CN105543322A - Detection method of promoted cell proliferation rate of freeze-dried powder - Google Patents
Detection method of promoted cell proliferation rate of freeze-dried powder Download PDFInfo
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Abstract
The invention relates to the technical field of tissue engineering, and in particular relates to a detection method of a promoted cell proliferation rate of freeze-dried powder. The method can be used for accurately detecting the promoted cell proliferation rate of freeze-dried powder. The embodiments of the invention provide a detection method of the promoted cell proliferation rate of freeze-dried powder. The detection method comprises the following steps: 1) dissolving to-be-detected freeze-dried powder in a complete culture medium to obtain a sample culture medium which has a first osmotic pressure; 2) by taking the complete culture medium without the to-be-detected freeze-dried powder as a control medium, adjusting the osmotic pressure of the control medium to be equal to the first osmotic pressure; 3) carrying out culture on the same number of cells under the same condition by using the obtained sample culture medium and the control medium respectively, and obtaining sample cells and control cells after a first preset time; and 4) carrying out MTT (methylthiazolyldiphenyl-tetrazolium bromide) dyeing and light absorption value detection on the obtained sample cells and control cells, and comparing the light absorption values of the sample cells and the control cells to obtain the promoted cell proliferation rate of a sample in respect to a control.
Description
Technical field
The present invention relates to tissue engineering technique field, particularly relate to the detection method that a kind of lyophilized powder urgees cell proliferation rate.
Background technology
Containing biologically active factors in lyophilized powder, when needing that in lyophilized powder, the activity of biologically active factors detects, lyophilized powder can be added in substratum and prepare sample cultivation base, and with do not add the control medium of lyophilized powder as benchmark, carry out cultivating the identical time to inoblast, thus the fibroblastic proliferative amount under reference substance and sample effect is detected, the short cell proliferation rate of sample relative to reference substance can be obtained, thus indirectly can characterize the biological activity of biologically active factors in lyophilized powder.
Because lyophilized powder needs to add lyophilized vaccine in freezing dry process; therefore, after lyophilized powder is added in substratum obtains sample cultivation base, compared with control medium; osmotic pressure increases greatly, accurately cannot detect the short cell proliferation rate of lyophilized powder.
Summary of the invention
Main purpose of the present invention is, provides a kind of lyophilized powder to urge the detection method of cell proliferation rate.Accurately can detect the short cell proliferation rate of lyophilized powder.
For achieving the above object, the present invention adopts following technical scheme:
The embodiment of the present invention provides a kind of lyophilized powder to urge the detection method of cell proliferation rate, comprising:
Step 1) lyophilized powder to be detected is dissolved in perfect medium obtains sample cultivation base, wherein, the sample cultivation base obtained has the first osmotic pressure;
Step 2) will the perfect medium substratum in contrast of lyophilized powder to be detected do not added, and regulate the osmotic pressure of described control medium to equal the first osmotic pressure;
Step 3) respectively the cell of equal amts is cultivated at identical conditions by the sample cultivation base that obtains and control medium, after the first Preset Time, to obtain under sample effect cell under cell and reference substance effect;
Step 4) MTT dyeing and light absorption value detection are carried out to the cell under cell under obtained sample effect and reference substance effect, by the light absorption value of both contrasts, obtain the short cell proliferation rate of sample relative to reference substance.
Preferably, in described perfect medium, the content of serum is 10%-20%.
Optionally, described lyophilized powder to be detected at least comprises: human epidermal growth factor hEGF, vascular endothelial growth factor VEGF, fibroblast growth factor FGF, transforming growth factor-beta 1 TGF-β 1, transforminggrowthfactor-β2 TGF-β 2, para-insulin growth factor IGF-1, para-insulin No. two growth factor IGF-2, trehalose, Dextran 40 and N.F,USP MANNITOL.
Preferably, described step 2) in regulate the osmotic pressure of described control medium equal the first osmotic pressure before also comprise: obtain the osmotic pressure Y of described control medium and the funtcional relationship of the addition X of osmotic pressure regulator in perfect medium.
Optionally, described step 2) described in regulate the osmotic pressure of described control medium to equal the first osmotic pressure to comprise: according to funtcional relationship and described first osmotic pressure Y1, calculate the addition X1 of described osmotic pressure regulator, and be that the osmotic pressure regulator of X1 is dissolved in described control medium by obtained addition.
Preferably, described osmotic pressure regulator is N.F,USP MANNITOL, and the osmotic pressure Y of described control medium and the N.F,USP MANNITOL addition X in perfect medium meets funtcional relationship: Y=5425.9X+336.87.
