CN112080466A - Bone marrow culture medium and preparation method and application thereof - Google Patents
Bone marrow culture medium and preparation method and application thereof Download PDFInfo
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- 229930182555 Penicillin Natural products 0.000 claims abstract description 23
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract description 23
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- 210000004027 cell Anatomy 0.000 claims description 34
- 239000006228 supernatant Substances 0.000 claims description 28
- 239000002609 medium Substances 0.000 claims description 25
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 239000007640 basal medium Substances 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 14
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- 238000004458 analytical method Methods 0.000 claims description 8
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- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 claims description 6
- 239000006143 cell culture medium Substances 0.000 claims description 6
- 108010019160 Pancreatin Proteins 0.000 claims description 5
- 229940055695 pancreatin Drugs 0.000 claims description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 5
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- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 description 3
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- 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
- C12N5/0634—Cells from the blood or the immune system
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
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- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention provides a bone marrow culture medium and a preparation method and application thereof. The bone marrow culture medium comprises a basic culture medium, human lymphocyte culture supernatant and NaHCO, wherein the human lymphocyte culture supernatant is cultured by adding fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite and rhu-EPO in the basic culture medium3. The human lymphocyte culture supernatant cultured by rhu-EPO provides growth factors required by bone marrow growth, is low in use amount, is easy to obtain the human lymphoma cell culture supernatant required by production, is low in cost, and is convenient for batch production. Moreover, the bone marrow cells cultured by the bone marrow culture medium provided by the invention are used for analyzing chromosome karyotype, and have multiple and clear division phases, good forms and good dispersity.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a bone marrow culture medium and a preparation method and application thereof.
Background
Chromosomal aberrations are closely related to hematological diseases. In recent years, with the development of techniques such as cell culture and chromosome banding, cytogenetics has become an essential part for analysis and research of blood system diseases. The bone marrow chromosome karyotype analysis is used for diagnosis, treatment, prognosis and pathogenesis research of blood system diseases and has very important function. However, the preparation process of bone marrow karyotype is complex, many influencing factors, low success rate and high cost, and the obtained mitotic phase is few, the chromosome is coarse and short, and the dispersion is not good.
In recent years, there have been some bone marrow media; for example, engineering soldiers, summer-grown plants, aged red plums and the like adopt RPMI1640 basic culture medium, penicillin/streptomycin, fetal calf serum and human lymphoma cell culture to prepare bone marrow culture medium; preparing a bone marrow culture medium by adopting a basal culture medium, penicillin, streptomycin, fetal calf serum, a human lymphoma cell culture and a compound Cephaloziellin N through the glutelin culture; the bone marrow cells cultured by the two bone marrow culture media are used for chromosome G slice production, and good effects are obtained. However, the above two bone marrow media contain human lymphoma cell cultures at a concentration of 60 to 140ul/ml, are high in concentration, are used for mass production, are difficult to obtain human lymphoma cell cultures required for production, and are also high in cost.
Therefore, establishing a bone marrow cell culture medium which has good growth of bone marrow cells, many and clear division phases, good form and dispersity, low cost and convenient batch production is particularly important.
Disclosure of Invention
The invention provides a marrow culture medium for overcoming the defects of the prior art, so that when the cultured marrow cells are used for G banding chromosome karyotype analysis, the background is clear, the chromosome split phases are many, the form is good, the dispersity is good, and the observation is easy; and the cost is low, and the batch production is convenient.
In addition, the invention also provides a preparation method and application of the bone marrow cell culture medium.
In order to solve the technical problems, the invention adopts the following technical scheme:
a bone marrow culture medium comprises a basic culture medium, human lymphocyte culture supernatant, NaHCO, sodium selenite, rhu-EPO, and fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite3。
Preferably, the basic culture medium is RPMI1640 culture medium.
The concentration of each component of the bone marrow culture medium is as follows:
the concentration of the fetal calf serum in the basic culture medium is 80-150 mL/L;
the concentration of the L-ascorbic acid in the basal culture medium is 5-20 mg/L;
the concentration of the vitamin E in the basal culture medium is 5-15 mg/L;
the concentration of penicillin in the basal medium was 1X 105U/L；
The concentration of streptomycin in the basic culture medium is 50 mg/L;
the concentration of the sodium pyruvate is 80-120 mg/L;
the concentration of sodium selenite in the basal culture medium is 10-20 nM;
rhu-EPO, the concentration of human lymphocyte culture supernatant in the basic culture medium is 0.5-5 mL/L;
NaHCO3the concentration in the basic culture medium is 1.5-2 g/L.
