CN109161515A - The isolated culture method of megalobrama amblycephala primary hepatocyte - Google Patents

The isolated culture method of megalobrama amblycephala primary hepatocyte Download PDF

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CN109161515A
CN109161515A CN201810886568.6A CN201810886568A CN109161515A CN 109161515 A CN109161515 A CN 109161515A CN 201810886568 A CN201810886568 A CN 201810886568A CN 109161515 A CN109161515 A CN 109161515A
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megalobrama amblycephala
liver
primary hepatocyte
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CN109161515B (en
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刘文斌
曹秀飞
蒋广震
戴永军
袁向阳
王聪聪
黄洋洋
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Nanjing Agricultural University
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Abstract

The invention discloses the isolated culture methods of megalobrama amblycephala primary hepatocyte.It chooses health, the megalobrama amblycephala juvenile fish that weight is 30-50g, carries out whole body disinfection with 1% potassium permanganate, after tail vein blood, liver is taken in sterile dissection.Cell suspension is obtained by filtration through 200 mesh cell sieves with after collagenase digesting, removes cell fragment through red blood cell extra in erythrocyte cracked liquid removal liver cell, then through gradient centrifugation.Appropriate complete medium suspension cell is added to obtained cell precipitation, then calculates and adjust cell concentration with cell counter, after bed board, is put into 28 DEG C, 5%CO2Cell incubator culture, adherent situation is observed after 48 hours.Novelty of the present invention combines the characteristics of research species to be separately cultured liver cell, and obtained Cell viability is up to 90% or more, meets originally culture requirement, provides theoretical foundation and technical support further to carry out the thin related experiment of the primary liver of megalobrama amblycephala.

Description

The isolated culture method of megalobrama amblycephala primary hepatocyte
Technical field
The invention belongs to cell biology and field of biotechnology, are related to a kind of fish local organization cell side of being separately cultured Method, in particular to a kind of megalobrama amblycephala primary hepatocyte isolated culture method.
Background technique
With the development of culture fishery, cultivation density is continuously improved, and feed formula is unreasonable and environmental pollution etc. is asked Topic causes serious damage to liver, and liver is most important Physiological and Biochemical Metabolism organ in fish body, so culture fish liver Cell has realistic meaning.In addition, there are many advantages that experiment in vivo can not replace in vitro culture primary hepatocyte.Primary liver is thin Born of the same parents can largely obtain in a short time, shorten experimental period, with strong points, and hepatocyte excludes its hetero-organization in vivo and mutually makees While influence, retain the original physiological function of liver.Therefore, liver cell is widely used in fish threpsology, pharmacology And toxicologic study.
Megalobrama amblycephala belongs to natural sciences class fish, is that a kind of medium-sized lake for being distributed mainly on lower Yangtze is miscellaneous Feeding habits fresh-water fishes, growth are pierced rapidly, between delicious meat, flesh less, and at low cost, its yield comes national fresh-water fishes the 6th, deeply Liked by China raiser and consumer.However, due to its body manage design feature, i.e., systemic ratio shared by head and visceral mass compared with It is small, cause megalobrama amblycephala intolerant to stress, excessive Fat Accumulation can also cause fatty liver as mammal.Therefore by being separately cultured Liver cell more preferably to carry out correlative study to megalobrama amblycephala.
Currently, the hepatocyte cultures of some fish have been studied, but the cell impurities obtained are more, and cell viability compared with Low, these methods are not suitable for the culture of megalobrama amblycephala primary hepatocyte.This experiment combines raw possessed by this species of megalobrama amblycephala Object feature, it is insufficient by improveing previous fish primitive cell culture method, establish megalobrama amblycephala primary hepatocyte isolated culture method.
