CN110229784A - A method of it removing the separating liquid of dead liver cell and removes dead liver cell using it - Google Patents
A method of it removing the separating liquid of dead liver cell and removes dead liver cell using it Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
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Abstract
The invention discloses a kind of separating liquid for removing dead liver cell and the methods for removing dead liver cell using it, belong to liver cell isolation and purification technical field.The separating liquid is to add following component in William ' sE culture medium: the universal Nycodenz for cultivating additive, the L-Glutamine of 1.5mmol/L-2.5mmol/L, the epidermal growth factor of 1 μ g/L-20 μ g/L, the hydrocortisone of 15mg/L-20mg/L, the dexamethasone of 35 μ g/L-45 μ g/L, the mycillin of 0.5%v/v-1%v/v, the fetal calf serum of 1%v/v-10%v/v and 0.25g/mL-0.5g/mL of the ITS of 0.1%v/v-1.5%v/v.The present invention has the advantages that maintain liver cell normal osmotic pressure, repairs to be partially damaged and liver cell and provides nutritional need for liver cell.
Description
Technical field
The present invention relates to a kind of separating liquid for removing dead liver cell and the methods for removing dead liver cell using it, and it is thin to belong to liver
Born of the same parents' isolation and purification technical field.
Background technique
Primary hepatocyte refers to the liver cell cultivated immediately after hepatic tissue taking-up, can be widely used for basic research and drug
Research and development, including liver research, hepatitis virus research, drug metabolism and toxicity research.However, when separation primary hepatocyte, cell
Vigor is influenced by a variety of conditions, including hepatic tissue freshness, infusion time, primer solution composition etc., and there are biggish batches
Difference.The cell viability of the primary hepatocyte of prior art separation is 45%-95%, this has been seriously affected based on primary hepatocyte
Experimental study and medicament research and development.
Currently, remove there are two types of the conventional methods of dead liver cell, it is specific as follows:
First method, low-speed centrifugation.The principle of this method is the matter based on alive liver cell and dead cell, cell fragment
Amount is different from density, and by low-speed centrifugal (about centrifugal acceleration 50g), alive liver cell is broken with precipitation form and the cell in supernatant
Piece and dead cell separate.The above method is step conventional in liver cell separation process, can remove the cell debris of a part
With a small amount of dead cell, but the method removal effect is limited and causes Activity of hepatocytes relatively low.
Second method utilizes centrifugation medium --- Percoll or Ficoll400, by cell suspension be added to Percoll or
On Ficoll400, by centrifugal force, living cells is deposited to gradient bottom, dead cell stays on medium.The above method
Principle is: only living cells has the ability across medium, however dead cell can form dead cell layer between sample and medium, resistance
Only living cells passes through.Sample big for cell concentration, Cell viability is low, the efficiency that this method removes dead liver cell are relatively low.
In consideration of it, removing the side of dead liver cell it is necessary to provide a kind of separating liquid of new dead liver cell of removal and using it
Method, so as to solve the deficiencies in the prior art.
Summary of the invention
An object of the present invention provides a kind of separating liquid for removing dead liver cell.The dead liver cell of removal of the invention
Separating liquid is that the universal culture additive of ITS, L-Glutamine, epidermal growth factor are added in William ' s E culture medium
Son, hydrocortisone, dexamethasone, mycillin, fetal calf serum and Nycodenz, have maintain liver cell normal osmotic pressure,
Reparation is partially damaged liver cell, the advantages of providing nutritional need for liver cell.
The technical scheme to solve the above technical problems is that a kind of separating liquid for removing dead liver cell, be
Following component is added in William ' s E culture medium: the universal culture additive of the ITS of 0.1%v/v-1.5%v/v,
The L-Glutamine of 1.5mmol/L-2.5mmol/L, the epidermal growth factor of 1 μ g/L-20 μ g/L, 15mg/L-20mg/L hydrogen
Change the tire of cortisone, the dexamethasone of 35 μ g/L-45 μ g/L, the mycillin of 0.5%v/v-1%v/v, 1%v/v-10%v/v
The Nycodenz of cow's serum and 0.25g/mL-0.5g/mL.
Each raw material introduction of the invention:
1, William ' s E culture medium, i.e. WILLIAMS-DARLING Ton E culture medium.In the prior art, William ' s E culture medium, it is main
The long term cell culture and differentiation state for being used for liver cell maintain.In the present invention, based on William ' s E culture medium
Culture medium.
The commercially available purchase of above-mentioned William ' s E culture medium is such as purchased from U.S. Thermo Fisher Scientific
Company, article No. 12551032；Or it is purchased from Sigma Co., USA, article No. W1878；Or it is (deep purchased from vertical fertile biotechnology
Ditch between fields) Co., Ltd, article No. LV-WE001.The above commercial product ingredient is similar, can achieve equivalent effect.
2, the universal culture additive of ITS
ITS is Insulin, and the abbreviation of Transferrin, SeleniumSolution are a kind of universal culture additions
Agent transports ferritin and selenous acid containing insulin, people, can promote increasing of the various kinds of cell under low serum or serum-free environment
It grows.
5L equivalent contains: 25mg insulin, 25mg transport protein and 25 μ g selenous acid.
20L equivalent contains: 100mg insulin, 100mg transport protein and 100 μ g selenous acid.The above-mentioned universal culture of ITS
The commercially available purchase of additive is such as purchased from U.S. Thermo Fisher Scientific company, article No. 41400045；Or
Purchased from Sigma Co., USA, article No. I3146-5mL；Or purchase, to vertical fertile biotechnology (Shenzhen) Co., Ltd, article No. is
LV-ITS001.The above commercial product ingredient is similar, can achieve equivalent effect.
3, L-Glutamine, English name L-Glutamine, abbreviation L-Gln are the amides of glutamic acid.Glutamine
It can be used for treating gastric and duodenal ulcer, gastritis and hyperhydrochloria, be also used for improving brain function.In the present invention, glutamy
Amine is important energy source, participates in albumen synthesis and nucleic acid metabolism, and Activity of hepatocytes can be improved and promote cell growth.
The above-mentioned commercially available purchase of glutamine, is such as purchased from U.S. ThermoFisher Scientific company, and article No. is
25030081；Or it is purchased from Sigma Co., USA, article No. G7513；Or the limited public affairs of biotechnology share are trained purchased from Shanghai source
Department, article No. S210JV.The above commercial product ingredient is similar, can achieve equivalent effect.
4, epidermal growth factor, English name are Epidermal Growth Factor, and abbreviation EGF has found earliest
Growth factor exchanges ganglion cell's growth, proliferation and differentiation and plays an important role.Epidermal growth factor is a kind of small peptide, by 53
Amino acid residue composition.In the present invention, epidermal growth factor, which is used as, promotes hepatic cell growth.
The above-mentioned commercially available purchase of epidermal growth factor is such as purchased from U.S. R&D Systems company, article No. 236-EG；
Or it is purchased from U.S. Thermo Fisher Scientific company, article No. PHG0311；Or purchased from vertical fertile biotechnology
(Shenzhen) Co., Ltd, article No. LV-EGF001.The above commercial product ingredient is similar, can achieve equivalent effect.
