CN102344904B - Porcine cells medium - Google Patents
Porcine cells medium Download PDFInfo
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- CN102344904B CN102344904B CN201010242482.3A CN201010242482A CN102344904B CN 102344904 B CN102344904 B CN 102344904B CN 201010242482 A CN201010242482 A CN 201010242482A CN 102344904 B CN102344904 B CN 102344904B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
Abstract
The invention belongs to the cell biology field, and relates to a porcine cells medium which is composed of a basic medium and porcine fetus serum. The invention also relates to a method for cultivating the porcine cells, a type of porcine fetus serum and an application of the porcine fetus serum in cultivation of the porcine cells or preparation of porcine cells medium.
Description
Technical field
The invention belongs to cytobiology field, relate to a kind of porcine cells medium, it comprises basic medium and tire porcine blood serum.The invention still further relates to a kind of method of cultivating pig cell, a kind of tire porcine blood serum and described tire porcine blood serum are being cultivated pig cell or are preparing the purposes in porcine cells medium.
Background technology
Substratum is the basic substance of supplying with cytotrophy in cell cultures and impelling germiparity propagation, is also the living environment of Growth of Cells and breeding.The kind of substratum is a lot, by its state of matter, is divided into semisolid medium and substratum two classes; By its source, be divided into synthetic medium and natural medium.Main use is the perfect medium that adds natural component to form in synthetic medium at present.
Perfect medium is comprised of basic medium (as MEM) and added ingredients (as serum, or for the substratum of serum-free, using hormone and somatomedin).
Basic medium usually will add serum, and serum final concentration mostly is 5%-20% (v/v).The serum source of special purpose must be definite by experience, and the serum kind of widespread use has horse serum and foetal calf serum.In foetal calf serum, be rich in mitotic factor, Chang Xuanqi does cell proliferation, and also for clone and former culture, and the neurone that horse serum is usually used for after mitotic division is cultivated.
At present, porcine cells medium is mainly the perfect medium consisting of basic medium DMEM and/or F12 and foetal calf serum, and meanwhile, this substratum is also widely used in the cultivation of other kind cell.Foetal calf serum source is comparatively easy, and output is larger, is the serum generally using.But the rate of propagation of cell growth state and cell is all comparatively limited when pig cell is cultivated on this kind of substratum, the passage number of cell has also been subject to very large impact.At present aspect the cell cultures of pig, still lacking comparatively desirable suitable substratum.
To this, the inventor has developed a kind of porcine cells medium, wherein in basic medium, adds tire porcine blood serum to substitute foetal calf serum, to get rid of the rejection of using xenogenesis composition that foetal calf serum causes to cause.The present invention, can also add in described pig cell and be selected from the interpolation factor of at least one as follows: Urogastron (EGF), Prostatropin (bFGF), human chorionic gonadotrophin (HCG), pregnant mare serum gonadotrop(h)in (PMSG) (PMSG).
Adopt porcine cells medium of the present invention can significantly improve the cultivation conditions of pig cell and the propagation level of raising pig cell, be conducive to carry out with the cell of pig correlative study and the application of transgene clone and inductive pluripotent stem cells.
Summary of the invention
One aspect of the present invention relates to a kind of porcine cells medium, and it comprises basic medium and tire porcine blood serum, and according to the cumulative volume of substratum, the content of described tire porcine blood serum is 5%-25% (v/v).In one embodiment of the invention, the content of the porcine blood serum of tire described in porcine cells medium is 8-12% (v/v).In yet another embodiment of the present invention, the content of the porcine blood serum of tire described in porcine cells medium is 10-12% (v/v).
In the present invention, pig cell refers to the cell of the various positions different growing stage that derives from pig individuality.Porcine cells medium of the present invention is applicable to the various cells from the individual different growing stage of pig, includes but not limited to: primary pig inoblast, porcine kidney cell, pig myocardium cell, porcine oocytes, pig vascular endothelial cell etc.Wherein, the strain of the pig in described " pig cell " and " tire porcine blood serum " is not particularly limited, commonly family pig.
