CN104818242B - Separation and preparation method of primary hepatocytes - Google Patents
Separation and preparation method of primary hepatocytes Download PDFInfo
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Abstract
The invention discloses a primary hepatocyte separation preparation method, which comprises the following steps of a, liver in-vitro perfusion, wherein isolated animal liver is taken and perfused with perfusion liquid I at the perfusion speed of 300-1000m L/min for 10-12 minutes, then perfused with perfusion liquid II at the perfusion speed of 300-1000m L/min for 2-3 minutes, finally perfused with perfusion liquid III at the perfusion speed of 400-1200m L/min for 25-40 minutes, and b, hepatocytes are separated, the liver treated in the step a is washed, hepatocyte suspension is collected, filtered and centrifuged, and sediment is collected.
Description
Technical field
The invention belongs to medical biotechnology fields, are related to the method for separating and preparing of primary hepatocyte.
Background technology
The liver metabolic organ important as body, plays a significant role in a variety of pathological processes.Liver cell row
The main function of liver is made, has such as removed toxin, participates in biosynthesis and bioconversion, a variety of nutriments of metabolism, storage Portugal
Grape sugar, secretion have the active material etc. for promoting hepatic cell growth.Isolated primary hepatocyte is applied to medicine in body
The in vitro studies such as toxicology, immunology, cell biology are managed, the application with bioartificial liver in treatment hepatic failure field,
Need a large amount of, high activity primary hepatocyte as biomaterial.Therefore, how to detach and high yield, high-purity, height is prepared
The primary hepatocyte of survival rate by researcher extensive concern.
Since Seglen in 1976 is successfully separated rat hepatocytes using two step perfusion in situ methods for the first time, two steps fill in situ
Stream method is proved to effective to separation many animals liver cell, becomes classical way prepared by current primary hepatocyte separation.It is many
More researchers improve hepatocyte isolation methods, improve liver cell to a certain extent on the basis of the method
Total amount and motility rate, but the needs of practical application cannot be met.
Such as：Publication No. CN1632107A, entitled " the bioartificial liver preparation side of pig and human liver cell
The patent of method ", it was recently reported that the method for two kinds of isolating hepatocytes, one is four step perfusions, and one is two step perfusions, are filled using four steps
When stream, the liver cell motility rate and liver cell sum of acquisition are higher, respectively 90-99% and 6.25 × 107A/gram hepatic tissue, but
Purity is relatively low, only 90-95%, and perfusion step is too many, of high cost；Although and two step perfusion steps are few, liver cell work
Rate, liver cell sum and purity are undesirable, respectively only 80-90%, 1.25 × 107A/gram hepatic tissue and 80-90%.
Zhang ZG etal.,An updated method for the isolation and culture of
primary calf hepatocytes,Vet J,2012,191:323-326 discloses the side of three step perfusion isolating hepatocytes
Method, but sum, the motility rate of the liver cell obtained are relatively low, respectively 1.12 × 107A/gram hepatic tissue, 85.7%.
Therefore, it is badly in need of being further improved the preparation method of primary hepatocyte, to prepare high yield, high-purity, high survival
The primary hepatocyte of rate.
Invention content
The purpose of the present invention is to provide a kind of method for separating and preparing efficiently separating primary hepatocyte.