5, hydrocortisone, it is also naturally occurring glucocorticoid that English name Cortisol, which is artificial synthesized, is resisted
Inflammation effect is 1.25 times of cortisone, it may have immunosuppressive action, antitoxic action, Hemorrhagic shock and certain mineralocorticoid are living
Property etc..In the present invention, hydrocortisone, which is used as, maintains hepatocyte differentiation state.
The above-mentioned commercially available purchase of hydrocortisone is such as purchased from MCE company, China, article No. HY-N0583；Or it is purchased from
Sigma Co., USA, article No. H0888.The above commercial product ingredient is the same, can achieve equivalent effect.
6, dexamethasone, English name are Dexamethasone, abbreviation DXMS also known as dexamethasone, fluorine methylprednisolone,
Dexamethasone is glucocorticosteroid hormone.Its derivative has hydrocortisone, prednisone etc., and pharmacological action is mainly anti-inflammatory, anti-
Poison, antiallergy, antirheumatic, clinical use are wide.In the present invention, dexamethasone, which is used as, maintains hepatocyte differentiation state.
The above-mentioned commercially available purchase of dexamethasone is such as purchased from MCE company, China, article No. HY-14648；Or purchased from beauty
Sigma company, state, article No. D4902.The above commercial product ingredient is the same, can achieve equivalent effect.
7, mycillin is the mixture of both penicillin and streptomysin.Penicillin and streptomysin can be respectively acting on leather
Blue positive and gram-negative bacteria, with the use of the germ contamination that can prevent cell culture.Penicillin is initially from Penicillium
It is purified in fungi, by the turnover of direct interference bacteria cell wall, triggers the release of enzyme indirectly, to further change thin
Cell wall.Streptomysin is initially purified from streptomycete.Streptomysin is by conjunction with bacterial ribosome 30S subunit, to inhibit
Protein synthesizes and leads to bacterial death.In the present invention, mycillin, which is used as, inhibits or kills bacterium.
The above-mentioned commercially available purchase of mycillin is such as purchased from U.S. Thermo Fisher Scientific company, article No.
It is 15140163；Or it is purchased from Sigma Co., USA, article No. V900929；Or it is purchased from Israel Biological
Industries company, article No. 03-031-1B.The above commercial product ingredient is similar, can achieve equivalent effect.
8, fetal calf serum, full name in English are fetal calf serum, and abbreviation FBS is one of serum, are mainly come
From the tire ox of caesarean birth.Fetal calf serum is to pass through the blood for the tire ox that cardiac puncture blood sampling obtains when butchering Pregnant cows
Clearly, newborn bovine serum picks up from the calf of birth 10 to 14 days.Fetal calf serum is that quality is highest, because tire ox is also not in contact with outer
Boundary, antibody, complement contained in serum etc. are minimum to the harmful ingredient of cell；Cow's serum is the maximum day of dosage in cell culture
Right culture medium, cell rich in grow necessary nutrition, are usually used in the in vitro culture of zooblast, have following make
With: the hormone grown to maintenance cell index (1) is provided, does not have or measure seldom nutrients in basal medium, and main
Low molecule nutrients.(2) binding protein is provided, vitamin, lipid, metal and other hormones etc. can be identified, can combine or adjust
Become the substance vitality that they are combined.(3) binding protein mass-energy plays solution in conjunction with toxic metals and pyrogen in some cases
Toxic action.(4) it is that cell is adherent, spreads over required Factor Source in plastic culture matrix.(5) play pH value buffer.
(6) protease inhibitors is provided, makes to make remaining trypsin inactivation when cell passes on, protection cell preserves from.In this hair
In bright, fetal calf serum is used as liver cell and provides of short duration nutritional support, protection or reparation liver cell.
The above-mentioned commercially available purchase of fetal calf serum is such as purchased from U.S. Thermo Fisher Scientific company, article No.
It is 10091148；Or it is purchased from Israel Biological Industries company, article No. 04-001-1A；Or purchased from beauty
Hyclone company, state, article No. SH30079.03.The above commercial product ingredient is similar, can achieve equivalent effect.
9、Nycodenz
The Chinese of Nycodenz is Ni Kedeng, and molecular weight 821 is non-ionic, to cytotoxic
Compound can be used for the separation or purification of the various biological substances such as cell, organelle, large biological molecule, virus.Nycodenz can
Into inside dead cell, thus increase the density of dead cell, and the cell membrane of living cells is complete, after centrifugation, dead cell is heavy
Centrifugation bottom of the tube is dropped to, living cells is arrived by exclusion to above centrifugation medium.It is only isolated and purified on a small quantity using Nycodenz at present
The method of living cells, but such method whether be suitable for alive liver cell separation and its efficiency there is no literature reported on either with or without more
Excellent separating liquid composition is still unknown.
The above-mentioned commercially available purchase of Nycodenz powder is such as purchased from Norway Axis-Shield company, article No. 1002424-
1。
The principle of the present invention is:
The present invention is added to the universal culture additive of ITS, L-Glutamine, epidermis in William ' s E culture medium
Growth factor, hydrocortisone, dexamethasone, mycillin, fetal calf serum and Nycodenz.Above-mentioned several components are according to corresponding
Proportion be combined, reach jointly efficiently remove dead liver cell, maintain liver cell osmotic pressure, of short duration battalion is provided for liver cell
It supports and supports, increase Activity of hepatocytes, promote cell growth, maintaining hepatocyte differentiation state, inhibition or kill bacterium, protect or repair
Multiple wounded hepatocytes and other effects.
The separating liquid of the dead liver cell of removal of the invention, low-speed centrifugation compared with the existing technology, Percoll method,
Ficoll400 method and routine Nycodenz method can be obviously improved recovery efficiency, cell viability and the liver cell training of alive liver cell
The state of supporting.
Based on the above technical solution, the present invention can also be improved as follows.
Further, the separating liquid of the dead liver cell of removal, is to add following component in William ' s E culture medium:
The universal culture additive of the ITS of 1%v/v, the L-Glutamine of 2mmol/L, the epidermal growth factor of 10 μ g/L, 18mg/L
Hydrocortisone, the dexamethasone of 40 μ g/L, the mycillin of 1%v/v, the fetal calf serum of 5%v/v and 0.3g/mL
Nycodenz。
Using above-mentioned further beneficial effect is: above-mentioned for optimal parameter.The separating liquid obtained using above-mentioned parameter
The effect for removing dead liver cell is best.
Further, William ' the s E culture medium prescription is specific as follows[1]:
Be using above-mentioned further beneficial effect: William ' s E culture medium had both been liver cell as basic culture medium
The nutriment on basis is provided, is also beneficial to maintain the osmotic pressure of liver cell.
Further, in the mycillin, the streptomysin of penicillin and 100mg/mL containing 100U/mL.
Further, the L-Glutamine of the 1.5mmol/L-2.5mmol/L by 1.5mmol/L-2.5mmol/L L- third
Aminoacyl-L-Glutamine dipeptides substitution.
It is using above-mentioned further beneficial effect: compared with L-Glutamine, the property of Ala-Gln dipeptides
It can be more stable.
The above-mentioned commercially available purchase of Ala-Gln dipeptides is such as purchased from U.S. Thermo Fisher
Scientific company, article No. 35050079.
