mM EDTA, 5.4mM CaCl 2And 0.4g/L
Figure 919716DEST_PATH_IMAGE009
The Collagen Type VI enzyme, and suitably clamp the postcaval vein of having cut off with mosquito forceps, perfusate is slowly flowed out, liver is digested gradually behind the 15min;
Figure 715503DEST_PATH_IMAGE010
touch liver surface appear organize softer after; Begin it is won and put into an aseptic plate; William Si (family name) the medium E of adding 50ml precooling (Williams ' medium E) basic culture solution; Reject blood vessel, fat and reticular tissue, cut off the incomplete part of liver periphery digestion;
Figure 623416DEST_PATH_IMAGE011
tears the liver coating with tweezers; Shred hepatic tissue; The heavy caliber suction pipe is blown and beaten gently; Cross 100 orders (150 μ m), 200 orders (75 μ m) and 400 orders (30 μ m) cell sieve subsequently respectively; The gained hepatocyte suspension cleans 2 times with William Si (family name) medium E (Williams ' medium E) basic culture solution, again at 4 ℃ of centrifugal 5min of 500rpm;
Figure 571780DEST_PATH_IMAGE012
Use adherent culture liquid, contain volume percent in this adherent culture liquid and be 10% NBCS, 10 -6M insulin human, 10 -6The basic culture solution of M DEXAMETHASONE BP98,10 μ g/ml HTrfs, 10 μ g/ml Vit C, two anti-(100U/ml penicillium mould and 100 μ g/ml Streptomycin sulphates, Gibco companies)) resuspended, trypan blue exclusion method detects cell motility rate, blood cell counting plate counting;
Two, the cultivation of livestock and poultry liver primary cell
After the cell counting, using adherent culture liquid is 2～5 * 10 with the cell suspension dilution 5Cell/ml inoculates 15ml in diameter is the culturing bottle of 60ml, inoculum density is 1 * 10 5Cell/cm 2, at 37 ℃, 5% CO 2With cultivate under the condition of saturated humidity; Behind the 4h cell attachment; Change liquid with grown cultures liquid full dose, contain volume percent in this grown cultures liquid and be 10% NBCS and two anti-basic culture solutions and continue to cultivate 20h, after this; With serum-free medium, 10 μ g/ml vitamins Cs, 0.15% BSA and two anti-Williams ' medium E basic culture solution) full dose changes liquid, contains 5 * 10 in this serum-free medium -7M DEXAMETHASONE BP98,1 * SITE (this be (insulin, transferrin, selenium, abbreviation ethanolamine) also can be write as ITES, Sigma-Aldrich company contains 10 μ g/ml Sigma I8405s, 5.5 μ g/ml HTrfs, 2.9 * 10 -8M Sodium Selenite, 2 μ g/ml thanomins), later every 24h changes nutrient solution, and inverted microscope is observed liver cell form and growing state down;
Three, the glycogen of livestock and poultry primary hepatocyte is identified
Getting above-mentioned serum-free medium cultivates the liver cell of 48 h (inoculation back the 3rd d) and carries out staining for glycogen and identify that the concrete operations step is following:
Figure 539736DEST_PATH_IMAGE001
inhales and removes nutrient solution; Abandon liquid after adding Carnoy ' s liquid chamber temperature fixed cell 15 min; Add 95% alcohol, 5 min;
Figure 324284DEST_PATH_IMAGE002
abandons liquid; Abandon liquid after adding 70% alcohol, 15 min; The adding mass percent is 0.8% periodic acid spirituous solution oxidation, 10 min;
Behind
Figure 35888DEST_PATH_IMAGE003
distillation washing 2 min; Add Schiff ' s alcohol blend, 10 min;
After
Figure 104338DEST_PATH_IMAGE004
abandons liquid; Add 0.5% sodium metabisulphite solution, zero(ppm) water is washed 2 min again behind 2 min;
Figure 430146DEST_PATH_IMAGE007
adds Ehrlich ' s haematoxylin redyeing nucleus, 20 min;
Figure 747995DEST_PATH_IMAGE010
with 0.5% hydrochloride alcohol color separation, tap water oil blackeite 10 min;
Figure 200973DEST_PATH_IMAGE011
95% alcohol and 100% alcohol 2 min that dewater respectively;
Figure 186247DEST_PATH_IMAGE012
YLENE is transparent; The natural gum mounting, inverted microscope is observed down.