Described tire porcine blood serum can make as follows:
1) serum is separated: the blood sample available from pig tire is carried out centrifugal, obtain serum;
2) serum freezing de-fibre (being de-fibering) at-20 ℃ that freezing de-fibre: by step 1), centrifugation obtains;
3) clarification filtration: by step 2), obtaining sample uses Cai Shi Mierocrystalline cellulose degree of depth filter to filter;
4) sample serum dialysis: by step 3) obtaining is dialysed in 0.15M NaCl solution with the dialysis membrane of molecular weight 12000-14000, until the glucose content < 0.5mg/mL of sample;
5) sample gammairradiation: by step 4) obtaining carries out gammairradiation, and irradiation dose is 1.2-4Gy, obtains rough serum; With
6) the rough serum obtaining Sterile Filtration: successively use 0.2 micron and 0.1 micron of filter membrane, by step 5) carries out filter membrane Sterile Filtration again.
Above-mentioned steps 1) in, the pig tire in the preferred 3-4 of described pig tire age in week.
In one embodiment of the invention, the preparation method of described tire porcine blood serum also comprises step 7) and 8):
7) uviolizing: use uviolizing before bottling;
8) encapsulation is preserved: by preceding step 7) the tire porcine blood serum bottling sealing that makes ,-20 ℃ of cryopreservation, before using, 4 ℃ are spent the night and thaw.
In one embodiment of the invention, the basic medium in described porcine cells medium is DMEM or F12 or the artificial medium that forms as shown in Table 1 below:
Table 1: artificial culture based formulas
| Compound title | Content (mg/L) |
| Calcium Chloride Powder Anhydrous | 200.00-240.00 |
| Serine | 42.00-55.00 |
| L-Trp | 16.00-20.00 |
| L-threonine | 95.00-110.00 |
| TYR sodium salt | 104.00-123.00 |
| Iron nitrate | 0.10-0.40 |
| Repone K | 400.00-600.00 |
| Valine | 94.00-112.00 |
| L-arginine hydrochloride | 84.00-98.00 |
| L-glutaminate | 584.00-628.00 |
| L-hydrochloric acid Gelucystine | 63.00-72.00 |
| L-Leu | 95.00-110.00 |
| ILE | 93.00-105.00 |
| L-histidine monohydrochloride | 32.00-52.00 |
| LYS | 126.00-156.00 |
| METHIONINE | 25.00-40.00 |
| L-Phe | 46.00-78.00 |
| Anhydrous magnesium sulfate | 87.67-99.60 |
| Sodium-chlor | 4400.00-6700.00 |
| AMSP | 105.00-145.00 |
| D-VB5 calcium | 2.00-6.00 |
| Choline chloride 60 | 2.00-5.50 |
| Folic acid | 3.00-5.50 |
| Inositol | 5.20-8.30 |
| Glycine | 20.00-40.00 |
| Niacinamide | 3.00-5.00 |
| Riboflavin | 0.20-0.60 |
| Thiamine hydrochloride | 2.00-5.00 |
| Pyridoxine hydrochloride | 3.00-6.00 |
| Glucose | 3500.00-5500.00 |
| Sodium.alpha.-ketopropionate | 100.00-120.00 |
| Phenol red | 14.00-16.00 |
In one embodiment of the invention, described porcine cells medium also comprises the interpolation factor.In the present invention, term " the interpolation factor " refers to and is selected from following at least one: 1) Urogastron (EGF), for example M-EGF (as: Sigma E1257); 2) Prostatropin (bFGF), for example bFGF-2 (as Sigma F5392); Human chorionic gonadotrophin (HCG) (as Sigma C0684) and pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) (as Sigma G4877).
In one embodiment of the invention, in described porcine cells medium, add kind and the content of the factor as shown in table 2 independently of one another.
