CN112080466B - Bone marrow culture medium and preparation method and application thereof - Google Patents
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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 basal culture medium, fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, rhu-EPO cultured human lymphocyte culture supernatant, naHCO 3 . The invention adopts rhu-EPO cultured human lymphocyte culture supernatant to provide growth factors required by bone marrow growth, and the human lymphocyte culture supernatant has low usage amount, is easy to obtain the human lymphoma cell culture supernatant meeting the production requirement, has low cost and is convenient for mass production. In addition, the bone marrow cells cultured by the bone marrow culture medium provided by the invention are used for chromosome karyotype analysis, and have the advantages of multiple and clear division phases, good morphology 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 have a close correlation with blood system diseases. In recent years, cytogenetics has become an essential element in blood system disease analysis and research with the development of techniques such as cell culture and chromosome banding. Bone marrow chromosome karyotype analysis plays a very important role in diagnosis, treatment, prognosis, pathogenesis research of blood system diseases. However, the preparation process of the bone marrow chromosome karyotype is complex, the influence factors are more, the success rate is low, the cost is high, the obtained split phase is less, the chromosome is rough and short, and the dispersion is poor.
In recent years, there have been some bone marrow media; for example Cheng Jianbing, xia Chengqing, chen Gongmei, etc., bone marrow medium was prepared using RPMI1640 basal medium, penicillin/streptomycin, fetal bovine serum, and human lymphoma cell cultures; the cereal super adopts basic culture medium, penicillin, streptomycin, fetal bovine serum, human lymphoma cell culture and compound Cephaloziellin N to prepare bone marrow culture medium; the bone marrow cells cultured by the two bone marrow culture media are used for chromosome G preparation, and have good effects. However, the above two kinds of bone marrow culture media contain human lymphoma cell cultures at a concentration of 60-140ul/ml, are high in concentration, are used for mass production, are difficult to obtain human lymphoma cell cultures satisfying production requirements, and are also high in cost.
Therefore, it is important to establish a marrow cell culture medium which has good growth of marrow cells, multiple and clear division phases, good morphology, good dispersity and low cost and is convenient for mass production.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the bone marrow culture medium, so that when the cultured bone marrow cells are used for G-band chromosome karyotype analysis, the background is clear, the chromosome splitting phases are multiple, the morphology is good, the dispersity is good, and the observation is easy; and the cost is low, and the mass 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 is realized by adopting the following technical scheme:
a bone marrow culture medium comprises basal medium, and human lymphocyte culture supernatant, naHCO, cultured with fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, rhu-EPO added to basal medium 3 。
Preferably, the basal medium is RPMI1640 medium.
The bone marrow culture medium comprises the following components in percentage by weight:
the concentration of the fetal bovine serum in the basal medium is 80-150mL/L;
the concentration of the L-ascorbic acid in the basal medium is 5-20mg/L;
the concentration of vitamin E in the basal medium is 5-15mg/L;
penicillin concentration in basal medium is 1×10 5 U/L；
The concentration of streptomycin in the basal medium is 50mg/L;
the concentration of sodium pyruvate is 80-120mg/L;
the concentration of sodium selenite in the basal medium is 10-20nM;
the concentration of rhu-EPO cultured human lymphocyte culture supernatant in the basal medium is 0.5-5mL/L;
NaHCO 3 the concentration in the basal medium is 1.5-2g/L.
Preferably, the preparation method of rhu-EPO-cultured human lymphocyte culture supernatant in bone marrow cell culture medium comprises the following steps: human lymphocytes 1×10 6 Inoculating 1-10 IU/L rhu-EPO in RPMI1640 culture medium, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10min, collecting supernatant, centrifuging at 4500rpm for 20 min, and collecting supernatant.
The invention provides a method for preparing the bone marrow culture medium, which comprises the following steps:
and (3) taking all the components of the bone marrow culture medium, adding the components into a basic culture medium according to the use amount of the respective concentration, fully dissolving and uniformly mixing, and then filtering and sterilizing by a 0.22uM filter membrane, and sub-packaging into 5 ml/bottle to obtain the bone marrow culture medium.