The invention has the beneficial effects as follows: the present invention integrates chelating method and in-situ two-step perfusion method, and promptly before digestion during perfusion, perfusion contains the no Ca of 5mM EDTA 2+, Mg 2+The HEPES damping fluid, liver cell degree of scatter that obtains behind the method improvement and purity are higher, and the cell motility rate can reach more than 90%.But its mechanism is the EDTA chelating blood that contains in the perfusate and the Ca in the liver cell interstitial fluid 2+, Mg 2+, play anticoagulation on the one hand, better red corpuscle is developed, can remove the Ca between liver cell on the other hand 2+The dependency adhesion factor promotes hepatocellular separation.
Present invention includes three aspects such as the separation of type perfusion in situ digestion method, primary hepatocyte serum-free culture and the evaluation of liver cell glycogen of livestock and poultry primary hepatocyte; The present invention can effectively improve former generation livestock and poultry liver cell quantity, motility rate and the purity that is separated to, and applicable to all kinds of animals of livestock and poultry, the primary hepatocyte that is obtained damage is few; Be prone to adherent; Active good, can be widely used in the hepatocellular physiological function adjusting of livestock and poultry, metabolic disease, pharmacology and toxicology mechanism; Set up the virus infected cell model, and the research in fields such as physics and chemistry and poisonous substance factor affecting.
Description of drawings
Fig. 1 is a technological process block-diagram of the present invention.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further specified.
Referring to Fig. 1,The method that a kind of livestock and poultry primary hepatocyte separates, cultivates and identify, its operation steps is: one, the separation of livestock and poultry primary hepatocyte
Fasting before the operation of
Figure 184421DEST_PATH_IMAGE001
livestock and poultry; Freely drink water and spend the night; The anti-freezing of neck injection 1400IU/kg dosage heparin sodium, abdominal injection vetanarcol (50mg/kg) anesthetized animal;
Figure 989566DEST_PATH_IMAGE002
with postanesthetic livestock and poultry dorsal position Baoding on the animalcule operating table; This operating table is placed on the big porcelain dish; With 75% alcohol wipe whole body, change in the Bechtop;
Open the abdominal cavity under
Figure 980655DEST_PATH_IMAGE003
aseptic condition; Pull to the body right side to enteron aisle gently; Expose mesenteric vein; Ligation postcava two ends; And importing hepatic vein direction intubate therein, this pipe is that the tubing passivity is gone the syringe needle part, unclamps an end that flows to liver then;
Figure 7386DEST_PATH_IMAGE004
Carry out perfusion and separate, at first digest preceding perfusion, be divided into two portions again: first part is with the perfusate of 37 ℃ of preheatings of speed perfusion of 50ml/min , this perfusate
Figure 983749DEST_PATH_IMAGE005
In contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O and 5mM EDTA treat that liver cuts off precava slightly during bulging, continue 20min, then with the perfusate of 37 ℃ of preheatings of same speed perfusion
Figure 575268DEST_PATH_IMAGE006
, this perfusate
Figure 235268DEST_PATH_IMAGE006
In contain 10mM HEPES, 137mM NaCl, 3mM KCl and 3mM Na 2HPO 4.12H 2O continues 5min;
Figure 824512DEST_PATH_IMAGE007
After treating that effusive liquid becomes clearly, carry out second step digestion perfusion, promptly pour into the perfusate of 37 ℃ of preheatings with 20ml/min speed , this perfusate
Figure 186409DEST_PATH_IMAGE008
In contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O, 5mM EDTA, 5.4mM CaCl 2And 0.4g/L
Figure 781DEST_PATH_IMAGE009
The Collagen Type VI enzyme, and suitably clamp the postcaval vein of having cut off with mosquito forceps, perfusate is slowly flowed out, liver is digested gradually behind the 15min;
Figure 760927DEST_PATH_IMAGE010
touch liver surface appear organize softer after; Begin it is won and put into an aseptic plate; William Si (family name) the medium E of adding 50ml precooling (Williams ' medium E) basic culture solution; Reject blood vessel, fat and reticular tissue, cut off the incomplete part of liver periphery digestion;
