CN104830766B - A kind of peripheral blood lymphocytes culture medium of serum-free people - Google Patents
A kind of peripheral blood lymphocytes culture medium of serum-free people Download PDFInfo
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- CN104830766B CN104830766B CN201510268603.4A CN201510268603A CN104830766B CN 104830766 B CN104830766 B CN 104830766B CN 201510268603 A CN201510268603 A CN 201510268603A CN 104830766 B CN104830766 B CN 104830766B
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Abstract
The present invention relates to a kind of biological agent, more particularly to a kind of lymphocytes culture medium.It includes agglutinin, basal medium and serum substitute, the agglutinin is L-type phytohemagglutinin, the basal medium is RPMI1640 basal mediums, and the serum substitute includes bovine serum albumin(BSA), rh-insulin, ironic citrate and span amino acid mother liquor.The peripheral blood lymphocytes culture medium of the serum-free people of the present invention does not have to animal or human serum, so can not only reduce culture medium cost, can also reduce pathophorous risk；Component determines used in the peripheral blood lymphocytes culture medium of the serum-free people of the present invention, avoids traditional serum composition complexity, the problem of having mass discrepancy between different batches, and cause culture effect to fluctuate；Cell culture medium lymphocyte proliferation test provided by the invention is high with lymphocytic cell division index, and reagent cost is low, steady quality, and the cell of culture can be used for the clinical diagnosis of chromosome karyotype analysis.
Description
Technical field
The present invention relates to a kind of biological agent, more particularly to a kind of lymphocytes culture medium.
Background technology
With cytogenetic development, Chromosome In Peripheral Blood Lymphocytes karyotyping has turned into the routine of pre-natal diagnosis
Detection project.Main material of the lymphocytes culture medium as this detection project, its product quality level directly affect detection
Success rate and accuracy.Conventional peripheral blood lymphocytes culture medium is added on the basis of basal medium (such as RPMI1640)
Add a certain amount of animal blood serum (such as cow's serum), as material necessary to providing cell growth, propagation.But utilize animal blood serum
The shortcomings that be also apparent from：Because serum composition is complicated, not only growth promoting substance, also containing potential growth inhibitory substance；And
Quality fluctuation existing for the serum of different batches, cause the differences between batches of culture medium cell culture effect.
The technical problems to be solved by the invention are that existing peripheral blood lymphocytes culture medium uses animal or people mostly
Serum as additive, not only cost is higher, and adds pathophorous risk, while quality between different batches be present
The problem of difference.
The content of the invention
The defects of in order to overcome prior art, the present invention provide a kind of PBLC of the serum-free composition of optimization
Culture medium, for chromosome karyotype analysis, there is the advantages of cheap cost, steady quality, good culture effect.
The present invention provide a kind of peripheral blood lymphocytes culture medium of serum-free people, including agglutinin, basal medium with
And serum substitute, the agglutinin are L-type phytohemagglutinin, the basal medium is the culture of RPMI1640 bases
Base, the serum substitute include bovine serum albumin(BSA), rh-insulin, ironic citrate and span amino acid mother liquor.
Preferably, the peripheral blood lymphocytes culture medium of serum-free people of the invention, including following component：RPMI1640 is trained
Foster backbone powder 10.4g/L, sodium acid carbonate 2.0g/L, bovine serum albumin(BSA) 4.0g/L, L-type phytohemagglutinin 20mg/L, ammonia
Base acid mother liquor 20ml/L, ironic citrate 1mg/L, rh-insulin 10mg/L.
Preferably, the peripheral blood lymphocytes culture medium of serum-free people of the invention, the span amino acid mother liquor include L- paddy
Propylhomoserin, L- glycine and L-Aspartic acid.
Preferably, the peripheral blood lymphocytes culture medium of serum-free people of the invention, Pidolidone in span amino acid mother liquor
Concentration is 300mg/L, and the concentration of L- glycine is 100mg/L, and the concentration of L-Aspartic acid is 400mg/L.
Preferably, the peripheral blood lymphocytes culture medium of serum-free people of the invention, the purity of the bovine serum albumin(BSA)
Not less than 98%.
Preferably, the peripheral blood lymphocytes culture medium of serum-free people of the invention, the L-type phytohemagglutinin
(PHA-L) the mitotic Pleurotus Ostreatus glycoprotein fraction of T lymphocytes is stimulated for having from red kidney beans.
