CN106754643A - A kind of serum free hepatocyte medium and preparation method thereof - Google Patents
A kind of serum free hepatocyte medium and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to cell culture culture medium, and in particular to a kind of serum free hepatocyte medium and preparation method thereof.Culture medium of the present invention includes basal medium and addO-on therapy, wherein the addO-on therapy includes：Insulin, transferrins, sodium selenite, EGF, HGF, laminin, dexamethasone, non-esterified fatty acid, sodium acid carbonate, tanshinone IIA and Cryptotanshinone.Culture medium of the present invention can promote the growth of cell, and be surprised to find that serum free hepatocyte medium of the present invention can largely promote the synthesis of albumin, can for a long time maintain cell viability.
Description
Technical field
The present invention relates to cell culture culture medium, and in particular to a kind of serum free medium for hepatocyte cultures and
Its preparation method.
Background technology
Liver carries the Various Complexes such as synthesis, secretion, metabolism, removing toxic substances as one of most important organ in human body
Function.Once causing a large amount of necrosis of liver cell by a variety of causes, it will hepatic failure occur, cause organism metabolic disorder and toxicity
The accumulation of material, and this has aggravated the damage of liver cell in turn, influences the regeneration of liver cell, and then form the evil of hepatic failure
Property circulation.Acute liver failure is a kind of serious liver diseases, and the death rate is up to 60~90%.At present, it is most effective to this
Treatment method be liver transplant.However, due to donor organ shortage, high cost, needing the originals such as long-term use immunodepressant
Cause, often during donor is waited, patient is rapid dead due to disease progression, significantly limit the wide of orthotopic liver transplantation
General development.Therefore, the Biotype artificial liver technology based on culture hepatocyte turns into End-stage liver disease patient and is moved to liver in situ
The important means for smoothly transitting and being provided to acute liver failure patient damaged liver and be regenerated with repair time is provided.
With liver cell separation, the development of culture technique, the liver cell of in vitro culture can maintain the metabolic activity of a few hours,
Perform metabolism and the function of detoxification of many livers.Due to the critical shortage of donor liver in liver transplantation, the liver cell using in vitro culture enters
Row cell transplantation treats acute or chronic liver with bioartificial liver (bioartificial liver systems, BAL) is built
The method of MSOF patient is just of increasing concern.
At present usually using the William ' s-E of 10% (v/v) hyclone (FBS) of addition in Hepatocytes culture in vetro
Culture medium.However, the composition in serum is sufficiently complex, unknown materials present in it and variable factor often influence result of study
With the accuracy and repeatability of therapeutic effect.In addition, after the animal such as the animal blood serum such as FBS or haemocyanin derived components enter human body
Various adverse reactions and disease can be triggered, be that hidden danger has been buried in the clinical practice that cell transplantation and BAL are treated.U.S. FDA will
Stopping accepts declaring with the medical technology and the biotechnology new drug of preparation for containing serum cell culture process.Free serum culture
Have become the general trend of biotechnological pharmaceuticses production and biomedical engineering clinical treatment.
Chinese patent application (CN105087465A) discloses a kind of hepatocyte serum-free medium, and it is included with the following group
Point：Basal medium 500mL；Sericin 0.05~0.5%；0.1~1000nmol/mL of dexamethasone；Hepatocyte growth factor
5~20ng/mL of son；10~50ng/mL of EGF；Mycillin 100U/mL, this patent application is mainly in combination with silk gum
The advantage of albumen.
The content of the invention
To solve the above problems, the invention provides a kind of serum free hepatocyte medium, it can promote the life of cell
Length, and can largely promote the synthesis of albumin, can for a long time maintain cell viability.Present invention also offers one kind without blood
The preparation method of clearing liver cell culture medium.
