CN106047798A - Aminotic cell culture medium for high-density cell culture system - Google Patents
Aminotic cell culture medium for high-density cell culture system Download PDFInfo
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Abstract
The present invention relates to an aminotic cell culture medium for a high-density cell culture system. The aminotic cell culture medium comprises a base culture medium and added components, wherein the base culture medium is a F12 culture medium, and the added components comprise fetal bovine serum with a concentration of 80-120 ml/L, human recombinant insulin with a concentration of 24-30 mg/L, human recombinant epidermal growth factor with a concentration of 20-25 [mu]g/L, human recombinant basic fibroblast growth factor with a concentration of 35-45 [mu]g/L, human albumin with a concentration of 10-15 g/L, transferring with a concentration of 4-16 mg/L, Hydrocortisone with a concentration of 0.2-1 mg/L, Testosterone with a concentration of 0.2-1 mg/L, progesterone with a concentration of 0.2-1 mg/L, vitamin E with a concentration of 1-5 mg/L, L-glutamine with a concentration of 12-20 g/L, HEPES with a concentration of 5-10 g/L, and a block polyether F-68 with a concentration of 0.5-2 g/L, wherein the concentrations of each added component adopt the total volume of the aminotic cell culture medium as the reference. Compared with the aminotic cell culture medium in the prior art, the aminotic cell culture medium of the present invention has advantages of rapid and large-scale aminotic cell proliferation, good proliferation effect and short culture time, and is particularly suitable for the high-density cell culture system of the aminotic cells.
Description
Technical field
The present invention relates to a kind of culture medium of amniotic fluid cells being applicable to concentration cultivation system, belong to cell and cultivate skill
Art field.
Background technology
Prenatal diagnosis is eugenic important component part, and it passes through the most right on the basis of being built upon genetic counselling
Embryo or the developmental condition of fetus, whether suffer from the aspects such as disease and carry out checkout and diagnosis.Prenatal diagnosis currently mainly has floss in early days
Hair tissue taking-up, second trimester amniotic fluid and the diagnostic method such as Cord blood, maternal blood isolation of fetal cells.Wherein second trimester amniotic fluid
Cell detection remains topmost diagnostic method.
Detect about second trimester amniocyte, be the amniotic fluid specimen by amniocentesis extraction anemia of pregnant woman, amniocyte is entered
Row is cultivated, or extracts amniocyte DNA, then carries out genetic diagnosis (such as, diagnosing fetal chromosomal disease, monogenic disease etc.).
In vitro culture about amniocyte is that amniocyte detects a most key link.
About Cell culture invitro system, except two dimensions such as conventional culture plate, culture dish, culture bottles in prior art
Cell culture system, has been developed that dimensional culture system, the most presently commercially available concentration cultivation bucket now, be one both
Go for attached cell, go for again the dimensional culture system of suspension cell, a large amount of cell can be cultivated rapidly,
And the long term cell culture of half a year by a definite date more than can be carried out, it is not necessary to often change consumptive material, with traditional culture plate, culture dish
Compare with culture bottle, more convenient, cost is lower, therefore suffers from the extensive concern of this area research staff.
But, traditional culture medium of amniotic fluid cells of the prior art is mostly applicable to medium scale cell and cultivates body
System, in general cannot be applicable to cell large-scale, highdensity and cultivate；On the other hand, existing serum-free medium is only
It is applicable to the cell culture system of the two dimensions such as culture plate, culture dish, culture bottle, it is impossible to be applicable to concentration cultivation bucket so
Dimensional culture system.
The most urgently exploitation is exclusively used in the culture medium of amniotic fluid cells of concentration cultivation system.