Preferably, the peripheral blood lymphocytes culture medium of serum-free people of the invention, the L-type phytohemagglutinin
(PHA-L) purity is not less than 95%.
Phytohemagglutinin (Phytohemagglutinin, PHA) used is to be present in red kidney beans in this case
The plant agglutinoid of (Phaseolus vulgaris).Different from the agglutinin such as ConA being made up of subunit of the same race, PHA is by two
The agglutinin of the different subunits compositions of kind.Its subunit can be divided into PHA-E and the classes of PHA-L two, and wherein PHA-E types subunit has more
Significant aggegation erythrocytic function and without lymphocyte stimulation activity, and PHA-L types subunit then has more significant stimulates leaching
The function of bar cell propagation.
Preferably, the peripheral blood lymphocytes culture medium of serum-free people of the invention, the rh-insulin are to use
Yeast or the insulin that Escherichia coli are the pharmaceutical grade that engineering bacteria is produced.
The preparation being related in present patent application can be bought except specified otherwise from market, be illustrated hereby.
The peripheral blood lymphocytes culture medium of the serum-free people of the present invention is used dynamic using serum substitute replacement tradition
Thing or human serum, culture medium cost so can be not only reduced, pathophorous risk can also be reduced；The nothing of the present invention
Component used in the peripheral blood lymphocytes culture medium of serum people determines, avoids traditional serum composition complexity, has between different batches
The problem of having mass discrepancy, and causing culture effect to fluctuate；Cell culture medium lymphocyte proliferation test provided by the invention and leaching
Bar cell division index height, reagent cost is low, steady quality, and the cell of culture can be used for the clinic of chromosome karyotype analysis to examine
It is disconnected.
Brief description of the drawings
Using accompanying drawing, the present invention is further illustrated, but the content in accompanying drawing does not form any limit to the present invention
System.
Fig. 1 is the PHA-L electrophoresis detection results of PHA extracts and purifying, wherein 1：PHA semifinished products；2：PHA-L；3：Egg
White molecule Markers.
Embodiment
The invention will be further described with the following Examples.
The present invention provide a kind of peripheral blood lymphocytes culture medium of serum-free people, including agglutinin, basal medium with
And serum substitute, the agglutinin are L-type phytohemagglutinin, the basal medium is the culture of RPMI1640 bases
Base, the serum substitute include bovine serum albumin(BSA), rh-insulin, ironic citrate and span amino acid mother liquor.
The peripheral blood lymphocytes culture medium of the serum-free people of the present invention, the purity of bovine serum albumin(BSA) used are not less than
98%.L-type phytohemagglutinin (PHA-L) used stimulates T lymphocytes mitotic for having from red kidney beans
Pleurotus Ostreatus glycoprotein fraction, purity are not less than 95%.It using yeast or Escherichia coli is engineering that the rh-insulin, which is,
The insulin for the pharmaceutical grade that bacterium is produced.
Embodiment one
(1) preparation of 50X span amino acid mother liquors
L-type amino acid used all comes from Chinese medicines group (BR levels), and 1L span amino acid mother liquors are specifically formulated as follows：
Pidolidone 300mg, L- glycine 100mg, L-Aspartic acid 400mg are weighed respectively, add 500mL ultra-pure waters
In, stir, add ultra-pure water to be settled to 1000mL, dispensed after mixing, it is standby in 4 DEG C of storages.
(2) PHA-L preparation method
(1) PHA crude extracts extract：
1. material：Red kidney beans (fresh, bright and lustrous without mouldy).
2. qualified red kidney bean powder is broken by examining, 100 eye mesh screens are crossed, red kidney beans bean powder is made.Take a certain amount of red
Kidney beans bean powder, soaked with the physiological saline measured again, put immersion 12 hours in refrigerator.Sample is centrifuged (20 DEG C, 10000rpm,
15min), supernatant is collected.Reset and add ethanol by to 30%, put in refrigerator 12 hours.Centrifugation, supernatant discarding, precipitation use 4# funnels
Filtering, precipitation is taken to put 4 DEG C of refrigerators 12 hours.Ethanol, ether are added in precipitation, solution ethanol concentration is reached 70%, puts 4 DEG C of ice
Case 12 hours.Supernatant discarding, pelleting centrifugation, washed with ethanol, centrifuge, take precipitation.Sample puts -20 DEG C of refrigerator precooling 1h, freezes,
Sample is stored in 4 DEG C of refrigerators and preserved, standby.