Figure 27960DEST_PATH_IMAGE011
tears the liver coating with tweezers; Shred hepatic tissue; The heavy caliber suction pipe is blown and beaten gently; Cross 100 orders (150 μ m), 200 orders (75 μ m) and 400 orders (30 μ m) cell sieve subsequently respectively; The gained hepatocyte suspension cleans 2 times with William Si (family name) medium E (Williams ' medium E) basic culture solution, again at 4 ℃ of centrifugal 5min of 500rpm;
Figure 915276DEST_PATH_IMAGE012
Use adherent culture liquid, contain volume percent in this adherent culture liquid and be 10% NBCS, 10 -6M insulin human, 10 -6The basic culture solution of M DEXAMETHASONE BP98,10 μ g/ml HTrfs, 10 μ g/ml Vit C, two anti-(100U/ml penicillium mould and 100 μ g/ml Streptomycin sulphates, Gibco companies)) resuspended, trypan blue exclusion method detects cell motility rate, blood cell counting plate counting;
Two, the cultivation of livestock and poultry liver primary cell
After the cell counting, using adherent culture liquid is 2～5 * 10 with the cell suspension dilution 5Cell/ml inoculates 15ml in diameter is the culturing bottle of 60ml, inoculum density is 1 * 10 5Cell/cm 2, at 37 ℃, 5% CO 2With cultivate under the condition of saturated humidity; Behind the 4h cell attachment; Change liquid with grown cultures liquid full dose, contain volume percent in this grown cultures liquid and be 10% NBCS and two anti-basic culture solutions and continue to cultivate 20h, after this; With serum-free medium, 10 μ g/ml vitamins Cs, 0.15% BSA and two anti-Williams ' medium E basic culture solution) full dose changes liquid, contains 5 * 10 in this serum-free medium -7(Sigma-Aldrich company contains 10 μ g/ml Sigma I8405s, 5.5 μ g/ml HTrfs, 2.9 * 10 for M DEXAMETHASONE BP98,1 * SITE -8M Sodium Selenite, 2 μ g/ml thanomins), later every 24h changes nutrient solution, and inverted microscope is observed liver cell form and growing state down;
Three, the glycogen of livestock and poultry primary hepatocyte is identified
Getting above-mentioned serum-free medium cultivates the liver cell of 48 h (inoculation back the 3rd d) and carries out staining for glycogen and identify that the concrete operations step is following:
Figure 521838DEST_PATH_IMAGE001
inhales and removes nutrient solution; Abandon liquid after adding Carnoy ' s liquid chamber temperature fixed cell 15 min; Add 95% alcohol, 5 min;
Figure 515202DEST_PATH_IMAGE002
abandons liquid; Abandon liquid after adding 70% alcohol, 15 min; Add 0.8% periodic acid spirituous solution oxidation, 10 min;
Behind
Figure 456482DEST_PATH_IMAGE003
distillation washing 2 min; Add Schiff ' s alcohol blend, 10 min;
After abandons liquid; Add 0.5% sodium metabisulphite solution, zero(ppm) water is washed 2 min again behind 2 min;
Figure 920141DEST_PATH_IMAGE007
adds Ehrlich ' s haematoxylin redyeing nucleus, 20 min;
Figure 84406DEST_PATH_IMAGE010
with 0.5% hydrochloride alcohol color separation, tap water oil blackeite 10 min;
95% alcohol and 100% alcohol 2 min that dewater respectively;
YLENE is transparent; The natural gum mounting, inverted microscope is observed down.
The invention has the advantages that chelating method and in-situ two-step perfusion method are integrated improvement, slough Ca in the liver organization in advance 2+With Mg 2+, make it as often as possible to discharge primary hepatocyte; And improved serum free culture system, expanded the science purposes of the primary hepatocyte that obtains; In addition, this has invented with the previous methods reduced in comparison running time, has improved the hepatocellular quantity of former generation livestock and poultry, motility rate and purity effectively, and damage is few, and easy adherent, cell characteristics is intact, good separation.
Agents useful for same of the present invention and through aseptically process, can not cause the pollution problem in the liver cell sepn process all within the safe dose scope, introduces ectogenic objectionable impurities to follow-up study.
Technical process of the present invention is simple relatively, and training equipment is easy to get, and is workable, is convenient to realize scientific experiment.