The present invention provides a kind of method for separating and preparing of primary hepatocyte, it includes the following steps：
A, the In vitro perfusion of liver：In vitro animal's liver is taken, I perfusion of perfusate, perfusion rate 300- are first used
1000mL/min, Hemoperfusion time are 10-12 minutes；II perfusion of perfusate, perfusion rate 300-1000mL/min, perfusion are used again
Time is 2-3 minutes；It is 25-40 points finally to use III perfusion of perfusate, perfusion rate 300-1200mL/min, Hemoperfusion time
Clock；
Wherein, every liter of the perfusate I contains following ingredient：8.1-8.5 grams of sodium chloride, 0.5 gram of potassium chloride, 2.2-2.6
Gram HEPES, 3.5-4.5 grams of NAC, 0.9-1.1 grams of EGTA, remaining is deionized water；
Every liter of the perfusate II contains following ingredient：8.1-8.5 grams of sodium chloride, 0.5 gram of potassium chloride, 2.2-2.6 grams
HEPES, remaining is deionized water；
Every liter of the perfusate III contains following ingredient：3.8-4 grams of sodium chloride, 0.5 gram of potassium chloride, 23-25 grams of HEPES,
0.07 gram of calcium chloride dihydrate, 3.5-4.5 grams of albumin, 162-260W ü nsch unit clostridiopetidase As, remaining is deionized water；
B, isolating hepatocytes：Washing step a treated livers, collect hepatocyte suspension, and precipitation is collected in filtering, centrifugation
.
HEPES：4- hydroxyethyl piperazineethanesulfonic acids.
NAC：N-acetylcysteine, n-acetyl-L-cysteine.
EGTA：Ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA).
W ü nsch units：It is the dedicated active unit of clostridiopetidase A.
Further, in step a, every liter of the perfusate I contains following ingredient：8.3 grams of sodium chloride, 0.5 gram of potassium chloride,
2.4 grams of HEPES, 3.5-4.5 grams of NAC, 0.9-1.1 grams of EGTA, remaining is deionized water；
Every liter of perfusate II contains following ingredient：8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of HEPES, remaining is to go
Ionized water；
Every liter of perfusate III contains following ingredient：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 clostridiopetidase As, remaining is deionized water.
Further, every liter of the perfusate I contains following ingredient：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 ingredient：8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of HEPES, remaining is to go
Ionized water；
Every liter of perfusate III contains following ingredient：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 clostridiopetidase As, remaining is deionized water.
Wherein, in the perfusate III, clostridiopetidase A is clostridiopetidase A SERVA NB8 or clostridiopetidase A Roche
LiberaseTM。
Wherein, the perfusion rate of the 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 a, perfusate I, perfusate II, perfusate III perfusion temperature be 37 ゜ C.
Wherein, in step a, the perfusion of perfusate III is cycle perfusion mode.
Wherein, in step a, the dissolving partial pressure of oxygen of perfusate I, perfusate II and/or perfusate III are 180-260mmHg.
Wherein, in step b, the filtering is filtered with four layers of absorbent gauze.
Wherein, in step b, the centrifugation is repeatedly to centrifuge, and is comprised the concrete steps that：
(1) it centrifuges, discards supernatant liquid.
(2) precipitation, centrifugation is resuspended；
(3) process (2) 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 (2), precipitation is resuspended and uses liver cell cleaning solution.
Wherein, in step (2), washing is washed using liver cell cleaning solution.
Wherein, following ingredient is contained in every liter of the liver cell cleaning solution：9.3-10.8 gram Williams'Medium E powder
End, 2.2 grams of sodium bicarbonates, 2.4-2.8 grams of HEPES, 0.1 gram of streptomysin, 100000 units of Penicillin, remaining is deionized water.
Further, following ingredient is contained in every liter of the liver cell cleaning solution：9.3-10.8 grams of Williams'Medium
E powder, 2.2 grams of sodium bicarbonates, 2.6 grams of HEPES, 0.1 gram of streptomysin, 100000 units of Penicillin, remaining is deionized water.
Further, following ingredient is contained in every liter of the liver cell cleaning solution：9.5 grams of Williams'Medium E
Powder, 2.2 grams of sodium bicarbonates, 2.6 grams of HEPES, 0.1 gram of streptomysin, 100000 units of Penicillin, remaining is deionized water.
The method of the present invention can be not only used for pig liver, and can be adapted for it is various with larger animal liver, as people,
The liver of pig, dog, ox has the popularity of application.
The method for separating and preparing of primary hepatocyte provided by the invention can efficiently separate primary hepatocyte, isolated
Liver cell quantity is more, motility rate and purity are high, be hepatocyte transplantation, biological artificial liver support system treat acute and chronic hepatitis with
And liver cell external application model provides potential liver cell resource, potential applicability in clinical practice is excellent.
