CN105602892A - Bone marrow cell culture medium - Google Patents
Bone marrow cell culture medium Download PDFInfo
- Publication number
- CN105602892A CN105602892A CN201610196367.4A CN201610196367A CN105602892A CN 105602892 A CN105602892 A CN 105602892A CN 201610196367 A CN201610196367 A CN 201610196367A CN 105602892 A CN105602892 A CN 105602892A
- Authority
- CN
- China
- Prior art keywords
- bone marrow
- culture medium
- cell culture
- marrow cell
- basal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0669—Bone marrow stromal cells; Whole bone marrow
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Abstract
The invention discloses a bone marrow cell culture medium which comprises a basal culture medium containing fetal calf serum, human lymphoma cell culture and a compound Cephaloziellin N. The invention also provides a method for preparing bone marrow cells used in bone marrow chromosome sectioning, comprising the steps of: inoculating bone marrow cells with density of 1-3*106/ml into the bone marrow cell culture medium, and putting into an incubator of 37 DEG C with 5% CO2 for culturing for 20-28 hours. When the bone marrow cells cultured by the bone marrow cell culture medium provided by the invention are used in chromosome G band sectioning, a clearer background, more division phases and better form and dispersion degree are obtained.
Description
Technical field
The invention belongs to field of cell culture, be specifically related to a kind of bone marrow cell culture medium.
Background technology
In recent years, along with molecular biology and cytogenetic development, the karyotyping of bone marrow stain body is in disease in the blood systemIn diagnosis, treatment and prognosis, bring into play more and more important effect. The preparation of bone marrow stain body is because there being significant quantities of fat particle in marrowInterference, in marrow the cell cycle of various clones fixing, disunity, is difficult to treat with a certain discrimination and makes bone marrow stain bodyDi is low, and chromosome is short and thick, and decentralization is poor, and cost is higher.
Therefore, set up a kind ofly have that background is more clear, the division marrow culture medium of many, form and the feature such as decentralization is good mutuallyFill a prescription particularly important.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of bone marrow cell culture medium is provided, make the marrow of turning out thinBorn of the same parents' background when the G-band chromosome film-making is more clear, division mutually more, form and decentralization better.
Above-mentioned purpose of the present invention is to be achieved by technical scheme below:
A kind of bone marrow cell culture medium, comprises basal medium, contains hyclone, human lymphoma in described basal mediumCell culture and Compound C ephaloziellinN.
Further, the preparation method of human lymphoma cell culture is: human lymphoma cell is recovered and gone down to posterity,When passage cell culture medium color becomes yellow, collecting cell centrifugal, centrifugal after, collect supernatant and get final product.
Further, the concentration of described human lymphoma cell culture in basal medium is 100～140 μ l/ml.
Further, the concentration of described hyclone in basal medium is 80～120 μ l/ml.
Further, the concentration of described Compound C ephaloziellinN in basal medium is 20～30 μ g/ml.
Further, also comprise antibiotic.
Further, described antibiotic is penicillin, and the concentration in basal medium is 4000～6000U/ml.
Further, described antibiotic is streptomysin, and the concentration in basal medium is 5000～7000 μ g/ml.
Further, described basal medium is RPMI1640 culture medium.
For the preparation of a method for the bone marrow cell of bone marrow stain system sheet, it is characterized in that, comprise the steps: marrowCell is with 1～3 × 106The density of individual/ml is inoculated in bone marrow cell culture medium as above, puts into 37 DEG C, 5%CO2CultivateCase is cultivated 20～28 hours.
Advantage of the present invention:
The bone marrow cell that bone marrow cell medium culture provided by the invention goes out during for G-band chromosome film-making background more clear,Division mutually more, form and decentralization better.
Detailed description of the invention
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit protection domain of the present invention with this. To the greatest extentPipe is explained in detail the present invention with reference to preferred embodiment, and those of ordinary skill in the art should be appreciated that can be to the present inventionTechnical scheme modify or be equal to replacement, and do not depart from essence and the scope of technical solution of the present invention.