Summary of the invention
In view of the problems referred to above and/or the other problems of correlation technique, one aspect of the present invention provides one and is applicable to highly dense
The culture medium of amniotic fluid cells of degree cell culture system, it includes basal medium and addO-on therapy, wherein, described basal medium
For F12 culture medium；Described addO-on therapy and concentration thereof are as follows: hyclone, 80-120ml/L；Biosynthetic human insulin, 24-
30mg/L；Recombinant human epidermal growth factor, 20-25 μ g/L；People's recombination basic fibroblast growth factor, 35-45 μ g/
L；Human albumin, 10-15g/L；Transferrins, 4-16mg/L；Hydrocortisone, 0.2-1mg/L；Testosterone, 0.2-1mg/L；
Progesterone, 0.2-1mg/L；Vitamin E, 1-5mg/L；L-glutaminate, 12-20g/L；HEPES, 5-10g/L；Blocked polyethers
F-68,0.5-2g/L；The concentration of each addO-on therapy is on the basis of the cumulative volume of described culture medium of amniotic fluid cells.
Preferably, on the basis of the cumulative volume of described culture medium of amniotic fluid cells, the dry powder amount of described F12 culture medium is 15-
18g/L。
Preferably, the concentration of described biosynthetic human insulin is 28mg/L, and the concentration of recombinant human epidermal growth factor is
24 μ g/L, the concentration of people's recombination basic fibroblast growth factor is 40 μ g/L, and the concentration of human albumin is 12g/L.
Preferably, the concentration of described L-glutaminate is 12-20g/L.
Preferably, the concentration of described transferrins is 12mg/L.
Preferably, the concentration of described blocked polyethers F-68 is 1g/L.
Preferably, the concentration of described hydrocortisone is 0.7mg/L, and the concentration of described Testosterone is 0.6mg/L, described Huang
The concentration of body ketone is 0.6mg/L, and the concentration of described vitamin E is 2mg/L.
Preferably, described addO-on therapy also includes penicillin and streptomycin；Concentration 100-400IU/mL of described penicillin,
Described streptomycin concentration is 100-400IU/mL.
Another aspect of the present invention provides the application in concentration cultivation system of the above-mentioned culture medium of amniotic fluid cells.
Preferably, described concentration cultivation system is concentration cultivation bucket.
The culture medium of amniotic fluid cells being applicable to concentration cultivation system of the present invention, uses F12 culture medium as base
Basal culture medium and the combination of specific addO-on therapy, especially biosynthetic human insulin, recombinant human epidermal growth factor, people
Recombination basic fibroblast growth factor and human albumin, four when meeting specific concentration range simultaneously, it is possible to makes sheep
Water cell propagation quick, substantial amounts of, cultivation effect is good, and incubation time is short, is particularly suitable for the concentration cultivation of amniocyte
System.
Detailed description of the invention
The present invention is further illustrated by the following examples, but the present invention is not limited to these specific embodiment parties
Formula.
In one embodiment of the invention, a kind of amniocyte training being applicable to concentration cultivation system
Supporting base, it includes basal medium and addO-on therapy, and wherein, described basal medium is F12 culture medium；Described addO-on therapy and
Its concentration is as follows: hyclone, 80-120ml/L；Biosynthetic human insulin, 24-30mg/L；Recombinant human epidermal growth factor,
20-25μg/L；People's recombination basic fibroblast growth factor, 35-45 μ g/L；Human albumin, 10-15g/L；Transferrins,
4-16mg/L；Hydrocortisone, 0.2-1mg/L；Testosterone, 0.2-1mg/L；Progesterone, 0.2-1mg/L；Vitamin E, 1-
5mg/L；L-glutaminate, 12-20g/L；HEPES, 5-10g/L；Blocked polyethers F-68,0.5-2g/L；Each addO-on therapy
Concentration is on the basis of the cumulative volume of described culture medium of amniotic fluid cells.
The present inventor finds through substantial amounts of development test, when culture medium of amniotic fluid cells uses F12 culture medium to make
Based on culture medium and the combination of above-mentioned specific addO-on therapy, especially biosynthetic human insulin, people epidermis cell of recombinating raw
The long factor, people's recombination basic fibroblast growth factor and human albumin, four when meeting above-mentioned concentration range simultaneously, energy
Enough make amniocyte propagation quick, substantial amounts of, and cell survival rate is higher, be particularly suitable for the high-density cells training of amniocyte
The system of supporting.