Obviously, the above according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific implementation mode of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on the above of the present invention
The technology realized all belongs to the scope of the present invention.
Specific implementation mode
It is described further below with embodiment, but the present invention is not limited to these embodiments.
Laboratory apparatus and reagent used in the present invention is as follows：
Surgical operating instrument is a set of, and self-control perfusion equipment is a set of, refrigerated centrifuge (Thermo Scientific, the U.S.),
Water purification machine (Millipore, Germany)；Electronic analytical balance (Sartorius, Germany)；Peristaltic pump (MasterRex L/S, it is beautiful
State)；Olympus biomicroscope (BHS types, Japan)；Carbon dioxide cell incubator (SANYO MCO-17AI, Japan)；It is super
Net workbench (NUAIRE-600E, the U.S.)；Grain count instrument (Multisizer 3Beckman Coulter counter, moral
State).
Ketamine, xylazine, heparin sodium injection are purchased from the pharmacy of Chengdu Cologne；EGTA；Williams'Medium E trainings
Foster based powders (abbreviation Williams'Medium E powder), Pen-Strep100 × (Penicillin 10,000U/ml,
Streptomycin 10,000ug/ml), NAC, trypan blue cell activity staining reagent, albumin is purchased from U.S. Sigma-
Aldrich；Clostridiopetidase A NB8 is purchased from SERVA companies of Germany.
The method for separating and preparing of the primary hepatocyte of 1 present invention of embodiment
One, test reagent
Perfusate I, perfusate II, perfusate III, liver cell cleaning solution；
Respective preparation method is as follows：
1. perfusate I：For preparing 1 liter of reagent：
(1) following ingredient is taken：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, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.In use, being preheated to 37 DEG C.
2. perfusate II：For preparing 1 liter of reagent：
(1) following ingredient is taken：8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of HEPES；
(2) mentioned component is mixed, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.In use, being preheated to 37 DEG C.
3. perfusate III：For preparing 1 liter of reagent：
(1) following ingredient is taken：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 clostridiopetidase A SERVA NB8 (1mg enzymes inside be 1.08 W ü nsch units) (i.e.
216W ü nsch units)；
(2) mentioned component is mixed, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.6, with 0.2 micron membrane filter filtration sterilization, is configured using first 15 minutes, in use, being preheated to 37 DEG C.
4. liver cell cleaning solution：For preparing 1 liter of reagent：
(1) following ingredient is taken：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, 100000 unit of penicillin；
(2) mentioned component is mixed, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.3, and with 0.2 micron membrane filter filtration sterilization, 4 DEG C preserve and use.
Two, test method
The normal bar of complete liver of horse pig is obtained, is detached so that full liver weighs 300 grams as an example and prepares primary porcine hepatocyte.
A, the In vitro perfusion of liver
The complete liver that will be won, is placed in sterile chamber, is intubated using internal diameter 6mm hose trans-portal veins, filtering is gone out
Bacterium is preheated to 37 DEG C of 4000mL perfusates I, through peristaltic pump with 400mL/min speed perfusion 10 minutes, discards perfusate；Then
With filtration sterilization, 37 DEG C of 800mL perfusates II are preheated to, with 400mL/min speed perfusion 2 minutes, discard perfusate；Later will
Filtration sterilization, the 2000mL perfusates III for being preheated to 37 DEG C, with 500mL/min speed loops perfusion 30 minutes.The above perfusion mistake
Cheng Zhong, all perfusates all pass through oxygenation process, ensure that dissolving partial pressure of oxygen is 200mmHg in perfusate.