Table 2 adds kind and the content thereof of the factor:
| Title | Content |
| Urogastron (EGF) | 3-15ng/mL |
| Prostatropin (bFGF) | 2-15ng/mL |
| Human chorionic gonadotrophin (HCG) | 4-30IU/mL |
| Pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) | 2-28IU/mL |
In one embodiment of the invention, in described porcine cells medium, add kind and the content of the factor as shown in table 3 independently of one another.
Table 3 adds kind and the content thereof of the factor
| Title | Content |
| Urogastron (EGF) | 8.5-12.6ng/mL |
| Prostatropin (bFGF) | 6.6-11.8ng/mL |
| Human chorionic gonadotrophin (HCG) | 16.7-23.4IU/mL |
| Pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) | 17.3-22.6IU/mL |
In the present invention, described pig cell can be to be selected from a kind of in following cell: primary pig inoblast, porcine kidney cell, pig myocardium cell, porcine oocytes, pig vascular endothelial cell.
Porcine cells medium of the present invention can for example, be prepared with reference to the method for conventional cell culture medium (DMEM cell culture medium), for example can be with reference to step below:
1) obtain solution
For example, to basic medium powder (DMEM substratum), add a part for institute's water requirement (aseptic ultrapure water), dissolve in advance, then according to the volume of final substratum, then add the tire porcine blood serum of 5%-25%, finally supply institute's water requirement.
If liquid D MEM substratum, directly, to the tire porcine blood serum that adds final volume 5%-25% in container, mix.For example: configuration 100mL DMEM perfect medium, can in 90mLDMEM substratum, add 10mL tire porcine blood serum, mix.
Then according to the final volume of substratum, add the interpolation factor, it is selected from least one in following material:
3-15ng/mL Urogastron (EGF), 2-15ng/mL Prostatropin (FGF), 4-30IU/mL human chorionic gonadotrophin (HCG), 2-28IU/mL pregnant mare serum gonadotrop(h)in (PMSG) (PMSG), mix.
2) regulate pH value
By the pH value of pH test paper (or pH potentiometer, hydrogen ion concentration colorimeter) test media, as do not meet needs, available 10%HCl or 10%NaOH regulate, until be adjusted to the pH value that formula requires, and generally between 6.9-7.5.
Optionally, the compound method of porcine cells medium of the present invention also comprises the steps:
3) packing
For the substratum configuring, should carry out packing according to experiment real needs situation, within general minute, install in 50mL centrifuge tube.After packing, from each centrifuge tube, extract 1mL and be placed in 35mm Tissue Culture Dish, put it in cell culture incubator and cultivate and within two days, observe whether there is the appearance of pollution, if do not had, can use; If there is pollution, need to reconfigure.
4) sealing
With sealed membrane, will the 50mL centrifuge tube mouth of pipe sealing of substratum be housed.
Another aspect of the present invention relates to a kind of method of cultivating pig cell, it is characterized in that using porcine cells medium of the present invention.Wherein, described pig cell includes but not limited to: primary pig inoblast, porcine kidney cell, pig myocardium cell, porcine oocytes, pig vascular endothelial cell etc.
Another aspect of the present invention relates to a kind of tire porcine blood serum being prepared as follows:
1) serum is separated: the blood sample available from pig tire is carried out centrifugal, obtain serum;
2) serum freezing de-fibre (being de-fibering) at-20 ℃ that freezing de-fibre: by step 1), centrifugation obtains;
3) clarification filtration: by step 2), obtaining sample uses Cai Shi Mierocrystalline cellulose degree of depth filter to filter;
4) sample serum dialysis: by step 3) obtaining is dialysed in 0.15M NaCl solution with the dialysis membrane of molecular weight 12000-14000, until the glucose content < 0.5mg/mL of sample;
5) sample gammairradiation: by step 4) obtaining carries out gammairradiation, and irradiation dose is 1.2-4Gy, obtains rough serum; With
6) the rough serum obtaining Sterile Filtration: successively use 0.2 micron and 0.1 micron of filter membrane, by step 5) carries out filter membrane Sterile Filtration again.