Optionally, described cell is l cell;
Described l cell obtains as follows:
By l cell Secondary Culture to growing logarithmic phase, suspending with perfect medium and being diluted to 1-10 × 10
4individual/ml, is prepared into cell suspension, and wherein, the serum content in described perfect medium is 10%-20%;
Described cell suspension is inoculated in the different holes of 96 orifice plates with identical density;
Described 96 orifice plates are cultivated under default culture condition the second Preset Time to cell attachment, obtain l cell.
Preferably, described step 3) in respectively cultivation is carried out at identical conditions to the inoblast of equal amts by described sample cultivation base and described control medium and comprises: suck the supernatant liquor in 96 orifice plates at obtained l cell place, described sample cultivation base is added at least 2 holes of described 96 orifice plates, described control medium is added in other at least 2 holes of described 96 orifice plates, the l cell in described orifice plate is cultivated.
Optionally, described step 4) in MTT dyeing and light absorption value are carried out to the cell in cell under obtained sample effect and reference substance detect and comprise:
Suck the supernatant liquor in each hole in described 96 orifice plates, MTT is added in each hole described and cell under cell under sample effect and reference substance effect is dyeed;
Suck the supernatant liquor in each hole after dyeing, methyl-sulphoxide is added in each hole, and be placed in microplate reader concussion the 3rd Preset Time, under test wavelength, to read under each sample effect cell light absorption value under cell light absorption value and reference substance effect.
Preferably, the described light absorption value by both contrasts, obtain sample to comprise relative to the short cell proliferation rate of reference substance: cell light absorption value under cell light absorption value under each obtained sample effect and reference substance effect is averaged respectively acquisition first light absorption value and the second light absorption value, the first light absorption value and the second light absorption value are substituted into formula and urge to calculate in cell proliferation rate=(the first light absorption value-the second light absorption value)/second light absorption value × 100% to obtain.
The embodiment of the present invention provides a kind of lyophilized powder to urge the detection method of cell proliferation rate.By will the substratum of lyophilized powder to be detected be added with as sample cultivation base, the substratum substratum in contrast of lyophilized powder to be detected will do not added, and regulate the osmotic pressure of described control medium equal with the osmotic pressure of described sample cultivation base, and, at identical conditions the cell of equal amts is cultivated, under making sample effect, cell is identical with the growing environment of cell under reference substance effect, thus can urge cell proliferation rate to lyophilized powder and accurately detect.Overcome the defect cannot carrying out accurately detection in prior art to the short cell proliferation rate of lyophilized powder.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 urgees the schema of the detection method of cell proliferation rate for a kind of lyophilized powder that the embodiment of the present invention provides;
The typical curve of the osmotic pressure Y of a kind of control medium that Fig. 2 provides for the embodiment of the present invention and the N.F,USP MANNITOL addition X in perfect medium.
Embodiment
Now will provide the reference of embodiment of the present invention in detail, one or more example is described in hereafter.The illustratively unrestricted the present invention of each example is provided.In fact, to those skilled in the art, it is evident that, can numerous modifications and variations be carried out to the present invention and not deviate from scope of the present invention or spirit.Such as, as an embodiment part and to illustrate or the feature that describes may be used in another embodiment, produce further embodiment.Therefore, based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Material involved by the embodiment of the present invention all can obtain by commercial sources or by applicant.
The embodiment of the present invention provides a kind of lyophilized powder to urge the detection method of cell proliferation rate, see Fig. 1, comprising:
Step 1) lyophilized powder to be detected is dissolved in perfect medium obtains sample cultivation base, wherein, the sample cultivation base obtained has the first osmotic pressure;
Step 2) will the perfect medium substratum in contrast of lyophilized powder to be detected do not added, and regulate the osmotic pressure of described control medium to equal the first osmotic pressure;
Step 3) respectively the cell of equal amts is cultivated at identical conditions by the sample cultivation base that obtains and control medium, after the first Preset Time, to obtain under sample effect cell under cell and reference substance effect;
Step 4) MTT dyeing and light absorption value detection are carried out to cell under cell under obtained sample effect and reference substance effect, by the light absorption value of both contrasts, obtain the short cell proliferation rate of sample relative to reference substance.
Wherein, MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt, commodity are called tetrazolium bromide) dyeing, also known as MTT colorimetry, is used to the method detecting cell survival and growth; Concrete principle is: the succinodehydrogenase in viable cell plastosome can make exogenous MTT be reduced to water-insoluble bluish voilet crystallization first a ceremonial jade-ladle, used in libation and be deposited in cell, and dead cell is without this function; By the first a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) dissolved cell, measure its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect viable cell quantity.Within the scope of certain cell count, the quantity of absorbance value and viable cell is proportional.