Preferably, the preparation method of the culture supernatant of rhu-EPO cultured human lymphocytes in the bone marrow cell culture medium comprises the following steps: human lymphocyte 1X 106Inoculating the cells/mL into an RPMI1640 culture medium containing 1-10 IU/L rhu-EPO, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10 minutes, collecting a supernatant, centrifuging the supernatant at 4500rpm for 20 minutes, and collecting the supernatant.
The invention provides a method for preparing the bone marrow medium, which comprises the following steps:
taking the components of the bone marrow culture medium, adding the components into a basic culture medium according to the respective concentration usage amount, fully dissolving and uniformly mixing, then filtering and sterilizing by a 0.22uM filter membrane, and subpackaging into 5 ml/bottle to obtain the bone marrow culture medium.
The invention also provides application of the bone marrow culture medium in bone marrow karyotype analysis, wherein bone marrow cells are subjected to 1.5-2 multiplied by 107Inoculating into the above bone marrow cell culture medium, and adding 5% CO at 37 deg.C2Culturing in the incubator for 24 hours or 48 hours, and adding colchicine 2 hours before terminating the culture; collecting cells, hypotonic, pre-fixing, dripping, baking, ageing, pancreatin, and Giemsa staining, and optionally stainingAnd (4) carrying out karyotype analysis on the color body.
The invention has the advantages that: the human lymphocyte culture supernatant cultured by rhu-EPO provides growth factors required by bone marrow growth, has low usage amount, is easy to obtain human lymphoma cell culture supernatant required by production, has low cost and is convenient for batch production. Moreover, the bone marrow cells cultured by the bone marrow culture medium provided by the invention are used for analyzing chromosome karyotype, and have multiple and clear division phases, good forms and good dispersity.
Detailed Description
In order that those skilled in the art will more clearly and intuitively understand the present invention, the present invention will be further described below.
The bone marrow culture medium comprises an RPMI1640 culture medium, and human lymphocyte culture supernatant and NaHCO which are added into the RPMI1640 culture medium, wherein the human lymphocyte culture supernatant is cultured by human lymphocytes, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, rhu-EPO3Wherein:
the concentration of the fetal calf serum in the basic culture medium is 80-150 mL/L; the concentration of the L-ascorbic acid in the basal culture medium is 5-20 mg/L; the concentration of the vitamin E in the basal culture medium is 5-15 mg/L; the concentration of penicillin in the basal medium was 1X 105U/L; the concentration of streptomycin in the basic culture medium is 50 mg/L; the concentration of the sodium pyruvate is 80-120 mg/L; the concentration of sodium selenite in the basal culture medium is 10-20 nM; rhu-EPO, the concentration of human lymphocyte culture supernatant in the basic culture medium is 0.5-5 mL/L; NaHCO 23The concentration in the basic culture medium is 1.5-2 g/L.
The following is a detailed description of the preferred embodiment.
Example 1
The bone marrow medium of this example comprises: basal medium RPMI1640 containing human lymphocyte culture supernatant and NaHCO, which is cultured with fetal calf serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, rhu-EPO3Wherein:
the concentration of fetal calf serum in the basic culture medium is 100 mL/L; the concentration of the L-ascorbic acid in the basic culture medium is 20 mg/L; the concentration of the vitamin E in the basic culture medium is 15 mg/L; the concentration of penicillin in the basal medium was 1X 105U/L; the concentration of streptomycin in the basic culture medium is 50 mg/L; the concentration of the sodium pyruvate is 120 mg/L; the concentration of sodium selenite in the basal medium is 10 nM; rhu-EPO, the concentration of human lymphocyte culture supernatant in the basic culture medium is 5 mL/L; NaHCO 23The concentration in the basal medium was 2 g/L.
The preparation method of the culture supernatant of the rhu-EPO cultured human lymphocytes comprises the following steps: human lymphocyte 1X 106Inoculating the cells/mL into RPMI1640 culture medium containing 10IU/L rhu-EPO, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10 minutes, collecting supernatant, centrifuging the supernatant at 4500rpm for 20 minutes, and collecting supernatant; the human lymphocyte culture supernatant was prepared in more detail as follows:
step 1, wiping an ultra-clean workbench by using 75% alcohol or benzalkonium bromide, and then irradiating the ultra-clean workbench for 30-50 minutes by using ultraviolet rays.