B, isolating hepatocytes
Perfusate is discarded, the liver cell cleaning solution that 600mL is cooled to 4 DEG C in advance in hepatic metastasis to new sterile basin, will be poured into,
Glisson's capsule is scratched, indigested hepatic tissue and connective tissue are removed, collects hepatocyte suspension.Above-mentioned hepatocyte suspension is de- through four layers
After fat filtered through gauze, in the sterile centrifuge bottles that average mark is 750mL loaded on two volumes, under the conditions of 4 DEG C, 50 × g of centrifugal force from
The heart 10 minutes.Centrifuged supernatant is discarded, the liver cell cleaning solution 500mL resuspensions for repeating 4 DEG C of the addition into each centrifugal bottle are heavy
The liver cell in shallow lake, under the conditions of 4 DEG C, 50 × g of centrifugal force is centrifuged 10 minutes.Centrifuged supernatant is discarded, is repeated to each centrifugal bottle
The middle liver cell cleaning solution 500mL for being added 4 DEG C is resuspended the liver cell of precipitation, and under the conditions of 4 DEG C, 50 × g of centrifugal force is centrifuged 10 minutes.
Liquid is discarded supernatant, precipitation, as isolated liver cell are collected.
Three, detection method
After precipitation liver cell is weighed, it is cooled to 4 DEG C of serum-free hepatocyte cultures liquid in advance with 500mL and is resuspended, calculate for the first time
Extension rate then takes out 10mL hepatocyte suspensions, is further diluted to 400 times with serum-free hepatocyte cultures liquid, to dilution
Trypan Blue is being carried out to 400 times of liver cells, with blood cell counting plate, is being counted under microscope, nucleus indigo plant dye is dead thin
Born of the same parents calculate Cell viability, and cell purity is calculated according to cellular morphology under microscope.Pass through grain count instrument simultaneously
(Multisizer 3Beckman Coulter counter) measures total number of cells and cell concentration.Remaining hepatocyte suspension is set
It is spare in ice bath, and cell concentration is finally adjusted according to the purposes of required cell and is further cultivated.
Four, test result
The results are shown in table below for liver cell prepared by the present invention：
| The primary porcine hepatocyte index of fresh separated | The method of the present invention |
| Liver cell motility rate (%) | 99-99.5 |
| Obtained liver cell is total (a/gram hepatic tissue) | 6.93-8.39×107 |
| Liver cell purity (%) | 95-98 |
The experiment results show that the isolated primary porcine hepatocyte of the method for the present invention in terms of motility rate, quantity and purity
It is very excellent.
The method for separating and preparing of the primary hepatocyte of 2 present invention of embodiment
One, test reagent
1. perfusate I：For preparing 1 liter of reagent：
(1) following ingredient is taken：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, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.In use, being preheated to 37 DEG C.
2. perfusate II：For preparing 1 liter of reagent：
(1) following ingredient is taken：8.1 grams of sodium chloride, 0.5 gram of potassium chloride, 2.6 grams of HEPES；
(2) mentioned component is mixed, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.In use, being preheated to 37 DEG C.
3. perfusate III：For preparing 1 liter of reagent：
(1) following ingredient is taken：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, clostridiopetidase A Roche LiberaseTM(being 5.2 W ü nsch units inside 1mg enzymes) 50 milli
Gram (i.e. 260W ü nsch units)；
(2) mentioned component is mixed, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.6, with 0.2 micron membrane filter filtration sterilization, is configured using first 15 minutes, in use, being preheated to 37 DEG C.
4. liver cell cleaning solution：For preparing 1 liter of reagent：
(1) following ingredient is taken：10.8 grams of Williams'Medium E powder, 2.2 grams of sodium bicarbonate, 2.8 grams of HEPES,
0.1 gram of streptomysin, 100000 unit of penicillin；
(2) mentioned component is mixed, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.3, is preserved and is used for 4 DEG C with 0.2 micron membrane filter filtration sterilization.
Two, test method
The normal bar of complete liver of horse pig is obtained, is detached so that full liver weighs 800 grams as an example and prepares primary porcine hepatocyte.
