CN106047806A - Peripheral blood cell culture medium for high-density cell culture system - Google Patents
Peripheral blood cell culture medium for high-density cell culture system Download PDFInfo
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Abstract
The present invention relates to a peripheral blood cell culture medium for a high-density cell culture system. The peripheral blood cell culture medium comprises a base culture medium and added components, wherein the base culture medium is a RMPI 1640 culture medium, and the added components comprise bovine serum with a concentration of 160-220 ml/L, PHA-plant hemagglutinin with a concentration of 16-30 g/L, L-glutamine with a concentration of 8-12 g/L, glutathione with a concentration of 8-14 g/L, linoleic acid with a concentration of 0.1-0.3 g/L, and lipoic acid with a concentration of 0.1-0.3 g/L, wherein the concentrations of each added component adopt the total volume of the peripheral blood cell culture medium as the reference. Compared with the peripheral blood cell culture medium in the prior art, the peripheral blood cell culture medium of the present invention has advantages of rapid and large-scale peripheral blood cell proliferation, good proliferation effect and short culture time, and is particularly suitable for the high-density cell culture system of the peripheral blood cells.
Description
Technical field
The present invention relates to a kind of peripheral blood cells culture medium being applicable to concentration cultivation system, belong to cell and cultivate
Technical field.
Background technology
Cell is cultivated and has been widely used in the every field such as biology, medical science, new drug development, becomes most important basis
One of science.
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 peripheral blood cells culture medium 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 peripheral blood cells culture medium 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 peripheral blood cells culture medium of degree cell culture system, it includes basal medium and addO-on therapy, wherein, the cultivation of described basis
Base is RMPI 1640 culture medium；Described addO-on therapy and concentration thereof are as follows: Ox blood serum, 160-220ml/L；PHA-plant blood cell
Agglutinin, 16-30g/L；L-glutaminate, 8-12g/L；Glutathion, 8-14g/L；Linoleic acid, 0.1-0.3g/L；Thioctic acid,
0.1-0.3g/L；The concentration of each addO-on therapy is on the basis of the cumulative volume of described peripheral blood cells culture medium.
Preferably, on the basis of the cumulative volume of described peripheral blood cells culture medium, the dry powder of described RMPI 1640 culture medium
Amount is 10-15g/L.It is furthermore preferred that on the basis of the cumulative volume of described peripheral blood cells culture medium, described RMPI 1640 cultivates
The dry powder amount of base is 13.6g/L.
Preferably, in described addO-on therapy, the concentration of described Ox blood serum is 200ml/L, described PHA-plant hemagglutination
The concentration of element is 24g/L, and the concentration of described L-glutaminate is 11g/L, and the concentration of described glutathion is 12g/L.
Preferably, in described addO-on therapy, described linoleic concentration is 0.2g/L, and the concentration of described thioctic acid is
0.2g/L。
Preferably, described addO-on therapy also includes that penicillin and streptomycin, the concentration of described penicillin are 1 × 105-4×
105IU/L, the concentration of described streptomycin is 1 × 105-4×105IU/L。
Preferably, described addO-on therapy also includes that phenol red sodium, the concentration of described phenol red sodium are 5-7mg/L.
Preferably, the pH value of described peripheral blood cells culture medium is 7.3～7.5.
Another aspect of the present invention provides above-mentioned peripheral blood cells culture medium answering in concentration cultivation system
With.
Preferably, described concentration cultivation system is concentration cultivation bucket.
The peripheral blood cells culture medium being applicable to concentration cultivation system of the present invention, uses RMPI1640 culture medium
Based on culture medium and the combination of specific addO-on therapy, especially Ox blood serum, PHA-phytohemagglutinin, L-paddy ammonia
Amide and glutathion, four when meeting above-mentioned concentration range simultaneously, it is possible to makes peripheral blood cells increasing quick, substantial amounts of
Grow, be particularly suitable for the concentration cultivation system of peripheral blood cells.
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 peripheral blood cells being applicable to concentration cultivation system
Culture medium, it includes basal medium and addO-on therapy, and wherein, described basal medium is RMPI 1640 culture medium；Described add
Add component and concentration is as follows: Ox blood serum, 160-220ml/L；PHA-phytohemagglutinin, 16-30g/L；L-glutaminate,
8-12g/L；Glutathion, 8-14g/L；Linoleic acid, 0.1-0.3g/L；Thioctic acid, 0.1-0.3g/L；The concentration of each addO-on therapy
On the basis of the cumulative volume of described peripheral blood cells culture medium.
The present inventor finds through substantial amounts of development test, when peripheral blood cells culture medium uses RMPI 1640
Culture medium and the combination of above-mentioned specific addO-on therapy based on culture medium, especially Ox blood serum, PHA-plant blood cell coagulate
Collection element, L-glutaminate and glutathion, four when meeting above-mentioned concentration range simultaneously, it is possible to makes peripheral blood cells fast
Propagation fast, substantial amounts of, is particularly suitable for the concentration cultivation system of peripheral blood cells.