The present invention is achieved through the following technical solutions：
A kind of serum free hepatocyte medium, comprising basal medium and addO-on therapy, addO-on therapy by following component and
Its concentration is constituted：Insulin 1-15mg/mL, transferrins 1-10mg/L, sodium selenite 6-12 μ g/L, EGF 10-
100 μ g/L, HGF 15-100ng/mL, laminin 0.5-0.85 μ g/mL, dexamethasone 1-10mmol/L,
Non-esterified fatty acid 0.1-0.5mmol/L, sodium acid carbonate 1-3g/L, tanshinone IIA 10-50 μ g/L and Cryptotanshinone 1-10 μ g/
L。
Preferably, the non-esterified fatty acid is by oleic acid, the composition of stearic acid and palmitoleic acid, weight shared by each composition
Than being oleic acid 35%, stearic acid 35%, palmitoleic acid 30%.
Preferably, the non-esterified fatty acid is linoleic acid, oleic acid, palmitic acid and stearic composition, each composition institute
Accounting example is linoleic acid 20%, oleic acid 45%, palmitic acid 25% and stearic acid 10%.
Preferably, the basal medium is DMEM/F12, DEME or RPMI1640 culture mediums.
A kind of serum free hepatocyte medium preparation method, it is characterised in that comprise the following steps：
(1) basal medium, insulin, transferrins, sodium selenite, EGF, liver cell life are accurately weighed
The factor long and laminin are dissolved in the ultra-pure water after sterilizing, are stirred, and obtain solution I；
(2) accurately weigh dexamethasone to be dissolved in 10mL absolute ethyl alcohols, then dilute 100 times with ultra-pure water, obtain solution II；
(3) after accurately weighing non-esterified fatty acid in proportion, it is dissolved in 10mL absolute ethyl alcohols, stirs, obtains solution
III；
(4) in solution II and solution III being added into solution I, and sodium acid carbonate is added, is stirred, obtain solution IV；
(5) solution IV is filtered with 0.22 μM of sterilised membrane filter, is obtained final product.
The each component that culture medium of the present invention is added is screened by a large amount of scientific experiments and obtained.Wherein, insulin master
It is to increase intracellular cAMP levels to act on, and promotes glucose, amino acid to utilize, while promoting RNA, the albumen in liver cell
The synthesis of matter and aliphatic acid, suppresses Apoptosis, so as to strengthen the vigor and function of liver cell.Transferrins generally and pancreas islet
Element, selenium element are added jointly, and abbreviation ITS additives are recruitment factors necessary to most of serum free medium.Transferrins
Main Function be the metabolism for adjusting internal ferro element, ferro element is transferred to by cell by the TfR of cell surface
It is interior.Sodium selenite plays a part of to protect cells from oxidative stress as the existence form of the essential trace element selenium of human body.
The Main Function of EGF and HGF be promote liver cell propagation, and regulation liver cell work(
Energy.The Main Function of laminin is the adhesion for promoting liver cell, and its adherent growth.Dexamethasone is important in vivo to swash
Element, participates in glycometabolism, lipid metabolism of liver cell etc., can adjust the propagation and functional expression of liver cell.
Non-esterified fatty acid is added with culture medium of the present invention, there are some researches prove non-esterified fatty acid has to liver cell
Regulating and controlling effect.In present invention culture, the oleic acid for filtering out, stearic acid and palmitoleic acid composition or linoleic acid, oleic acid, palmitin
Sour and stearic composition is added in culture medium the growth that can be obviously promoted stem cell.
Tanshinone IIA is a kind of Alcohol soluble composition of the red sage root, is natural antioxidant.Tanshinone IIA has as one kind
The inhibitor that the lipid within endothelial cells peroxide of effect interacts with DNA, can eliminate generation during mitochondrial lipid peroxidation
Lipid free radical, so as to eliminate mitochondrial absorption function.At the same time, there are some researches prove tanshinone IIA can improve
TNF-α and H2O2The damage of caused Cultured Hepatocytes in vitro strain.It is an unexpected discovery of the invention that 10-50 μ ought be added in the medium
The Cryptotanshinone in tanshinone IIA and 1-10 μ g/L concentration ranges in g/L concentration ranges can not only effectively suppress culture
Microorganism ground grows and infects virally in base, and can be obviously promoted the synthesis of albumin.
This hair serum free hepatocyte medium compared with existing culture medium, with the growth that can promote cell, and can
The synthesis of a large amount of promotion albumin, can for a long time maintain the advantage of cell viability.
