CN105087465B

CN105087465B - Hepatocyte serum-free medium

Info

Publication number: CN105087465B
Application number: CN201510531097.3A
Authority: CN
Inventors: 黄耘; 高毅
Original assignee: Southern Medical University Zhujiang Hospital
Current assignee: Southern Medical University Zhujiang Hospital
Priority date: 2015-08-26
Filing date: 2015-08-26
Publication date: 2019-09-17
Anticipated expiration: 2035-08-26
Also published as: CN105087465A

Abstract

The present invention discloses a kind of hepatocyte serum-free medium comprising following components: basal medium 500mL；Sericin 0.05~0.5%；0.1~1000nmol/mL of dexamethasone；5~20ng/mL of hepatocyte growth factor；10~50ng/mL of epidermal growth factor；Mycillin 100U/mL.Hepatocyte serum-free medium of the present invention is suitble to growth and the Function of liver cell, can be used for the preparation of Biotype artificial liver.

Description

Hepatocyte serum-free medium

Technical field

The present invention relates to a kind of animal cell culture technology more particularly to a kind of hepatocyte serum-free mediums.

Background technique

It is to rely on that biological artificial liver support system (BALSS), which is with the liver cell with certain function, can be certain The device for being similar to liver function is played human body in degree, on the one hand can be provided in short term for invertibity hepatic disorder patient Support, smoothly transit to liver function recovery, on the other hand can be to irreversibility liver failure patient in waiting liver transfer operation Period, which is given, to be supported, the function served as bridge between liver failure and liver transfer operation is played.Liver cell is the core of BALSS, because This Cultured Hepatocytes in vitro simultaneously makes it play original physiological action, becomes the emphasis of BALSS research.Previous liver cell makes more Employment or animal blood serum are cultivated, but since it is with immunological rejection and sensitization, vulnerable to inoculating microbe pollution etc. Disadvantage, and it is not suitable for human body application.

General cell culture needs that 5-10% animal blood serum is added in basal medium, and basal medium contains suitable Energy matter and microelement etc., and various growth factors necessary to being grown in serum containing cell, hormone and protein etc. Ingredient.Basal medium currently used for hepatocyte cultures mainly includes William ' s E culture medium, DMEM, DMEM/F12, RPMI-1640, M-199, ML-15 etc..The major defect of traditional serum-containing media is 1. due to species specificity, and xenogenesis is dynamic Object serum has immunological rejection and sensitization；2. easy to carry prion etc. in serum, and vulnerable to microbiological contamination； 3. there may be differences between different batches, it is easy to cause experimental result nonrepeatability；4. certain protein ingredients may in serum Experimental result is had an impact.

Serum free medium exploitation thinking it is critical that added in basal medium suitable growth factor, hormone, Microelement, rush coherent substance etc., make the effect of serum free medium close to serum-containing media.The liver being commercialized at present Cell non-serum culture medium is less, wherein that representative is HepatoZYME-SFM.HepatoZYME-SFM is current liver One of representative in cell non-serum culture medium, but different liver cancer cell lines, demand to ingredient each in culture medium not phase Together, therefore the optimum culture medium prescription of different cell lines is not identical, and HepatoZYME-SFM does not have extensive versatility.

Sericin (sericin) is the protein extracted from silk, the amino acid containing a large amount of side chains with hydrophilic radical Such as serine, aspartic acid, molecular weight be 10-400kDa, it is soluble easily in water in, play adhesive effect in silkworm cocooning.At present Sericin has been widely used in the cell culture of various mammals, and experiment confirms sericin to a variety of external trainings The proliferation and adhesive capacity of feeding cell have apparent facilitation [2].In the research of Nayak S [3] etc., in 2% seaweed 0.125% sericin is added in acid salt solution, HepG2 cell is resuspended by 1 × 106/ml density with this solution, uses note It penetrates pump and cell suspension is squeezed out into different size of microballon with different rates, microballon is collected in the glue containing 0.2MCaCl2 solution Change in bath and stand 15min, package 30min is then carried out to microballon with 0.5% chitosan solution, microballon is finally immersed into capital Buddhist nun It is crosslinked in flat solution (2.5mg/ml, 37 DEG C, 4h), so that micro-capsule be made.Cell through this microencapsulation technology, cell viability And albumin, urea synthesizing function are significantly better than that the control group for not adding sericin.