And 2, taking the cryopreserved tube of the human lymphocytes out of the liquid nitrogen tank, immediately putting the cryopreserved tube into a water bath at 37 ℃ for 2-5 minutes, and melting the content.
Step 3, human lymphocyte suspension is according to 1 × 106Each/mL of the cells was transferred to RPMI1640 medium containing 10IU/L rhu-EPO, and cultured at 37 ℃ in 5% CO 2. (in other embodiments, rhu-EPO can be used to prepare human lymphocyte culture supernatants from other concentrations)
And 4, culturing for 3 days, collecting cells, transferring the cells into a centrifuge tube, and centrifuging at 1500rpm for 10 minutes.
Step 5, the supernatant was collected (without any pellet encountered during collection), and then centrifuged at 4500rpm for 10 minutes to collect the supernatant (without any pellet encountered during collection), which was the culture supernatant of human lymphocytes. The obtained human lymphocyte culture supernatant can be used immediately or filtered by a 0.22uM filter membrane and stored below 20 ℃ for later use.
The bone marrow medium of this example was prepared by the following method: the components are added into a basic culture medium according to the respective concentration and use amount, fully dissolved and uniformly mixed, and then filtered, sterilized and subpackaged into 5 ml/bottle by a 0.22uM filter membrane.
Example 2
Bone marrow culture medium of culture supernatant of human lymphocytes without rhu-EPO culture
Bone marrow medium containing no rhu-EPO-cultured human lymphocyte culture supernatant was prepared according to the method of example 1. The bone marrow culture medium of human lymphocyte culture supernatant without rhu-EPO culture is basal culture medium RPMI1640 containing fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, human lymphocyte culture supernatant, NaHCO3Wherein:
the concentration of fetal calf serum in the basic culture medium is 100 mL/L; the concentration of the L-ascorbic acid in the basic culture medium is 20 mg/L; the concentration of the vitamin E in the basic culture medium is 15 mg/L; the concentration of penicillin in the basal medium was 1X 105U/L; the concentration of streptomycin in the basic culture medium is 50 mg/L; the concentration of the sodium pyruvate is 120 mg/L; the concentration of sodium selenite in the basal medium is 10 nM; the concentration of the human lymphocyte culture supernatant in a basic culture medium is 5 mL/L; NaHCO 23The concentration in the basal medium was 2 g/L.
The preparation method of the human lymphocyte culture supernatant comprises the following steps: human lymphocyte 1X 106Inoculating each/mL of the cells into an RPMI1640 culture medium, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10 minutes, collecting a supernatant, centrifuging the supernatant at 4500rpm for 20 minutes, and collecting the supernatant; the human lymphocyte culture supernatant was prepared in more detail as follows:
step 1, wiping an ultra-clean workbench by using 75% alcohol or benzalkonium bromide, and then irradiating the ultra-clean workbench for 30-50 minutes by using ultraviolet rays.
And 2, taking the cryopreserved tube of the human lymphocytes out of the liquid nitrogen tank, immediately putting the cryopreserved tube into a water bath at 37 ℃ for 2-5 minutes, and melting the content.
Step 3, human lymphocyte suspension is according to 1 × 106Transferring each/mL of the cells into RPMI1640 medium, and culturing at 37 deg.C with 5% CO2And (5) culturing.
And 4, culturing for 3 days, collecting cells, transferring the cells into a centrifuge tube, and centrifuging at 1500rpm for 10 minutes.
Step 5, the supernatant was collected (without any pellet encountered during collection), and then centrifuged at 4500rpm for 10 minutes to collect the supernatant (without any pellet encountered during collection), which was the culture supernatant of human lymphocytes. The obtained human lymphocyte culture supernatant can be used immediately or filtered by a 0.22uM filter membrane and stored below 20 ℃ for later use.
The bone marrow medium of this example was prepared by the following method: the components are added into a basic culture medium according to the respective concentration and use amount, fully dissolved and uniformly mixed, and then filtered, sterilized and subpackaged into 5 ml/bottle by a 0.22uM filter membrane.