(2) PHA-L purifying：
PHA is separated using anion-exchange chromatography method, method is as follows：With 15mmol Na2HPO4(pH6.5) it is molten
Liquid balance DEAE-Sepharose chromatographic columns (1.6cm × 20cm), it is standby.PHA crude extract 10g are taken, it is molten with above-mentioned equilibrium liquid
Solution, 15min is centrifuged with 10000r/min, supernatant is used for DEAE-Sepharose ion-exchange chromatographies, then with 15mmol
Na2HPO4(pH6.5) equilibrium liquid washes post, and 276nm wavelength detectings, peak to be penetrated is all after outflow, and use 15mmol Na instead2HPO4
The cleaning buffer solution of (including 50mmol/L NaCl, pH6.5) is cleaned, flow velocity 5ml/min.15mmol Na are used again2HPO4
The elution buffer of (including 90mmol/L NaCl, pH6.5) is eluted, flow velocity 5ml/min, collects eluting peak, is
PHA-L solution.The PHA-L purity of gained is more than 95% (see Fig. 1 No. 2 swimming lanes), and content determines (purifying with 276nm wavelength
1.174) milligram extinction coefficient corresponding to PHA-L is.
(3) preparation of 10L serum-frees lymphocytes culture medium：
Add the rising amount of RPMI1640 dry powder 10 (104.0g, 3g containing glutamine, from GIBCO companies) to 5.0L ultra-pure waters
In, sodium acid carbonate (AR levels) 20g, span amino acid mother liquor (50X) 200mL, bovine serum albumin(BSA) are sequentially added after stirring clarification, and (MP is public
Department, stem cell culture level) 40g, rh-insulin (Recombinant protein expression, from LIFE companies) 100mg, ironic citrate
10mg,PHA-L 200mg.Ultra-pure water is added to 9.5L or so, 1M HCl is added thereafter and adjusts pH value to 7.2 and add ultra-pure water constant volume
As for 10.0L.0.2um PVDF membrane filtrations are degerming, are sub-packed in 20mL cillin bottles (5.0ml/ bottles), and -20 DEG C of storages are standby.
Comparative experiments
The serum-free peripheral blood lymphocytes culture medium of the present invention is imitated with traditional peripheral blood lymphocytes culture medium culture
Fruit is compared.
Traditional lymphocytes culture medium containing calf serum is prepared by table 1, and wherein RPMI1640 is with example 3, newly
Serum origin calve in Hangzhou Chinese holly company.It is degerming with 0.2um PVDF membrane filtrations to filter wild Oryza species, is sub-packed in
In 20mL cillin bottles (5.0ml/ bottles), -20 DEG C of storages are standby.
Cell culture effect detection is carried out by the following method：
(1) on aseptic manipulation work table, 1.0mL human peripherals are added into the blake bottle of the culture medium containing 5mL；
(2) by sample blending, put and cultivated in 37 DEG C of insulating boxs about 72 hours, jiggle a blake bottle within every 24 hours；
(3) colchicinamide was added before culture terminates before 2-4 hours to final concentration of 0.5ug/mL；
(4) cell suspension is added into 10mL centrifuge tubes, centrifuged 10 minutes with 1000 revs/min；
(5) supernatant is removed, stays cell mass；
(6) it is hypotonic：Add 37 DEG C of 0.075M KCl 4-6ml, suction pipe piping and druming, 37 DEG C of water-bath 3-5 minutes；
(7) pre-fix：Add 1.5mL methanol：Glacial acetic acid=3：1 fixer, is gently mixed, 37 DEG C of water-baths 5 minutes, with
1000 revs/min centrifuge 10 minutes.Go honest and upright and thrifty to stay 0.5mL；
(8) it is fixed：Add fixative 8mL, 37 DEG C of water-baths 5 minutes, centrifuged 10 minutes with 1000 revs/min.Go honest and upright and thrifty
Stay 0.5mL；
(9) once, 1000 revs/min centrifuge 10 minutes repeat step (8) thereafter, remove supernatant, are added depending on ttom of pipe cell concentration
Appropriate a few drop fixatives；
(10) piece, roasting piece are dripped：Every 1-2 drop, 75 DEG C are toasted 3 hours；
(11) lymphocyte transformation rate and division Phase Proportion are detected after GIEMSA dyeing.