Brief description of the drawings
Activity of hepatocytes is determined in Fig. 1 different culture medias：Culture medium is what embodiment 1-2 and comparative example 1-3 was prepared
Culture medium.
Albumin (ALB) content in Fig. 2 different culture medias：Culture medium is what embodiment 1-2 and comparative example 1-3 was prepared
Culture medium.
ALT (alanine aminotransferase) content in Fig. 3 different culture medias：Culture medium is prepared by embodiment 1-2 and comparative example 1-3
The culture medium for obtaining.
Specific embodiment
The present invention is described in further details with reference to the accompanying drawings and examples, but this is not to limit of the invention
System, those skilled in the art's basic thought of the invention, various modifications may be made or improves, but without departing from this
The basic thought of invention, within the scope of the present invention.
Medicine is conventional medication needed for of the invention, and tanshinone IIA, Cryptotanshinone are purchased from the Nanjing scape limited public affairs of bamboo medical sci-tech
Department.
A kind of serum free hepatocyte medium of embodiment 1
A kind of serum free hepatocyte medium, is made up of following component and its concentration：DMEM/F1235g/L, insulin
8.65mg/mL, transferrins 5.74mg/L, the μ g/L of sodium selenite 8.2, the μ g/L of EGF 69, HGF
78ng/mL, laminin 0.71 μ g/mL, dexamethasone 7.9mmol/L, oleic acid 0.112mmol/L, stearic acid
0.112mmol/L, palmitoleic acid 0.096mmol/L, sodium acid carbonate 1.5g/L, the μ g/L of tanshinone IIA 45 and the μ of Cryptotanshinone 4.3
g/L。
Serum free hepatocyte medium preparation method of the present invention, is divided into following steps：
(1) basal medium, insulin, transferrins, sodium selenite, EGF, liver cell life are accurately weighed
The factor long and laminin are dissolved in the ultra-pure water after sterilizing, are stirred, and obtain solution I；
(2) accurately weigh dexamethasone to be dissolved in 10mL absolute ethyl alcohols, then dilute 100 times with ultra-pure water, obtain solution II；
(3) after accurately weighing non-esterified fatty acid in proportion, it is dissolved in 10mL absolute ethyl alcohols, stirs, obtains solution
III；
(4) in solution II and solution III being added into solution I, and sodium acid carbonate is added, is stirred, obtain solution IV；
(5) solution IV is filtered with 0.22 μM of sterilised membrane filter, is obtained final product.
A kind of serum free hepatocyte medium of embodiment 2
A kind of serum free hepatocyte medium, is made up of following component and its concentration：DMEM/F1235g/L, insulin
8.65mg/mL, transferrins 5.74mg/L, the μ g/L of sodium selenite 8.2, the μ g/L of EGF 69, HGF
78ng/mL, laminin 0.71 μ g/mL, dexamethasone 7.9mmol/L, linoleic acid 0.064mmol/L, oleic acid
0.144mmol/L, palmitic acid 0.08mmol/L, stearic acid 0.032mmol/L, sodium acid carbonate 1.5g/L, the μ g/L of tanshinone IIA 45
With the μ g/L of Cryptotanshinone 4.3.
The culture medium preparation method is similar to Example 1.
A kind of serum free hepatocyte medium of embodiment 3
A kind of serum free hepatocyte medium, is made up of following component and its concentration：DMEM/F1242g/L, insulin
1mg/mL, transferrins 1mg/L, the μ g/L of sodium selenite 6, EGF 10 μ g/L, HGF 15ng/mL,
Laminin 0.5 μ g/mL, dexamethasone 1mmol/L, oleic acid 0.035mmol/L, stearic acid 0.035mmol/L, palmitoleic acid
0.03mmol/L, sodium acid carbonate 1g/L, the μ g/L of tanshinone IIA 10 and the μ g/L of Cryptotanshinone 1.
The culture medium preparation method is similar with embodiment 1.