Example 3
Bone marrow culture medium without addition of human lymphocyte culture supernatant
Bone marrow medium without addition of human lymphocyte culture supernatant was prepared according to the method of example 1. The basic culture medium of the bone marrow culture medium without adding human lymphocyte culture supernatant is RPMI1640 containing fetal calf serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, NaHCO3Wherein:
the concentration of fetal calf serum in the basic culture medium is 100 mL/L; the concentration of the L-ascorbic acid in the basic culture medium is 20 mg/L; the concentration of the vitamin E in the basic culture medium is 15 mg/L; the concentration of penicillin in the basal medium was 1X 105U/L; the concentration of streptomycin in the basic culture medium is 50 mg/L; the concentration of the sodium pyruvate is 120 mg/L; the concentration of sodium selenite in the basal medium is 10 nM; NaHCO 23The concentration in the basal medium was 2 g/L.
The bone marrow medium of this example was prepared by the following method: the components are added into a basic culture medium according to the respective concentration and use amount, fully dissolved and uniformly mixed, and then filtered, sterilized and subpackaged into 5 ml/bottle by a 0.22uM filter membrane.
Example 4
Bone marrow control medium
The bone marrow culture medium invented by engineering soldiers, summer green and old red plum is used as a bone marrow contrast culture medium for test effect comparison. The bone marrow contrast culture medium is added with penicillin/streptomycin, fetal calf serum and human lymphoma cell culture in a basal culture medium. Wherein the basic culture medium is RPMI1640 culture medium, and the dosage of each additive component is as follows:
penicillin/streptomycin 8 ul/ml;
fetal bovine serum 96 ul/ml;
human lymphoma cell cultures 96 ul/ml;
wherein, penicillin can be selected from 10000U/ml, and streptomycin can be selected from 10000 mug/ml. In other embodiments, penicillin and streptomycin at other concentrations may be selected for use in formulating the medium.
Wherein, the human lymphoma cell culture is prepared by the following method: the cells are revived and passaged, and when the color of the culture medium of the passaged cells turns to yellow, the cells are collected and centrifuged, and after centrifugation, the supernatant is collected and filtered to obtain the cell culture.
More specifically, the method for preparing the human lymphoma cell culture comprises the following steps:
a. the clean bench was wiped clean with benzalkonium bromide and irradiated with ultraviolet light for 30 minutes.
b. The vial was removed from the liquid nitrogen tank and immediately placed in a 37 ℃ water bath and shaken to cause the contents to rapidly melt within 2 minutes.
c. Transferring the cell suspension into 10ml RPMI-1640 medium containing 9.6% fetal calf serum at 37 deg.C and 5% CO2And (5) culturing.
d. When the cells reached approximately 50% confluence, 4ml of trypsin (containing EDTA) was added, ensuring that the bottom of the cell culture flask was covered with a thin layer of trypsin.
e. The flask was placed in an incubator and incubated for 5min, examined microscopically, and if all cells had fallen off, 10ml of medium was added and the cells were dispersed with a pipette.
f. Then 30ml of fresh medium was added, the cell suspension was transferred one by one to a cell culture flask containing 10ml of medium, and the cells were gently mixed.
g. The flask was placed in an incubator (the lid was unscrewed) and the medium was changed to a final concentration of about 40ml when the cell density reached about 50% confluency.
h. The medium was ready for collection when it became yellow in color. The cultured cells were transferred to a 50ml round-bottom polypropylene centrifuge tube (labeled with the corresponding label) using a serum pipette and centrifuged at 2000rpm for 10 min.
i. The supernatant was collected and filtered through a 0.22 μm sterile filter (without hitting the cell pellet at the time of collection), which was the human lymphoma cell culture. If not used, the filtered culture can be stored in aliquots at-20 ℃.
The components are added into a basic culture medium according to the respective concentration and use amount, fully dissolved and uniformly mixed, and then filtered, sterilized and subpackaged into 5 ml/bottle by a 0.22uM filter membrane.
In other embodiments, cultures from other concentrations of penicillin/streptomycin, fetal bovine serum, and human lymphoma cells may be used to formulate the media.
Example 5
Preparation of control Medium
A control medium was prepared by the method of reference example 1 to 4. The control culture medium only contains a basal culture medium, fetal calf serum, penicillin and streptomycin; the basic culture medium of the control culture medium is RPMI1640, the concentration of fetal calf serum in the basic culture medium is 100mL/L, and the concentration of penicillin in the basic culture medium is 1 multiplied by 105U/L; the concentration of streptomycin in the basic culture medium is 50mg/L, NaHCO3The concentration in the basal medium was 2 g/L.