The invention also provides the application of the marrow culture medium in the karyotype analysis of marrow chromosome, and the marrow cells are mixed with 1.5-2 x 10 7 Inoculating into the above bone marrow cell culture medium, and placing into 5% CO at 37deg.C 2 Culturing in an incubator for 24 hours or 48 hours, and adding colchicine 2 hours before stopping culturing; cells were collected, hypotonic, pre-fixed, drip-plated, roast-plated, pancreatin digested, giemsa stained, and then subjected to chromosome karyotyping.
The invention has the advantages that: the human lymphocyte culture supernatant cultured by rhu-EPO provides the growth factors required by bone marrow growth, and the human lymphocyte culture supernatant has low use level, is easy to obtain the human lymphoma cell culture supernatant meeting the production requirement, has low cost and is convenient for mass production. In addition, the bone marrow cells cultured by the bone marrow culture medium provided by the invention are used for chromosome karyotype analysis, and have the advantages of multiple and clear division phases, good morphology and good dispersity.
Detailed Description
The present invention will be further described below for the sake of clarity and intuition to those skilled in the art.
The bone marrow culture medium of the invention, saidThe marrow culture medium comprises RPMI1640 culture medium, fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, rhu-EPO cultured human lymphocyte culture supernatant, naHCO, and other components added into the RPMI1640 culture medium 3 Wherein:
the concentration of the fetal bovine serum in the basal medium is 80-150mL/L; the concentration of the L-ascorbic acid in the basal medium is 5-20mg/L; the concentration of vitamin E in the basal medium is 5-15mg/L; penicillin concentration in basal medium is 1×10 5 U/L; the concentration of streptomycin in the basal medium is 50mg/L; the concentration of sodium pyruvate is 80-120mg/L; the concentration of sodium selenite in the basal medium is 10-20nM; the concentration of rhu-EPO cultured human lymphocyte culture supernatant in the basal medium is 0.5-5mL/L; naHCO (NaHCO) 3 The concentration in the basal medium is 1.5-2g/L.
The following examples are given with a preferred choice of amounts.
Example 1
The bone marrow medium of this example includes: basic culture medium RPMI1640 containing fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, rhu-EPO cultured human lymphocyte culture supernatant, naHCO 3 Wherein:
the concentration of the fetal bovine serum in the basal medium is 100mL/L; the concentration of the L-ascorbic acid in the basal medium is 20mg/L; the concentration of vitamin E in the basal medium is 15mg/L; penicillin concentration in basal medium is 1×10 5 U/L; the concentration of streptomycin in the basal medium is 50mg/L; the concentration of sodium pyruvate is 120mg/L; the concentration of sodium selenite in basal medium was 10nM; the concentration of rhu-EPO cultured human lymphocyte culture supernatant in the basal medium is 5mL/L; naHCO (NaHCO) 3 The concentration in the basal medium was 2g/L.
The preparation method of the rhu-EPO cultured human lymphocyte culture supernatant comprises the following steps: human lymphocytes 1×10 6 Inoculating 10IU/L rhu-EPO-containing RPMI1640 medium, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10 daysAfter the minutes, collecting the supernatant, centrifuging the supernatant at 4500rpm for 20 minutes, and collecting the supernatant; the more detailed preparation steps of human lymphocyte culture supernatants are as follows:
step 1, wiping the ultra-clean workbench with 75% alcohol or benzalkonium bromide, and then irradiating with ultraviolet rays for 30-50 minutes.
And 2, taking out the frozen tube of the human lymphocyte from the liquid nitrogen tank, and immediately putting the frozen tube into a 37 ℃ water bath for 2-5 minutes to melt the content.
Step 3, human lymphocyte suspensions were prepared according to 1X 10 6 Each mL was transferred to RPMI1640 medium containing 10IU/L rhu-EPO, and cultured at 37℃with 5% CO 2. (in other embodiments, rhu-EPO from other concentrations can be used to prepare human lymphocyte culture supernatants)
Step 4, after 3 days of culture, the cells were collected and transferred into a centrifuge tube and centrifuged at 1500rpm for 10 minutes.
Step 5, collecting supernatant (no cell sediment is encountered during collection), centrifuging at 4500rpm for 10 minutes, and collecting supernatant (no sediment is encountered during collection) to obtain human lymphocyte culture supernatant. The obtained human lymphocyte culture supernatant can be used immediately or after being filtered by a 0.22uM filter membrane, stored below-20 ℃ for standby.