In a preferred embodiment of the invention, on the basis of the cumulative volume of described culture medium of amniotic fluid cells, described
The dry powder amount of F12 culture medium is 15-18g/L.It is furthermore preferred that the concentration of biosynthetic human insulin is 28mg/L, people's epidermis of recombinating is thin
The concentration of the intracellular growth factor is 24 μ g/L, and the concentration of people's recombination basic fibroblast growth factor is 40 μ g/L, and the white egg of people
White concentration is 12g/L.When in the culture medium of amniotic fluid cells of the present invention, the concentration of F12 culture medium, and biosynthetic human insulin,
Recombinant human epidermal growth factor, people's recombination basic fibroblast growth factor and human albumin use above-mentioned specific simultaneously
Concentration time, be particularly suitable for the concentration cultivation system of amniocyte, the most such as concentration cultivation bucket etc
Dimensional culture system, there is preferable cultivation effect and cell survival rate.
In a further preferred embodiment of the present invention, the concentration of described L-glutaminate is 12-20g/L.
In another further preferred embodiment of the present invention, the concentration of described transferrins is 12mg/L.
In a preferred embodiment of the invention, the concentration of described blocked polyethers F-68 is 1g/L.Blocked gathers
Ether F-68, i.e. Pluronic F-68, it is a kind of surfactant, is typically commonly used for emulsifying agent and solubilizing agent；But, invention
People is found surprisingly that, adds the Pluronic F-68 of certain concentration, for high density in the culture medium of amniotic fluid cells of the present invention
The growth of the amniocyte in cell system has Stabilization, it is possible to increase the survival rate of amniocyte.
In a preferred embodiment of the invention, the addition of the hormonal substance of culture medium of amniotic fluid cells is as follows:
The concentration of hydrocortisone is 0.7mg/L, and the concentration of described Testosterone is 0.6mg/L, and the concentration of described Progesterone is 0.6mg/
L, and the concentration of described vitamin E is 2mg/L；The growth demand of amniocyte in high-density cells system can be met.
In a preferred embodiment of the invention, described addO-on therapy can such as include penicillin with antibiotic
And streptomycin；The concentration of described penicillin is 100-400IU/mL, and described streptomycin concentration is 100-400IU/mL.More preferably
, the concentration of penicillin is 200IU/mL, and streptomycin concentration is 200IU/mL.Both antibiotic combinations and the denseest
Degree, is particularly suitable for the concentration cultivation system of amniocyte, and antibacterial effect is excellent, and cell survival rate is high.
In a preferred embodiment of the invention, the concentration of described hyclone is 100ml/L.
In a preferred embodiment of the invention, the concentration of described HEPES is 6.4g/L.
In another preferred embodiment of the present invention, the pH value of this serum-free medium is controlled 7.3～7.5.
In a preferred embodiment of the invention, the culture medium of amniotic fluid cells of the present invention is to be exclusively used in high-density cells
Cultivate the culture medium of amniotic fluid cells of bucket.
Embodiment 1
The process for preparation of the culture medium of amniotic fluid cells being applicable to concentration cultivation system of embodiment 1 is as follows:
Step 1): aseptically the F12 dry powder of synthesis is weighed 168g on electronic balance and be placed on disinfecting container
In, stir after adding the dilution of 8.5L redistilled water acquisition basal medium；
Step 2): by addO-on therapy: 280mg biosynthetic human insulin, 240 μ g recombinant human epidermal growth factor, 400 μ g
People's recombination basic fibroblast growth factor, 120g human albumin, 120mg transferrins, 7mg hydrocortisone, 6mg testis
Element, 6mg Progesterone, 2mg vitamin E, 1.2g penicillin (are equivalent to 2 × 105IU/L), 2g streptomycin (is equivalent to 2 × 105IU/
L), 64g HEPES, 10g Pluronic F-68, and 1000ml hyclone is sequentially added into step 1) the basis cultivation that obtains
In base, fully mix；
Step 3): filtration sterilization, then regulate pH value to 7.4 ± 0.1, it is eventually adding redistilled water by volume regulation to 10L；
Step 4): after culture medium has been prepared, filtration sterilization, subpackage, sealing, last cold preservation (-20 DEG C of environment) is standby.