In embodiments of the present invention, the viable cell quantity in sample and reference substance indirectly can be reflected by mtt assay, and, pass through obtained sample light absorption value and reference substance light absorption value can calculate the short cell proliferation rate of sample relative to reference substance, thus the short cell proliferation rate of lyophilized powder can be obtained.
A kind of lyophilized powder that the embodiment of the present invention provides urgees the detection method of cell proliferation rate.By will the substratum of lyophilized powder to be detected be added with as sample cultivation base, the substratum substratum in contrast of lyophilized powder to be detected will do not added, and regulate the osmotic pressure of described control medium equal with the osmotic pressure of described sample cultivation base, and, at identical conditions the cell of equal amts is cultivated, under making sample effect, cell is identical with the growing environment of cell under reference substance effect, thus can urge cell proliferation rate to lyophilized powder and accurately detect.Overcome the defect cannot carrying out accurately detection in prior art to the short cell proliferation rate of lyophilized powder.
Wherein, the empirical value of osmotic pressure that has of substratum per sample, step 1) and step 2) can exchange, do not limit at this, as long as make the osmotic pressure of described control medium equal with described sample cultivation base.
Wherein, described perfect medium possesses the condition making cell Growth and reproduction to greatest extent, described perfect medium according to add serum amount number can be divided into cell growth medium and cell maintain base; Cell growth medium is the use maintaining growth and proliferation of cell, larger containing serum proportion, it consists of: basic medium 80% ~ 90%, serum (multiplex calf serum) 10% ~ 20%, microbiotic (multiplex penicillin 100U/mL and Streptomycin sulphate 100 μ g/mL); Cell maintain base is maintain cell slowly to grow or not dead nutrient solution, and serum content is less, and it consists of: minimum medium 95%, serum 2% ~ 5%, microbiotic (multiplex penicillin 100U/mL and Streptomycin sulphate 100 μ g/mL).
Wherein, the content of serum in described perfect medium is not limited.
In one embodiment of the invention, in described perfect medium, the content of serum is 10%-20%.Due to when adopting described perfect medium to cultivate cell, cell can be caused when serum content is lower in sample not breed even dead, thus make final the cell quantity obtained in sample and reference substance inaccurate, be difficult to the short cell proliferation rate accurately detecting lyophilized powder, therefore, in embodiments of the present invention, cell growth medium is adopted, can avoid cell in culturing process, do not breed even dead phenomenon, thus accurately can detect the short cell proliferation rate of lyophilized powder.
Wherein, the composition of described lyophilized powder to be detected is not limited.
In one embodiment of the invention, described lyophilized powder to be detected at least comprises: human epidermal growth factor hEGF, vascular endothelial growth factor VEGF, fibroblast growth factor FGF, transforming growth factor-beta 1 TGF-β 1, transforminggrowthfactor-β2 TGF-β 2, para-insulin growth factor IGF-1, para-insulin No. two growth factor IGF-2, trehalose, Dextran 40 and N.F,USP MANNITOL.
In actual applications, described lyophilized powder can be the lyophilized powder through concentrating or after uf processing, by detecting the short cell proliferation rate of lyophilized powder after treatment, can obtain the impact of different treatment condition on the short cell proliferation rate of lyophilized powder.
In another embodiment of the present invention, described step 2) in regulate the osmotic pressure of described control medium equal the first osmotic pressure before also comprise: obtain the osmotic pressure Y of described control medium and the funtcional relationship of the addition X of osmotic pressure regulator in described perfect medium.
Wherein, the funtcional relationship of the osmotic pressure Y and the addition X of osmotic pressure regulator in perfect medium that obtain described control medium can obtain by experiment, also can consult related data and obtain, not limit at this.
In one embodiment of the invention, described step 2) in regulate the osmotic pressure of described control medium to equal the first osmotic pressure to comprise: according to funtcional relationship and described first osmotic pressure Y1, calculate the addition X1 of described osmotic pressure regulator, and be that the osmotic pressure regulator of X1 is dissolved in described control medium by obtained addition.
Wherein, do not limit described osmotic pressure regulator, described osmotic pressure regulator can be various salt, also can be lyophilized vaccine.
In one embodiment of the invention, described osmotic pressure regulator is N.F,USP MANNITOL, and the osmotic pressure Y of described control medium and the N.F,USP MANNITOL addition X in perfect medium meets funtcional relationship: Y=5425.9X+336.87.Because described lyophilized powder is in freeze-drying process; in order to reduce the bioactive influence degree of cold condition to biologically active factors; usually in advance in lyophilized powder, lyophilized vaccine is added; and lyophilized vaccine is generally N.F,USP MANNITOL, therefore, in embodiments of the present invention; select N.F,USP MANNITOL as osmotic pressure regulator; can avoid introducing other interfering substances, thus interfering factors in cell cultivation process can be reduced further, improve the accuracy of detected result.