In one embodiment of the invention, the preparation method of described tire porcine blood serum also comprises step 7) and 8):
7) uviolizing: use uviolizing before bottling;
8) encapsulation is preserved: by preceding step 7) the tire porcine blood serum bottling sealing that makes ,-20 ℃ of cryopreservation, before using, 4 ℃ are spent the night and thaw.
In the present invention, described pig tire is the pig tire of putting to death, and the preferred pig tire in 3-4 age in week, and blood sample can be collected with reference to the acquisition mode of conventional foetal calf serum.
The tire porcine blood serum prepared as aforesaid method of also relating in one aspect to of the present invention is cultivated and/or prepares the purposes in porcine cells medium at pig cell.Wherein, described pig cell includes but not limited to: primary pig inoblast, porcine kidney cell, pig myocardium cell, porcine oocytes, pig vascular endothelial cell etc.
The beneficial effect of the invention
1) improve cell growth state:
No matter whether use the interpolation factor, adopt porcine cells medium of the present invention (containing basic medium and tire porcine blood serum) to cultivate pig cell, the growthhabit of pig cell is obviously better than adopting the situation of the traditional substratum that contains basic medium and foetal calf serum.
2) improve cell proliferation rate:
No matter whether use the interpolation factor, adopt porcine cells medium of the present invention (containing basic medium and tire porcine blood serum) to cultivate pig cell, the rate of propagation of pig cell is obviously better than adopting the situation of the traditional substratum that contains basic medium and foetal calf serum.
This illustrates that porcine cells medium of the present invention is being better than the tradition system substratum that contains basic medium and foetal calf serum aspect the rate of propagation of raising pig cell.
Accompanying drawing explanation
Fig. 1: the fibroblastic aspect graph of primary pig (200X) that culture medium A is cultivated.
Fig. 2: the fibroblastic aspect graph of primary pig (200X) that substratum B cultivates.
Fig. 3: the fibroblastic aspect graph of primary pig (200X) that culture medium C is cultivated.
Fig. 4: the fibroblastic growth curve chart of primary pig that culture medium A-C cultivates.
Fig. 5: the fibroblastic karyotyping figure of primary pig (200X) that culture medium A is cultivated
Fig. 6: the fibroblastic karyotyping figure of primary pig (200X) that substratum B cultivates
Fig. 7: the fibroblastic karyotyping figure of primary pig (200X) that culture medium C is cultivated
Fig. 8: the fibroblastic growth curve chart of primary pig that substratum D-G cultivates.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that the following examples are only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition (such as with reference to works such as J. Pehanorm Brookers, the < < molecular cloning experiment guide > > that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1: the formula of porcine cells medium A, B, C
1) formula of porcine cells medium A:
Porcine cells medium A is comprised of artificial medium 1 (component is as shown in table 4), 5% (v/v) tire porcine blood serum and interpolation combinations of factors 1 (component is as shown in table 5).