A kind of serum free hepatocyte medium of embodiment 4
A kind of serum free hepatocyte medium, is made up of following component and its concentration：DMEM/F1225g/L, insulin
15mg/mL, transferrins 10mg/L, the μ g/L of sodium selenite 12, the μ g/L of EGF 100, HGF
100ng/mL, laminin 0.85 μ g/mL, dexamethasone 10mmol/L, linoleic acid 0.1mmol/L, oleic acid 0.225mmol/
L, palmitic acid 0.125mmol/L, stearic acid 0.05mmol/L, sodium acid carbonate 3g/L, the μ g/L of tanshinone IIA 50, Cryptotanshinone 10
μg/L。
The culture medium preparation method is similar to Example 1.
A kind of serum free hepatocyte medium of comparative example 1
A kind of serum free hepatocyte medium, is made up of following component and its concentration：DMEM/F1235g/L, insulin
8.65mg/mL, transferrins 5.74mg/L, the μ g/L of sodium selenite 8.2, the μ g/L of EGF 69, HGF
78ng/mL, laminin 0.71 μ g/mL, dexamethasone 7.9mmol/L, oleic acid 0.112mmol/L, stearic acid
0.112mmol/L, palmitoleic acid 0.096mmol/L, sodium acid carbonate 1.5g/L, the μ g/L of Cryptotanshinone 49.3.
The culture medium preparation method is similar to Example 1.
It is not add tanshinone IIA with the difference of embodiment 1, and it is the red sage root in embodiment to add the amount of Cryptotanshinone
The total amount of the A of ketone II and Cryptotanshinone.
A kind of serum free hepatocyte medium of comparative example 2
A kind of serum free hepatocyte medium, is made up of following component and its concentration：DMEM/F1235g/L, insulin
8.65mg/mL, transferrins 5.74mg/L, the μ g/L of sodium selenite 8.2, the μ g/L of EGF 69, HGF
78ng/mL, laminin 0.71 μ g/mL, dexamethasone 7.9mmol/L, oleic acid 0.224mmol/L, palmitoleic acid
0.096mmol/L, sodium acid carbonate 1.5g/L, the μ g/L of tanshinone IIA 45, the μ g/L of Cryptotanshinone 4.3.
The culture medium preparation method is similar to Example 1.
It is not add stearic acid with the difference of embodiment 1, and the addition of oleic acid is stearic acid and oil in embodiment 1
The total amount of acid.
A kind of serum free hepatocyte medium of comparative example 3
A kind of serum free hepatocyte medium, is made up of following component and its concentration：DMEM/F1235g/L, insulin
8.65mg/mL, transferrins 5.74mg/L, the μ g/L of sodium selenite 8.2, the μ g/L of EGF 69, HGF
78ng/mL, laminin 0.71 μ g/mL, dexamethasone 7.9mmol/L, linoleic acid 0.064mmol/L, oleic acid
0.144mmol/L, palmitic acid 0.08mmol/L, arachidonic acid 0.032mmol/L, sodium acid carbonate 1.5g/L, tanshinone IIA 45
μ g/L, the μ g/L of Cryptotanshinone 4.3.
The culture medium preparation method is similar to Example 1.
It is that stearic acid is replaced with into arachidonic acid with the difference of embodiment 2.
The culture medium quality test of test example 1
Test media：The culture medium that the culture medium and comparative example 1-2 that embodiment 1-4 is prepared are prepared.
Test content：
(1) Sterility testing
Detected using flat band method, take 1 piece of 1 piece of SBA flat board and Sabouraud's agar flat board, balance to room temperature.Take above-mentioned
Test media is from sample bottom of the tube pipette samples, every piece of μ L of flat board 100, with inoculation after sample is centrifuged 15min through 4000rpm
Pin is rule.Flat board is put into 37 DEG C of incubators, it is feminine gender, sample passes that result is observed after culture 48h.
(2) endotoxin detection
Detected using gel method, take above-mentioned test media for sample through dilution after, same to negative control, positive control, confession
Test product positive control is separately added into the TAL after melting again through sterility test water, every kind of parallel two pipe.Result is negative right
It is feminine gender to look after, and positive control pipe is the positive, and test sample positive control pipe is feminine gender, sample passes for positive and sample cell.