Embodiment 2
The process for preparation of the culture medium of amniotic fluid cells being applicable to concentration cultivation system of embodiment 2 is as follows:
Step 1): aseptically the F12 dry powder of synthesis is weighed 160g on electronic balance and be placed on disinfecting container
In, stir after adding the dilution of 8.5L redistilled water acquisition basal medium；
Step 2): by addO-on therapy: 240mg biosynthetic human insulin, 220 μ g recombinant human epidermal growth factor, 360 μ g
People's recombination basic fibroblast growth factor, 110g human albumin, 100mg transferrins, 6mg hydrocortisone, 5mg testis
Element, 5mg Progesterone, 3mg vitamin E, 1g penicillin (are equivalent to 1.67 × 105IU/L), 1g streptomycin (is equivalent to 1 × 105IU/
L), 60g HEPES, 12g Pluronic F-68, and 8000ml hyclone is sequentially added into step 1) the basis cultivation that obtains
In base, fully mix；
Step 3): filtration sterilization, then regulate pH value to 7.4 ± 0.1, it is eventually adding redistilled water by volume regulation to 10L；
Step 4): after culture medium has been prepared, filtration sterilization, subpackage, sealing, last cold preservation (-20 DEG C of environment) is standby.
Application examples 1 and application examples 2
Conventionally obtain amniocyte: in general, take amniotic fluid, aseptically centrifugal acquisition amniocyte
Agglomerate.
Application examples 1
Use the culture medium of amniotic fluid cells Eddy diffusion amniocyte agglomerate that above-described embodiment 1 obtains, and by amniocyte
Concentration dilution is to 1 × 106Individual/ml.
The concentration cultivation bucket using commercially available FiberCell brand C2025 model (sees FiberCell cell
Culture systems production marketing webpage), and the embodiment of the present invention 1 obtain culture medium of amniotic fluid cells cultivate.
It is 1 × 10 by 2ml amniocyte concentration6The culture medium syringe of individual/ml is loaded into above-mentioned high-density cells training
Support in bucket, then the culture medium of amniotic fluid cells of 18ml embodiment 1 is joined (amniotic fluid in 50ml concentration cultivation bucket liquid containing bottle
The inoculum density of cell is 1 × 105Individual/ml)；Specifically, the culture medium in liquid containing bottle cultivates bucket even by silica gel tube and cell
Connecing, under the effect of peristaltic pump, persistent loop flows, and the doughnut that culture medium cultivates bucket by cell swaps, and gives thin
Born of the same parents cultivate the cell in bucket and constantly provide nutrient substance；Whole concentration cultivation bucket is put into 5%CO2, the training of 37 DEG C
Support in case and cultivate.
Application examples 2
Detailed process is identical with application examples 1, and difference is that the culture medium of amniotic fluid cells using embodiment 2 to obtain carries out amniotic fluid
The cultivation of cell.
Effect data
Comparative example 1: use the concentration cultivation bucket of commercially available FiberCell brand C2025 model (to see
FiberCell cell culture system production marketing webpage), and commercially available conventional culture medium of amniotic fluid cells (Community in Baiyunshan, Guangzhou life
" fine horse throughout one's life " the F12 culture medium produced), incubation is identical with application examples 1, specifically repeats no more.
Comparative example A: use the Tissue Culture Flask of commercially available Corning brand T25 model, and commercially available conventional amniotic fluid is thin
Born of the same parents' culture medium (" fine horse throughout one's life " F12 culture medium that Community in Baiyunshan, Guangzhou produces)；Specifically, by 4.5ml routine amniotic fluid cell culture
Base pipet is transferred in T25 culture bottle, and adding 0.5ml amniocyte concentration is 1 × 106The conventional medium of individual/ml is entered
(inoculum density of amniocyte is 1 × 10 to row suspension culture5Individual/ml), culture bottle is put into 5%CO2, in the incubator of 37 DEG C
Cultivate 72 hours.
Detection application examples 1, application examples 2, comparative example 1 and the amniotic fluid cell culture effect of comparative example A respectively.