Embodiment 1
1, the hepatocellular isolation and purification of piglet
Figure 870725DEST_PATH_IMAGE001
chooses 1 of the healthy long white piglet of 7 ages in days; Freely drink water and overnight fasting before the art, abdominal injection vetanarcol (50mg/kg) are anaesthetized;
Figure 658421DEST_PATH_IMAGE002
adopts 75% gauze wiping piglet whole body after alcohol-pickled; To anaesthetize piglet dorsal position Baoding (operating table is placed on the big porcelain dish) on the animalcule operating table, change in the Bechtop;
Open the abdominal cavity under aseptic condition; Pull to the body right side to enteron aisle gently; Expose mesenteric vein; Appropriateness ligation postcava two ends; And import hepatic vein direction intubate (the tubing passivity is gone the syringe needle part) therein, then intubate and vein tighten are formed path;
Figure 403840DEST_PATH_IMAGE004
Carrying out perfusion separates: the first step is the preceding perfusion of digestion, and be divided into two portions: first part sucks the 50ml syringe with 1400IU/kg dosage heparin sodium, and sneaks into perfusate
Figure 636239DEST_PATH_IMAGE005
(contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O and 5mM EDTA) inject liver organization and carry out blood anticoagulant, second section is with the perfusate of 37 ℃ of preheatings then
Figure 96301DEST_PATH_IMAGE005
Press the speed perfusion of 50ml/min, treat that liver cuts off postcava chest section during bulging a little, continue about 15min (not having or few red corpuscle is advisable), then with the perfusate of 37 ℃ of preheatings of same speed perfusion with elute
Figure 109256DEST_PATH_IMAGE006
(contain 10mM HEPES, 137mM NaCl, 3mM KCl and 3mM Na 2HPO 4.12H 2O), continue 5min (become yellowish-white, and organize comparatively loose being advisable) with liver;
Figure 617860DEST_PATH_IMAGE007
After treating that effusive liquid becomes clearly, carry out second step digestion perfusion, promptly pour into the perfusate of 37 ℃ of preheatings with 20ml/min speed
Figure 970344DEST_PATH_IMAGE008
(contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O, 5mM EDTA, 5.4mM CaCl 2And 0.4g/L
Figure 99843DEST_PATH_IMAGE009
The Collagen Type VI enzyme), and suitably clamp the postcava chest section of having cut off with mosquito forceps, make perfusate through the slowly outflow of liver organization circulation back, liver is digested gradually behind the 15min;
Figure 475461DEST_PATH_IMAGE010
slightly touches the liver tunicle; Show softer after, begin to win liver, remove gall-bladder; Put into an aseptic plate; The basic culture solution that adds the 50ml precooling is rejected blood vessel, fat and reticular tissue, cuts off the incomplete part of liver periphery digestion;
Figure 161657DEST_PATH_IMAGE011
Tear the liver coating with tweezers; Shred hepatic tissue; The heavy caliber suction pipe is blown and beaten gently, crosses 100 orders (150 μ m), 200 orders (75 μ m) and 400 orders (30 μ m) cell sieve subsequently respectively, and the gained hepatocyte suspension cleans 2 times (4 ℃ of centrifugal 5min of 500rpm) with basic culture solution; It is 96% that the trypan blue exclusion method detects the cell motility rate, TCS (3.2 ± 1.8) * 10 10
Figure 57063DEST_PATH_IMAGE012
With adherent culture liquid [contain 10% NBCS, (Sigma-Aldrich company contains 10 μ g/ml Sigma I8405s, 5.5 μ g/ml HTrfs, 2.9 * 10 to 1 * SITE -8M Sodium Selenite, 2 μ g/ml thanomins), 10 -6M insulin human, 10 -6The basic culture solution of M DEXAMETHASONE BP98,10 μ g/ml HTrfs, 10 μ g/ml vitamins Cs, two anti-(100IU/ml penicillium mould and 100IU/ml Streptomycin sulphate, Gibco companies)] resuspended, be diluted to the required cell concn inoculation of experiment.
2, piglet hepatocellular former be commissioned to train foster
After the cell counting, using adherent culture liquid is 3 * 10 with the cell suspension dilution 5Individual/ml, in 6 orifice plates, inoculate 2ml; Inoculation 100 μ l place 37 ℃, 5%CO in 96 orifice plates 2With cultivate in the cell culture incubator under the condition of saturated humidity.Behind the 4h cell attachment, change liquid, remove dead cell and fragment of tissue as far as possible, continue to cultivate 20h with grown cultures liquid (containing the two anti-basic culture solutions of 5% NBCS and 100IU/ml) full dose.After this, (contain 5 * 10 with serum-free medium -7(Sigma-Aldrich company contains 10 μ g/ml Sigma I8405s, 5.5 μ g/ml HTrfs, 2.9 * 10 for M DEXAMETHASONE BP98,1 * SITE -8M Sodium Selenite, 2 μ g/ml thanomins), 10 μ g/ml vitamins Cs, 0.15%BSA and two anti-Williams ' the medium E basic culture solutions of 100IU/ml) full dose changes liquid; Later every 24h changes nutrient solution; Regularly carry out observing liver cell form and growing state under the inverted microscope, and Taking Pictures recording.