In a preferred embodiment of the invention, on the basis of the cumulative volume of described peripheral blood cells culture medium, institute
The dry powder amount stating RMPI 1640 culture medium is 10-15g/L.
In a further preferred embodiment of the present invention, the dry powder amount of described RMPI 1640 culture medium is 13.6g/
L。
In another further preferred embodiment of the present invention, in described addO-on therapy, the concentration of described Ox blood serum is
200ml/L, the concentration of described PHA-phytohemagglutinin is 24g/L, and the concentration of described L-glutaminate is 11g/L, and institute
The concentration stating glutathion is 12g/L.When in the peripheral blood cells culture medium of the present invention, the concentration of RMPI 1640 culture medium, with
And Ox blood serum, PHA-phytohemagglutinin, L-glutaminate and glutathion are when using above-mentioned specific concentration simultaneously, meet
The nutritional need of the High Density Cultivation of peripheral blood cells, is particularly suitable for the concentration cultivation system of peripheral blood cells, especially
It is the dimensional culture system of such as concentration cultivation bucket etc, there is preferable cultivation effect.
In a preferred embodiment in accordance with this invention, in described addO-on therapy, described linoleic concentration is 0.2g/
L, the concentration of described thioctic acid is 0.2g/L.The combination of both materials and certain concentration rises for the growth of peripheral blood cells
To protective effect, particularly with the High Density Cultivation system of peripheral blood cells, by increasing capacitance it is possible to increase the survival rate of peripheral blood cells.
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 1 × 105-4×105IU/L, described streptomycin concentration is 1 × 105-4×
105IU/L.It is furthermore preferred that the concentration of penicillin is 2 × 105IU/L, streptomycin concentration is 2 × 105IU/L.Both antibiotic
Combination and specific concentration thereof, be particularly suitable for the concentration cultivation system of peripheral blood cells, and antibacterial effect is excellent, and cell is deposited
Motility rate is high.
In a preferred embodiment in accordance with this invention, described addO-on therapy also includes phenol red sodium, described phenol red sodium
Concentration is 5-7mg/L.
In a preferred embodiment of the invention, the pH value of this serum-free medium is controlled 7.3～7.5.
In a preferred embodiment of the invention, the peripheral cells culture medium of the present invention is to be exclusively used in high-density cells
Cultivate the peripheral cells culture medium of bucket.
Embodiment 1
The process for preparation of the peripheral blood cells culture medium being applicable to concentration cultivation system of embodiment 1 is as follows:
Step 1): aseptically the RPMI1640 dry powder of synthesis is weighed 136g on electronic balance and be placed on sterilization
In container, stir after adding the dilution of 7.5L redistilled water acquisition basal medium；
Step 2): by addO-on therapy: 2L Ox blood serum, 240g PHA-phytohemagglutinin, 110g L-glutaminate,
120g glutathion, 1.2g penicillin (are equivalent to 2 × 106IU), 2g streptomycin (is equivalent to 2 × 106IU), 2g linoleic acid, 2g sulfur
Octanoic acid and the phenol red sodium of 60mg are sequentially added into step 1) in the basal medium that obtains, 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 peripheral blood cells culture medium being applicable to concentration cultivation system of embodiment 2 is as follows:
Step 1): aseptically the RPMI1640 dry powder of synthesis is weighed 140g on electronic balance and be placed on sterilization
In container, stir after adding the dilution of 7.5L redistilled water acquisition basal medium；
Step 2): by addO-on therapy: 1.7L Ox blood serum, 260g PHA-phytohemagglutinin, 100g L-glutaminate,
110g glutathion, 2g penicillin (are equivalent to 3.3 × 106IU), 3g streptomycin (is equivalent to 3 × 106IU), 1.8g linoleic acid,
1.8g thioctic acid and the phenol red sodium of 66mg are sequentially added into step 1) in the basal medium that obtains, 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
Disinfect finger skin in alcohol, with blood-taking needle broken skin skin, gather peripheral blood with the hollow blood taking tube containing heparin sodium,
Jiggle mixing, in the peripheral blood cells culture medium obtain 5ml peripheral blood addition to above-described embodiment 1, and by peripheral blood
Cell concentration is diluted to 1.2 × 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 peripheral blood cells culture medium cultivate.
It is 1.2 × 10 by 5ml peripheral blood cells concentration6It is thin that the culture medium syringe of individual/ml is loaded into above-mentioned high density
Born of the same parents cultivate in bucket, then 25ml Peripheral blood culture base joins (peripheral blood cells in 50ml concentration cultivation bucket liquid containing bottle
Inoculum density be 2 × 105Individual/ml)；Specifically, the culture medium in liquid containing bottle is cultivated bucket by silica gel tube with cell and is connected,
Under the effect of peristaltic pump, persistent loop flows, and the doughnut that culture medium cultivates bucket by cell swaps, and cultivates to cell
Cell in Tong constantly provides nutrient substance；Whole concentration cultivation bucket is put into 5%CO2, in the incubator of 37 DEG C
Cultivate.