The traditional lymphocytes culture medium formula (5L) containing calf serum of table 1
| Tradition has blood serum medium | |
| Step 1：Ultra-pure water (ml) | 3800 |
| Step 2：RPMI1640 dry powder (g) | 41 |
| Step 3：NaHCO3(g) | 10.0 |
| Step 4：Calf serum (ml) | 800 |
| Step 5：PHA-L mother liquors (mg) | 100 |
| Step 6：Add ultra-pure water to (L) | 4.5 |
| Step 6:Add 1MHCl and adjust pH value extremely | 7.2 |
| Step 7：Ultra-pure water is added to be settled to (L) | 5.0 |
Cell culture effect comparative result is relatively shown in Table 2.As seen from Table 2, serum free medium is in conversion ratio and split coil method
Will be better than in terms of index has blood serum medium, and is fluctuated between having less batch.
The serum-free of table 2 is compared with the peripheral blood lymphocytes culture medium culture effect for having serum
Note：The NBCS lot number for having blood serum medium to use of different lot numbers is respectively 20130103,20130108,
20140201、20140205、20140404。
The peripheral blood lymphocytes culture medium of the serum-free people of the present invention is used dynamic using serum substitute replacement tradition
Thing or human serum, culture medium cost so can be not only reduced, pathophorous risk can also be reduced；The nothing of the present invention
Component used in the peripheral blood lymphocytes culture medium of serum people determines, avoids traditional serum composition complexity, has between different batches
The problem of having mass discrepancy, and causing culture effect to fluctuate；Cell culture medium lymphocyte proliferation test provided by the invention and leaching
Bar cell division index height, reagent cost is low, steady quality, and the cell of culture can be used for the clinic of chromosome karyotype analysis to examine
It is disconnected.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should manage
Solution, can modify or equivalent substitution to technical scheme, without departing from technical solution of the present invention essence and
Scope.
Claims (1)
1. a kind of peripheral blood lymphocytes culture medium of serum-free people, the content of the culture medium each component are：
Wherein, the L-type phytohemagglutinin stimulates the mitotic aggegation of T lymphocytes for having from red kidney beans
Plain class glycoprotein fraction, the rh-insulin are the pharmaceutical grades produced using yeast or Escherichia coli for engineering bacteria
Insulin；
Wherein, the span amino acid mother liquor includes Pidolidone, L- glycine and L-Aspartic acid, and Pidolidone is in span amino acid mother liquor
In concentration be 300mg/L, concentration of the L- glycine in span amino acid mother liquor is 100mg/L, and L-Aspartic acid is female in amino acid
Concentration in liquid is 400mg/L；Wherein, the purity of the bovine serum albumin(BSA) is not less than 98%；
Wherein, the purity of the L-type phytohemagglutinin is not less than 95%.
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| WO2016201657A1 (en) * | 2015-06-17 | 2016-12-22 | 深圳市达科为生物工程有限公司 | Serum replacement for culturing lymphocytes, and preparation method therefor |
| CN106754690A (en) * | 2016-12-06 | 2017-05-31 | 河北意和医学检验所有限公司 | A kind of chromosome culture medium of quick results medium cell and application |
| CN106520694A (en) * | 2016-12-24 | 2017-03-22 | 叶宗耀 | Human peripheral blood lymphocyte medium and preparation method thereof |
| 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 |
| CN109055309B (en) * | 2018-08-02 | 2021-07-02 | 广州白云山拜迪生物医药有限公司 | Serum-free cell culture medium and application thereof |
| CN109799123A (en) * | 2018-12-29 | 2019-05-24 | 广州和能生物科技有限公司 | A kind of method of quick carry out Rui Shi Giemsa staining |
| CN112795537A (en) * | 2019-11-13 | 2021-05-14 | 苏州易迈吉生物医药科技有限公司 | Serum-free medium for culturing human peripheral blood dendritic cells |
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| CN100432218C (en) * | 2005-01-05 | 2008-11-12 | 上海尚优生物科技有限公司 | Embryo cattle serum substitute without serum for animal cell culture |
| CN102352343B (en) * | 2011-09-28 | 2013-04-24 | 上海柯莱逊生物技术有限公司 | Lymphocyte culture medium and use method thereof |
| CN102816735B (en) * | 2012-09-12 | 2014-08-13 | 山东省齐鲁细胞治疗工程技术有限公司 | Method for culturing autologous peripheral blood lymphocytes |
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