Further, the fetal calf serum of the 1%v/v-10%v/v is substituted by the human serum albumin of 1%v/v-10%v/v.
It is using above-mentioned further beneficial effect: using human serum albumin, the effect of fetal calf serum can be substituted, played
Steady seepage pressure, protects the effect of cell, at the same exclude it is some using fetal calf serum introduce insecurity factors, as prion,
PERV virus, infecting both domestic animals and human pathogen, mycoplasma, anaphylactogen etc..
The above-mentioned commercially available purchase of human serum albumin is such as purchased from Shenzhen Weiguang Biological Products Co., Ltd., traditional Chinese medicines
Quasi- word S20033032.
The second object of the present invention provides the method for removing dead liver cell using the separating liquid of the dead liver cell of above-mentioned removal.
The present invention utilizes the separating liquid of the dead liver cell of above-mentioned removal, and the method for being removed dead liver cell has cell viability height, cell
The advantage that yield is high, processing cell concentration is big, the adherent situation of cell is excellent, liver cell form has maintained.
The technical scheme to solve the above technical problems is that a kind of separating liquid using the dead liver cell of above-mentioned removal
The method for removing dead liver cell, includes the following steps:
Step 1: two step collagenase perfusion methods separate primary hepatocyte
Fresh hepatic tissue is taken, 10min-30min is perfused with primer solution I, until rinse the blood in hepatic tissue well,
Then 15min-30min is perfused in the primer solution II preheated again with 37 DEG C, until hepatic tissue follows the string, obtains the liver digested
Tissue；
Step 2: digestion is terminated to be dispersed with cell
The hepatic tissue digested that step 1 is obtained is transferred in the culture dish containing room temperature primer solution II, is carefully trembled
The cell digested is fallen, cell suspension is formed and crosses cell sieve, obtain single cell suspension and is centrifuged for the first time；
Step 3: the cleaning of cell washing lotion
Supernatant after the first time that step 2 obtains is centrifuged removes, and the cell washing lotion of pre-cooling is added, and pasteur pipet is resuspended thin
Born of the same parents, second of centrifugation, repeat this step 2 times, obtain cleaned single cell suspension；
Step 4: the detection of cell viability
The cleaned single cell suspension and 0.4%m/v platform phenol indigo plant dyeing liquor for taking step 3 to obtain are mixed with volume ratio 1:1,
Then cell viability is detected；
Step 5: the purifying of primary hepatocyte living cells
Single cell suspension liver cell of the cell viability less than 80% is chosen, third time centrifugation is carried out, removes supernatant, is added
The separating liquid of the described in any item dead liver cells of removal of 10mL claim 1-5, is gently resuspended, then the training of 5mL bed board is gently added
Base is supported, forms apparent two layers of solution, the 4th centrifugation takes white cellular layer between separating liquid and bed board culture medium layer extremely
In 50mL centrifuge tube, plate culture medium is overlayed to 50mL, is gently mixed by inversion, the 5th centrifugation takes precipitating, work as after purification
Liver cell.
In step 4 of the invention, the method for detecting cell viability is: take that 20 μ L steps 3 obtain is cleaned unicellular
Suspension and 0.4%m/v platform phenol indigo plant dyeing liquor are obtained mixed liquor, 20 μ L mixed liquors are added to meter rapidly with volume ratio 1:1 mixing
In number plate, then the tally is inserted into calculating instrument, corresponding cell density and cell viability are read on calculating instrument.Using
The principle of platform phenol indigo plant dyeing liquor is: the cell membrane of dead cell is imperfect, and platform phenol indigo plant can enter, and dead cell is dyed blue；And
The cell membrane of living cells is complete, and platform phenol indigo plant not can enter, therefore living cells is not colored.
Above-mentioned calculating instrument and commercially available purchase are such as purchased from U.S. ALIT Life Science company, modelBioTech.Above-mentioned tally is above-mentionedThe mating consumptive material of BioTech.
Blood counting chamber progress can also be used in above-mentioned counting step, and the commercially available purchase of blood counting chamber is such as purchased from Shanghai
Refinement biochemical reagents Instrument Ltd., city, model amount Shanghai word 02270113.
Based on the above technical solution, the present invention can also be improved as follows.
Further, in step 1, the hepatic tissue from fish, birds, rodent, rabbit, pig, dog, tree shrew,
Monkey or human body contribute one of material or a variety of.
Further, the fish are zebra fish, and the birds are chicken, duck, goose, and the rodent is rat, small
Mouse or cavy.
Further, in step 1, the primer solution I is by 8.00g/L sodium chloride, 0.40g/L potassium chloride, 3.57g/L hydroxyl
Ethyl piperazidine ethanesulfonic acid, 0.06g/L phosphate dihydrate disodium hydrogen, 0.06g/L potassium dihydrogen phosphate, 1g/L glucose, 1mM pyruvic acid are received
With bis- (2- amino ethyl ether) the tetraacethyl compositions of 2mM ethylene glycol, pH value 7.4, with 0.22 μm of millipore filter filtration sterilization, 4 DEG C of guarantors
Deposit gained.
Further, in step 1, the primer solution II be in William ' s E culture medium add 2mM calcium chloride,
15mM 4- hydroxyethyl piperazineethanesulfonic acid and 0.3g/L type Ⅳ collagenase, 37 DEG C of water-baths dissolve 30min-60min, and pH value is
7.0-7.4, with 0.22 μm of millipore filter filtration sterilization, matching while using.
The above-mentioned commercially available purchase of type Ⅳ collagenase is such as purchased from Sigma Co., USA, article No. C5138.
Further, in step 2, the aperture of the cell sieve is 70 μm；First time acceleration of gravity of centrifugation is
50g, temperature are 4 DEG C, time 5min.
Be using above-mentioned further beneficial effect: cell sieve uses above-mentioned aperture, available single cell suspension.Using
Above-mentioned centrifugal condition can settle alive liver cell, remove the dead liver cell of a part, liver non-parenchymal cell and cell fragment.
Further, in step 3, the cell washing lotion be in DMEM/F12 culture medium be added 1%v/v mycillin and
5%v/v-10%v/v fetal calf serum；The acceleration of gravity of second of centrifugation is 50g, and temperature is 4 DEG C, time 5min.
Further, in the mycillin, the streptomysin of penicillin and 100mg/mL containing 100U/mL.
Further, the 5%v/v-10%v/v fetal calf serum is substituted by 1%v/v-10%v/v human serum albumin.
Further, in step 4, the 0.4% phenol indigo plant dyeing liquor is that 0.4g platform phenol indigo plant solid is dissolved into 100mL phosphoric acid
Gained in buffer.
Further, the phosphate buffer is by 8.0g/L sodium chloride, 0.2g/L potassium chloride, 12 water phosphorus of 3.58g/L
Sour disodium hydrogen and 0.24g/L potassium dihydrogen phosphate composition, pH value 7.2-7.4, high pressure steam sterilization, 4 DEG C of preservations gained.
Above-mentioned phosphate buffered solutions english abbreviation is PBS, and commercially available purchase is such as purchased from U.S. Hyclone company, goods
Number be SH30256.01.
