CN103667183B - Bone marrow cell cultures - Google Patents
Bone marrow cell cultures Download PDFInfo
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- CN103667183B CN103667183B CN201310591742.1A CN201310591742A CN103667183B CN 103667183 B CN103667183 B CN 103667183B CN 201310591742 A CN201310591742 A CN 201310591742A CN 103667183 B CN103667183 B CN 103667183B
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Abstract
The invention discloses the bone marrow cell cultures formulas that chromosome karyotype analysis is used in a kind of field of medical examination, and penicillin/streptomycin, fetal calf serum and human lymphoma cell culture are added in basal medium.The G-band chromosome that the bone marrow cell prepared using the culture medium of the present invention is produced, background are more clear, and split coil method is more, and form and dispersion degree are better, convenient for analysis.When such formula is for Marrow chromosome karyotyping, there is time saving, labour-saving advantage, and accuracy higher, there is wider application prospect in field of medical examination.
Description
Technical field
The invention belongs to biotechnologies, thin for the marrow of chromosome karyotype analysis more particularly in field of medical examination
Born of the same parents' culture medium prescription.
Background technology
G shows band because chromosome is mainly that band is shown after Giemsa dyeings, therefore referred to as G banding techniques, institute
The band line of display is distributed in whole chromosome.People will handle dyeing with a variety of different methods and with different dyestuffs
After body sample, make to occur light and dark or depth different band line technology on every chromosome to be known as banding technique（banding
technique）.Since 1970's, banding technique has obtained developing on a large scale very much, and in numerous banding techniques（Q bands, G
Band, C bands, R bands, T bands）, G bands are a kind of banding patterns being widely used at present.
The study found that human chromosome sample then is used after the processing of the reagents such as trypsase, Na0H, citrate or urea
Giemsa is dyed, and can make to show the alternate band of the depth on every chromosome, here it is the G bands of chromosome.Every chromosome
There is its more constant band line feature, so after G shows band, can accurately identify every chromosome, and can find to contaminate
Subtleer structural aberration on colour solid.
In recent years, with molecular biology and cytogenetic development, Marrow chromosome karyotyping is in hematological system
Increasingly important role has been played in diagnosis, treatment and the prognosis of disease.The preparation of Marrow chromosome is a large amount of because having in marrow
The interference of lipochondrion, in marrow the cell cycle of various cell lines be not fixed, disunity, it becomes difficult to distinguish treat and cause
Marrow chromosome splitting index is low, and chromosome is short and thick, and dispersion degree is poor, and cost is higher.
Therefore, a kind of marrow training for having many characteristics, such as that background is more clear, split coil method is more, form and dispersion degree are good is established
It is particularly important to support based formulas.
Invention content
The defects of the purpose of the present invention is overcoming the prior art, provides a kind of bone marrow cell cultures, is cultivated on basis
Penicillin/streptomycin, fetal calf serum and human lymphoma cell culture are added in base；
Basal medium：RPMI1640 culture mediums
The dosage of each adding ingredient is as follows：
Penicillin/streptomycin 5-15ul/ml
Fetal calf serum 60-140 ul/ml
Human lymphoma cell culture 60-140 ul/ml
Further, penicillin is selected from 10000U/ml, and streptomysin is selected from 10000 μ g/ml.
Further, human lymphoma cell culture is produced by following methods：Cell recovery and passage, work as passage cell
It when culture medium color becomes yellow, collects cell and centrifuges, after centrifugation, collect supernatant and filter to obtain cell culture.
Further, each adding ingredient dosage is as follows：
Penicillin/streptomycin 8ul/ml
Fetal calf serum 96ul/ml
Human lymphoma cell culture 96ul/ml
The present invention also provides application of the bone marrow cell cultures in Marrow chromosome film-making, by bone marrow cell with 1~3
×106The density of a/ml is inoculated into the marrow medium, is put into 37 DEG C, 5.0%CO2Incubator culture 24 hours.
The beneficial effects of the invention are as follows：This laboratory adds on the basis of studying for a long period of time in traditional infrastructure culture medium
Human lymphoma cell culture.The culture can provide more nutrients for bone marrow cell（Including growth factor
Deng）, thus the cell turned out is after demecolcine terminates culture, the G that produces out band background is more clear, split coil method is more,
Form and dispersion degree are better.
Description of the drawings
Fig. 1 is the microscopy result using 1 gained of culture medium
Fig. 2 is the microscopy result using 2 gained of culture medium
Fig. 3 is the microscopy result using 3 gained of culture medium
Fig. 4 is the microscopy result using 4 gained of culture medium
Specific embodiment
With reference to specific embodiment, the present invention is further explained.