A, the In vitro perfusion of liver
The complete liver that will be won, is placed in sterile chamber, is intubated using internal diameter 10mm hose trans-portal veins, filtering is gone out
Bacterium, I 12000mL of perfusate for being preheated to 37 DEG C discard perfusate through peristaltic pump with 1000mL/min speed perfusion 12 minutes；
Then with filtration sterilization, be preheated to 37 DEG C of II 3000mL of perfusate, with 1000mL/min speed perfusion 3 minutes, discard perfusion
Liquid；Later by filtration sterilization, be preheated to 37 DEG C of III 4000mL of perfusate, with 1200mL/min speed loops perfusion 40 minutes.
In the above perfusing course, all perfusates all pass through oxygenation process, ensure that dissolving partial pressure of oxygen is 200mmHg in perfusate.
B, isolating hepatocytes
Above-mentioned hepatocyte suspension after four layers of absorbent gauze filter, average mark loaded on four volumes be 750mL it is sterile from
In heart bottle, under the conditions of 4 DEG C, 50 × g of centrifugal force is centrifuged 10 minutes.Centrifuged supernatant is discarded, then 4 DEG C are added into each centrifugal bottle
Liver cell cleaning solution 500mL be resuspended precipitation, under the conditions of 4 DEG C, 50 × g of centrifugal force is centrifuged 10 minutes.Centrifuged supernatant is discarded, then
4 DEG C of liver cell cleaning solution 500mL resuspensions are added into each centrifugal bottle to precipitate, under the conditions of 4 DEG C, 50 × g of centrifugal force centrifugations 10
Minute.Liquid is discarded supernatant, precipitation, as primary hepatocyte are collected.
Three, detection method
With embodiment 1.
Four, test result
The results are shown in table below for liver cell prepared by the present invention：
| The primary porcine hepatocyte index of fresh separated | The method of the present invention |
| Liver cell motility rate (%) | 96-99 |
| Obtained liver cell is total (a/gram hepatic tissue) | 6.55-7.28×107 |
| Liver cell purity (%) | 95-98 |
The experiment results show that the isolated primary porcine hepatocyte of the method for the present invention in terms of motility rate, quantity and purity
It is very excellent.
The method for separating and preparing of the primary hepatocyte of 3 present invention of embodiment
One, test reagent
1. perfusate I：For preparing 1 liter of reagent：
(1) following ingredient is taken：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, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.In use, being preheated to 37 DEG C.
2. perfusate II：For preparing 1 liter of reagent：
(1) following ingredient is taken：8.5 grams of sodium chloride, 0.5 gram of potassium chloride, 2.2 grams of HEPES；
(2) mentioned component is mixed, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.4, and with 0.2 micron membrane filter filtration sterilization, room temperature preserves.In use, being preheated to 37 DEG C.
3. perfusate III：For preparing 1 liter of reagent：
(1) following ingredient is taken：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 clostridiopetidase A SERVA NB8 (1mg enzymes inside be 1.08 W ü nsch units)
ü nsch units)；
(2) mentioned component is mixed, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.6, with 0.2 micron membrane filter filtration sterilization, is configured using first 15 minutes, in use, being preheated to 37 DEG C.
4. liver cell cleaning solution：For preparing 1 liter of reagent：
(1) following ingredient is taken：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, 100000 unit of penicillin；
(2) mentioned component is mixed, is dissolved in deionized water, stirred 10 minutes at room temperature, so that it is fully dissolved, be settled to
1L adjusts pH to 7.3, and with 0.2 micron membrane filter filtration sterilization, 4 DEG C preserve and use.
Two, test method
The normal bar of complete liver of horse pig is obtained, is detached so that full liver weighs 200 grams as an example and prepares primary porcine hepatocyte.
A, the In vitro perfusion of liver
The complete liver that will be won, is placed in sterile chamber, is intubated using internal diameter 4mm hose trans-portal veins, filtering is gone out
Bacterium, I 3300mL of perfusate for being preheated to 37 DEG C discard perfusate through peristaltic pump with 300mL/min speed perfusion 11 minutes；It connects
It with filtration sterilization, be preheated to 37 DEG C of II 750mL of perfusate, with 300mL/min speed perfusion 2.5 minutes, discard perfusate；
Later by filtration sterilization, be preheated to 37 DEG C of III 1500mL of perfusate, with 300mL/min speed loops perfusion 25 minutes.More than
In perfusing course, all perfusates all pass through oxygenation process, ensure that dissolving partial pressure of oxygen is 200mmHg in perfusate.