Further, in step 5, the bed board culture medium is that following component is added in William ' s E culture medium: 1%
The universal hydrogen for cultivating additive, the L-Glutamine of 2mmol/L, the epidermal growth factor of 10 μ g/L, 18mg/L of the ITS of v/v
Change cortisone, the dexamethasone of 40 μ g/L, the mycillin of 1%v/v and the fetal calf serum of 5%v/v；The third time centrifugation
Acceleration of gravity is 50g, and temperature is 4 DEG C, time 5min；The acceleration of gravity of 4th centrifugation is 300g-800g, temperature
Degree is 4 DEG C, time 20min；The acceleration of gravity of 5th centrifugation is 50g-300g, and temperature is 4 DEG C, and the time is
5min。
Further, the L-Glutamine of the 1.5mmol/L-2.5mmol/L by 1.5mmol/L-2.5mmol/L L-
Alanyl-L-glutamine dipeptides substitution.
Further, the fetal calf serum of the 1%v/v-10%v/v is replaced by the human serum albumin of 1%v/v-10%v/v
Generation.
Beneficial effects of the present invention:
(1) separating liquid of the dead liver cell of removal of the invention is that be added to ITS in William ' s E culture medium general
Type culture additive, L-Glutamine, epidermal growth factor, hydrocortisone, dexamethasone, mycillin, fetal calf serum and
Nycodenz.Above-mentioned several components are combined according to corresponding proportion, are reached jointly and are efficiently removed dead liver cell, maintain liver thin
Born of the same parents' osmotic pressure provides of short duration nutritional support for liver cell, increases Activity of hepatocytes, promote cell growth, maintain liver cell point
Change state, inhibition kill bacterium, protection or repair wounded hepatocytes and other effects.
(2) present invention carries out the removal of dead liver cell using the separating liquid of the dead liver cell of above-mentioned removal, has cell viability
Advantage high, cell yield is high, processing cell concentration is big, the adherent situation of cell is excellent, liver cell form has maintained.
(3) separating liquid of the dead liver cell of removal of the invention, relative to Percoll method, Ficoll400 method and routine
Nycodenz method can be obviously improved recovery efficiency, cell viability and the hepatocyte cultures state of alive liver cell.
(4) separating liquid of the dead liver cell of removal of the invention, low in cost, wide market are suitble to large-scale promotion
Using.
Detailed description of the invention
Fig. 1 is using the method for the dead liver cell of removal of the embodiment of the present invention 2, the adherent figure of obtained liver cell.Wherein, carefully
Born of the same parents' scale bar is 50 μm.
Fig. 2 be comparative example 1 in, using the Ficoll400 method of the prior art, the adherent figure of obtained liver cell.Wherein, cell
Scale bar is 50 μm.
Fig. 3 be comparative example 2 in, using the Percoll method of the prior art, the adherent figure of obtained liver cell.Wherein, cell ratio
Example ruler is 50 μm.
Fig. 4 is in comparative example 3, using the conventional Nycodenz method of the prior art, the adherent figure of obtained liver cell.Wherein,
Cell proportion ruler is 50 μm.
Specific embodiment
Principles and features of the present invention are described below in conjunction with specific attached drawing, example is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.
Embodiment 1
The separating liquid of the dead liver cell of removal of the present embodiment, is to add following component in William ' s E culture medium:
The universal culture additive of the ITS of 0.1%v/v, the L-Glutamine of 2.5mmol/L, the epidermal growth factor of 1 μ g/L, 20mg/
The hydrocortisone of L, the dexamethasone of 35 μ g/L, the mycillin of 1%v/v and the Nycodenz of 0.25g/mL, the blueness chain
In mycin, the streptomysin of penicillin and 100mg/mL containing 100U/mL.
The method for removing dead liver cell using the separating liquid of the dead liver cell of above-mentioned removal of the present embodiment, including walk as follows
It is rapid:
Step 1: two step collagenase perfusion methods separate primary hepatocyte
Fresh tree shrew hepatic tissue is taken, 10min is perfused with primer solution I, until rinsing the blood in hepatic tissue well, so
15min is perfused in the primer solution II preheated again with 37 DEG C afterwards, until hepatic tissue follows the string, obtains the hepatic tissue digested.Its
In, the primer solution I be by 8.00g/L sodium chloride, 0.40g/L potassium chloride, 3.57g/L hydroxyethyl piperazineethanesulfonic acid,
0.06g/L phosphate dihydrate disodium hydrogen, 0.06g/L potassium dihydrogen phosphate, 1g/L glucose, 1mM pyruvic acid are received double with 2mM ethylene glycol
(2- amino ethyl ether) tetraacethyl composition, pH value 7.4, with 0.22 μm of millipore filter filtration sterilization, 4 DEG C of preservations gained.The filling
Note solution II is that 2mM calcium chloride, 15mM 4- hydroxyethyl piperazineethanesulfonic acid and 0.3g/L are added in William ' s E culture medium
Type Ⅳ collagenase, 37 DEG C of water-baths dissolve 30min-60min, and pH value 7.0 is current with 0.22 μm of millipore filter filtration sterilization
Now match.
Step 2: digestion is terminated to be dispersed with cell
The hepatic tissue digested that step 1 is obtained is transferred in the culture dish containing room temperature primer solution II, is carefully trembled
The cell digested is fallen, cell suspension is formed and crosses the cell sieve that aperture is 70 μm, obtain single cell suspension and is centrifuged for the first time.
Wherein, the acceleration of gravity of the first time centrifugation is 50g, and temperature is 4 DEG C, time 5min.
Step 3: the cleaning of cell washing lotion
Supernatant after the first time that step 2 obtains is centrifuged removes, and the cell washing lotion of pre-cooling is added, and pasteur pipet is resuspended thin
Born of the same parents, second of centrifugation, repeat this step 2 times, obtain cleaned single cell suspension.Wherein, the cell washing lotion is in DMEM/
1%v/v mycillin and 5%v/v fetal calf serum are added in F12 culture medium；In the mycillin, the blueness containing 100U/mL
The streptomysin of mycin and 100mg/mL；The acceleration of gravity of second of centrifugation is 50g, and temperature is 4 DEG C, time 5min.
Step 4: the detection of cell viability
The cleaned single cell suspension and 0.4%m/v platform phenol indigo plant dyeing liquor for taking step 3 to obtain are mixed with volume ratio 1:1,
Mixed liquor is obtained, 20 μ L mixed liquors are added in tally rapidly, then the tally is inserted into calculating instrument, in calculating instrument
It is upper to read corresponding cell density and cell viability.
Step 5: the purifying of primary hepatocyte living cells
The single cell suspension liver cell that cell viability is about 70% is chosen respectively, is carried out third time centrifugation, is removed supernatant, add
The separating liquid for entering the dead liver cell of the above-mentioned removal of 10mL, is gently resuspended, then 5mL bed board culture medium is gently added, and forms apparent two
Layer solution, the 4th centrifugation, takes the white cellular layer between separating liquid and bed board culture medium layer into 50mL centrifuge tube, overlays plate
Culture medium is gently mixed by inversion, the 5th centrifugation takes precipitating, alive liver cell as after purification to 50mL.