Application examples 1 and application examples 2, and comparative example 1 and comparative example A, all after cultivating 7 days, separately sampled cell is cultivated
Liquid, carries out mitotic figure clone's number and total clone's number detection to often organizing cell culture fluid.
Detection process approximately as:
After cultivating 7 days, separately sampled 5ml；In each sample, add 0.1ml Demecolcine solution, hatch for 37 DEG C
30min, is centrifuged 5 minutes with 1000rpm, abandoning supernatant；Addition 10ml 0.075M KCl solution in each sample again, 37
DEG C hatching 10 minutes.It is centrifuged 5 minutes, abandoning supernatant with 1000rpm；It is added dropwise over Fresh again in each centrifuge tube
, fixative (fixative is acetic acid: methanol=1: the 3) 5ml of pre-cooling, limit edged rocks；Again it is centrifuged 5 minutes with 1000rpm,
Abandoning supernatant；Suspension made by the fixative adding 1ml ice.Taking 35mm culture dish, dropping 0.5ml cell suspension is to culture dish
Coverslip central authorities.It is put in hothouse or chemical hood.Dyeing, carries out chromosome analysis on request, calculates the clone of mitotic figure
Number sees table 1 below with always cloning number (often group detection 3 times, averages) testing result.
Table 1
| Mitotic figure is had to clone number | Number is cloned without mitotic figure | Always clone number | |
| Application examples 1 | 32 | 3 | 35 |
| Application examples 2 | 26 | 5 | 31 |
| Comparative example 1 | 18 | 8 | 26 |
| Comparative example A | 20 | 7 | 27 |
Can be seen that from the result of above-mentioned table 1, on the one hand, the mitotic figure that has of comparative example 1 is cloned number and always clones number (amniotic fluid
Cell culture effect) result be slightly inferior to the result of comparative example A, this explanation: conventional culture medium of amniotic fluid cells is more suitable for all
Such as the tradition on a small scale cultivating system of cell bottle etc, unsuitable for concentration cultivation system；On the other hand, the present invention should
The having mitotic figure clone's number and always clone number (amniotic fluid cell culture effect) far above comparative example 1 of use-case 1, this illustrates: this
The culture medium of amniotic fluid cells of bright embodiment is compared the culture medium of amniotic fluid cells of routine and is more suitable for concentration cultivation system.
It is to be understood that, although this specification is been described by according to embodiment, but the most each embodiment only comprises one
Individual independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should will say
Bright book is as an entirety, and the technical scheme in each embodiment can also be through appropriately combined, and forming those skilled in the art can
With other embodiments understood.
The a series of detailed description of those listed above is only for the feasibility embodiment of the present invention specifically
Bright, they also are not used to limit the scope of the invention, all equivalent implementations made without departing from skill of the present invention spirit
Or change should be included within the scope of the present invention.
Claims (10)
1. being applicable to a culture medium of amniotic fluid cells for concentration cultivation system, it includes basal medium and interpolation group
Point, wherein,
Described basal medium is F12 culture medium；
Described addO-on therapy and concentration thereof are as follows:
Hyclone, 80-120ml/L；
Biosynthetic human insulin, 24-30mg/L；
Recombinant human epidermal growth factor, 20-25 μ g/L；
People's recombination basic fibroblast growth factor, 35-45 μ g/L；
Human albumin, 10-15g/L；
Transferrins, 4-16mg/L；
Hydrocortisone, 0.2-1mg/L；
Testosterone, 0.2-1mg/L；
Progesterone, 0.2-1mg/L；
Vitamin E, 1-5mg/L；
L-glutaminate, 12-20g/L；
HEPES, 5-10g/L；
Blocked polyethers F-68,0.5-2g/L；
The concentration of each addO-on therapy is on the basis of the cumulative volume of described culture medium of amniotic fluid cells.
2. culture medium of amniotic fluid cells as claimed in claim 1, it is characterised in that:
On the basis of the cumulative volume of described culture medium of amniotic fluid cells, the dry powder amount of described F12 culture medium is 15-18g/L.
3. culture medium of amniotic fluid cells as claimed in claim 2, it is characterised in that:
The concentration of described biosynthetic human insulin is 28mg/L, and the concentration of described recombinant human epidermal growth factor is 24 μ g/L,
The concentration of described people's recombination basic fibroblast growth factor is 40 μ g/L, and the concentration of described human albumin is 12g/L.