The preparation method of the compounds of this invention CephaloziellinN is referring to document: SecondaryMetabolitesfromtheChineseLiverwortCephaloziellakiaeri,J.Nat.Prod.2013,76,1700-1708。
Embodiment 1: the preparation of bone marrow cell culture medium
Basal medium is RPMI1640 culture medium, contains hyclone, human lymphoma cell culture and compoundCephaloziellinN; The concentration of hyclone in basal medium is 100 μ l/ml, and Compound C ephaloziellinN is at baseConcentration in basal culture medium is 25 μ g/ml. Also contain penicillin, concentration is 5000U/ml. Penicillin can replace with streptomysin,Concentration is 6000 μ g/ml. The concentration of human lymphoma cell culture in basal medium is 120 μ l/ml, and human lymphoma is thinBorn of the same parents' culture preparation method is: human lymphoma cell recovered and gone down to posterity, and when passage cell culture medium color becomes yellow,Collecting cell centrifugal, centrifugal after, collect supernatant and get final product. More detailed preparation process is as follows:
Step S1, by super-clean bench bromogeramine wiped clean, ultraviolet ray is irradiated 30 minutes;
Step S2 takes out cryopreservation tube from liquid nitrogen container, puts into immediately 37 DEG C of short its contents of water-bath shake and melts rapidly for 2 minutes;
Step S3, cell suspension proceeds to 10ml containing in 8% hyclone RPMI-1640 culture medium, 37 DEG C, 5%CO2Cultivate;
Step S4, in the time that cell reaches approximately 50% fusion rate, adds the trypsase (containing EDTA) of 4ml, guarantees cellBlake bottle bottom covers skim trypsase;
Step S5, puts into incubator by blake bottle and hatches 5min, and microscopy, if all cells all comes off, adds 10ml'sCulture medium, and cell is broken up with pipette;
Step S6, and then add 30ml fresh culture, cell suspension one is passed to four, proceed to and contain the thin of 10ml culture mediumIn born of the same parents' blake bottle, leniently mix cell;
Step S7, puts into incubator (unscrewing bottle cap) by blake bottle, in the time that cell density reaches about 50% fusion rate, moreChange culture medium, to final concentration 40ml left and right;
Step S8 prepares to collect in the time that culture medium color becomes yellow; Cultured cell is proceeded to 50ml with serum pipetteIn poly-the third ethene centrifuge tube of round bottom (sticking respective labels), the centrifugal 10min of 2000rpm;
Step S9, collects supernatant, and filters (when collection, being sure not to encounter cell precipitation) through 0.22 μ m sterile filters, isHuman lymphoma cell culture; Culture packing after filtering can be stored in to-20 DEG C if do not used.
The contrast of embodiment 2: embodiment 1, does not add CephaloziellinN
Basal medium is RPMI1640 culture medium, contains hyclone and human lymphoma cell culture; Hyclone existsConcentration in basal medium is 100 μ l/ml. Also contain penicillin, concentration is 5000U/ml. Penicillin can be used streptomysin generationReplace, concentration is 6000 μ g/ml. The concentration of human lymphoma cell culture in basal medium is 120 μ l/ml, mankind's lymphOncocyte culture preparation method is: human lymphoma cell is recovered and gone down to posterity, and passage cell culture medium color becomes HuangWhen look, collecting cell centrifugal, centrifugal after, collect supernatant and get final product. More detailed preparation process is as follows:
Step S1, by super-clean bench bromogeramine wiped clean, ultraviolet ray is irradiated 30 minutes;
Step S2 takes out cryopreservation tube from liquid nitrogen container, puts into immediately 37 DEG C of short its contents of water-bath shake and melts rapidly for 2 minutes;
Step S3, cell suspension proceeds to 10ml containing in 8% hyclone RPMI-1640 culture medium, 37 DEG C, 5%CO2Cultivate;
Step S4, in the time that cell reaches approximately 50% fusion rate, adds the trypsase (containing EDTA) of 4ml, guarantees cellBlake bottle bottom covers skim trypsase;
Step S5, puts into incubator by blake bottle and hatches 5min, and microscopy, if all cells all comes off, adds 10ml'sCulture medium, and cell is broken up with pipette;
Step S6, and then add 30ml fresh culture, cell suspension one is passed to four, proceed to and contain the thin of 10ml culture mediumIn born of the same parents' blake bottle, leniently mix cell;
Step S7, puts into incubator (unscrewing bottle cap) by blake bottle, in the time that cell density reaches about 50% fusion rate, moreChange culture medium, to final concentration 40ml left and right;
Step S8 prepares to collect in the time that culture medium color becomes yellow; Cultured cell is proceeded to 50ml with serum pipetteIn poly-the third ethene centrifuge tube of round bottom (sticking respective labels), the centrifugal 10min of 2000rpm;
Step S9, collects supernatant, and filters (when collection, being sure not to encounter cell precipitation) through 0.22 μ m sterile filters, isHuman lymphoma cell culture; Culture packing after filtering can be stored in to-20 DEG C if do not used.