Summary of the invention
The object of the present invention is to provide a kind of separation of megalobrama amblycephala primary hepatocyte and cultural methods, obtain purity is high, vigor High liver cell, in order to be further research nutrition relevant to fish, immunology, toxicology, pharmacology and cell Biology etc. provides theoretical foundation and technical support.This method improves existing fish primary cultured hepatocyt method, The cell purity of acquisition is high, and high survival rate is up to 90%.
In order to achieve the above-mentioned object of the invention, the technical solution of implementation is as follows:
A kind of isolated culture method of megalobrama amblycephala primary hepatocyte comprising the steps of:
(1) culture plate pre-processes: before bed board, with 1-2 μ g/cm2The fiber laminins of concentration are coated with culture plate；
(2) liver separation and digestion: winning megalobrama amblycephala liver, and DPBS cleans liver, removes impurity part, is cut into 1-3mm3 Size is collected in liver to sterile centrifugation tube, and 28 DEG C of digestion 30- of 5~6 times of volume type Ⅳ collagenases or II Collagenase Type are added 50min is added isometric complete medium and terminates digestion, digestive juice is crossed 200 mesh cell sieves, 1000rmp centrifugation 10min removes Supernatant obtains cell precipitation；
(3) cell suspension preparation and purifying: being added erythrocyte cracked liquid to the cell precipitation obtained in the step b, Suspension is blown and beaten into repeatedly with liquid-transfering gun, stands 5 minutes, and 800rmp centrifugation 10min removes supernatant and obtains cell precipitation, adds pre- The complete medium that temperature is crossed blows and beats into suspension repeatedly with liquid-transfering gun to dilute remaining erythrocyte cracked liquid, 500rmp centrifugation 10min removes supernatant and obtains cell precipitation, by obtaining purifying cells after erythrocyte cracked liquid and gradient centrifugation；
(4) before bed board, with the DMEM/F12 culture medium suspension cell for being free of serum, 20 μ megalobrama amblycephala hepatocyte cultures: are taken Cell viability is counted and counted through cell counter after the isometric Trypan Blue of l suspension, with 106-107Cell/ml concentration Repopulating cell makes megalobrama amblycephala liver cell in 28 DEG C, 5%CO2Under the conditions of cultivate, be put into after incubator 30min minutes again to culture 15% fetal calf serum and 5% megalobrama amblycephala serum are added in plate, carries out cell culture with the complete medium of formation, liver is increased with this Adherence rate finally obtains in vitro megalobrama amblycephala primary hepatocyte.
As a kind of the preferred of megalobrama amblycephala primary hepatocyte isolated culture method, step (1) is by the fibre of 1mg/ml Laminins are tieed up to use without Ca2+,Mg2+The phosphate buffer of ion dilutes 100 times, is then added in culture plate, 37 DEG C incubate It is stand-by after washing culture plate 2 times with sterilizing distilled water after educating 1h or 4 DEG C of refrigerator overnight.
As a kind of the preferred of megalobrama amblycephala primary hepatocyte isolated culture method, megalobrama amblycephala is won in step (2) Megalobrama amblycephala is pre-processed before liver: choosing the healthy megalobrama amblycephala juvenile fish that weight is 30-50g, is disappeared with the immersion of 1% potassium permanganate Malicious 10min, it is then micro- white from tail vein blood to the fish gill with asepsis injector, then fish body is wiped with cotton ball soaked in alcohol.
As a kind of the preferred of megalobrama amblycephala primary hepatocyte isolated culture method, with sterilized in step (2) Eye scissors are cut across from megalobrama amblycephala anus, and fin, side line and gill cover edge cut off body wall on the left of fish under side line, are opened abdominal cavity and are used Sterilizing ophthalmic tweezers win liver.
As a kind of the preferred of megalobrama amblycephala primary hepatocyte isolated culture method, the middle liver obtained of step (2) It is cleaned 4-5 times with DPBS, liver shreds into 1-3mm3Fritter, type Ⅳ collagenase or II Collagenase Type concentration are 0.1% (quality Volume ratio), type Ⅳ collagenase is added or II Collagen Type VI enzyme amount is 5 times of liver volume, cell sieve used is 200 mesh, centrifugal rotational speed For 1000rmp, time 10min.