Table 4 artificial medium 1 formula:
| Title | Content (mg/L) |
| Calcium Chloride Powder Anhydrous | 200.00 |
| Serine | 42.00 |
| L-Trp | 16.00 |
| L-threonine | 95.00 |
| TYR sodium salt | 104.00 |
| Iron nitrate | 0.10 |
| Repone K | 400.00 |
| Valine | 94.00 |
| L-arginine hydrochloride | 84.00 |
| L-glutaminate | 584.00 |
| L-hydrochloric acid Gelucystine | 63.00 |
| L-Leu | 95.00 |
| ILE | 93.00 |
| L-histidine monohydrochloride | 32.00 |
| LYS | 126.00 |
| METHIONINE | 25.00 |
| L-Phe | 46.00 |
| Anhydrous magnesium sulfate | 87.67 |
| Sodium-chlor | 4400.00 |
| AMSP | 105.00 |
| D-VB5 calcium | 2.00 |
| Choline chloride 60 | 2.00 |
| Folic acid | 3.00 |
| Inositol | 5.20 |
| Glycine | 20.00 |
| Niacinamide | 3.00 |
| Riboflavin | 0.20 |
| Thiamine hydrochloride | 2.00 |
| Pyridoxine hydrochloride | 3.00 |
| Glucose | 3500.00 |
| Sodium.alpha.-ketopropionate | 100.00 |
| Phenol red | 14.00 |
Table 5 adds combinations of factors 1 formula
| Title | Content |
| M-EGF (EGF) (Sigma E1257) | 3ng/mL |
| BFGF-2 (bFGF) (Sigma F5392) | 2ng/mL |
| Human chorionic gonadotrophin (HCG) (Sigma C0684) | 4IU/mL |
| Pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) (Sigma G4877) | 2IU/mL |
The preparation method who partly introduces according to specification sheets summary of the invention, dissolves artificial medium 1 use sterile purified water, according to formula rate, adds tire porcine blood serum, after adding sterile purified water extremely volume required, then adds interpolation combinations of factors 1.So make porcine cells medium A.
2) formula of porcine cells medium B:
Composition and the culture medium A of porcine cells medium B are basic identical, just wherein use the foetal calf serum of same concentrations to replace tire porcine blood serum.
3) formula of porcine cells medium C:
Formula and the culture medium A of porcine cells medium C are basic identical, just wherein do not contain and add factor EGF, bFGF, HCG and PMSG.
Embodiment 2: the formula of porcine cells medium D, E, F, G
1) formula of porcine cells medium D:
Porcine cells medium D is comprised of artificial medium 2 (component is as shown in table 6), 25% (v/v) tire porcine blood serum and interpolation combinations of factors 2 (component is as shown in table 7).
Table 6 artificial medium 2 formulas:
| Compound title | Content (mg/L) |
| Calcium Chloride Powder Anhydrous | 240.00 |
| Serine | 55.00 |
| L-Trp | 20.00 |
| L-threonine | 110.00 |
| TYR sodium salt | 123.00 |
| Iron nitrate | 0.40 |
| Repone K | 600.00 |
| Valine | 112.00 |
| L-arginine hydrochloride | 98.00 |
| L-glutaminate | 628.00 |
| L-hydrochloric acid Gelucystine | 72.00 |
| L-Leu | 110.00 |
| ILE | 105.00 |
| L-histidine monohydrochloride | 52.00 |
| LYS | 156.00 |
| METHIONINE | 40.00 |
| L-Phe | 78.00 |
| Anhydrous magnesium sulfate | 99.60 |
| Sodium-chlor | 6700.00 |
| AMSP | 145.00 |
| D-VB5 calcium | 6.00 |
| Choline chloride 60 | 5.50 |
| Folic acid | 5.50 |
| Inositol | 8.30 |
| Glycine | 40.00 |
| Niacinamide | 5.00 |
| Riboflavin | 0.60 |
| Thiamine hydrochloride | 5.00 |
| Pyridoxine hydrochloride | 6.00 |
| Glucose | 5500.00 |
| Sodium.alpha.-ketopropionate | 120.00 |
| Phenol red | 16.00 |
Table 7 adds combinations of factors 2 formulas
| Title | Content |
| M-EGF (EGF) (Sigma E1257) | 15ng/mL |
| BFGF-2 (bFGF) (Sigma F5392) | 15ng/mL |
| Human chorionic gonadotrophin (HCG) (Sigma C0684) | 18IU/mL |
| Pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) (Sigma G4877) | 20IU/mL |
2) formula of substratum E:
The formula of substratum E and substratum D are basic identical, just wherein use the foetal calf serum of same concentrations to replace tire porcine blood serum.