B, isolating hepatocytes
Above-mentioned hepatocyte suspension after four layers of absorbent gauze filter, average mark loaded on two volumes be 750mL it is sterile from
In heart bottle, under the conditions of 4 DEG C, 50 × g of centrifugal force is centrifuged 10 minutes.Centrifuged supernatant is discarded, then 4 DEG C are added into each centrifugal bottle
Liver cell cleaning solution 500mL be resuspended precipitation, under the conditions of 4 DEG C, 50 × g of centrifugal force is centrifuged 10 minutes.Centrifuged supernatant is discarded, then
4 DEG C of liver cell cleaning solution 500mL resuspensions are added into each centrifugal bottle to precipitate, under the conditions of 4 DEG C, 50 × g of centrifugal force centrifugations 10
Minute.Liquid is discarded supernatant, precipitation, as primary hepatocyte are collected.
Three, detection method
With embodiment 1.
Four, test result
The results are shown in table below for liver cell prepared by the present invention：
| The primary porcine hepatocyte index of fresh separated | The method of the present invention |
| Liver cell motility rate (%) | 99-99.5 |
| Obtained liver cell is total (a/gram hepatic tissue) | 7.33-8.55×107 |
| Liver cell purity (%) | 95-98 |
The experiment results show that the isolated primary porcine hepatocyte of the method for the present invention in terms of motility rate, quantity and purity
It is very excellent.
Beneficial effects of the present invention are illustrated below by way of test example.
The method for separating and preparing of the primary hepatocyte of 1 present invention of test example is compared with other methods
One, existing method prepares liver cell
Existing 1：Four step perfusion methods disclosed in CN1632107A prepare liver cell, and the obtained liver cell sum of this method 6.25 ×
107A/gram hepatic tissue, Cell viability 90-99%, cell purity 90-95%.
Existing 2：Two step perfusion methods disclosed in CN1632107A prepare liver cell, and the obtained liver cell sum of this method 1.25 ×
107A/gram hepatic tissue, Cell viability 80-90%, cell purity 80-90%.
Existing 3：Zhang ZG etal.,An updated method for the isolation and culture
of primary calf hepatocytes,Vet J,2012,191:Method disclosed in 323-326 prepares liver cell, this method
Obtained liver cell sum 1.12 × 107A/gram hepatic tissue, Cell viability 85.7%.
Two, the method for the present invention prepares liver cell
The method of the present invention prepares liver cell：Liver cell, the obtained liver cell sum 6.93- of this method are prepared according to embodiment 1
8.39×107A/gram hepatic tissue, Cell viability 99-99.5%, cell purity 95-98%.
Three, control methods prepares liver cell
The step of separation method is not in addition to containing II perfusion of perfusate shown in the embodiment of the present invention 1, remaining is the same as the present invention
Experimental example 1, as a result obtained liver cell sum 2.62-4.19 × 107A/gram hepatic tissue, Cell viability 90-95%, cell are pure
Degree is 86-92%.
The effect of five kinds of connection agents is summarized as follows table：
As can be seen from the above table：
1, compared with existing three steps perfusion method and two step perfusion methods, three step perfusion methods of the invention are thin for detaching primary liver
When born of the same parents, liver cell sum, Cell viability and the cell purity of acquisition are significantly increased.
2, compared with existing four steps perfusion method, when three step perfusion methods of the invention are used to detach primary hepatocyte, in acquisition
It is slightly excellent on liver cell sum, Cell viability, it is significantly improved in liver cell purity.
3 and control methods, that is, the two step perfusion methods for lacking II perfusion of perfusate compare, three step perfusion methods of the invention are used for
When detaching primary hepatocyte, liver cell sum, Cell viability and the cell purity of acquisition have significantly more excellent.