Embodiment 3: effect embodiment
Use respectively the bone marrow cell culture medium of embodiment 1 and 2 to cultivate as follows bone marrow cell, after results for chromosomeG is with film-making:
(1) preparation marrow culture medium: the method preparation of pressing embodiment 1 and 2;
(2) inoculation: bone marrow cell is inoculated in the culture medium described in step 1;
(3) stop cultivating: cell stops cultivating through demecolcine;
(4) collect bone marrow cell culture;
(5) chromosome specimen film-making: obtain the chromosome specimen with the aobvious band of G after utilizing dyeing.
Wherein, by bone marrow cell with 1～3 × 106The density of individual/ml is inoculated in marrow culture medium, puts into 37 DEG C, 5%CO2TrainingSupporting case cultivates 24 hours. Gained marrow culture can be used for bone marrow stain system sheet.
Wherein, collect bone marrow cell culture and can collect as follows, also can collect by other conventional method of this area.
(1) the gentle blake bottle that rocks, and culture is proceeded in corresponding 15ml centrifuge tube. Tighten cultivation bottle cap, and reallyProtect sample not mixed mutually. The centrifugal 10min of 1000rpm. Suck supernatant, leave approximately 0.5 and mix to 1.0ml vortex.
(2) hypotonic: the 0.075MKCl solution that adds 10ml37 DEG C of incubator preheating. Vortex or repeatedly put upside down makes itself and sample several timesProduct mix, and 20～30min is hatched in 37 DEG C of water-baths, centrifuge tube are rolled three times during this time so that cell is hypotonic evenly.
(3) pre-fix: after hypotonic end, add the fixer (methyl alcohol: glacial acetic acid=3:1) of 1ml, tighten lid and repeatedly runFall three times. The centrifugal 10min of 1000rpm. Suck supernatant, leave approximately 0.5 to 1.0ml.
(4) after sediment vortex is mixed, dropwise add the fresh fixer of 8ml.
(5) vortex mixes the centrifugal 10min of rear 1000rpm. Suck supernatant, leave approximately 0.5 to 1.0ml.
(6) add the fresh fixer of 8ml after mixing cell precipitation.
(7) with 5 and 6.
(8) vortex mixes the centrifugal 10min of rear 1000rpm. Suck supernatant, stay appropriate fixer, and add several ice vinegarSour to make the cell suspension that concentration is suitable, film-making after standing 15min.
Wherein the chromosome specimen flaking method in the present embodiment is as follows:
(1) preparation of slide: in advance slide is used to 1%HCl soaked overnight, be dipped in 95% after rinsing with a large amount of clear waterFor subsequent use in ethanol. Before using, the slide soaking is taken out, after cleaning with a large amount of clear water, be positioned in 2～8 DEG C of refrigerators stand-by.
(2) drip sheet: draw after cell suspension, dropper is placed in to the height of 15～20cm, drip 4～5 cell suspensions in slideUpper, cell is flowed to slide mark end distally. Suitably overdo, help Chromosome spread. A general patient is dripped 1～2 of sheet.
(3) roasting sheet is aging: be placed in that 60 DEG C of oven for baking are spent the night or 80 DEG C of bakings 1 hour.
(4) preparation pancreatin: fresh preparation 50ml0.3% trypsase (with the dilution of HANKS buffer solution) solution is placed in and dyes sheetIn cylinder, in 37 DEG C of water baths more than preheating half an hour. Trypsase and HANKS buffer solution are all purchased from Invitrogen company.