Adding the components into a basic culture medium according to the respective concentration and use amount, fully dissolving and uniformly mixing, and then filtering, sterilizing and subpackaging into 5 ml/bottle by using a 0.22uM filter membrane;
example 6
Application effects of the embodiments
Bone marrow cells of the same sample were cultured using the bone marrow medium of example 1, the bone marrow medium of the culture supernatant of human lymphocytes cultured without rhu-EPO of example 2, the bone marrow medium of the culture supernatant of human lymphocytes not added to example 3, the bone marrow control medium of example 4 and the control medium of example 5, respectively, according to the following procedures, and then harvested for analysis of G-banding karyotype, as follows:
(1) preparing a culture medium: the culture medium was prepared according to the methods of examples 1, 2, 3, 4 and 5.
(2) Inoculation: the bone marrow cells are treated at a ratio of 1.5-2X 107And (2) inoculating into each culture medium described in step (1).
(3) Culturing: 37 ℃ and 5% CO2For 24 hours or 48 hours.
(4) Terminating the culture: colchicine was added about 2 hours before termination of the culture.
(5) Harvesting cells: the flask was gently shaken and the culture was transferred to a 15mL centrifuge tube, centrifuged, and centrifuged at 1500rpm for 10 min. The supernatant was aspirated, leaving about 0.5 to 1.0mL to mix.
(6) Hypotonic: slowly adding 8-10 ml of 0.075mol/L KCL solution preheated to 37 ℃ along the tube wall, blowing, uniformly mixing, placing in a 37 ℃ water bath box for 25-30 minutes, and shaking the centrifugal tube left and right for three times during the process to enable the cells to be hypotonic uniformly.
(7) Pre-fixing: adding 1ml of fixation solution prepared from methanol and glacial acetic acid with the ratio of 3:1, blowing, beating and mixing uniformly, and carrying out water bath at 37 ℃ for 10 minutes. Centrifuge at 1500rpm for 5 minutes. The supernatant was aspirated, leaving about 0.5 to 1.0mL to mix.
(8) Fixing: adding 6-8 ml of freshly prepared 3:1 methanol and glacial acetic acid fixing solution, blowing, beating and uniformly mixing, and carrying out water bath at 37 ℃ for 10 minutes. Centrifuge at 1500rpm for 10 minutes. The supernatant was aspirated, leaving about 0.5 to 1.0mL to mix.
(9) And (5) repeating the step 8, sucking the supernatant, leaving about 0.5 to 1.0mL of the supernatant, uniformly mixing, centrifuging at 1500rpm for 10 minutes, sucking the supernatant, reserving a proper amount of fixing solution, and adding the fixing solution to prepare a cell suspension with proper concentration.
(10) Preparation and preservation of cell suspension. And (3) lightly beating the cell suspension uniformly by using a suction pipe, sucking a small amount of the cell suspension, dripping the cell suspension onto a clean grease-free glass slide with one end inclined at 15 ℃ and soaked by ice water or 20% ethanol, dripping 2-3 drops of the cell suspension onto each glass slide, and then baking the glass slide in a drying air box at 75 ℃ for 2-3 hours.
Wherein, the glass slide is soaked in 1% HCl overnight in advance, washed by a large amount of clear water and then soaked in 95% ethanol for standby. The soaked glass slide is taken out before use, cleaned by a large amount of clear water and placed in a refrigerator at the temperature of 2-8 ℃ for later use.
(11) Displaying a band: the slides were first digested in 0.025% pancreatin (pH 7.2-7.4) at 37 ℃ for about 1 minute and 30 seconds, but the duration of each action was not exactly the same. The method comprises the steps of firstly testing a piece of the pancreatin, adjusting the action time of the pancreatin according to the banding effect, rinsing twice in 0.85% NaCl solution pre-warmed to 37 ℃, dyeing for 10 minutes in Giemsa solution pre-warmed to 37 ℃ (Giemsa stock solution is mixed with phosphate buffer solution with pH 6.8 according to a ratio of 1: 10-20), washing with tap water, blow-drying by a blower, observing the form under a microscope, counting the division phase, selecting 5 visual fields for counting cells and the division phase, calculating the division phase index, and finding the statistical result in table 1.