The shortcomings that sericin-alginates-chitosan microcapsules culture, is also very prominent, and it is more multiple to essentially consist in production process It is miscellaneous, it is unfavorable for daily use, is widely popularized and the industrialization culture of liver cell, and solution composition, osmotic pressure in encapsulation process Acute variation, be easy to cause cell irreversible damage.

Summary of the invention

The purpose of the present invention is combining the advantage of sericin, a kind of fitting for sericin for being added to debita spissitudo is provided Hepatocyte serum-free medium for bioartificial liver.

To reach the above technical purpose, The technical solution adopted by the invention is as follows:

A kind of hepatocyte serum-free medium comprising following components:

Basal medium 500mL；

Sericin 0.05~0.5%；

0.1~1000nmol/mL of dexamethasone；

5~20ng/mL of hepatocyte growth factor；

10~50ng/mL of epidermal growth factor；

Mycillin 100U/mL.

Selectively, the basal medium is RPMI-1640, DMEM, MEM, M199, F-10, F-12, DMEM/F-12 1:1, McCoy ' s 5A, Wi lliam ' s E, any one in ML-15.Preferably, the basal medium is DMEM.

It more preferably, further include Insulin-Transferrin-sodium selenite mixture.Insulin-Transferrin-the selenous acid The concentration of sodium mixture is 1%.

It more preferably, further include linoleic acid.The linoleic concentration is 11ng/mL.

It preferably, further include hydroxyethyl piperazine second thiosulfonic acid.The concentration of the hydroxyethyl piperazine second thiosulfonic acid is 10mmol/ L。

Compared with prior art, the present invention has the advantage that

(1) hepatocyte serum-free medium of the invention makes it be more suitable for liver thin with an improved free serum culture based formulas The growth of born of the same parents and Function.

(2) hepatocyte serum-free medium of the invention can carry out serum-free training to liver cell without complicated operation It supports.

(3) hepatocyte serum-free medium of the invention can be directly used for Biotype artificial liver.

Detailed description of the invention

Fig. 1 is that the influence of hepatocyte serum-free medium of the invention and DMEM high glucose medium to cell viability compares knot Fruit figure.

Fig. 2 is hepatocyte serum-free medium of the invention and DMEM high glucose medium to white in cell culture supernatant The comparing result figure of protein content.

Fig. 3 is hepatocyte serum-free medium of the invention and DMEM high glucose medium to urinating in cell culture supernatant The comparing result figure of cellulose content.

Fig. 4 is hepatocyte serum-free medium of the invention and DMEM high glucose medium to paddy in cell culture supernatant The comparing result figure of careless transaminase content.

Specific embodiment

Below in conjunction with the drawings and specific embodiments, present invention is further described in detail.

Embodiment one

The concentration screening of each component of hepatocyte serum-free medium of the invention

1. the concentration of each component in Orthogonal Optimum Design screening and culturing medium:

Orthogonal test scheme: this research includes sericin (A), dexamethasone (B), hepatocyte growth factor (HGF (C)) And 4 factors of epidermal growth factor (EGF (D)), each factor selects 3 levels (being shown in Table 1), therefore selects L9 (3⁴) orthogonal Table, testing program are shown in Table 2.It is basic culture medium that DEME high glycoform is selected in research, and 5 μ g/ml of insulin, 5 μ g/ of transferrins is added Ml, sodium selenite 10 μ g/L, linoleic acid 11ng/ml, HEPES 10mmol/L, mycillin 100U/ml, and by 2 side of experiment of table Case is separately added into 4 kinds of ingredients of corresponding dosage, is prepared by mixing into 9 groups of culture mediums.

1 orthogonal design factor level table of table

2 orthogonal test scheme table of table

2. liver cell is inoculated in each group respectively, in 5%CO₂And secondary culture in 37 DEG C of environment, it uses in succeeding generations After 0.25% trypsase-EDTA is digested, soybean trypsin inhibitor (1mg/ml) is added by 1:1 volume ratio and lays equal stress on It is outstanding, 5min is then centrifuged with 1000rpm, is discarded supernatant, culture bottle is added after being resuspended with respective culture medium and relays continuous secondary culture, It is every to replace culture medium for 24 hours.After cell growth is stablized, 6 orifice plates or 96 orifice plates are inoculated in, continue culture 8 days, are used for subsequent reality It tests.