3) formula of substratum F:
The formula of substratum F and substratum D are basic identical, just wherein do not contain and add factor EGF, bFGF, HCG and PMSG.
4) formula of substratum G:
The formula of substratum G is identical with substratum F, just wherein uses the foetal calf serum of same concentrations to replace tire porcine blood serum.
Embodiment 3: the fibroblastic preparation of primary pig
Primary pig inoblast can be prepared with traditional enzyme digestion, and step is as follows:
1) will put to death the pig tire of (putting to death in latter 10 hours), with 70% alcohol, clean three times fast, put into the dual anti-solution of PBS+;
2) in Bechtop, use PBS rinsing clean on pig tire, remove head, four limbs and the internal organ of pig tire;
3) in diameter 60mm plate by step 2) pig tire tissue chopping after processing, transfer organizes fragment to 50mL Erlenmeyer flask, further shreds and becomes 1mm
3the fragment of size, takes a morsel and organizes fragment to be laid in 100mm culture dish, adds 10mL nutrient solution (DMEM+15%FBS+ is dual anti-), puts into CO
2incubator is cultivated (tissue block attaching method); Remaining tissue fragment adds 0.25% tryptic digestive juice of 39 ℃ of preheatings of 3-5mL, puts into CO
2incubator digestion 15-30 minute, took out and stirs every 5 minutes, and took a morsel in micro-Microscopic observation digestion situation;
4) after digestion, add the dual anti-termination digestion of 15mL DMEM+15%FBS+, 1000rpm, centrifugal 10 minutes, abandons supernatant;
5) cell getting off with the resuspended digestion of DMEM and indigested tissue block;
6) with 100 eye mesh screens or with two layers of sterile gauze, filter digestion product, collect filtrate in 50mL centrifuge tube, 1000rpm, centrifugal 5 minutes, collecting cell;
7) use nutrient solution (DMEM+15%FBS) centrifuge washing once, diluting cells to concentration is about 10 again
7individual cell/mL.
In the present embodiment, described dual anti-concentration 100IU/mL penicillin+100mg/mL Streptomycin sulphate that all refers to.
Embodiment 4: the primary fibroblastic form of pig, growth curve and karyotyping that porcine cells medium A, B, C cultivate
Use respectively culture medium A, B, the C of preparation in embodiment 1 to cultivate the primary pig inoblast of preparation in embodiment 3, with identical inoculum density, seed cells in the culture dish of diameter 100mm, be placed in 39 ℃, 5%CO
2in incubator, cultivate.Different except substratum, other culture condition is all identical.
After pig cell converges to 80%, go down to posterity, go down to posterity 15 times, and each clone (the primary inoblast of pig of cultivating with culture medium A, B, C respectively) is carried out to form, growth curve and karyotyping.
Conclusion is as follows:
1) morphological analysis:
Cultivation results shows: under identical culture condition, adopt porcine cells medium A cultured cells density higher than traditional substratum B cultured cells density, illustrate and adopt porcine cells medium of the present invention to be better than traditional porcine cells medium.
Simultaneously, adopt porcine cells medium A cultured cells to be spindle shape, adherent ability is better, growth conditions is significantly better than adopting porcine cells medium C cultured cells, illustrate the cell effect of the porcine cells medium of the present invention that adopts tire porcine blood serum and the interpolation factor to be better than the porcine cells medium of the present invention (referring to accompanying drawing 1-3) that only adopts tire porcine blood serum.
2) growth curve analysis (MTT colorimetric determination):
For adopting above-mentioned three kinds of porcine cells mediums to cultivate the pig cell growth curve of 0-16 days as described in accompanying drawing 4, wherein the growth conditions optical density value of mtt assay detection porcine cells medium A, B, C cultured cells is as shown in table 8.