(5) preparation Giemsa dye liquor: face the used time Gimesa stoste is mixed according to 1:20 with the phosphate buffer of pH6.8.
(6) with after trypsinization dyeing for chromosome karyotype analysis.
The bone marrow cell of medium culture prepared by result: embodiment 1 during for G-band chromosome film-making background more clear, pointSplit that mutually more, form and decentralization are better; In the microscopy photo that embodiment 2 cultivates, form is imperfect, and impurity is more,Division mutually less and group, outside wasting time and energy, very easily cause mistaken diagnosis.
The effect of above-described embodiment is to illustrate essentiality content of the present invention, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art should be appreciated that and can modify or be equal to replacement technical scheme of the present invention, and de-From essence and the protection domain of technical solution of the present invention.
Claims (10)
1. a bone marrow cell culture medium, comprises basal medium, it is characterized in that: in described basal medium, contain tire ox bloodHuman lymphoma cell culture and Compound C ephaloziellinN clearly.
2. bone marrow cell culture medium according to claim 1, is characterized in that, the preparation of human lymphoma cell cultureMethod is: human lymphoma cell is recovered and gone down to posterity, and when passage cell culture medium color becomes yellow, collecting cell alsoCentrifugal, centrifugal after, collect supernatant and get final product.
3. bone marrow cell culture medium according to claim 2, is characterized in that: described human lymphoma cell culture existsConcentration in basal medium is 100～140 μ l/ml.
4. bone marrow cell culture medium according to claim 1, is characterized in that: described hyclone is in basal mediumConcentration be 80～120 μ l/ml.
5. bone marrow cell culture medium according to claim 1, is characterized in that: described Compound C ephaloziellinN existsConcentration in basal medium is 20～30 μ g/ml.
6. bone marrow cell culture medium according to claim 1, is characterized in that: also comprise antibiotic.
7. bone marrow cell culture medium according to claim 6, is characterized in that: described antibiotic is penicillin, on basisConcentration in culture medium is 4000～6000U/ml.
8. bone marrow cell culture medium according to claim 6, is characterized in that: described antibiotic is streptomysin, on basisConcentration in culture medium is 5000～7000 μ g/ml.
9. according to the arbitrary described bone marrow cell culture medium of claim 1～8, it is characterized in that: described basal medium is RPMI1640 culture mediums.
10. for the preparation of a method for the bone marrow cell of bone marrow stain system sheet, it is characterized in that, comprise the steps: byBone marrow cell is with 1～3 × 106The density of individual/ml is inoculated in bone marrow cell culture medium as claimed in claim 9, put into 37 DEG C,5％CO2Incubator is cultivated 20～28 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610196367.4A CN105602892A (en) | 2016-03-31 | 2016-03-31 | Bone marrow cell culture medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610196367.4A CN105602892A (en) | 2016-03-31 | 2016-03-31 | Bone marrow cell culture medium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105602892A true CN105602892A (en) | 2016-05-25 |
Family
ID=55983240
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610196367.4A Pending CN105602892A (en) | 2016-03-31 | 2016-03-31 | Bone marrow cell culture medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105602892A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109580299A (en) * | 2017-09-28 | 2019-04-05 | 上海新培晶医学检验所有限公司 | Bone marrow cell chromosome flaking method |
| CN112080466A (en) * | 2020-09-16 | 2020-12-15 | 上海培晖生物科技发展有限公司 | Bone marrow culture medium and preparation method and application thereof |
| CN112063579B (en) * | 2020-09-16 | 2023-08-25 | 上海培晖生物科技发展有限公司 | Serum-free bone marrow culture medium and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667183A (en) * | 2013-11-22 | 2014-03-26 | 南昌艾迪康临床检验所有限公司 | Bone marrow cell culture medium |
| CN103710435A (en) * | 2013-11-22 | 2014-04-09 | 福州艾迪康医学检验所有限公司 | Marrow chromosome extraction kit |