Wherein, do not limit described cell, described cell can be various cell, such as, can be human fibroblasts, also can be animal fibroblast cell.
In one embodiment of the invention, described cell is l cell (L929 cell);
Described l cell obtains as follows:
By l cell Secondary Culture to growing logarithmic phase, suspending with perfect medium and being diluted to 1-10 × 10
4individual/ml, is prepared into cell suspension, and wherein, the serum content in described perfect medium is 10%-20%;
By described cell suspension inoculation in the different holes of 96 orifice plates;
Described 96 orifice plates are cultivated under default culture condition the second Preset Time to cell attachment, obtain l cell.
In embodiments of the present invention, by the l cell of logarithmic phase is seeded in 96 orifice plates, the l cell that quality is homogeneous can be obtained, thus the function cells that quality can be provided homogeneous for sample cultivation base and control medium, improve the accuracy of detected result.
Wherein, do not limit the inoculum density of described l cell on 96 orifice plates, the inoculum density of described l cell is-5000,2000/hole/hole.
Described default culture condition is not limited.In one embodiment of the invention, 96 orifice plates of l cell there are is to be placed in 35-37 DEG C, 3%-5%CO inoculation
2incubator in carry out cultivation 12-24h.
Wherein, described step 3) in respectively cultivation is carried out at identical conditions to the inoblast of equal amts by described sample cultivation base and described control medium and has multiple implementation.
In one embodiment of the invention, described step 3) in respectively cultivation is carried out at identical conditions to the inoblast of equal amts by described sample cultivation base and described control medium and comprises: suck the supernatant liquor in 96 orifice plates at l cell place respectively, described sample cultivation base is added at least 2 holes of described 96 orifice plates, described control medium is added in other at least 2 holes of described 96 orifice plates, the l cell in described 96 orifice plates is cultivated.
In embodiments of the present invention, by cultivating quantity and inoblast identical in quality respectively in direct different holes sample cultivation base and control medium being added described 96 orifice plates, make to drop to minimum to the interfering factors of detected result in whole culturing process, improve the accuracy of detected result; Meanwhile, the fibroblastic operating process obtaining equal amts and quality is simple and convenient, can avoid shifting inoblast and causing cell damage or death.
Wherein, it should be noted that, in actual applications, because 96 orifice plates comprise: 12 holes that 8 rows are laterally arranged in order, therefore, a laterally row of 96 orifice plates is 12 holes, longitudinally one is classified as 8 holes; In order to make whole operating process simply be easy to identification, in one embodiment of the invention, sample cultivation base being added in 8 holes of any row in described 96 orifice plates, control medium being added in 8 holes of another row in described 96 orifice plates.In embodiments of the present invention, sample cultivation base and control medium are all cultivated the cell in 8 holes, carry out compared with cultivation, can reducing metrical error respectively with sample cultivation base and control medium to the cell in 1 hole.
Wherein, do not limit described first Preset Time, in one embodiment of the invention, described first Preset Time is 24-36h.
Wherein, described step 4) in MTT dyeing and light absorption value are carried out to cell under cell under obtained sample effect and reference substance effect detect and do not limit.
In one embodiment of the invention, described step 4) in MTT dyeing and light absorption value are carried out to cell under cell under obtained sample effect and reference substance effect detect and comprise:
Suck the supernatant liquor in each hole in described 96 orifice plates, MTT is added in each hole described and cell under cell under sample effect and reference substance effect is dyeed;
Suck the supernatant liquor in each hole after dyeing, methyl-sulphoxide is added in each hole, and be placed in microplate reader concussion the 3rd Preset Time, under test wavelength, to read under sample effect cell light absorption value under cell light absorption value and reference substance effect.
Wherein, to MTT being added in each hole described, the specific operation process that cell under cell under sample effect and reference substance effect dyes is not limited.
In one embodiment of this invention, MTT is added respectively in each hole described, at 35-37 DEG C, 3%-5%CO
2incubator in hatch 3-5h.
Wherein, do not limit described second Preset Time, preferably, described second Preset Time is 10-20min.
Wherein, do not limit described test wavelength, preferably, determined wavelength is 492nm.