3, the glycogen of piglet primary hepatocyte is identified
Getting above-mentioned serum-free medium cultivates the liver cell of 48 h (inoculation back the 3rd d) and carries out staining for glycogen and identify.The concrete operations step is following:
Figure 170513DEST_PATH_IMAGE001
inhales and removes nutrient solution; Abandon liquid after adding Carnoy ' s liquid chamber temperature fixed cell 15 min; Add 95% alcohol, 5 min;
Figure 33426DEST_PATH_IMAGE002
abandons liquid; Abandon liquid after adding 70% alcohol, 15 min; Add 0.8% periodic acid spirituous solution oxidation, 10 min;
Behind
Figure 257734DEST_PATH_IMAGE003
distillation washing 2 min; Add Schiff ' s alcohol blend, 10 min;
After
Figure 771761DEST_PATH_IMAGE004
abandons liquid; Add 0.5% sodium metabisulphite solution, zero(ppm) water is washed 2 min again behind 2 min;
Figure 728216DEST_PATH_IMAGE007
adds Ehrlich ' s haematoxylin redyeing nucleus, 20 min;
Figure 140743DEST_PATH_IMAGE010
with 0.5% hydrochloride alcohol color separation, tap water oil blackeite 10 min;
Figure 857157DEST_PATH_IMAGE011
95% alcohol and 100% alcohol 2 min that dewater respectively;
Figure 38740DEST_PATH_IMAGE012
YLENE is transparent; The natural gum mounting, inverted microscope is observed down.
Embodiment 2
1, the isolation and purification of chicken primary hepatocyte
Figure 431675DEST_PATH_IMAGE001
chooses yellow chicken 1 plumage in the Healthy Youth south of the Five Ridges; Fasting 3h before the art; The anti-freezing of wing intravenous injection 1750IU/kg dosage heparin sodium, abdominal injection vetanarcol (50mg/kg) carry out anesthetized animal;
Figure 252869DEST_PATH_IMAGE002
will anaesthetize chicken dorsal position Baoding (operating table is placed on the big porcelain dish) on the animalcule operating table; 75% alcohol wipe chicken whole body; Change in the Bechtop; Open the abdominal cavity under the aseptic condition, pull to the body left side to enteron aisle gently, expose mesenteric vein; One directly imports intubate (5 number sword-shaped needles remove syringe needle) in the hepatoportal vena mesenterica posterior therein, and another flows to the vein of liver ligation;
Figure 818980DEST_PATH_IMAGE003
The first step is the preceding perfusion of digestion, is divided into two portions: first part is with the perfusate of 37 ℃ of preheatings of speed perfusion of 50ml/min (contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O and 5mM EDTA), treat that liver cuts off postcaval vein slightly during bulging, continue 9min, second section is with the perfusate of 37 ℃ of preheatings of same speed perfusion
Figure 684485DEST_PATH_IMAGE006
(contain 10mM HEPES, 137mM NaCl, 3mM KCl and 3mM Na 2HPO 4.12H 2O), continue 3min; After treating that effusive liquid becomes clearly, carry out second step digestion perfusion, promptly pour into the perfusate of 37 ℃ of preheatings with 20ml/min speed
Figure 494440DEST_PATH_IMAGE008
(contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O, 5mM EDTA, 5.4mM CaCl 2And 0.4g/L
Figure 864241DEST_PATH_IMAGE009
The Collagen Type VI enzyme), and suitably clamp the postcaval vein of having cut off with mosquito forceps, perfusate is slowly flowed out, cover livers with 37 ℃ of warm sterile gauzes, and rub liver gently and be beneficial to digestion, liver digests gradually behind the 15min, depressing depression and being difficult for recovering;
Figure 958099DEST_PATH_IMAGE004