Embodiment 1 prepares bone marrow cell cultures
Penicillin/streptomycin, fetal calf serum and human lymphoma cell culture are added in basal medium.Wherein
Basal medium is RPMI1640 culture mediums, and the dosage of each adding ingredient is as follows：
Penicillin/streptomycin 5-15ul/ml
Fetal calf serum 60-140 ul/ml
Human lymphoma cell culture 60-140 ul/ml
Wherein, penicillin may be selected from 10000U/ml, and streptomysin may be selected from 10000 μ g/ml.In other embodiments, may be used
It is used to prepare culture medium selected from other concentration penicillin and streptomysin.
Wherein, human lymphoma cell culture is produced by the following method：Cell is through recovering and passing on, when passage cell is trained
It when foster base color becomes yellow, collects cell and simultaneously centrifuges, after centrifugation, collect supernatant and simultaneously filter to obtain cell culture.
The method for more specifically producing human lymphoma cell culture includes the following steps：
A. by super-clean bench bromogeramine wiped clean, ultraviolet light irradiates 30 minutes.
B. cryopreservation tube is taken out from liquid nitrogen container, 37 DEG C of water-baths is immediately placed in and shakes and promote its content in 2 minutes and melt rapidly.
C. cell suspension is transferred to 10ml containing in 9.6% fetal calf serum RPMI-1640 culture mediums, 37 DEG C, 5%CO2 is cultivated.
D. when cell reaches about 50% fusion rate, the trypsase of 4ml is added in（Containing EDTA）, it is ensured that Tissue Culture Flask
Bottom covers a thin layer trypsase.
E. culture bottle is put into incubator and is incubated 5min, microscopy if all cells have all come off, adds in the culture of 10ml
Base, and broken up cell with pipette.
F. and then 30ml fresh cultures are added, cell suspension one is passed four, is transferred to the cell containing 10ml culture mediums
In culture bottle, leniently mixing cell.
G. culture bottle is put into incubator（Unscrew bottle cap）, when cell density reaches about 50% fusion rate, replace training
Base is supported, until final concentration 40ml or so.
H. prepare to collect when culture medium color becomes yellow.Cultured cell is transferred to 50ml circles with serum pipette
In the polystyrene centrifuge tube of bottom（Stick respective labels）, 2000 rpm centrifugations 10min.
I. supernatant is collected, and is filtered through 0.22 μm of sterile filters（It is sure not to encounter cell precipitation during collection）, the as mankind
Lymphoma cell culture.If it is stored in -20 DEG C without using the culture after filtering can be dispensed.
2 medullary microeirculation of embodiment and chromosome sectioning
The present embodiment cultivates bone marrow cell as follows, and G-band chromosome film-making is used for after harvest.In other embodiment
In, other method culture bone marrow cells and chromosome sectioning can also be used.The present embodiment the method is：
（1）Prepare marrow medium：It prepares as described in Example 1；
（2）Inoculation：Bone marrow cell is inoculated into the culture medium described in step 1；
（3）Terminate culture：Cell is terminated through demecolcine and is cultivated；
（4）Collect bone marrow cell cultures；
（5）Chromosome specimen film-making：The chromosome specimen of band is shown with G using acquisition after dyeing.
Wherein by bone marrow cell with 1~3 × 106The density of a/ml is inoculated into marrow medium, is put into 37 DEG C, and 5.0%
CO2Incubator culture 24 hours.Gained bone-marrow cultures can be used for Marrow chromosome film-making.
Wherein, collecting bone marrow cell cultures can collect as follows, can also be received by other methods commonly used in the art
Collection.
(1) it is mild to rock culture bottle, and culture is transferred in corresponding 15ml centrifuge tubes.Culture bottle cap is tightened, and
Ensure sample without mutually mixed.1,000 rpm centrifuge 10min.Supernatant is sucked, leaves about 0.5 to 1.0 ml vortex mixings.
(2) it is hypotonic：Add in the 0.075M KCl solution of 37 DEG C of incubator preheatings of 10ml.Being vortexed or overturning repeatedly makes it several times
With sample blending, 37 DEG C of water-baths are incubated 20-30min, and during which centrifuge tube rolls three times so that cell is hypotonic uniformly.
(3) it pre-fixes：The fixer of 1ml is added in after hypotonic（Methanol：Glacial acetic acid=3：1）, tighten lid and repeatedly
It overturns three times.1,000 rpm centrifuge 10min.Supernatant is sucked, leaves about 0.5 to 1.0 ml.
(4) by after sediment vortex mixing, 8ml Fresh fixatives are added dropwise.
(5) 1,000 rpm centrifuge 10min after vortex mixing.Supernatant is sucked, leaves about 0.5 to 1.0 ml.
(6) 8ml Fresh fixatives are added in after mixing cell precipitation.
(7) with 5 and 6.
(8) 1,000 rpm centrifuge 10min after vortex mixing.Supernatant is sucked, stays appropriate fixer, and add in few drops of ice
Acetic acid stands film-making after 15min so that the suitable cell suspension of concentration is made.