In one embodiment of the invention, the described light absorption value by both contrasts, obtain sample to comprise relative to the short cell proliferation rate of reference substance: cell light absorption value under cell light absorption value under obtained sample effect and reference substance effect is averaged respectively acquisition first light absorption value and the second light absorption value, the first light absorption value and the second light absorption value are substituted into formula and urge to calculate in cell proliferation rate=(the first light absorption value-the second light absorption value)/second light absorption value × 100% to obtain.
Next, by specific embodiment, the present invention will be described.Following examples are only described for the actual interpolation concentration of component each in specific implementation process, and during actual use, the concentration of each component does not form impact to the realization of the object of the invention.
Embodiment 1
1, the preparation of sample cultivation base and control medium:
The preparation of sample cultivation base:
Be dissolved in DMEM substratum by lyophilized powder after concentration, and to be diluted to protein concentration be 100ppm, carry out filtration sterilization with 0.2 μm of millipore filtration, the serum adding 10% wherein obtains sample cultivation base, by described sample cultivation base 4 DEG C of preservations.
The preparation of control medium:
Respectively to the N.F,USP MANNITOL adding 0.5%, 0.92%, 1.5%, 2%, 3%, 5%, 6%, 8% and 10% in DMEM substratum, and survey corresponding osmotic pressure value, specifically see table 1, with the addition of N.F,USP MANNITOL for X-coordinate, osmotic pressure is ordinate zou drawing standard curve, see Fig. 2, obtain the funtcional relationship between osmotic pressure and the addition X of N.F,USP MANNITOL: Y=5425.8X+336.87; In described DMEM substratum, dissolve a certain amount of N.F,USP MANNITOL makes the osmotic pressure of DMEM substratum identical with sample cultivation base, filtration sterilization is carried out with 0.2 μm of millipore filtration, the serum adding 10% wherein obtains control medium, by described control medium 4 DEG C of preservations.
Table 1
2, with the sample cultivation base obtained and control medium, inoblast is cultivated:
Fibroblastic acquisition:
By l cell (L929 cell) Secondary Culture to growing logarithmic phase, being suspended in and being diluted to concentration in DMEM substratum (serum containing 10%) is 1 × 10
4individual/mL, is inoculated in 96 orifice plates, every hole 200 μ L, and every hole contains 2000 cells, at 37 DEG C, 5%CO
2incubator in cultivate 12h, cell attachment is grown, obtain inoblast.
Culturing process:
Suck the supernatant liquor in the 96 each holes of orifice plate, added by sample cultivation base in secondary series 8 holes of described 96 orifice plates, control medium added in the 3rd row 8 holes of described 96 orifice plates, every hole 200 μ L, at 37 DEG C, 5%CO
2incubator in cultivate 24h, to obtain under sample effect cell under cell and reference substance effect.
3, the proliferative conditions of cell under cell under sample effect and reference substance effect is detected:
Suck the secondary series in described 96 orifice plates and the supernatant liquor in tertial 16 holes, added by MTT in 16 holes of described 96 orifice plates respectively, every hole 20 μ L, at 37 DEG C, 5%CO
2incubator in hatch 5h, suck supernatant liquor, added by DMSO in 16 holes of described 96 orifice plates, every hole 150 μ L, is placed in microplate reader and shakes 15min, to detect under sample effect the light absorption value of cell at 492nm place under cell and reference substance effect.
4, repeat above-mentioned steps and obtain three groups of sample light absorption values and reference substance light absorption value three times, the mean value calculating sample light absorption value and reference substance light absorption value in each group respectively obtains the first light absorption value in each group and the second light absorption value, and the first light absorption value in each group and the second light absorption value are substituted into formula respectively: short cell proliferation rate=(the first light absorption value-the second light absorption value)/second light absorption value × 100%, in first group, the first light absorption value is the 1.0819 ± 6.54%, second light absorption value is 1.1406 ± 5.09%; In second group, the first light absorption value is the 0.9862 ± 4.01%, second light absorption value be that in the 1.0276 ± 9.84%, three group, the first light absorption value is the 1.1163 ± 5.31%, second light absorption value is 1.0940 ± 4.93%; Concrete outcome is see table 2:
Table 2
Conclusion: make the osmotic pressure of control medium identical with the osmotic pressure of sample cultivation base by adding N.F,USP MANNITOL in control medium, control medium can be avoided different with sample cultivation base osmotic pressure and the cell culture condition that causes is different, take off data accurately can be obtained, simultaneously, from above testing data: the short cell proliferation rate of the lyophilized powder after concentration is lower, without significantly urging cell-proliferation activity.