wins liver; Put into an aseptic plate; The basic culture solution that contains 0.2%BSA that adds the 50ml precooling is rejected blood vessel, fat and reticular tissue, cuts off the incomplete part of liver periphery digestion; Tear the liver coating with tweezers; Shred hepatic tissue, the heavy caliber suction pipe is blown and beaten gently, crosses 100 orders (150 μ m), 200 orders (75 μ m) and 500 orders (30 μ m) cell sieve subsequently respectively;
The gained hepatocyte suspension is cleaned 2 times (4 ℃ of centrifugal 2min of 500rpm) with basic culture solution, (contain 10% NBCS, 10 with adherent culture liquid -6M insulin human, 10 -6M DEXAMETHASONE BP98,10 μ g/ml HTrfs, 10 μ g/ml Vit C, two anti-(100U/ml penicillium mould and 100 μ g/ml Streptomycin sulphates; Gibco company) basic culture solution) resuspended; It is 94% that the trypan blue exclusion method detects the cell motility rate, TCS (4.3 ± 0.7) * 10 8
2, the cultivation of chicken primary hepatocyte
After the cell counting, using adherent culture liquid is 5 * 10 with the cell suspension dilution 5Individual/ml, (inoculum density is 1 * 10 in diameter is the plate of 60mm, to inoculate 4ml 5Individual/cm 2), at 37 ℃, 5%CO 2With cultivate under the condition of saturated humidity.Behind the 4h cell attachment, change liquid, continue to cultivate 20h with grown cultures liquid (containing 7% NBCS and two anti-basic culture solution) full dose.After this, (contain 5 * 10 with serum-free medium -7(Sigma-Aldrich company contains 10 μ g/ml Sigma I8405s, 5.5 μ g/ml HTrfs, 2.9 * 10 for M DEXAMETHASONE BP98,1 * SITE -8M Sodium Selenite, 2 μ g/ml thanomins), 10 μ g/ml vitamins Cs, 0.15%BSA and two anti-basic culture solution) full dose changes liquid, later every 24h changes nutrient solution, inverted microscope is observed liver cell form and growing state down.
3, the chicken primary hepatocyte is cultivated back staining for glycogen evaluation
Figure 550941DEST_PATH_IMAGE001
gets the liver cell sample that serum-free medium is cultivated 72 h (inoculation back 4 d); The nutrient solution in the plate is removed in suction, adds fixed cell 15min under Carnoy ' the s liquid chamber temperature;
Figure 396537DEST_PATH_IMAGE002
abandons liquid; Add 95% alcohol 5min, 70% alcohol 15min backwater;
Figure 407218DEST_PATH_IMAGE003
abandons liquid; Add 0.8% periodic acid spirituous solution oxidation 10min, zero(ppm) water speed is washed;
Figure 51871DEST_PATH_IMAGE004
adds Schiff ' s alcohol blend (measure 2.9ml Schiff ' s reagent, 0.125ml 1N HCl and 7ml 100% alcohol and mix, join with preceding at present) effect 10min and carries out staining for glycogen;
Figure 148003DEST_PATH_IMAGE007
abandons liquid; Add the 0.5% sodium metabisulphite solution 2min that discolors, wash with tap water and zero(ppm) water successively speed;
Figure 797290DEST_PATH_IMAGE010
adds Ehrlich ' s haematoxylin redyeing nucleus 20min; With 0.5% hydrochloride alcohol color separation, tap water oil blackeite 10min;
Figure 849429DEST_PATH_IMAGE011
95% alcohol and the 100% alcohol 1min that dewaters respectively; YLENE is transparent; The natural gum mounting is observed under the inverted microscope and is taken pictures.

Claims (2)

1. the method that the livestock and poultry primary hepatocyte separates, cultivates and identify is characterized in that chelating method and in-situ two-step perfusion method are integrated, and sloughs Ca in the livestock and poultry liver organization in advance 2+With Mg 2+, making it as often as possible to discharge primary hepatocyte, the additive prescription through the adjustment nutrient solution improves its serum free culture system, and carries out glycogen with regard to obtaining primary hepatocyte and identify.