Wherein, the viable count of described 70% single cell suspension liver cell is no more than 1 × 108It is a；Viable count is more than 1
×108When a, separating liquid volume equal proportion increases.The bed board culture medium is below adding in William ' s E culture medium
Ingredient: the universal culture additive of the ITS of 1%v/v, the L-Glutamine of 2mmol/L, the epidermal growth factor of 10 μ g/L,
The hydrocortisone of 18mg/L, the dexamethasone of 40 μ g/L, the mycillin of 1%v/v and the fetal calf serum of 5%v/v；Described
The acceleration of gravity being centrifuged three times is 50g, and temperature is 4 DEG C, time 5min；It is described 4th time centrifugation acceleration of gravity be
300g, temperature are 4 DEG C, time 20min；The acceleration of gravity of 5th centrifugation is 50g, and temperature is 4 DEG C, and the time is
5min。
Embodiment 2
The separating liquid of the dead liver cell of removal of the present embodiment, is to add following component in William ' s E culture medium:
The universal culture additive of the ITS of 1%v/v, the L-Glutamine of 2mmol/l, the epidermal growth factor of 10 μ g/l, 18mg/l
Hydrocortisone, the dexamethasone of 40 μ g/l, the mycillin of 1%v/v, the fetal calf serum of 5%v/v and 0.3g/mL
Nycodenz, in the mycillin, the streptomysin of penicillin and 100mg/mL containing 100U/mL.
The method for removing dead liver cell using the separating liquid of the dead liver cell of above-mentioned removal of the present embodiment, including walk as follows
It is rapid:
Step 1: two step collagenase perfusion methods separate primary hepatocyte
Fresh liver tissues of rats is taken, 20min is perfused with primer solution I, until rinsing the blood in hepatic tissue well, so
22min is perfused in the primer solution II preheated again with 37 DEG C afterwards, until hepatic tissue follows the string, obtains the hepatic tissue digested.Its
In, the primer solution I be by 8.00g/L sodium chloride, 0.40g/L potassium chloride, 3.57g/L hydroxyethyl piperazineethanesulfonic acid,
0.06g/L phosphate dihydrate disodium hydrogen, 0.06g/L potassium dihydrogen phosphate, 1g/L glucose, 1mM pyruvic acid are received double with 2mM ethylene glycol
(2- amino ethyl ether) tetraacethyl composition, pH value 7.4, with 0.22 μm of millipore filter filtration sterilization, 4 DEG C of preservations gained.The filling
Note solution II is that 2mM calcium chloride, 15mM 4- hydroxyethyl piperazineethanesulfonic acid and 0.3g/L are added in William ' s E culture medium
Type Ⅳ collagenase, 37 DEG C of water-baths dissolve 30min-60min, pH value 7.0-7.4, with 0.22 μm of millipore filter filtration sterilization,
Matching while using.
Step 2: digestion is terminated to be dispersed with cell
The hepatic tissue digested that step 1 is obtained is transferred in the culture dish containing room temperature primer solution II, is carefully trembled
The cell digested is fallen, cell suspension is formed and crosses the cell sieve that aperture is 70 μm, obtain single cell suspension and is centrifuged for the first time.
Wherein, the acceleration of gravity of the first time centrifugation is 50g, and temperature is 4 DEG C, time 5min.
Step 3: the cleaning of cell washing lotion
Supernatant after the first time that step 2 obtains is centrifuged removes, and the cell washing lotion of pre-cooling is added, and pasteur pipet is resuspended thin
Born of the same parents, second of centrifugation, repeat this step 2 times, obtain cleaned single cell suspension.Wherein, the cell washing lotion is in DMEM/
1%v/v mycillin and 8%v/v fetal calf serum are added in F12 culture medium；In the mycillin, the blueness containing 100U/mL
The streptomysin of mycin and 100mg/mL；The acceleration of gravity of second of centrifugation is 50g, and temperature is 4 DEG C, time 5min.
Step 4: the detection of cell viability
The cleaned single cell suspension and 0.4%m/v platform phenol indigo plant dyeing liquor for taking step 3 to obtain are mixed with volume ratio 1:1,
Mixed liquor is obtained, 20 μ L mixed liquors are added in tally rapidly, then the tally is inserted into calculating instrument, in calculating instrument
It is upper to read corresponding cell density and cell viability.
Step 5: the purifying of primary hepatocyte living cells
The single cell suspension liver cell that cell viability is about 60% is chosen, third time centrifugation is carried out, removes supernatant, is added
The separating liquid of the dead liver cell of the above-mentioned removal of 10mL, is gently resuspended, then 5mL bed board culture medium is gently added, and is formed apparent two layers
Solution, the 4th centrifugation, takes the white cellular layer between separating liquid and bed board culture medium layer into 50mL centrifuge tube, overlays plate training
Base is supported to 50mL, is gently mixed by inversion, the 5th centrifugation takes precipitating, alive liver cell as after purification.
Wherein, the viable count of described 70% single cell suspension liver cell is no more than 1 × 108It is a；Viable count is more than 1
×108When a, separating liquid volume equal proportion increases.The bed board culture medium is below adding in William ' s E culture medium
Ingredient: the universal culture additive of the ITS of 1%v/v, the L-Glutamine of 2mmol/L, the epidermal growth factor of 10 μ g/L,
The hydrocortisone of 18mg/L, the dexamethasone of 40 μ g/L, the mycillin of 0.8%v/v and the fetal calf serum of 5%v/v；It is described
The acceleration of gravity of third time centrifugation is 50g, and temperature is 4 DEG C, time 5min；It is described 4th time centrifugation acceleration of gravity be
500g, temperature are 4 DEG C, time 20min；The acceleration of gravity of 5th centrifugation is 200g, and temperature is 4 DEG C, and the time is
5min。
Embodiment 3
The separating liquid of the dead liver cell of removal of the present embodiment, is to add following component in William ' s E culture medium:
The universal culture additive of the ITS of 1.5%v/v, the L-Glutamine of 1.5mmol/l, the epidermal growth factor of 20 μ g/l,
The hydrocortisone of 15mg/l, the dexamethasone of 45 μ g/l, the mycillin of 0.5%v/v, the fetal calf serum of 10%v/v and
The Nycodenz of 0.5g/mL, in the mycillin, the streptomysin of penicillin and 100mg/mL containing 100U/mL.
The method for removing dead liver cell using the separating liquid of the dead liver cell of above-mentioned removal of the present embodiment, including walk as follows
It is rapid:
Step 1: two step collagenase perfusion methods separate primary hepatocyte
Fresh chicken gizzard tissue is taken, 30min is perfused with primer solution I, until rinsing the blood in hepatic tissue well, then
30min is perfused in the primer solution II preheated again with 37 DEG C, until hepatic tissue follows the string, obtains the hepatic tissue digested.Wherein,
The primer solution I is by 8.00g/L sodium chloride, 0.40g/L potassium chloride, 3.57g/L hydroxyethyl piperazineethanesulfonic acid, 0.06g/L
Phosphate dihydrate disodium hydrogen, 0.06g/L potassium dihydrogen phosphate, 1g/L glucose, 1mM pyruvic acid are received and bis- (the 2- amino second of 2mM ethylene glycol
Ether) tetraacethyl composition, pH value 7.4, with 0.22 μm of millipore filter filtration sterilization, 4 DEG C of preservations gained.The primer solution II is
2mM calcium chloride, 15mM 4- hydroxyethyl piperazineethanesulfonic acid and 0.3g/L type Ⅳ collagenase are added in William ' s E culture medium,
37 DEG C of water-baths dissolve 30min-60min, pH value 7.2, with 0.22 μm of millipore filter filtration sterilization, matching while using.