Embodiment 2
1, the preparation of sample cultivation base and control medium:
The preparation of sample cultivation base:
Permeate lyophilized powder is dissolved in DMEM substratum, and to be diluted to protein concentration be 50ppm, filtration sterilization is carried out with 0.2 μm of millipore filtration, the serum adding 20% wherein obtains sample cultivation base, the osmotic pressure recording obtained sample cultivation base is 615mOsm/kg, by described sample cultivation base 4 DEG C of preservations.
The preparation of control medium:
Funtcional relationship according between the addition X of osmotic pressure Y and N.F,USP MANNITOL in Fig. 2: Y=5425.8X+336.87; The N.F,USP MANNITOL dissolving 5.13% in described DMEM substratum makes the osmotic pressure of DMEM substratum identical with sample cultivation base, filtration sterilization is carried out with 0.2 μm of millipore filtration, the serum adding 20% wherein obtains control medium, by described control medium 4 DEG C of preservations.
2, with the sample cultivation base obtained and control medium, inoblast is cultivated:
Fibroblastic acquisition:
By l cell (L929 cell) Secondary Culture to growing logarithmic phase, being suspended in and being diluted to concentration in DMEM substratum (serum containing 10%) is 10 × 10
4individual/mL, is inoculated in 96 orifice plates respectively, every hole 300 μ L, and every hole contains 5000 cells, at 35 DEG C, 3%CO
2incubator in cultivate 24h, cell attachment is grown, obtain inoblast.
Culturing process:
Suck the supernatant liquor in the 96 each holes of orifice plate, added by sample cultivation base in first row 8 holes of described 96 orifice plates, control medium added in the 5th row 8 holes of described 96 orifice plates, every hole 300 μ L, at 35 DEG C, 3%CO
2incubator in cultivate 36h, to obtain under sample effect cell under cell and reference substance effect.
3, the proliferative conditions of cell under cell under sample effect and reference substance effect is detected:
Suck the first row in described 96 orifice plates and the supernatant liquor in the 5th 16 holes arranged, added by MTT in 16 holes of described 96 orifice plates respectively, every hole 20 μ L, at 35 DEG C, 3%CO
2incubator in hatch 3h, suck supernatant liquor, added by DMSO in 16 holes of described 96 orifice plates, every hole 150 μ L, is placed in microplate reader and shakes 15min, to detect under sample effect the light absorption value of cell at 492nm place under cell and reference substance effect.
4, repeat above-mentioned steps and obtain three groups of sample light absorption values and reference substance light absorption value three times, the mean value calculating sample light absorption value and reference substance light absorption value in each group respectively obtains the first light absorption value in each group and the second light absorption value, and the first light absorption value in each group and the second light absorption value are substituted into formula respectively: short cell proliferation rate=(the first light absorption value-the second light absorption value)/second light absorption value × 100%, in first group, the first light absorption value is the 0.8299 ± 1.22%, second light absorption value is 0.9014 ± 2.99%; In second group, the first light absorption value is the 0.8204 ± 2.22%, second light absorption value be that in the 0.7698 ± 2.46%, three group, the first light absorption value is the 0.7988 ± 1.35%, second light absorption value is 0.7585 ± 1.42%; Concrete outcome is see table 3:
Table 3
| Experiment number | First light absorption value | Second light absorption value | Short cell proliferation rate |
| First group | 0.8299±1.22％ | 0.9014±2.99％ | -7.93％ |
| Second group | 0.8204±2.22％ | 0.7698±2.46％ | +6.57％ |
| 3rd group | 0.7988±1.35％ | 0.7585±1.42％ | +5.31％ |
Conclusion: make the osmotic pressure of control medium identical with the osmotic pressure of sample cultivation base by adding N.F,USP MANNITOL in control medium, control medium can be avoided different with sample cultivation base osmotic pressure and the cell culture condition that causes is different, take off data accurately can be obtained, simultaneously, lower from the short cell proliferation rate of the permeate lyophilized powder of above testing data: 50ppm, without significantly urging cell-proliferation activity.
Embodiment 3
1, the preparation of sample cultivation base and control medium:
The preparation of sample cultivation base:
Permeate lyophilized powder is dissolved in DMEM substratum, and to be diluted to protein concentration be 100ppm, filtration sterilization is carried out with 0.2 μm of millipore filtration, the serum adding 15% wherein obtains sample cultivation base, the osmotic pressure recording obtained sample cultivation base is 887mOsm/kg, by described sample cultivation base 4 DEG C of preservations.
The preparation of control medium:
Funtcional relationship according between the addition X of osmotic pressure Y and N.F,USP MANNITOL in Fig. 2: Y=5425.8X+336.87; The N.F,USP MANNITOL dissolving 10.14% in described DMEM substratum makes the osmotic pressure of DMEM substratum identical with sample cultivation base, filtration sterilization is carried out with 0.2 μm of millipore filtration, the serum adding 15% wherein obtains control medium, by described control medium 4 DEG C of preservations.