Chromosome specimen flaking method wherein in the present embodiment is as follows：
A. the preparation of slide：In advance by 1% HCl soaked overnights of slide, 95% is dipped in after being rinsed with a large amount of clear water
It is spare in ethyl alcohol.It is taken out, is positioned over after being cleaned with a large amount of clear water in 2-8 DEG C of refrigerator for use using the preceding slide by immersion.
B. piece is dripped：After drawing cell suspension, dropper is placed in the height of 15~20cm, drop 4-5 drop cell suspensions are in slide
On, cell is made to be flowed to slide marker end distal side.It suitably overdoes, helps Chromosome spread.A general patient drips piece 1-2.
C. piece aging is baked：It is placed in 60 DEG C of oven for baking overnight or 80 DEG C is toasted 1 hour.
D. pancreatin is prepared:0.3% trypsase of Fresh 50ml（It is diluted with HANKS buffer solutions）Solution is placed in dye piece
In cylinder, preheating is more than half an hour in 37 DEG C of water baths.Trypsase and HANKS buffer solutions are purchased from Invitrogen companies.
E. Giemsa dye liquors are prepared:Face the used time by the phosphate buffer of Gimesa stostes and pH6.8 according to 1:20 mixing.
F. chromosome karyotype analysis is used for after being dyed with pancreatin digestion.
3 contrast experiment 1 of embodiment
Three groups of culture mediums are prepared according to the method for embodiment 1, are culture medium 1, culture medium 2, culture medium 3 respectively, and set up
Control group carries out marrow culture, the culture medium of the control group（Culture medium 4）In do not contain human lymphoma cell culture.It takes
The bone marrow cell of same sample carries out cell culture, and by real in culture medium 1 to culture medium 4 respectively as described in Example 2
The method for applying example 2 makes G-band chromosome, and in micro- Microscopic observation film-making result.The microscope model used：Leica
DM2500。
Wherein：
The formula of culture medium 1 is：
Basal medium：RPMI1640 culture mediums
Penicillin/streptomycin 5ul/ml
Fetal calf serum 60ul/ml
60 ul/ml of human lymphoma cell culture
The formula of culture medium 2 is：
Basal medium：RPMI1640 culture mediums
Penicillin/streptomycin 15ul/ml
140 ul/ml of fetal calf serum
140 ul/ml of human lymphoma cell culture
The formula of culture medium 3 is：
Basal medium：RPMI1640 culture mediums
Penicillin/streptomycin 8ul/ml
96 ul/ml of fetal calf serum
96 ul/ml of human lymphoma cell culture
The formula of culture medium 4 is：
Basal medium：RPMI1640 culture mediums
Penicillin/streptomycin 10ul/ml
100 ul/ml of fetal calf serum
Microscopy result as shown in Figures 1 to 4：Fig. 1 is the experimental result after bone marrow cell is cultivated in culture medium 1, and Fig. 2 is bone
Myelocyte cultivated in culture medium 2 after experimental result, Fig. 3 is the experimental result after bone marrow cell is cultivated in culture medium 3, figure
4 be the experimental result that bone marrow cell is cultivated in culture medium 4.See with being apparent from microscopy photo from Fig. 1 to 3, use this
Inventive method carries out G band colour developings, and background is more clear, split coil method is more, form and dispersion degree are better.When thus diagnosing,
More time saving and energy saving, diagnostic result is more accurate.And in the microscopy photo of Fig. 4 conventional method cultures, form is imperfect, impurity compared with
More, split coil method is few and rolls into a ball, time-consuming and laborious outer, easily causes mistaken diagnosis, wherein arrow show marrow split coil method in figure.
Claims (1)
1. application of a kind of bone marrow cell cultures in Marrow chromosome film-making, which is characterized in that provide one kind for dyeing
The bone marrow cell cultures of body karyotyping are added to penicillin/streptomycin, fetal calf serum and the mankind in basal medium
Lymphoma cell culture；
Basal medium：RPMI1640 culture mediums
The dosage of each adding ingredient is as follows：
Penicillin/streptomycin 5-15ul/ml
Fetal calf serum 60-140ul/ml
Human lymphoma cell culture 60-140ul/ml；
By bone marrow cell with 1~3 × 106The density of a/ml is inoculated into the marrow medium, is put into 37 DEG C, 5.0%CO2
Incubator culture 24 hours.
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| CN111411074A (en) * | 2019-01-05 | 2020-07-14 | 杭州可典生物科技有限公司 | Bone marrow cell culture medium |
| 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 |
| CN112577803B (en) * | 2020-12-10 | 2021-11-09 | 中国水产科学研究院珠江水产研究所 | Method for preparing turtle chromosome specimen by using peripheral blood cells |
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