As a kind of the preferred of megalobrama amblycephala primary hepatocyte isolated culture method, red blood cell is added in step (3) Centrifugal speed is 800rmp, time 10min after lysate, and centrifugal speed is 500rmp after complete medium is added, and the time is 10min。
As a kind of the preferred of megalobrama amblycephala primary hepatocyte isolated culture method, basis training used in step (4) Support base be DMEM/F12, final complete medium formula are as follows: DMEM/F12,15% fetal calf serum, 5% megalobrama amblycephala serum, 100IU/ml penicillin, 100 μ g/ml streptomysins, hepatocyte growth factor (20ng/ml), bovine insulin (10 μ g/ml) and paddy Glutamine (2mM).Plating cells density is 106-107Cell/ml, cell culture temperature are 28 DEG C, CO2Content is 5%.
The utility model has the advantages that
In conclusion this method is simple and easy, materials are convenient, and cell survival rate is high.Megalobrama amblycephala is carried out after sterilizing in vitro, Liver is taken out in an aseptic environment, suspension liver cell is obtained using collagenase digestion, with erythrocyte cracked liquid removal more than red Cell, then other impurities are removed through gradient centrifugation, with the complete medium diluting cells for containing 20% serum, cell counter is shown Cell density is 106-107Cell/ml, Cell viability are 90% or more.
Detailed description of the invention
The megalobrama amblycephala primary hepatocyte that Fig. 1 has just been separated, cell are spherical (amplification factor 10 × 20).
Megalobrama amblycephala primary hepatocyte after Fig. 2 culture 12h, cell start adherent situation (amplification factor 10 × 20) occur.
Megalobrama amblycephala primary hepatocyte after Fig. 3 culture for 24 hours, cell tensile deformation are in island shape, secured adherent (times magnification Number 10 × 20).
The megalobrama amblycephala liver cell motility rate that Fig. 4 method 1,2,3,4 is separately cultured
The megalobrama amblycephala liver cell 48h adherent rate that Fig. 5 method 1,2,3,4 is separately cultured
Ldh Activity in the supernatant that the liver cell that Fig. 6 method 1,2,3,4 is separately cultured obtains
Albumin content in the supernatant that the liver cell that Fig. 7 method 1,2,3,4 is separately cultured obtains
Urea nitrogen content in the supernatant that the liver cell that Fig. 8 method 1,2,3,4 is separately cultured obtains
Specific implementation method
Below with reference to embodiment, the invention will be further described.It should be pointed out that for those of ordinary skill in the art For, without departing from the principle of the present invention, several variations and modifications can also be made, these also should be regarded as of the invention In protection scope.1 megalobrama amblycephala primary hepatocyte isolation and culture method of embodiment:
1 main material, source and formula:
1) fetal calf serum purchase is in sigma company of the U.S..
2) DMEM/F12, glutamine and dual anti-(penicillin and streptomysin) purchase are in gibco company.
3) DPBS purchase is in HyClone company.
4) purchase of erythrocyte cracked liquid familial combined hyperlipidemia clostridiopetidase A is in biosharp company.
5) trypan blue purchase is in company of Nanjing Keygen Biotech.
6) complete medium formula: DMEM/F12,15% fetal calf serum, 5% megalobrama amblycephala serum, dual anti-(100IU/ml is green Mycin and 100 μ g/ml streptomysins), hepatocyte growth factor (20ng/ml), bovine insulin (10 μ g/ml) and glutamine (2mM)。
7) hepatocyte growth factor purchase is in R&D Systems company.
8) purchase of fiber laminins is in Sciencell company.
9) bovine insulin purchase is in Solarbio company.