2. livestock and poultry primary hepatocyte separation according to claim 1, the method for cultivating and identifying is characterized in that operation steps is:
One, the separation of livestock and poultry primary hepatocyte
Fasting before the operation of
Figure 2012101459595100001DEST_PATH_IMAGE002
livestock and poultry; Freely drink water and spend the night; The anti-freezing of neck injection 1400IU/kg dosage heparin sodium, abdominal injection vetanarcol (50mg/kg) anesthetized animal;
Figure 2012101459595100001DEST_PATH_IMAGE004
with postanesthetic livestock and poultry dorsal position Baoding on the animalcule operating table; This operating table is placed on the big porcelain dish; With 75% alcohol wipe whole body, change in the Bechtop;
Open the abdominal cavity under
Figure 2012101459595100001DEST_PATH_IMAGE006
aseptic condition; Pull to the body right side to enteron aisle gently; Expose mesenteric vein; Ligation postcava two ends; And import hepatic vein direction intubate therein, unclamp an end that flows to liver then;
Figure 2012101459595100001DEST_PATH_IMAGE008
Carry out perfusion and separate, at first digest preceding perfusion, be divided into two portions again: first part is with the perfusate of 37 ℃ of preheatings of speed perfusion of 50ml/min , this perfusate
Figure 895292DEST_PATH_IMAGE010
In contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O and 5mM EDTA treat that liver cuts off precava slightly during bulging, continue 20min, then with the perfusate of 37 ℃ of preheatings of same speed perfusion
Figure 2012101459595100001DEST_PATH_IMAGE012
, this perfusate
Figure 936191DEST_PATH_IMAGE012
In contain 10mM HEPES, 137mM NaCl, 3mM KCl and 3mM Na 2HPO 4.12H 2O continues 5min;
Figure 2012101459595100001DEST_PATH_IMAGE014
After treating that effusive liquid becomes clearly, carry out second step digestion perfusion, promptly pour into the perfusate of 37 ℃ of preheatings with 20ml/min speed
Figure 2012101459595100001DEST_PATH_IMAGE016
, this perfusate
Figure 737794DEST_PATH_IMAGE016
In contain 10mM HEPES, 137mM NaCl, 3mM KCl, 3mM Na 2HPO 4.12H 2O, 5mM EDTA, 5.4mM CaCl 2And 0.4g/L
Figure 2012101459595100001DEST_PATH_IMAGE018
The Collagen Type VI enzyme, and suitably clamp the postcaval vein of having cut off with mosquito forceps, perfusate is slowly flowed out, liver is digested gradually behind the 15min;
Figure 2012101459595100001DEST_PATH_IMAGE020
touch liver surface appear organize softer after; Begin it is won and put into an aseptic plate; William Si (family name) the medium E of adding 50ml precooling (Williams ' medium E) basic culture solution; Reject blood vessel, fat and reticular tissue, cut off the incomplete part of liver periphery digestion;
Figure 2012101459595100001DEST_PATH_IMAGE022
tears the liver coating with tweezers; Shred hepatic tissue; The heavy caliber suction pipe is blown and beaten gently; Cross 100 orders (150 μ m), 200 orders (75 μ m) and 400 orders (30 μ m) cell sieve subsequently respectively; The gained hepatocyte suspension cleans 2 times with William Si (family name) medium E (Williams ' medium E) basic culture solution, again at 4 ℃ of centrifugal 5min of 500rpm;
Figure 2012101459595100001DEST_PATH_IMAGE024
Use adherent culture liquid, contain volume percent in this adherent culture liquid and be 10% NBCS, 10 -6M insulin human, 10 -6The basic culture solution of M DEXAMETHASONE BP98,10 μ g/ml HTrfs, 10 μ g/ml Vit C, two anti-(100U/ml penicillium mould and 100 μ g/ml Streptomycin sulphates, Gibco companies)) resuspended, trypan blue exclusion method detects cell motility rate, blood cell counting plate counting;
Two, the cultivation of livestock and poultry liver primary cell
After the cell counting, using adherent culture liquid is 2～5 * 10 with the cell suspension dilution 5Cell/ml inoculates 15ml in diameter is the culturing bottle of 60ml, inoculum density is 1 * 10 5Cell/cm 2, at 37 ℃, 5% CO 2With cultivate under the condition of saturated humidity; Behind the 4h cell attachment; Change liquid with grown cultures liquid full dose, contain volume percent in this grown cultures liquid and be 10% NBCS and two anti-basic culture solutions and continue to cultivate 20h, after this; With serum-free medium, 10 μ g/ml vitamins Cs, 0.15% BSA and two anti-Williams ' medium E basic culture solution) full dose changes liquid, contains 5 * 10 in this serum-free medium -7M DEXAMETHASONE BP98,1 * SITE (this be (insulin, transferrin, selenium, abbreviation ethanolamine) also can be write as ITES, Sigma-Aldrich company contains 10 μ g/ml Sigma I8405s, 5.5 μ g/ml HTrfs, 2.9 * 10 -8M Sodium Selenite, 2 μ g/ml thanomins), later every 24h changes nutrient solution, and inverted microscope is observed liver cell form and growing state down;
Three, the glycogen of livestock and poultry primary hepatocyte is identified
Getting above-mentioned serum-free medium cultivates the liver cell of 48 h (inoculation back the 3rd d) and carries out staining for glycogen and identify that the concrete operations step is following:
Figure 739510DEST_PATH_IMAGE002
inhales and removes nutrient solution; Abandon liquid after adding Carnoy ' s liquid chamber temperature fixed cell 15 min; Add 95% alcohol, 5 min;
Figure 425707DEST_PATH_IMAGE004
abandons liquid; Abandon liquid after adding 70% alcohol, 15 min; The adding mass percent is 0.8% periodic acid spirituous solution oxidation, 10 min;
Behind
Figure 819648DEST_PATH_IMAGE006
distillation washing 2 min; Add Schiff ' s alcohol blend, 10 min;
After
Figure 933097DEST_PATH_IMAGE008
abandons liquid; Add 0.5% sodium metabisulphite solution, zero(ppm) water is washed 2 min again behind 2 min;
adds Ehrlich ' s haematoxylin redyeing nucleus, 20 min;
Figure 20319DEST_PATH_IMAGE020
with 0.5% hydrochloride alcohol color separation, tap water oil blackeite 10 min;
Figure 35811DEST_PATH_IMAGE022
95% alcohol and 100% alcohol 2 min that dewater respectively;
Figure 257845DEST_PATH_IMAGE024
YLENE is transparent; The natural gum mounting, inverted microscope is observed down.