Table 8:MTT method detects the growth conditions optical density value of porcine cells medium A, B, C cultured cells
| The 2nd day | The 4th day | The 6th day | The 8th day | The 10th day | The 12nd day | The 14th day | The 16th day | |
| Culture medium A | 0.35 | 0.46 | 0.55 | 0.46 | 0.41 | 0.43 | 0.33 | 0.36 |
| Substratum B | 0.29 | 0.39 | 0.49 | 0.37 | 0.35 | 0.36 | 0.29 | 0.29 |
| Culture medium C | 0.12 | 0.28 | 0.21 | 0.22 | 0.21 | 0.23 | 0.19 | 0.23 |
Remarks: start to detect once for every 2 days from cell cultures, detect altogether 8 times.
As seen from Figure 4:
The primary pig inoblast that adopts porcine cells medium C to cultivate, its growth conditions optical density value is all lower than adopting porcine cells medium A, B cultured cells, the porcine cells medium of the present invention that only adopts tire porcine blood serum and do not adopt the interpolation factor is described, it is the traditional substratum not as employing foetal calf serum and the interpolation factor to the growth result of pig cell;
And although employing porcine cells medium A cultured cells is similar to the growth tendency of porcine cells medium B cultured cells growth curve, but the primary pig inoblast that adopts porcine cells medium A to cultivate, its growth conditions optical density value all in the identical time all higher than substratum B cultured cells (referring to accompanying drawing 4).The porcine cells medium of the present invention that adopts tire porcine blood serum and add the factor is described, traditional substratum that its growth result to pig cell is better than adopting foetal calf serum and adds the factor.
3) karyotyping:
The primary pig inoblast that adopts porcine cells medium A, B, C to cultivate, its caryogram is all comparatively normal, does not occur the phenomenons (referring to accompanying drawing 5-7) such as shortening, adhesion.
To sum up, employing porcine cells medium A cultured cells growth conditions is good, the speed of growth very fast, adherent rate is higher, and cell chromosome caryogram is normal, and porcine cells medium A of the present invention is the new and improved substratum that is better than traditional substratum.
Embodiment 5: the primary fibroblastic form of pig, growth curve and karyotyping that porcine cells medium D, E, F, G cultivate
Use respectively substratum D, E, F, the G of preparation in embodiment 2 to cultivate the primary pig inoblast of preparation in embodiment 3, different except substratum, other condition of 4 cell cultures samples is identical.After cell converges to 80%, go down to posterity, go down to posterity 15 times, and each clone is carried out to form, growth curve and karyotyping.
Experimental procedure reference example 4, different except used medium, all the other operations are identical.
Result is as shown in table 9 below.
Table 9:MTT method detects the growth conditions optical density value of substratum D, E, F, G cultured cells
| The 2nd day | The 4th day | The 6th day | The 8th day | The 10th day | The 12nd day | The 14th day | The 16th day | |
| Substratum D | 0.36 | 0.48 | 0.56 | 0.42 | 0.38 | 0.33 | 0.315 | 0.31 |
| Substratum E | 0.31 | 0.41 | 0.51 | 0.39 | 0.32 | 0.31 | 0.28 | 0.25 |
| Substratum F | 0.11 | 0.35 | 0.22 | 0.12 | 0.18 | 0.19 | 0.16 | 0.21 |
| Substratum G | 0.15 | 0.28 | 0.21 | 0.15 | 0.17 | 0.13 | 0.12 | 0.18 |
Remarks: start to detect once for every 2 days from cell cultures, detect altogether 8 times.
Under identical culture condition, porcine cells medium D cultured cells, its growth conditions optical density value is all higher than the growth curve of the cell of substratum E, the porcine cells medium of the present invention that adopts tire porcine blood serum and add the factor is described, traditional substratum that its growth result to pig cell is better than adopting foetal calf serum and adds the factor.