TABLE 1
Remarking: division index ═ number of dividing cells/(total number of cells-number of dividing cells) × 100%
CV (coefficient of variation) represents the degree of dispersion of bone marrow cell culture
The results from table 1 show that: after the human lymphocyte culture supernatant is added, the bone marrow cell culture effect is better, and the mitotic phase is more, but rhu-EPO needs to be added in the process of obtaining the human lymphocyte culture supernatant. If the marrow culture medium is prepared from the human lymphocyte supernatant obtained by culturing without adding rhu-EPO, the effect is equivalent to that of the marrow culture medium without adding human lymphocyte culture supernatant, and no obvious difference exists. Compared with a bone marrow control culture medium, the bone marrow cells cultured by the bone marrow cell culture medium of the invention are G-sliced, have high division index and lower coefficient of variation although no statistical difference exists. When the kit is used for analyzing and diagnosing the karyotype of the bone marrow cell chromosome, the time and the labor are saved, and the diagnosis result is more accurate. Therefore, when the bone marrow culture medium and the preparation thereof are used for G banding chromosome karyotype analysis, the cell division phases are more, the chromosomes are clear, the shape is good, and the dispersion is better.
In addition, the preparation method of the bone marrow culture medium is simple, the used amount of the cell culture supernatant is lower than that of a bone marrow contrast culture medium, the human lymphoma cell culture supernatant meeting the production requirement is easy to obtain, the batch production is convenient, the cost is low, and the popularization and the use are facilitated.
In other embodiments, other concentrations of fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, human lymphocyte culture supernatant, NaHCO, in basal medium RPMI16403The present invention is not shown and described, and since the technical effect of the culture medium depends on the types of the components in the culture medium, the change of the amounts of the components mainly affects the quality of the technical effect, and therefore, it should be understood by those skilled in the art that the present invention can be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (8)
1. A bone marrow culture medium is characterized in that,
the bone marrow culture medium comprises a basic culture medium, human lymphocyte culture supernatant and NaHCO, wherein the human lymphocyte culture supernatant is cultured by adding fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite and rhu-EPO in the basic culture medium3。
2. The bone marrow medium of claim 1,
the basic culture medium is RPMI1640 culture medium.
3. The bone marrow medium of claim 1,
the concentration of the fetal calf serum in the basic culture medium is 80-150 mL/L;
the concentration of the L-ascorbic acid in the basal culture medium is 5-20 mg/L;
the concentration of the vitamin E in the basal culture medium is 5-15 mg/L;
the concentration of penicillin in the basal medium was 1X 105U/L；
The concentration of streptomycin in the basic culture medium is 50 mg/L;
the concentration of the sodium pyruvate is 80-120 mg/L;
the concentration of sodium selenite in the basal culture medium is 10-20 nM;
rhu-EPO, the concentration of human lymphocyte culture supernatant in the basic culture medium is 0.5-5 mL/L;
NaHCO3the concentration in the basic culture medium is 1.5-2 g/L.
4. The bone marrow medium of claim 1, wherein the rhu-EPO cultured human lymphocyte culture supernatant is prepared by the following process:
human lymphocyte 1X 106Inoculating the cells/mL into an RPMI1640 culture medium containing 1-10 IU/L rhu-EPO, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10 minutes, collecting a supernatant, centrifuging the supernatant at 4500rpm for 20 minutes, and collecting the supernatant.
5. The method for preparing bone marrow medium according to claim 3, comprising the steps of:
taking the components of the bone marrow culture medium, adding the components into a basic culture medium according to the respective concentration usage amount, fully dissolving and uniformly mixing, then filtering and sterilizing by a 0.22uM filter membrane, and subpackaging into 5 ml/bottle to obtain the bone marrow culture medium.
6. The method of claim 5, wherein the rhu-EPO-cultured human lymphocyte culture supernatant is prepared by:
human lymphocyte 1X 106Inoculating the cells/mL into an RPMI1640 culture medium containing 1-10 IU/L rhu-EPO, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10 minutes, collecting a supernatant, centrifuging the supernatant at 4500rpm for 20 minutes, and collecting the supernatant.
7. Use of a bone marrow medium according to any one of claims 1 to 4 for karyotyping bone marrow chromosomes.
8. The application of claim 7, wherein the step of applying comprises:
the bone marrow cells are treated at a ratio of 1.5-2X 107Inoculating into the above bone marrow cell culture medium, and adding 5% CO at 37 deg.C2Culturing in the incubator for 24 hours or 48 hours, and adding colchicine 2 hours before terminating the culture; cells were collected, hypotonic, pre-fixed, dripped, baked sheet aged, pancreatin, Giemsa stained, and then karyotype analysis could be performed.
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