Step 2: digestion is terminated to be dispersed with cell
The hepatic tissue digested that step 1 is obtained is transferred in the culture dish containing room temperature primer solution II, is carefully trembled
The cell digested is fallen, cell suspension is formed and crosses the cell sieve that aperture is 70 μm, obtain single cell suspension and is centrifuged for the first time.
Wherein, the acceleration of gravity of the first time centrifugation is 50g, and temperature is 4 DEG C, time 5min.
Step 3: the cleaning of cell washing lotion
Supernatant after the first time that step 2 obtains is centrifuged removes, and the cell washing lotion of pre-cooling is added, and pasteur pipet is resuspended thin
Born of the same parents, second of centrifugation, repeat this step 2 times, obtain cleaned single cell suspension.Wherein, the cell washing lotion is in DMEM/
1%v/v mycillin and 10%v/v fetal calf serum are added in F12 culture medium；In the mycillin, the blueness containing 100U/mL
The streptomysin of mycin and 100mg/mL；The acceleration of gravity of second of centrifugation is 50g, and temperature is 4 DEG C, time 5min.
Step 4: the detection of cell viability
The cleaned single cell suspension and 0.4%m/v platform phenol indigo plant dyeing liquor for taking step 3 to obtain are mixed with volume ratio 1:1,
Mixed liquor is obtained, 20 μ L mixed liquors are added in tally rapidly, then the tally is inserted into calculating instrument, in calculating instrument
It is upper to read corresponding cell density and cell viability.
Step 5: the purifying of primary hepatocyte living cells
The single cell suspension liver cell that cell viability is about 50% is chosen, third time centrifugation is carried out, removes supernatant, is added
The separating liquid of the dead liver cell of the above-mentioned removal of 10mL, is gently resuspended, then 5mL bed board culture medium is gently added, and is formed apparent two layers
Solution, the 4th centrifugation, takes the white cellular layer between separating liquid and bed board culture medium layer into 50mL centrifuge tube, overlays plate training
Base is supported to 50mL, is gently mixed by inversion, the 5th centrifugation takes precipitating, alive liver cell as after purification.
Wherein, the viable count of described 70% single cell suspension liver cell is no more than 1 × 108It is a；Viable count is more than 1
×108When a, separating liquid volume equal proportion increases.The bed board culture medium is below adding in William ' s E culture medium
Ingredient: the universal epidermal growth factor for cultivating additive, the L-Glutamine of 2.5mmol/L, 20 μ g/L of the ITS of 1.5%v/v
Son, the hydrocortisone of 20mg/L, the dexamethasone of 45 μ g/L, the mycillin of 1%v/v and the fetal calf serum of 10%v/v；Institute
The acceleration of gravity for stating third time centrifugation is 50g, and temperature is 4 DEG C, time 5min；The acceleration of gravity of 4th centrifugation
For 800g, temperature is 4 DEG C, time 20min；The acceleration of gravity of 5th centrifugation is 300g, and temperature is 4 DEG C, the time
For 5min.
Comparative example 1: it is compared with the Ficoll400 method of the prior art
The Ficoll400 method of the prior art and the method for the embodiment of the present invention 2 is respectively adopted, to primary hepatocyte living cells
It is purified, carries out cell viability compared with cell yield.
Unlike the embodiment of the present invention 2, in the step 5 of the Ficoll400 method of the prior art, separating liquid is to use
Ficoll400 solution, other steps are all the same.The Ficoll400 solution is to use the mass percent of PBS preparation for 15%
Ficoll400 solution.
The step 5 of the Ficoll400 method of the prior art is specifically: choosing 10mL cell viability respectively is about 60% and cell
Density is 2 × 105A/mL, 2 × 106A/mL, 2 × 107The single cell suspension of a/mL, be gently added to respectively 9mL, 15mL,
On the Ficoll400 solution that the mass percent of 30mL is 15%, apparent two layers of solution is formed, is centrifuged, takes precipitating, as
Alive liver cell after purification.Wherein, the acceleration of gravity of the centrifugation is 75g, and temperature is 4 DEG C, time 5min.Wherein, institute
State the configuration method of 15%Ficoll400 solution are as follows: weigh 15gFicoll400 powder (purchasing Sigma Co., USA), dissolve
Into the PBS solution of 100mL, filtration sterilization, 4 DEG C of preservations.
The step 5 of the embodiment of the present invention 2 is specifically: selection 10mL cell viability is about 60% respectively and cell density is 2
×105A/mL, 2 × 106A/mL, 2 × 107The single cell suspension liver cell of a/mL carries out third time centrifugation, removes supernatant,
The separating liquid of the dead liver cell of the above-mentioned removal of 10mL is added, is gently resuspended, then 5mL bed board culture medium is gently added, is formed apparent
Two layers of solution, the 4th centrifugation, takes the white cellular layer between separating liquid and bed board culture medium layer into 50mL centrifuge tube, overlays
Plate culture medium is gently mixed by inversion, the 5th centrifugation takes precipitating, alive liver cell as after purification to 50mL.
The alive liver cell after purification that will be obtained using the Ficoll400 method of embodiment 2 and the prior art, respectively with 1.5
×105A total number of cells/cm2, it is inoculated into the coated Tissue Culture Dish of collagen, after culture 6 hours, bed board culture solution is removed,
Liver cell is added and maintains culture solution, takes pictures.
The cell that the method for the dead liver cell of removal of embodiment 2 handles different cell concentrations from the Ficoll method of the prior art is living
Shown in the comparison of power, cell yield and cell culture state, as shown in table 1, Fig. 1 and Fig. 2.
As shown in Table 1, the Ficoll method of the method for the dead liver cell of removal of the embodiment of the present invention 2 compared with the prior art,
On cell treating capacity, final cell vigor, cell yield, there is significant effect.
As shown in Fig. 1 and Fig. 2, the method for the dead liver cell of removal of the embodiment of the present invention 2 is compared with the prior art
Ficoll method, cell debris less (see cell-free blank space), the adherent ability of cell significantly improve, and the polarization of liver cell form is special
Property becomes apparent from.
The method of the dead liver cell of removal of 1 embodiment 2 of table and the Ficoll method of the prior art
The cell viability of different cell concentrations is handled compared with cell yield
Remarks: 1, cell viability: the ratio of living cells and total cell；2, cell yield: living cells and purifying before purification
Live cell fraction afterwards；3, all numerical value are 3 independent mean+SDs repeated in experiment.
Comparative example 2: it is compared with the Percoll method of the prior art
The Percoll method of the prior art and the method for the embodiment of the present invention 2 is respectively adopted, to primary hepatocyte living cells into
Row purifying carries out cell viability compared with cell yield.
Unlike the embodiment of the present invention 2, in the step 5 of the Percoll method of the prior art, separating liquid is to use
Percoll solution, other steps are all the same.The Percoll solution is to use the mass percent of PBS preparation for 33%
Percoll solution.