2 operating procedures:
A. culture plate pre-processes: before bed board, needing with 2 μ g/cm2The fiber laminins of concentration are coated with culture plate, can The fiber laminins of 1mg/ml are used and are free of Ca2+,Mg2+The phosphate buffer of ion dilutes 100 times, is then added to training It supports in plate, it is stand-by after washing culture plate 2 times with sterilizing distilled water after 1h or 4 DEG C of refrigerator overnight of 37 DEG C of incubations.
B. pretreatment before the separation of megalobrama amblycephala liver: the healthy megalobrama amblycephala juvenile fish that weight is 30-50g is chosen, with 1% permanganic acid Potassium soaking disinfection 10min, it is then micro- white from tail vein blood to the fish gill with asepsis injector, then fish body is wiped with cotton ball soaked in alcohol.
C. liver separation and digestion: cut across from megalobrama amblycephala anus with sterilized eye scissors, under side line fin, side line with And gill cover edge cuts off body wall on the left of fish, opens abdominal cavity with sterilizing ophthalmic tweezers and wins liver, be put into fill pre-cooling DPBS it is sterile Culture dish cleans liver 4-5 times with DPBS again in superclean bench, while removing impurity part, later with eye scissors by liver It is dirty to be cut into 1-3mm3Left and right size.It collects in liver to sterile centrifugation tube, 5 times of volume type Ⅳ collagenases is added, in 28 DEG C of water-baths 30min is constantly shaken in pot, isometric complete medium is added and terminates digestion.Digestive juice is crossed into 200 mesh cell sieves, 1000rmp Centrifugation 10min removes supernatant and obtains cell precipitation.
D. 5ml erythrocyte splitting cell suspension preparation and purifying: is added to the cell precipitation obtained in the step b Liquid blows and beats into suspension with liquid-transfering gun repeatedly, stands 5 minutes, and 800rmp centrifugation 10min removes supernatant and obtains cell precipitation.Again plus Enter the complete medium that pre-temperature is crossed, blow and beat into suspension repeatedly with liquid-transfering gun to dilute remaining erythrocyte cracked liquid, 500rmp from Heart 10min removes supernatant and obtains cell precipitation, by obtaining purifying cells after erythrocyte cracked liquid and gradient centrifugation.
E. it megalobrama amblycephala hepatocyte cultures: before bed board, with containing the DMEM/F12 culture medium suspension cell without serum, takes Cell viability is counted and counted through cell counter after the isometric Trypan Blue of 20 μ l suspension, with 106-107cell/ml Concentration repopulating cell makes megalobrama amblycephala liver cell in 28 DEG C, 5%CO2Under the conditions of cultivate, be put into after incubator 30min minutes again to 15% fetal calf serum and 5% megalobrama amblycephala serum are added in culture plate, carries out cell culture with the complete medium of formation, is increased with this Add liver cell adherent rate, finally obtains in vitro megalobrama amblycephala primary hepatocyte, observe adherent situation after 48h.
Test analysis
1. cell count and survival rate test: using the method for Trypan Blue, then being counted in cell counter It is measured with motility rate, cell density 106-107Cell/ml, motility rate is up to 90% or more.
2. morphological observation: observation is separately cultured the cellular morphology of acquisition, the liver that just separation obtains in inverted microscope Cell is spherical (0h, following Fig. 1), and cell state is good；Cell starts to deform after culture for 24 hours, adherent situation occurs (for 24 hours, Following Fig. 2)；Cell tensile deforms after cultivating 48h, in threadiness, secured adherent (48h, following Fig. 3).