(3) detection of mycoplasma is the detection of PCR methods
Used after 12000rpm centrifugations 3min after above-mentioned test media is carried out into boiling water bath 10min for sample, used
25 μ L systems enter performing PCR amplification to testing sample, positive control, negative control；Amplified production enters row agarose gel electrophoresis,
Testing result is observed in gel imaging instrument.Testing result is feminine gender, sample passes.
Hepatic cell growth and cell biological Function detection in the different culture media of test example 2
Experiment is to culture medium：The culture medium that embodiment 1-4 and comparative example 1-3 are prepared
Content of the test：
Cell culture：By 107The C3A cells of individual/mL are seeded to 25cm2In blake bottle, first using 5mL containing serum training
Support base and (hyclone, the penicillin of HEPES, 10000U of 5mmol of 55mL are added in the DMEM high glucose mediums per 500mL
With the streptomysin of 10000U) culture, change a not good liquor within two days.After after C3A cell growths stabilization, the side for gradually reducing serum is taken
Method, with the culture of the suddenly change of acclimatizing culture medium and serum free medium.First, added using the serum-containing media of 2.5mL
It is 5% to enter the test medium co-incubation C3A cells of 2.5mL, i.e. serum-concentration, changes a not good liquor within two days.Treat that C3A cells pass 5
After generation and stabilization, the concentration of serum is further reduced, the experiment culture of 3.75mL is added using the serum-containing media of 1.25mL
Base culture medium co-incubation, i.e. serum-concentration are 2.5%, change a not good liquor within two days.After C3A cells pass 5 generations and stabilization, it is transitioned into
Culture medium is individually cultivated in 100% test medium, without any serum composition, i.e., using the free serum culture of 5mL
Base Long Term Passages culture.C3A cells after stabilization are inoculated in 6 orifice plates and 96 orifice plates, continue to cultivate 8 days.
Cell count：Cell count, experiment knot are carried out using desk tray indigo plant counting method to being sampled in cultured cells different time
Fruit is as shown in table 1.
Desk tray indigo plant counting method：It is centrifuged after trypsin digestion and cell, prepares single cell suspension, and make appropriate dilution (106Carefully
Born of the same parents/mL), take during 1mL cell suspensions move into EP, plus 0.1mL0.4% trypan blue solutions are mixed, and add blood cell counting plate, are seen
Examine counting.If cell membrane is complete, cell is not the blue dyeing of desk tray, then be normal cell；If cell membrane is imperfect, rupture,
Desk tray indigo plant dyestuff enters cell, and cell becomes blue, as necrosis.
Different time sections cell count situation in each culture medium of table 1
Test example 3 is using the detection of MTT decoration methods cell viability
Collection embodiment 1, embodiment 2, comparative example 1, the C3A cells of logarithmic phase in comparative example 2 and the culture medium of comparative example 3,
Adjustment C3A concentration of cell suspension, each hole adds the detection liquid of 100 μ L, make C3A cell densities to be measured for 1000-10000/
Hole, and in 5%CO2, it is incubated in 37 DEG C of environment.Then it is the MTT solution of 5mg/mL that 20uL concentration is added per hole, continues to cultivate
4h.Then terminate culture, suck nutrient solution in hole, then the dimethyl sulfoxide (DMSO) (DMSO) of 150uL is added in every hole, put low on shaking table
Speed vibration 10min, makes crystal fully dissolve.In enzyme-linked immunosorbent assay instrument OD490The light absorption value in each hole, record knot are measured at nm
Really, with the time as abscissa, light absorption value is that ordinate draws curve, as shown in Figure 1.
As shown in Figure 1, hepatic cell growth speed is significantly faster than that comparative example, and embodiment culture medium in embodiment culture medium
The middle cytotostatic phase is long, and Apoptosis speed is slow, it is possible thereby to illustrate that culture medium of the present invention can promote the growth of liver cell.