Application examples 2: detailed process is identical with application examples 1, difference is that the peripheral blood cells using embodiment 2 to obtain is cultivated
Base carries out the cultivation of peripheral blood cells.
Effect data
Comparative example 1: use the concentration cultivation bucket of commercially available FiberCell brand C2025 model and commercially available
The RPMI 1640 peripheral blood cells culture medium of Qingdao Lay Buddhist brand, 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 Qingdao Lay Buddhist product
The RPMI 1640 peripheral blood cells culture medium of board, is transferred to the peripheral blood cells culture medium pipet of 5ml Lay Buddhist brand
In T25 culture bottle, adding 1ml peripheral blood cells concentration is 1.2 × 106(inoculation of peripheral blood cells is close for the culture medium of individual/ml
Degree is 2 × 105Individual/ml) carry out suspension culture, culture bottle is put into 5%CO2, the incubator of 37 DEG C is cultivated 72 hours.
Detection application examples 1, application examples 2, comparative example 1 and the cell proliferation rate of comparative example A and cell survival rate respectively.
Detection cell proliferation rate
Application examples 1 and application examples 2, and comparative example 1 and comparative example A, all after cultivating 1,3 and 6 days, separately sampled cultivation
Liquid, carries out the detection of cell concentration, and testing result sees table 1 below.
Table 1
Detection cell survival rate
After cultivating 6 days, collect the culture fluid of each group, carry out flow cytometer detection.Testing result is as follows:
The cell survival rate of application examples 1: 98.4%；The cell survival rate of application examples 2: 96.7%；The cell of comparative example 1 is deposited
Motility rate: 84.2%；The cell survival rate of comparative example A: 88.6%.
Result in table 1 is carried out contrast can be seen that, on the one hand, cell proliferation rate and the cell of comparative example 1 are deposited
The result of motility rate (cell culture effect) is slightly inferior to the result of comparative example A, this explanation: conventional medium is more suitable for such as cell
The tradition of bottle etc cultivating system on a small scale, is not suitable for concentration cultivation system；On the other hand, application examples 1 of the present invention
Cell proliferation rate and cell survival rate are far above comparative example 1, and this illustrates: the peripheral blood cells culture medium of the embodiment of the present invention
The RMPI peripheral blood cells culture medium comparing routine 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 peripheral blood cells culture medium for concentration cultivation system, it includes basal medium and interpolation group
Point, wherein,
Described basal medium is RMPI 1640 culture medium；
Described addO-on therapy and concentration thereof are as follows:
Ox blood serum, 160-220ml/L；
PHA-phytohemagglutinin, 16-30g/L；
L-glutaminate, 8-12g/L；
Glutathion, 8-14g/L；
Linoleic acid, 0.1-0.3g/L；
Thioctic acid, 0.1-0.3g/L；
The concentration of each addO-on therapy is on the basis of the cumulative volume of described peripheral blood cells culture medium.
2. peripheral blood cells culture medium as claimed in claim 1, it is characterised in that:
On the basis of the cumulative volume of described peripheral blood cells culture medium, the dry powder amount of described RMPI 1640 culture medium is 10-15g/
L。
3. peripheral blood cells culture medium as claimed in claim 2, it is characterised in that:
On the basis of the cumulative volume of described peripheral blood cells culture medium, the dry powder amount of described RMPI 1640 culture medium is 13.6g/
L。
4. peripheral blood cells culture medium as claimed in claim 2, it is characterised in that:
In described addO-on therapy, the concentration of described Ox blood serum is 200ml/L, and the concentration of described PHA-phytohemagglutinin is
24g/L, the concentration of described L-glutaminate is 11g/L, and the concentration of described glutathion is 12g/L.
5. peripheral blood cells culture medium as claimed in claim 2, it is characterised in that:
In described addO-on therapy, described linoleic concentration is 0.2g/L, and the concentration of described thioctic acid is 0.2g/L.
6. the peripheral blood cells culture medium as described in any one in claim 1 to 5, it is characterised in that:
Described addO-on therapy also includes that penicillin and streptomycin, the concentration of described penicillin are 1 × 105-4×105IU/L, described
The concentration of streptomycin is 1 × 105-4×105IU/L。
7. the peripheral blood cells culture medium as described in any one in claim 1 to 5, it is characterised in that:
Described addO-on therapy also includes that phenol red sodium, the concentration of described phenol red sodium are 5-7mg/L.
8. peripheral blood cells culture medium as claimed in claim 7, it is characterised in that:
The pH value of described peripheral blood cells culture medium is 7.3～7.5.
9. peripheral blood cells culture medium as claimed in any of claims 1 to 8 in one of claims is in concentration cultivation system
Application.
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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Cited By (1)
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| CN107043748A (en) * | 2017-04-05 | 2017-08-15 | 上海逍鹏生物科技有限公司 | The serum free medium of peripheral blood cells |
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