2, with the sample cultivation base obtained and control medium, inoblast is cultivated:
Fibroblastic acquisition:
To l cell (L929 cell) Secondary Culture to growing logarithmic phase, being suspended in and being diluted to concentration in DMEM substratum (serum containing 10%) is 5 × 10
4individual/mL, is inoculated in 96 orifice plates respectively, every hole 250 μ L, and every hole contains 2500 cells, at 36 DEG C, 4%CO
2incubator in cultivate 16h, cell attachment is grown, obtain inoblast.
Culturing process:
Suck the supernatant liquor in the 96 each holes of orifice plate, added by sample cultivation base in the 3rd row 8 holes of described 96 orifice plates, control medium added in the 4th row 8 holes of described 96 orifice plates, every hole 250 μ L, at 36 DEG C, 4%CO
2incubator in cultivate 32h, to obtain under sample effect cell under cell and reference substance effect.
3, the proliferative conditions of cell under cell under sample effect and reference substance effect is detected:
Suck the 3rd row in described 96 orifice plates and the supernatant liquor in the 4th 16 holes arranged, added by MTT in 16 holes of described 96 orifice plates respectively, every hole 20 μ L, at 36 DEG C, 4%CO
2incubator in hatch 4h, suck supernatant liquor, added by DMSO in 16 holes of described 96 orifice plates, every hole 150 μ L, is placed in microplate reader and shakes 15min, to detect under sample effect the light absorption value of cell at 492nm place under cell and reference substance effect.
4, repeat above-mentioned steps and obtain three groups of sample light absorption values and reference substance light absorption value three times, the mean value calculating sample light absorption value and reference substance light absorption value in each group respectively obtains the first light absorption value in each group and the second light absorption value, and the first light absorption value in each group and the second light absorption value are substituted into formula respectively: short cell proliferation rate=(the first light absorption value-the second light absorption value)/second light absorption value × 100%, in first group, the first light absorption value is the 0.3643 ± 2.59%, second light absorption value is 0.2652 ± 2.98%; In second group, the first light absorption value is the 0.3222 ± 2.10%, second light absorption value be that in the 0.2424 ± 0.84%, three group, the first light absorption value is the 0.3029 ± 1.32%, second light absorption value is 0.2286 ± 1.02%; Concrete outcome is see table 4:
Table 4
| Experiment number | First light absorption value | Second light absorption value | Short cell proliferation rate |
| First group | 0.3643±2.59％ | 0.2652±2.98％ | +37.37％ |
| Second group | 0.3222±2.10％ | 0.2424±0.84％ | +32.92％ |
| 3rd group | 0.3029±1.32％ | 0.2286±1.02％ | +32.50％ |
Conclusion: make the osmotic pressure of control medium identical with the osmotic pressure of sample cultivation base by adding N.F,USP MANNITOL in control medium, control medium can be avoided different with sample cultivation base osmotic pressure and the cell culture condition that causes is different, take off data accurately can be obtained, simultaneously, higher from the short cell proliferation rate of the permeate lyophilized powder of above testing data: 100ppm, there is short cell-proliferation activity preferably.
In sum, by will the substratum of lyophilized powder to be detected be added with as sample cultivation base, the substratum substratum in contrast of lyophilized powder to be detected will do not added, and regulate the osmotic pressure of described control medium equal with the osmotic pressure of described sample cultivation base, and, cultivate the cell of equal amts at identical conditions, under making sample effect, cell is identical with the growing environment of cell under reference substance effect, thus can urge cell proliferation rate to lyophilized powder and accurately detect.Overcome the defect cannot carrying out accurately detection in prior art to the short cell proliferation rate of lyophilized powder.
The above; be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; change can be expected easily or replace, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of described claim.
Claims (10)
1. lyophilized powder urgees a detection method for cell proliferation rate, it is characterized in that, comprising:
Step 1) lyophilized powder to be detected is dissolved in perfect medium obtains sample cultivation base, wherein, described sample cultivation base has the first osmotic pressure;
Step 2) will the perfect medium substratum in contrast of lyophilized powder to be detected do not added, and regulate the osmotic pressure of described control medium to equal the first osmotic pressure;
Step 3) respectively the cell of equal amts is cultivated at identical conditions by the sample cultivation base that obtains and control medium, after the first Preset Time, to obtain under sample effect cell under cell and reference substance effect;
Step 4) MTT dyeing and light absorption value detection are carried out to cell under cell under obtained sample effect and reference substance effect, by the light absorption value of both contrasts, obtain the short cell proliferation rate of sample relative to reference substance.