4. culture medium of amniotic fluid cells as claimed in claim 3, it is characterised in that:
The concentration of described L-glutaminate is 12-20g/L.
5. culture medium of amniotic fluid cells as claimed in claim 3, it is characterised in that:
The concentration of described transferrins is 12mg/L.
6. culture medium of amniotic fluid cells as claimed in claim 2, it is characterised in that:
The concentration of described blocked polyethers F-68 is 1g/L.
7. culture medium of amniotic fluid cells as claimed in claim 2, it is characterised in that:
The concentration of described hydrocortisone is 0.7mg/L, and the concentration of described Testosterone is 0.6mg/L, the concentration of described Progesterone
For 0.6mg/L, and the concentration of described vitamin E is 2mg/L.
8. culture medium of amniotic fluid cells as claimed in any of claims 1 to 7 in one of claims, it is characterised in that:
Described addO-on therapy also includes penicillin and streptomycin；The concentration of described penicillin is 1 × 105-4×105IU/L, described
Streptomycin concentration be 1 × 105-4×105IU/L。
9. culture medium of amniotic fluid cells as claimed in any of claims 1 to 8 in one of claims answering in concentration cultivation system
With.
Apply the most as claimed in claim 9, it is characterised in that: described concentration cultivation system is concentration cultivation
Bucket.
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| CN106635964A (en) * | 2016-12-23 | 2017-05-10 | 江西宜信堂医疗科技有限公司 | Serum-free culture medium for culturing amniocytes and preparation method thereof |
| CN106834215A (en) * | 2016-12-24 | 2017-06-13 | 严志海 | A kind of culture medium of amniotic fluid cells |
| CN108060118A (en) * | 2017-12-26 | 2018-05-22 | 深圳市龙岗区妇幼保健院 | The application of culture medium of amniotic fluid cells, amniotic fluid cell culture method and culture medium |
| CN110747161A (en) * | 2019-11-28 | 2020-02-04 | 河南赛诺特生物技术有限公司 | Culture medium for amniotic fluid cells |
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| CN101705207A (en) * | 2009-11-10 | 2010-05-12 | 广州拜迪生物医药有限公司 | Culture medium of amniotic fluid cells |
| CN102409022A (en) * | 2010-09-25 | 2012-04-11 | 李凌松 | Method for culturing human artificially induced pluripotent stem cells by using human amnion mesenchyme cells as culture layer |
| CN105039244A (en) * | 2015-09-09 | 2015-11-11 | 广州和能生物科技有限公司 | Low serum amniotic fluid cell culture medium |
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| CN101705207A (en) * | 2009-11-10 | 2010-05-12 | 广州拜迪生物医药有限公司 | Culture medium of amniotic fluid cells |
| CN102409022A (en) * | 2010-09-25 | 2012-04-11 | 李凌松 | Method for culturing human artificially induced pluripotent stem cells by using human amnion mesenchyme cells as culture layer |
| CN105039244A (en) * | 2015-09-09 | 2015-11-11 | 广州和能生物科技有限公司 | Low serum amniotic fluid cell culture medium |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106635964A (en) * | 2016-12-23 | 2017-05-10 | 江西宜信堂医疗科技有限公司 | Serum-free culture medium for culturing amniocytes and preparation method thereof |
| CN106834215A (en) * | 2016-12-24 | 2017-06-13 | 严志海 | A kind of culture medium of amniotic fluid cells |
| CN108060118A (en) * | 2017-12-26 | 2018-05-22 | 深圳市龙岗区妇幼保健院 | The application of culture medium of amniotic fluid cells, amniotic fluid cell culture method and culture medium |
| CN110747161A (en) * | 2019-11-28 | 2020-02-04 | 河南赛诺特生物技术有限公司 | Culture medium for amniotic fluid cells |
| CN110747161B (en) * | 2019-11-28 | 2021-03-19 | 河南赛诺特生物技术有限公司 | Culture medium for amniotic fluid cells |
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