The step 5 of the Percoll method of the prior art is specifically: selection 10mL cell viability is about 60% respectively and cell is close
Degree is 2 × 105A/mL, 2 × 106A/mL, 2 × 107The single cell suspension of a/mL, is gently added to 9mL, 15mL, 30mL respectively
On the Percoll solution that mass percent is 33%, apparent two layers of solution is formed, is centrifuged, takes precipitating, as after purification
Alive liver cell.Wherein, the acceleration of gravity of the centrifugation is 350g, and temperature is 4 DEG C, time 25min.Described 33%
The configuration method of Percoll solution is: it measures 33mL100%Percoll mother liquor (purchasing in GE company, the U.S.), it is molten using PBS
Liquid is settled in 100mL, filtration sterilization, 4 DEG C of preservations.
The step 5 of the embodiment of the present invention 2 is specifically: selection 10mL cell viability is about 60% respectively and cell density is 2
×105A/mL, 2 × 106A/mL, 2 × 107The single cell suspension liver cell of a/mL carries out third time centrifugation, removes supernatant,
The separating liquid of the dead liver cell of the above-mentioned removal of 10mL is added, is gently resuspended, then 5mL bed board culture medium is gently added, is formed apparent
Two layers of solution, the 4th centrifugation, takes the white cellular layer between separating liquid and bed board culture medium layer into 50mL centrifuge tube, overlays
Plate culture medium is gently mixed by inversion, the 5th centrifugation takes precipitating, alive liver cell as after purification to 50mL.
The alive liver cell after purification that will be obtained using the Percoll method of embodiment 2 and the prior art, respectively with 1.5 ×
105A total number of cells/cm2, it is inoculated into the coated Tissue Culture Dish of collagen, after culture 6 hours, bed board culture solution is removed, is added
Enter liver cell and maintain culture solution, takes pictures.
The method of the dead liver cell of removal of embodiment 2 handles the cell of different cell concentrations from the Percoll method of the prior art
Shown in the comparison of vigor, cell yield and cell culture state, as shown in table 2, Fig. 1 and Fig. 3.
As shown in Table 2, the Percoll method of the method for the dead liver cell of removal of the embodiment of the present invention 2 compared with the prior art,
On cell treating capacity, final cell vigor, cell yield, there is significant improvement effect.
As shown in Fig. 1 and Fig. 3, the method for the dead liver cell of removal of the embodiment of the present invention 2 is compared with the prior art
Percoll method, cell debris less (see cell-free blank space), the adherent ability of cell significantly improve, the polarization of liver cell form
Characteristic becomes apparent from.
The method of the dead liver cell of removal of 2 embodiment 2 of table and the Percoll method of the prior art
The cell viability of different cell concentrations is handled compared with cell yield
Remarks: 1, cell viability: the ratio of living cells and total cell；2, cell yield: living cells and purifying before purification
Live cell fraction afterwards；3, all numerical value are 3 independent mean+SDs repeated in experiment.
Comparative example 3: it is compared with the conventional Nycodenz method (Buddhist nun can step on method) of the prior art
The method that conventional the Nycodenz method and the embodiment of the present invention 2 of the prior art is respectively adopted, it is living to primary hepatocyte
Cell is purified, and carries out cell viability compared with cell yield.
Unlike the embodiment of the present invention 2, in the step 5 of the Nycodenz of the prior art, separating liquid is to use
Nycodenz solution, other steps are all the same.The Nycodenz solution is: it is fixed after 15gNycodenz is dissolved with dilution to weigh
Hold 50mL (30%Nycodenz), 0.22 μm of filtering, it can 4 DEG C of preservations.The diluent ingredient are as follows: 0.85% (w/v) NaCl,
10mM Tris-HCl,pH7.4。。
The step 5 of the conventional Nycodenz method of the prior art is specifically: respectively choose 10mL cell viability be about 60% and
Cell density is 2 × 105A/mL, 2 × 106A/mL, 2 × 107The single cell suspension liver cell of a/mL, carry out third time from
The heart removes supernatant, it is added 10mL 0.3g/mL Nycodenz solution (30% Nycodenz solution), is gently resuspended, then gently
It is light that 5mL bed board culture medium is added, apparent two layers of solution is formed, the 4th centrifugation takes 0.3g/mL Nycodenz solution and paving
White cellular layer between plate culture medium layer overlays plate culture medium to 50mL, is gently mixed by inversion, the 5th into 50mL centrifuge tube
Secondary centrifugation takes precipitating, alive liver cell as after purification.The acceleration of gravity of the third time centrifugation is 50g, and temperature is 4 DEG C,
Time is 5min；The acceleration of gravity of 4th centrifugation is 800g, and temperature is 4 DEG C, time 20min；Described 5th time
The acceleration of gravity of centrifugation is 300g, and temperature is 4 DEG C, time 5min.
The step 5 of the embodiment of the present invention 2 is specifically: selection 10mL cell viability is about 60% respectively and cell density is 2
×105A/mL, 2 × 106A/mL, 2 × 107The single cell suspension liver cell of a/mL carries out third time centrifugation, removes supernatant,
The separating liquid of the dead liver cell of the above-mentioned removal of 10mL is added, is gently resuspended, then 5mL bed board culture medium is gently added, is formed apparent
Two layers of solution, the 4th centrifugation, takes the white cellular layer between separating liquid and bed board culture medium layer into 50mL centrifuge tube, overlays
Plate culture medium is gently mixed by inversion, the 5th centrifugation takes precipitating, alive liver cell as after purification to 50mL.
The alive liver cell after purification that will be obtained using the conventional Nycodenz method of embodiment 2 and the prior art, respectively with
1.5×105A total number of cells/cm2, it is inoculated into the coated Tissue Culture Dish of collagen, after culture 6 hours, bed board culture solution is gone
Fall, liver cell is added and maintains culture solution, takes pictures.
The method of the dead liver cell of removal of embodiment 2 handles different cell concentrations from the conventional Nycodenz method of the prior art
Shown in the comparison of cell viability, cell yield and cell culture state, as shown in table 3, Fig. 1 and Fig. 4.
As shown in Table 3, the method for the dead liver cell of removal of the embodiment of the present invention 2 is compared to Nycodenz method, final thin
On born of the same parents' vigor, cell yield, there is significant improvement effect.
As shown in Fig. 1 and Fig. 4, the method for the dead liver cell of removal of the embodiment of the present invention 2 can step on method, cell debris compared to Buddhist nun
Less (see cell-free blank space), illustrate that clasmatosis is few.
In addition, the method for only isolating and purifying living cells using Nycodenz on a small quantity at present, but it is thin whether to be suitable for liver living
Born of the same parents separation and its efficiency there is no literature reported on.It can refer to that Baidu's science, pubmed, Soopat etc. is academic and patent search engine
Search result there is no the method for being used for alive liver cell purifying using Nycodenz at present.And the embodiment of the present invention 2 is in William '
Be added in s E culture medium the universal culture additive of ITS, L-Glutamine, epidermal growth factor, hydrocortisone, fill in
Meter Song, mycillin, fetal calf serum and Nycodenz.Above-mentioned several components are combined according to corresponding proportion, are reached jointly
Efficiently remove dead liver cell, maintenance liver cell osmotic pressure, of short duration nutritional support is provided for liver cell, increasing Activity of hepatocytes,
Promote cell growth, maintain hepatocyte differentiation state, inhibition or kill bacterium, protection or repair wounded hepatocytes and other effects.