3. distinct methods culture megalobrama amblycephala primary hepatocyte compares: respectively using culture carp[1], crucian[2], jian carp[3]It is former It carries out megalobrama amblycephala primary hepatocyte with the method for liver cell method to be separately cultured, from adherent rate after Cell viability, 48h and carefully It is compared in terms of born of the same parents' metabolism state three.Method 1,2,3,4 respectively represents the side that culture carp, crucian, jian carp liver cell use Method and the method for the present invention.As shown in Figure 4, the megalobrama amblycephala liver cell motility rate that the method for the present invention is separately cultured is significantly higher than it He is three groups, and the method for the present invention obtains Cell viability and is up to 91.93%.As shown in Figure 5, after hepatocyte cultures 48h, method 4 is obtained Highest adherent rate is obtained, other three kinds of methods have lower adherent rate, are unfavorable for subsequent experimental development.It will be appreciated from fig. 6 that method 4 Ldh Activity is substantially less than other three kinds of methods in the supernatant that the liver cell being separately cultured obtains.Lactic dehydrogenase is deposited It is in liver cell, when liver cell damaged cell membrane permeability increases, lactic dehydrogenase escapes to extracellular loop from intracellular In border, therefore the liver cell extent of damage can be appreciated that by Ldh Activity in detection medium supernatant.As it can be seen that other three Kind separation method is larger to hepatocellular injury.By Fig. 7,8 it is found that after cell culture 48h, albumin and urine in 4 supernatant of method Plain nitrogen content is significantly higher than other three groups.More urea nitrogen is generated if cell growth metabolism is vigorous, therefore, urea nitrogen can be with As an index for determining cell survival.Liver is the unique place for synthesizing albumin, by white in detection culture supernatant Protein content may determine that whether liver cell synthesis secreting function is normal, can be used as the early stage index of hepatocellular injury.From obtaining Result it is found that application method 4 obtain megalobrama amblycephala hepatic cell growth it is vigorous and its synthesis and secreting function it is normal.
Compared with existing fish primary cultured hepatocyt method, the method for the present invention is more suitable for the separation training of megalobrama amblycephala liver cell It supports.Present invention incorporates the characteristics of studying this species and on the basis of existing method it is innovative improve be separately cultured it is some in step Deficiency, so as to obtain, motility rate is good, adherent rate is high, cellular damage degree is small and the normal liver cell of metabolic function.
Bibliography
[1] Li Yuehong, Ji Shanglei, Wu Dongming wait originally culture research [J] the Agriculture of Anhui science of carp liver cell, 2012, 40(22):11278-11279.
[2] Jia Rui, Cao Liping, Ding Weidong wait optimization [J] the North China agriculture of crucian liver cell separation and primary culture method Journal, 2011,26 (s2): 206-212.
[3] Liu Yingjuan, Du Jinliang, Jia Rui, the guarantor for waiting polysaccharides to damage tetrachloro-methane induction jian carp primary hepatocyte Protect Effect study [J] Shanghai Ocean University journal, 2014,23 (5): 718-725.

Claims (7)

1. a kind of isolated culture method of megalobrama amblycephala primary hepatocyte, it is characterised in that comprise the steps of:
(1) culture plate pre-processes: before bed board, with 1-2 μ g/cm2The fiber laminins of concentration are coated with culture plate；
(2) liver separation and digestion: winning megalobrama amblycephala liver, and DPBS cleans liver, removes impurity part, is cut into 1-3mm3Size, It collects in liver to sterile centrifugation tube, 28 DEG C of digestion 30-50min of 5~6 times of volume type Ⅳ collagenases or II Collagenase Type is added, Isometric complete medium is added and terminates digestion, digestive juice is crossed into 200 mesh cell sieves, 1000rmp centrifugation 10min removes supernatant and obtains Cell precipitation；
(3) cell suspension preparation and purifying: erythrocyte cracked liquid is added to the cell precipitation obtained in the step (2), uses Liquid-transfering gun blows and beats into suspension repeatedly, stands 5 minutes, and 800rmp centrifugation 10min removes supernatant and obtains cell precipitation, adds pre-temperature The complete medium crossed blows and beats into suspension repeatedly with liquid-transfering gun to dilute remaining erythrocyte cracked liquid, and 500rmp is centrifuged 10min Remove supernatant and obtain cell precipitation, by obtaining purifying cells after erythrocyte cracked liquid and gradient centrifugation；
(4) megalobrama amblycephala hepatocyte cultures: before bed board, with the DMEM/F12 culture medium suspension cell for being free of serum, take 20 μ l outstanding Cell viability is counted and counted through cell counter after the isometric Trypan Blue of supernatant liquid, with 106-107Cell/ml concentration kind Cell is planted, makes megalobrama amblycephala liver cell in 28 DEG C, 5%CO2Under the conditions of cultivate, be put into after incubator 30min minutes again to culture plate 15% fetal calf serum of middle addition and 5% megalobrama amblycephala serum, carry out cell culture with the complete medium of formation, and it is thin to increase liver with this Born of the same parents' adherent rate finally obtains in vitro megalobrama amblycephala primary hepatocyte.