Comparison result illustrates that the method for the present invention can effectively be divided by the combination of specific perfusate and perfusion step
From primary hepatocyte, separating effect is superior to existing perfusion, and only three kinds of perfusate, cost is not high.
To sum up, the method for the present invention can efficiently separate primary hepatocyte, the quantity of isolated liver cell is more, motility rate with
Purity is high, treats acute and chronic hepatitis for hepatocyte transplantation, biological artificial liver support system and liver cell external application model carries
For potential liver cell resource, potential applicability in clinical practice is excellent.
Claims (18)
1. a kind of method for separating and preparing of primary hepatocyte, it is characterised in that：It includes the following steps：
A, the In vitro perfusion of liver：In vitro animal's liver is taken, it is 300-1000 mL/ first to use I perfusion of perfusate, perfusion rate
Min, Hemoperfusion time are 10-12 minutes；It is 300-1000 mL/min, Hemoperfusion time to use II perfusion of perfusate, perfusion rate again
It is 2-3 minutes；It is 300-1200 mL/min finally to use III perfusion of perfusate, perfusion rate, and Hemoperfusion time is 25-40 minutes；
Wherein, every liter of the perfusate I is by as follows at being grouped as：8.1-8.5 grams of sodium chloride, 0.5 gram of potassium chloride, 2.2-2.6 grams
HEPES, 3.5-4.5 grams of NAC, 0.9-1.1 grams of EGTA, remaining is deionized water；
Every liter of the perfusate II is by as follows at being grouped as：8.1-8.5 grams of sodium chloride, 0.5 gram of potassium chloride, 2.2-2.6 grams
HEPES, remaining is deionized water；
Every liter of the perfusate III is by as follows at being grouped as：3.8-4 grams of sodium chloride, 0.5 gram of potassium chloride, 23-25 grams of HEPES,
0.07 gram of calcium chloride dihydrate, 3.5-4.5 grams of albumin, 162-260 W ü nsch unit clostridiopetidase As, remaining is deionized water；
B, isolating hepatocytes：Washing step a treated livers, collect hepatocyte suspension, and precipitation is collected in filtering, centrifugation；
The wherein described primary hepatocyte is the primary hepatocyte of pig liver.
2. method for separating and preparing according to claim 1, it is characterised in that：In step a,
Every liter of the perfusate I is by as follows at being grouped as：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 grams of EGTA, remaining is deionized water；
Every liter of perfusate II is by as follows at being grouped as：8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of HEPES, remaining for go from
Sub- water；
Every liter of perfusate III is by as follows at being grouped as：3.9 grams of sodium chloride, 0.5 gram of potassium chloride, 24 grams of HEPES, 0.07 gram of two water
Calcium chloride, 4 grams of albumin, 162-260 W ü nsch unit clostridiopetidase As, remaining is deionized water.
3. method for separating and preparing according to claim 2, it is characterised in that：
Every liter of the perfusate I is by as follows at being grouped as：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 is by as follows at being grouped as：8.3 grams of sodium chloride, 0.5 gram of potassium chloride, 2.4 grams of HEPES, remaining for go from
Sub- water；
Every liter of perfusate III is by as follows at being grouped as：3.9 grams of sodium chloride, 0.5 gram of potassium chloride, 24 grams of HEPES, 0.07 gram of two water
Calcium chloride, 4 grams of albumin, 216 W ü nsch unit clostridiopetidase As, remaining is deionized water.
4. method for separating and preparing according to claim 3, it is characterised in that：In the perfusate III, clostridiopetidase A is collagen
Enzyme SERVA NB8 or clostridiopetidase A Roche LiberaseTM。
5. method for separating and preparing according to any one of claims 1-4, it is characterised in that：
The perfusion rate of the perfusate I is 400 mL/min, and Hemoperfusion time is 10 minutes；
The perfusion rate of perfusate II is 400 mL/min, and Hemoperfusion time is 2 minutes；
The perfusion rate of perfusate III is 500 mL/min, and Hemoperfusion time is 30 minutes.