The method of the dead liver cell of removal of 3 embodiment 2 of table and the conventional Nycodenz method of the prior art
The cell viability of different cell concentrations is handled compared with cell yield
Remarks: 1, cell viability: the ratio of living cells and total cell；2, cell yield: living cells and purifying before purification
Live cell fraction afterwards；3, all numerical value are 3 independent mean+SDs repeated in experiment.
It summarizes: by comparative example 1-3 it is found that the separating liquid of the dead liver cell of removal of the invention, compared with the existing technology
Percoll method, Ficoll400 method and routine Nycodenz method, can be obviously improved recovery efficiency, the cell viability of alive liver cell with
And hepatocyte cultures state.
Bibliography:
[1].Williams,G.M.and Gunn,J.M.(1974)Long term cell culture of adult
rat liver epithelial cells.Exp.Cell Res.,89:139.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of separating liquid for removing dead liver cell, which is characterized in that be added in William ' s E culture medium it is following at
Point: the universal culture additive, the L-Glutamine of 1.5mmol/L-2.5mmol/L, 1 μ of the ITS of 0.1%v/v-1.5%v/v
The epidermal growth factor of g/L-20 μ g/L, the hydrocortisone of 15mg/L-20mg/L, 35 μ g/L-45 μ g/L dexamethasone,
The mycillin of 0.5%v/v-1%v/v, the fetal calf serum of 1%v/v-10%v/v and 0.25g/mL-0.5g/mL
Nycodenz。
2. the separating liquid of the dead liver cell of removal according to claim 1, which is characterized in that point of the dead liver cell of removal
Chaotropic is to add following component in William ' s E culture medium: the universal culture additive of the ITS of 1%v/v, 2mmol/L
L-Glutamine, the epidermal growth factor of 10 μ g/L, the hydrocortisone of 18mg/L, 40 μ g/L dexamethasone, 1%v/v
Mycillin, the fetal calf serum of 5%v/v and the Nycodenz of 0.3g/mL.
3. the separating liquid of the dead liver cell of removal according to claim 1 or 2, which is characterized in that in the mycillin, contain
There are the penicillin of 100U/mL and the streptomysin of 100mg/mL.
4. the separating liquid of the dead liver cell of removal according to claim 1, which is characterized in that the 1.5mmol/L-
The L-Glutamine of 2.5mmol/L is substituted by the Ala-Gln dipeptides of 1.5mmol/L-2.5mmol/L.
5. the separating liquid of the dead liver cell of removal according to claim 1, which is characterized in that the 1%v/v-10%v/v's
Fetal calf serum is substituted by the human serum albumin of 1%v/v-10%v/v.
6. a kind of method that the separating liquid using the described in any item dead liver cells of removal of claim 1-5 removes dead liver cell,
It is characterized by comprising the following steps:
Step 1: two step collagenase perfusion methods separate primary hepatocyte
Fresh hepatic tissue is taken, 10min-30min is perfused with primer solution I, until rinsing the blood in hepatic tissue well, then
15min-30min is perfused in the primer solution II preheated again with 37 DEG C, until hepatic tissue follows the string, obtains the liver group digested
It knits；
Step 2: digestion is terminated to be dispersed with cell
The hepatic tissue digested that step 1 is obtained is transferred in the culture dish containing room temperature primer solution II, carefully shakes off to disappear
The cell changed forms cell suspension and crosses cell sieve, obtains single cell suspension and is centrifuged for the first time；
Step 3: the cleaning of cell washing lotion
Supernatant after the first time that step 2 obtains is centrifuged removes, and the cell washing lotion of pre-cooling is added, and cell is resuspended in pasteur pipet,
Second of centrifugation, repeats this step 2 times, obtains cleaned single cell suspension；
Step 4: the detection of cell viability
The cleaned single cell suspension and 0.4%m/v platform phenol indigo plant dyeing liquor for taking step 3 to obtain are with volume ratio 1:1 mixing, then
Detect cell viability；
Step 5: the purifying of primary hepatocyte living cells
Single cell suspension liver cell of the cell viability less than 80% is chosen, third time centrifugation is carried out, removes supernatant, 10mL power is added
Benefit requires the separating liquid of the described in any item dead liver cells of removal of 1-5, is gently resuspended, then 5mL bed board culture medium, shape is gently added
At apparent two layers of solution, the 4th centrifugation takes white cellular layer between separating liquid and bed board culture medium layer to be centrifuged to 50mL
Guan Zhong overlays plate culture medium to 50mL, is gently mixed by inversion, the 5th centrifugation takes precipitating, alive liver cell as after purification.
7. the method for the dead liver cell of removal according to claim 6, which is characterized in that in step 1, the hepatic tissue source
One of material or a variety of is contributed in fish, birds, rodent, rabbit, pig, dog, tree shrew, monkey or human body.
8. the method for the dead liver cell of removal according to claim 6, which is characterized in that in step 1, the primer solution I
It is by 8.00g/L sodium chloride, 0.40g/L potassium chloride, 3.57g/L hydroxyethyl piperazineethanesulfonic acid, 0.06g/L hydrogen phosphate dihydrate two
Sodium, 0.06g/L potassium dihydrogen phosphate, 1g/L glucose, 1mM pyruvic acid are received and bis- (2- amino ethyl ether) the tetraacethyl groups of 2mM ethylene glycol
At pH value 7.4, with 0.22 μm of millipore filter filtration sterilization, 4 DEG C of preservations gained；The primer solution II is in William ' s
It is molten that 2mM calcium chloride, 15mM4- hydroxyethyl piperazineethanesulfonic acid and 0.3g/L type Ⅳ collagenase, 37 DEG C of water-baths are added in E culture medium
30min-60min, pH value 7.0-7.4 are solved, with 0.22 μm of millipore filter filtration sterilization, matching while using.
9. the method for the dead liver cell of removal according to claim 6, which is characterized in that in step 2, the hole of the cell sieve
Diameter is 70 μm；The acceleration of gravity of the first time centrifugation is 50g, and temperature is 4 DEG C, time 5min；It is described thin in step 3
Born of the same parents' washing lotion is that 1%v/v mycillin and 5%v/v-10%v/v fetal calf serum are added in DMEM/F12 culture medium；Described second
The acceleration of gravity of secondary centrifugation is 50g, and temperature is 4 DEG C, time 5min.
10. the method for the dead liver cell of removal according to claim 6, which is characterized in that in step 5, the bed board culture
Base is to add following component in William ' s E culture medium: the L- of the universal culture additive of the ITS of 1%v/v, 2mmol/L
Glutamine, the epidermal growth factor of 10 μ g/L, the hydrocortisone of 18mg/L, the dexamethasone of 40 μ g/L, 1%v/v blueness
The fetal calf serum of streptomysin and 5%v/v；The acceleration of gravity of third time centrifugation is 50g, and temperature is 4 DEG C, and the time is
5min；The acceleration of gravity of 4th centrifugation is 300g-800g, and temperature is 4 DEG C, time 20min；Described 5th time from
The acceleration of gravity of the heart is 50g-300g, and temperature is 4 DEG C, time 5min.
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