| CN103710434A (en) * | 2013-11-22 | 2014-04-09 | 长沙艾迪康医学检验所有限公司 | Manufacturing method for marrow chromosome G band |
-
2016
- 2016-03-31 CN CN201610196367.4A patent/CN105602892A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667183A (en) * | 2013-11-22 | 2014-03-26 | 南昌艾迪康临床检验所有限公司 | Bone marrow cell culture medium |
| CN103710435A (en) * | 2013-11-22 | 2014-04-09 | 福州艾迪康医学检验所有限公司 | Marrow chromosome extraction kit |
| CN103710434A (en) * | 2013-11-22 | 2014-04-09 | 长沙艾迪康医学检验所有限公司 | Manufacturing method for marrow chromosome G band |
Non-Patent Citations (1)
| Title |
|---|
| RUI-JUAN LI,等: "Secondary Metabolites from the Chinese Liverwort Cephaloziella kiaeri", 《J. NAT. PROD.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109580299A (en) * | 2017-09-28 | 2019-04-05 | 上海新培晶医学检验所有限公司 | Bone marrow cell chromosome flaking method |
| CN112080466A (en) * | 2020-09-16 | 2020-12-15 | 上海培晖生物科技发展有限公司 | Bone marrow culture medium and preparation method and application thereof |
| CN112080466B (en) * | 2020-09-16 | 2023-08-08 | 上海培晖生物科技发展有限公司 | Bone marrow culture medium and preparation method and application thereof |
| CN112063579B (en) * | 2020-09-16 | 2023-08-25 | 上海培晖生物科技发展有限公司 | Serum-free bone marrow culture medium and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103710434B (en) | A kind of making method of marrow chromosome G band | |
| CN105602892A (en) | Bone marrow cell culture medium | |
| CN105420184A (en) | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue | |
| CN107164319A (en) | A kind of method of the mescenchymal stem cell in original cuiture dog umbilical cord source | |
| CN109486753A (en) | A kind of fat stem cell extracting method | |
| CN108384745A (en) | A kind of method that improved two steps enzyme is separately cultured sustentacular cell of testis | |
| CN103667183B (en) | Bone marrow cell cultures | |
| CN102286532A (en) | Method for obtaining inductive pluripotent stem cell | |
| CN106119191A (en) | A kind of placental villi plate mescenchymal stem cell and clinicization preparation method | |
| CN114606186A (en) | Method for improving proliferation of umbilical cord mesenchymal stem cells | |
| CN106434536A (en) | Three-dimensional inducing method for inducing spermatogonia stem cell to differentiate into functional sperm cell in vitro | |
| CN107589002A (en) | A kind of preparation method of induced multi-potent stem cell chromosome | |
| CN102250841A (en) | Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof | |
| CN106497863A (en) | A kind of separation of cornea of rats endothelial cell, purifying and cultural method | |
| CN103756970B (en) | Optimize episomal technology and set up the method for people ips | |
| CN105624115B (en) | Culture medium for inducing human umbilical cord mesenchymal stem cells to differentiate into nerve-like cells and induction method thereof | |
| CN104928319B (en) | The method of hTERT slow virus recombinant immortal human periodontal ligament stem cells system | |
| Yokoyama | Production of monoclonal antibody supernatant and ascites fluid | |
| CN207294806U (en) | For dyeing the Tissue Culture Dish of observation | |
| CN106754672A (en) | A kind of cultural method of attached cell | |
| CN106566802A (en) | Preparation method for preparing culture kit of umbilical cord blood mesenchemal stem cells | |
| CN105816872A (en) | Preparation method of mink parvoviral enteritis inactivated vaccine and vaccine prepared by using same | |
| CN104498431B (en) | A kind of method of slide Culture in situ amniocyte and caryogram Treatment Analysis | |
| CN103710435B (en) | marrow chromosome extraction kit | |
| CN103667458B (en) | Bone marrow cell is cultivated stop buffer and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: Jinzhai road in Baohe District of Hefei city of Anhui Province, University of Science & Technology China No. 96 230026 Applicant after: Gu Chao Address before: Tong Xiang, Gulou District of Nanjing city of Jiangsu Province, China Medicine University No. 24 210009 Applicant before: Gu Chao |
|
| COR | Change of bibliographic data | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160525 |
|
| WD01 | Invention patent application deemed withdrawn after publication |