To sum up, adopt that porcine cells medium D cultured cells growth conditions is good, the speed of growth very fast (referring to accompanying drawing 8), adherent rate be higher, cell chromosome caryogram is normal, and porcine cells medium D is the new and improved substratum that is better than traditional substratum.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (5)
1. a porcine cells medium, it comprises basic medium and tire porcine blood serum, and according to the cumulative volume of porcine cells medium, the content of described tire porcine blood serum is 5%-25% (v/v);
Wherein, described tire porcine blood serum makes as follows:
1) serum is separated: the blood sample available from pig tire is carried out centrifugal, obtain serum;
2) serum freezing de-fibre at-20 ℃ that freezing de-fibre: by step 1), centrifugation obtains;
3) clarification filtration: by step 2), obtaining sample uses Cai Shi Mierocrystalline cellulose degree of depth filter to filter;
4) sample serum dialysis: by step 3) obtaining is dialysed in 0.15M NaCl solution with the dialysis membrane of molecular weight 12000-14000, until the glucose content < 0.5mg/mL of sample;
5) sample gammairradiation: by step 4) obtaining carries out gammairradiation, and irradiation dose is 1.2-4Gy, obtains rough serum; With
6) the rough serum obtaining Sterile Filtration: successively use 0.2 micron and 0.1 micron of filter membrane, by step 5) carries out filter membrane Sterile Filtration again;
Described porcine cells medium also contains following 4 kinds and adds the factor:
M-EGF,
BFGF-2,
Human chorionic gonadotrophin and
Pregnant mare serum gonadotrop(h)in (PMSG);
And according to the volume of porcine cells medium, the content that adds the factor is:
2. porcine cells medium according to claim 1, wherein, the content of described tire porcine blood serum is 8%-12% (v/v).
3. porcine cells medium according to claim 1, wherein, the content of described tire porcine blood serum is 10%-12% (v/v).
4. porcine cells medium according to claim 1, wherein, described basic medium is DMEM or F12 or the following artificial medium (mg/L) forming:
5. a method of cultivating pig cell, it is characterized in that, right to use requires the porcine cells medium described in any one in 1-4, and wherein, described pig cell is selected from: primary pig inoblast, porcine kidney cell, pig myocardium cell, porcine oocytes and pig vascular endothelial cell.
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| CN201010242482.3A CN102344904B (en) | 2010-08-02 | 2010-08-02 | Porcine cells medium |
| PCT/CN2011/001253 WO2012016427A1 (en) | 2010-08-02 | 2011-08-01 | Culture medium of pig cells |
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| CN201010242482.3A CN102344904B (en) | 2010-08-02 | 2010-08-02 | Porcine cells medium |
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| CN105087461A (en) * | 2015-06-18 | 2015-11-25 | 上海源培生物科技股份有限公司 | PK cell culture medium |
| CN105647848A (en) * | 2016-02-25 | 2016-06-08 | 苏州红冠庄药物研究有限公司 | Separation and preparation method of deer serum |
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| WO1999024556A1 (en) * | 1997-11-12 | 1999-05-20 | Vts Holdings Llc | Novel culture medium which comprises fetal porcine serum (fps) |
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| EP0882127A4 (en) * | 1996-01-09 | 2002-09-11 | Univ California | Ungulate embryonic stem-like cells, methods of making and using the cells to produce transgenic ungulates |
| US6700037B2 (en) * | 1998-11-24 | 2004-03-02 | Infigen, Inc. | Method of cloning porcine animals |
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| CN1217745A (en) * | 1996-03-18 | 1999-05-26 | 匹兹堡大学 | Cell culture media for mammalian cells |
| WO1999024556A1 (en) * | 1997-11-12 | 1999-05-20 | Vts Holdings Llc | Novel culture medium which comprises fetal porcine serum (fps) |
| US20070178074A1 (en) * | 2004-04-21 | 2007-08-02 | Dan-Ning Hu | Chondrocyte Culture Formulations |
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| WO2012016427A1 (en) | 2012-02-09 |
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