2. a kind of megalobrama amblycephala primary hepatocyte isolated culture method according to claim 1, which is characterized in that step (1) The fiber laminins of 1mg/ml are used and are free of Ca2+,Mg2+The phosphate buffer of ion dilutes 100 times, is then added to training It supports in plate, it is stand-by after washing culture plate 2 times with sterilizing distilled water after 1h or 4 DEG C of refrigerator overnight of 37 DEG C of incubations.
3. a kind of megalobrama amblycephala primary hepatocyte isolated culture method according to claim 1, it is characterised in that in step (2) Megalobrama amblycephala is pre-processed before winning megalobrama amblycephala liver: the healthy megalobrama amblycephala juvenile fish that weight is 30-50g is chosen, with 1% Gao Meng Sour potassium soaking disinfection 10min, it is then micro- white from tail vein blood to the fish gill with asepsis injector, then fish is wiped with cotton ball soaked in alcohol Body.
4. a kind of megalobrama amblycephala primary hepatocyte isolated culture method according to claim 3, it is characterised in that in step (2) It cutting across from megalobrama amblycephala anus with sterilized eye scissors, fin, side line and gill cover edge cut off body wall on the left of fish under side line, It opens abdominal cavity and wins liver with sterilizing ophthalmic tweezers.
5. a kind of megalobrama amblycephala primary hepatocyte isolated culture method according to claim 4, it is characterised in that in step (2) The liver of acquisition is cleaned 4-5 times with DPBS, and liver shreds into 1-3mm3Fritter, type Ⅳ collagenase or II Collagenase Type concentration are 0.1% (mass volume ratio), is added type Ⅳ collagenase or II Collagen Type VI enzyme amount is 5 times of liver volume, and cell sieve used is 200 Mesh, centrifugal rotational speed 1000rmp, time 10min.
6. a kind of megalobrama amblycephala primary hepatocyte isolated culture method according to claim 1, it is characterised in that in step (3) Centrifugal speed is 800rmp, time 10min after erythrocyte cracked liquid is added, and centrifugal speed is after complete medium is added 500rmp, time 10min.
7. a kind of megalobrama amblycephala primary hepatocyte isolated culture method according to claim 1, which is characterized in that step (4) In basal medium used be DMEM/F12, final complete medium formula are as follows: DMEM/F12,15% fetal calf serum, 5% Head triangular bream serum, 100IU/ml penicillin, 100 μ g/ml streptomysins, 20ng/ml hepatocyte growth factor, 10 μ g/ml bovine insulins With 2mM glutamine, plating cells density is 106-107Cell/ml, cell culture temperature are 28 DEG C, CO2Content is 5%.
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CN110684718A (en) * 2019-10-30 2020-01-14 南京师范大学 Method for efficiently separating and culturing primary hepatocytes of fugu obscurus
CN114586768A (en) * 2020-12-03 2022-06-07 江苏齐氏生物科技有限公司 Liver tissue preservation solution and preservation method and application thereof
CN115109745A (en) * 2022-07-19 2022-09-27 南京师范大学 Method for efficiently separating and primarily culturing pelteobagrus fulvidraco hepatocytes

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