Embodiment two

The culture effect of hepatocyte serum-free medium of the invention to liver cell

1. the detection method of indices

(1) method of cell viability detection: logarithmic phase cell is taken to be made 2 × 10⁴The cell suspension of/ml is inoculated in 96 holes Plate (100 hole μ l/), in 5%CO₂And cultivated for 24 hours in 37 DEG C of environment, 5 multiple holes are taken daily, 10 μ l CCK-8 reagents are added in every hole, After being incubated for 1 hour in above-mentioned environment, light absorption value is measured at 450nm wavelength with microplate reader, and it is bent to draw light absorption value-time Line takes maximum value in every group of absorbance.

(2) method of cell function detection: every hole inoculation 1 × 10 in 6 orifice plates⁵Cell, and 3ml culture medium is added, often Culture medium is replaced for 24 hours, in 5%CO₂And cultivated in 37 DEG C of environment, cell culture supernatant is collected daily, is measured using ELISA method Albumin content uses microplate reader colorimetric method for determining AST, LDH and urea content.

(3) Real-time qPCR is detected: taking logarithmic phase cell, Trizol is added and extracts total serum IgE, is scored with spectrophotometric Not Ce Liang absorbance at 260nm and 280nm wavelength, determine whether RNA purity and concentration meet experiment and want by A260/A280 It asks, and is saved backup in -20 DEG C.Compare rna expression difference by fluorescence quantitative PCR method, to reflect AFP, cytokeratin CK18, CK19, uridine diphosphate glucuronatetransferase (UDP-UGT), glutathione sulfydryl transferase (GST), glutamy Amine synzyme (GS), G-6-Pase, ALB, CYP3A4, the isogenic expression feelings of liver cell nuclear transcription factor HNF-4 α Condition draws melt curve analysis, records cycle threshold Ct.

(4) statistical analysis: after collecting indices, carrying out range analysis and variance analysis using 20.0 software of SPSS, Obtain preferred plan.

2. the check experiment of hepatocyte serum-free medium and complete medium of the invention

(1) each group culture medium is prepared by table 3.

3 experimental group of table and culture medium prescription

* dexamethasone, sericin, HGF, EGF dosage are determined according to after above-mentioned orthogonal test

(2) A group and the comparison of D group cell viability

With reference to Fig. 1, A group cell viability was gradually increasing since the 1st day, until the 4th day peaks, and D group (DMEM high sugar Group) cell viability the 1st day it is close with A group, be then decreased obviously since the 2nd day, be far below A group (P=0.000), and be gradually in Downward trend.

(3) albumin (ALB) content balance in A group and D group culture solution supernatant

With reference to Fig. 2, in cell culture overall process, ALB content in A group supernatant obviously higher than D group (P=0.000), And the ALB content in two groups of supernatants is as the time gradually decreases.

(4) urea content comparison in A group and D group culture solution supernatant

With reference to Fig. 3, the urea content in A group supernatant starts to significantly reduce on day 2, and keeps relative stability, but by It is gradually on a declining curve；Urea content in D group supernatant is on a declining curve with the time, and is significantly lower than A group (P=0.000).

(5) glutamic-oxalacetic transaminease (AST) content balance in A group and D group culture solution supernatant

With reference to Fig. 4, glutamic-oxalacetic transaminease (AST) content is gradually increased with the time in two groups of cell culture supernatants, A group Significantly lower than D group (P=0.000).

(6) conclusion

Cell viability, albumin and the urea synthesizing amount of A group are significantly better than that D group, and the HepGL cellular damage of A group is bright It is aobvious to be lighter than D group.

In conclusion hepatocyte serum-free medium of the present invention is suitble to growth and the Function of liver cell, can be used for giving birth to The preparation of object type artificial liver.

The above embodiment is a preferred embodiment of the present invention, but is not merely restricted to the described embodiments, other Any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, should be equivalent Substitute mode is all included in the scope of protection of the present invention.

Claims

1. a kind of HepGL cell non-serum culture medium, which is characterized in that include the following components:

DEME high glycoform basal medium 500mL；

Sericin 0.05~0.5%；

0.1~1000nmol/mL of dexamethasone；

5~20ng/mL of hepatocyte growth factor；

10~50ng/mL of epidermal growth factor；

Mycillin 100U/mL；

Insulin-Transferrin-sodium selenite mixture 1%；

Linoleic acid 11ng/mL；

The concentration of hydroxyethyl piperazine second thiosulfonic acid is 10mmol/L.