CN 201210145959 2012-05-13 2012-05-13 Method for separating, culturing and identifying primary hepatic cells of livestock and fowl Pending CN102643778A (en)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104630130A (en) * 2014-12-05 2015-05-20 新乡医学院 Method for separating rat hepatocytes
CN105505883A (en) * 2016-01-12 2016-04-20 河南师范大学 Optimized culture method for primary rat hepatocytes
CN107043738A (en) * 2017-02-07 2017-08-15 韶关学院 A kind of porcine hepatocyte serum free medium and preparation method thereof
CN108728398A (en) * 2017-04-19 2018-11-02 江苏齐氏生物科技有限公司 A kind of isolation and culture method of primary duck hepatocyte
CN109022348A (en) * 2017-06-09 2018-12-18 江苏齐氏生物科技有限公司 A kind of isolation and culture method of primary porcine hepatocyte
CN109055302A (en) * 2018-08-28 2018-12-21 重庆医科大学附属第医院 The method of separation, culture and the identification of HBV transgenic mice primary hepatocyte
CN109182247A (en) * 2018-09-04 2019-01-11 江苏省家禽科学研究所 A kind of Embryo liver cell Isolation and culture and steatosis method for establishing model
CN110724661A (en) * 2019-07-17 2020-01-24 湖南农业大学 Separation method of mouse primary hepatocytes, mouse primary hepatocytes prepared by separation method and application of mouse primary hepatocytes
CN111979179A (en) * 2020-08-24 2020-11-24 扬州大学 Pig liver tissue organoid model and in-vitro construction method thereof

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104630130A (en) * 2014-12-05 2015-05-20 新乡医学院 Method for separating rat hepatocytes
CN105505883A (en) * 2016-01-12 2016-04-20 河南师范大学 Optimized culture method for primary rat hepatocytes
CN107043738A (en) * 2017-02-07 2017-08-15 韶关学院 A kind of porcine hepatocyte serum free medium and preparation method thereof
CN108728398A (en) * 2017-04-19 2018-11-02 江苏齐氏生物科技有限公司 A kind of isolation and culture method of primary duck hepatocyte
CN109022348A (en) * 2017-06-09 2018-12-18 江苏齐氏生物科技有限公司 A kind of isolation and culture method of primary porcine hepatocyte
CN109055302A (en) * 2018-08-28 2018-12-21 重庆医科大学附属第医院 The method of separation, culture and the identification of HBV transgenic mice primary hepatocyte
CN109182247A (en) * 2018-09-04 2019-01-11 江苏省家禽科学研究所 A kind of Embryo liver cell Isolation and culture and steatosis method for establishing model
CN109182247B (en) * 2018-09-04 2021-06-04 江苏省家禽科学研究所 In-vitro isolation culture and steatosis model establishment method for chick embryo hepatocytes
CN110724661A (en) * 2019-07-17 2020-01-24 湖南农业大学 Separation method of mouse primary hepatocytes, mouse primary hepatocytes prepared by separation method and application of mouse primary hepatocytes
CN111979179A (en) * 2020-08-24 2020-11-24 扬州大学 Pig liver tissue organoid model and in-vitro construction method thereof

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