6. method for separating and preparing according to any one of claims 1-4, it is characterised in that：In step a, perfusate I, fill
Flow liquid II, perfusate III perfusion temperature be 37 DEG C.
7. method for separating and preparing according to any one of claims 1-4, it is characterised in that：In step a, perfusate III
Perfusion mode be cycle perfusion.
8. method for separating and preparing according to any one of claims 1-4, it is characterised in that：In step a, perfusate I, fill
The dissolving partial pressure of oxygen of flow liquid II and/or perfusate III is 180-260mmHg.
9. method for separating and preparing according to any one of claims 1-4, it is characterised in that：In step b, the filtering
It is to be filtered with four layers of absorbent gauze.
10. method for separating and preparing according to any one of claims 1-4, it is characterised in that：In step b, the centrifugation
It is repeatedly to centrifuge, comprises the concrete steps that：
（1）Centrifugation, discards supernatant liquid；
（2）Precipitation, centrifugation is resuspended；
（3）By process（2）It is repeated once.
11. method for separating and preparing according to claim 1, it is characterised in that：The condition of centrifugation is：Centrifuging temperature is 4 DEG C,
Centrifugal force is 50-55 × g, and the time of centrifugation is 10 minutes.
12. method for separating and preparing according to claim 10, it is characterised in that：The condition of centrifugation is：Centrifuging temperature is 4
DEG C, centrifugal force is 50-55 × g, and the time of centrifugation is 10 minutes.
13. method for separating and preparing according to claim 10, it is characterised in that：Step（2）In, it is thin using liver that precipitation is resuspended
Born of the same parents' cleaning solution.
14. according to the method for separating and preparing described in claim 1-4,11 any one, it is characterised in that：Step（2）In, washing
It is washed using liver cell cleaning solution.
15. method for separating and preparing according to claim 13, it is characterised in that：Every liter of the liver cell cleaning solution is by as follows
At being grouped as：9.3-10.8 grams of Williams'Medium E powder, 2.2 grams of sodium bicarbonates, 2.4-2.8 grams of HEPES, 0.1 gram
Streptomysin, 100000 units of Penicillin, remaining is deionized water.
16. method for separating and preparing according to claim 14, it is characterised in that：Every liter of the liver cell cleaning solution is by as follows
At being grouped as：9.3-10.8 grams of Williams'Medium E powder, 2.2 grams of sodium bicarbonates, 2.4-2.8 grams of HEPES, 0.1 gram
Streptomysin, 100000 units of Penicillin, remaining is deionized water.
17. method for separating and preparing according to claim 15 or 16, it is characterised in that：Every liter of the liver cell cleaning solution by
As follows at being grouped as：9.3-10.8 grams of Williams'Medium E powder, 2.2 grams of sodium bicarbonates, 2.6 grams of HEPES, 0.1 gram
Streptomysin, 100000 units of Penicillin, remaining is deionized water.
18. method for separating and preparing according to claim 17, it is characterised in that：Every liter of the liver cell cleaning solution is by as follows
At being grouped as：9.5 grams of Williams'Medium E powder, 2.2 grams of sodium bicarbonates, 2.6 grams of HEPES, 0.1 gram of streptomysin,
100000 units of Penicillin, remaining is deionized water.
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| CN106978388A (en) * | 2017-04-10 | 2017-07-25 | 安徽农业大学 | A kind of separation of dog liver cell and cultural method |
| 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 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103525756A (en) * | 2013-10-23 | 2014-01-22 | 南京农业大学 | Method for separating and culturing primary chicken hepatocytes |
| CN104630130A (en) * | 2014-12-05 | 2015-05-20 | 新乡医学院 | Method for separating rat hepatocytes |
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| CN104630130A (en) * | 2014-12-05 | 2015-05-20 | 新乡医学院 | Method for separating rat hepatocytes |
Non-Patent Citations (1)
| Title |
|---|
| "肝细胞培养方法研究进展";陈慧梅 等;《细胞生物学杂志》;20021231;第24卷(第3期);第163-166页 * |
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