Liver cell biological function detection in the different culture media of test example 4
C3A cells in different culture media are inoculated in 6 orifice plates respectively, respectively with the culture medium of each group of 3mL each
Culture, culture medium is changed daily, and Supernatant samples are left and taken in collection simultaneously, and determining albumin (ALB) using radio immunoassay contains
Amount, while detecting supernatant ALT (alanine aminotransferase) using automatic clinical chemistry analyzer, result of the test is as shown in Figure 2 and Figure 3.
As shown in Figure 2, albumin in the hepatocyte cultures liquid that embodiment medium culture is obtained in the different incubation times
Content is consistently higher than comparative example, and when cultivating 5, albumin content reaches maximum in embodiment 1,2 nutrient solutions, respectively
10.54 μ g/mL and 10.02 μ g/mL.It follows that culture medium of the present invention is conducive to dividing for liver cell own growth facilitative proteins
Secrete expression.
From the figure 3, it may be seen that ALT contents are substantially low in the hepatocyte cultures liquid that embodiment culture is obtained in the different incubation times
In comparative example such that it is able to promote hepatic cell growth.
Claims (5)
1. a kind of serum free hepatocyte medium, it is characterised in that：Comprising basal medium and addO-on therapy, addO-on therapy by with
Lower composition and its concentration are constituted：Insulin 1-15mg/mL, transferrins 1-10mg/L, sodium selenite 6-12 μ g/L, epidermal growth
The factor 10-100 μ g/L, HGF 15-100ng/mL, laminin 0.5-0.85 μ g/mL, dexamethasone 1-
10mmol/L, non-esterified fatty acid 0.1-0.5mmol/L, sodium acid carbonate 1-3g/L, tanshinone IIA 10-50 μ g/L and the hidden red sage root
Ketone 1-10 μ g/L.
2. serum free hepatocyte medium according to claim 1, it is characterised in that the non-esterified fatty acid by oleic acid,
The composition of stearic acid and palmitoleic acid, weight ratio shared by each composition is oleic acid 35%, stearic acid 35%, palmitoleic acid 30%.
3. serum free hepatocyte medium according to claim 1, it is characterised in that the non-esterified fatty acid is sub- oil
Acid, oleic acid, palmitic acid and stearic composition, each composition proportion are linoleic acid 20%, oleic acid 45%, palmitic acid 25%
With stearic acid 10%.
4. serum free hepatocyte medium according to claim 1 or claim 2, it is characterised in that the basal medium is DMEM/
F12, DEME or RPMI1640 culture medium.
5. according to any serum free hepatocyte medium preparation methods of claim 1-4, it is characterised in that be divided into following step
Suddenly：
(1) basal medium, insulin, transferrins, sodium selenite, EGF, hepatocyte growth factor are accurately weighed
Son and laminin are dissolved in the ultra-pure water after sterilizing, are stirred, and obtain solution I；
(2) accurately weigh dexamethasone to be dissolved in 10mL absolute ethyl alcohols, then dilute 100 times with ultra-pure water, obtain solution II；
(3) after accurately weighing non-esterified fatty acid in proportion, it is dissolved in 10mL absolute ethyl alcohols, stirs, obtains solution III；
(4) in solution II and solution III being added into solution I, and sodium acid carbonate is added, is stirred, obtain solution IV；
(5) solution IV is filtered with 0.22 μM of sterilised membrane filter, is obtained final product.
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| CN107227291A (en) * | 2017-06-20 | 2017-10-03 | 青岛金典生化器材有限公司 | A kind of culture medium of culture hepatocyte and preparation method thereof |
| CN110358683A (en) * | 2019-07-25 | 2019-10-22 | 中国科学院电工研究所 | A kind of bioreactor automatic control device |
| CN111733134A (en) * | 2020-04-13 | 2020-10-02 | 天津医科大学第二医院 | Novel culture solution for pancreatic cancer cells |
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| CN107227291A (en) * | 2017-06-20 | 2017-10-03 | 青岛金典生化器材有限公司 | A kind of culture medium of culture hepatocyte and preparation method thereof |
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| CN111733134A (en) * | 2020-04-13 | 2020-10-02 | 天津医科大学第二医院 | Novel culture solution for pancreatic cancer cells |
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