2. detection method according to claim 1, is characterized in that, the serum content in described perfect medium is 10%-20%.
3. detection method according to claim 1, it is characterized in that, described lyophilized powder to be detected at least comprises: human epidermal growth factor hEGF, vascular endothelial growth factor VEGF, fibroblast growth factor FGF, transforming growth factor-beta 1 TGF-β 1, transforminggrowthfactor-β2 TGF-β 2, para-insulin growth factor IGF-1, para-insulin No. two growth factor IGF-2, trehalose, Dextran 40 and N.F,USP MANNITOL.
4. detection method according to claim 1, it is characterized in that, described step 2) in regulate the osmotic pressure of described control medium equal the first osmotic pressure before also comprise: obtain the osmotic pressure Y of described control medium and the funtcional relationship of the addition X of osmotic pressure regulator in perfect medium.
5. detection method according to claim 4, it is characterized in that, described step 2) in regulate the osmotic pressure of described control medium to equal the first osmotic pressure to comprise: according to obtained funtcional relationship and described first osmotic pressure Y1, calculate the addition X1 of described osmotic pressure regulator, and be that the osmotic pressure regulator of X1 is dissolved in described control medium by obtained addition.
6. detection method according to claim 4, is characterized in that, described osmotic pressure regulator is N.F,USP MANNITOL, and the osmotic pressure Y of described control medium and the N.F,USP MANNITOL addition X in perfect medium meets funtcional relationship: Y=5425.9X+336.87.
7. detection method according to claim 1, is characterized in that, described cell is l cell;
Described l cell obtains as follows:
By l cell Secondary Culture to growing logarithmic phase, suspending with perfect medium and being diluted to 1-10 × 10
4individual/ml, is prepared into cell suspension, and wherein, the serum content in described perfect medium is 10%-20%;
Described cell suspension is inoculated in identical density in the different holes of described 96 orifice plates;
Described 96 orifice plates are cultivated under default culture condition the second Preset Time to cell attachment, obtain l cell.
8. detection method according to claim 7, it is characterized in that, described step 3) in respectively cultivation is carried out at identical conditions to the cell of equal amts by described sample cultivation base and described control medium and comprises: suck the supernatant liquor in 96 orifice plates at obtained l cell place, described sample cultivation base is added at least 2 holes of described 96 orifice plates, described control medium is added in other at least 2 holes of described 96 orifice plates, the l cell in described 96 orifice plates is cultivated.
9. detection method according to claim 8, is characterized in that, described step 4) in MTT dyeing and light absorption value are carried out to cell under cell under obtained sample effect and reference substance effect detect and comprise:
Suck the supernatant liquor in each hole in described 96 orifice plates, MTT is added in each hole described and cell under cell under sample effect and reference substance effect is dyeed;
Suck dyed after each hole in supernatant liquor, methyl-sulphoxide is added in each hole, and is placed in microplate reader concussion the 3rd Preset Time, under test wavelength, to read under sample effect cell light absorption value under cell light absorption value and reference substance effect.
10. detection method according to claim 9, it is characterized in that, the described light absorption value by both contrasts, obtain sample to comprise relative to the short cell proliferation rate of reference substance: cell light absorption value under cell light absorption value under obtained sample effect and reference substance effect is averaged respectively acquisition first light absorption value and the second light absorption value, the first light absorption value and the second light absorption value are substituted into formula and urge to calculate in cell proliferation rate=(the first light absorption value-the second light absorption value)/second light absorption value × 100% to obtain.
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| CN109554431A (en) * | 2017-09-26 | 2019-04-02 | 东莞自然衡健康科技有限公司 | One kind evaluating the active method of hair tonic freeze-dried powder by hair follicle stem cells in-vitro multiplication |
| CN111562210A (en) * | 2020-06-16 | 2020-08-21 | 北京挑战农业科技有限公司 | Method for detecting viable count in pre-coated forage microecological preparation product |
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| CN109554431A (en) * | 2017-09-26 | 2019-04-02 | 东莞自然衡健康科技有限公司 | One kind evaluating the active method of hair tonic freeze-dried powder by hair follicle stem cells in-vitro multiplication |
| CN111562210A (en) * | 2020-06-16 | 2020-08-21 | 北京挑战农业科技有限公司 | Method for detecting viable count in pre-coated forage microecological preparation product |
| CN111562210B (en) * | 2020-06-16 | 2023-01-03 | 北京挑战农业科技有限公司 | Method for detecting number of viable bacteria in pre-coated feed microecological preparation product |
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