The preparation method of the bone marrow culture medium of the embodiment comprises the following steps: the components are added into a basic culture medium according to the respective concentration, fully dissolved and uniformly mixed, and then filtered and sterilized by a 0.22uM filter membrane and packaged into 5 ml/bottle.
Example 2
Bone marrow medium without rhu-EPO cultured human lymphocyte culture supernatant
Bone marrow medium without rhu-EPO-cultured human lymphocyte culture supernatant was prepared according to the method of example 1. The bone marrow culture medium without rhu-EPO culture supernatant is basic culture medium RPMI1640 containing fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, human lymphocyte culture supernatant, naHCO 3 Wherein:
the concentration of the fetal bovine serum in the basal medium is 100mL/L; l-antibodyThe concentration of the bad blood acid in the basal medium is 20mg/L; the concentration of vitamin E in the basal medium is 15mg/L; penicillin concentration in basal medium is 1×10 5 U/L; the concentration of streptomycin in the basal medium is 50mg/L; the concentration of sodium pyruvate is 120mg/L; the concentration of sodium selenite in basal medium was 10nM; the concentration of the human lymphocyte culture supernatant in the basal medium is 5mL/L; naHCO (NaHCO) 3 The concentration in the basal medium was 2g/L.
The preparation method of the human lymphocyte culture supernatant comprises the following steps: human lymphocytes 1×10 6 Inoculating the cells/mL into RPMI1640 medium, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10 minutes, collecting supernatant, centrifuging at 4500rpm for 20 minutes, and collecting supernatant; the more detailed preparation steps of human lymphocyte culture supernatants are as follows:
step 1, wiping the ultra-clean workbench with 75% alcohol or benzalkonium bromide, and then irradiating with ultraviolet rays for 30-50 minutes.
And 2, taking out the frozen tube of the human lymphocyte from the liquid nitrogen tank, and immediately putting the frozen tube into a 37 ℃ water bath for 2-5 minutes to melt the content.
Step 3, human lymphocyte suspensions were prepared according to 1X 10 6 Transferring the mixture/mL into RPMI1640 medium, and transferring the mixture into 5% CO at 37 DEG C 2 Culturing.
Step 4, after 3 days of culture, the cells were collected and transferred into a centrifuge tube and centrifuged at 1500rpm for 10 minutes.
Step 5, collecting supernatant (no cell sediment is encountered during collection), centrifuging at 4500rpm for 10 minutes, and collecting supernatant (no sediment is encountered during collection) to obtain human lymphocyte culture supernatant. The obtained human lymphocyte culture supernatant can be used immediately or after being filtered by a 0.22uM filter membrane, stored below-20 ℃ for standby.
The preparation method of the bone marrow culture medium of the embodiment comprises the following steps: the components are added into a basic culture medium according to the respective concentration, fully dissolved and uniformly mixed, and then filtered and sterilized by a 0.22uM filter membrane and packaged into 5 ml/bottle.
Example 3
Bone marrow 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 case 1. The basic culture medium of the bone marrow culture medium without adding human lymphocyte culture supernatant is RPMI1640, and contains fetal bovine serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, naHCO 3 Wherein:
the concentration of the fetal bovine serum in the basal medium is 100mL/L; the concentration of the L-ascorbic acid in the basal medium is 20mg/L; the concentration of vitamin E in the basal medium is 15mg/L; penicillin concentration in basal medium is 1×10 5 U/L; the concentration of streptomycin in the basal medium is 50mg/L; the concentration of sodium pyruvate is 120mg/L; the concentration of sodium selenite in basal medium was 10nM; naHCO (NaHCO) 3 The concentration in the basal medium was 2g/L.
The preparation method of the bone marrow culture medium of the embodiment comprises the following steps: the components are added into a basic culture medium according to the respective concentration, fully dissolved and uniformly mixed, and then filtered and sterilized by a 0.22uM filter membrane and packaged into 5 ml/bottle.
Example 4
Bone marrow control medium
The bone marrow culture medium of the invention of Cheng Jianbing, xia Chengqing, chen Gongmei, etc. was used as a bone marrow control medium for comparison of test effects. The bone marrow control medium is supplemented with penicillin/streptomycin, fetal bovine serum and human lymphoma cell cultures in basal medium. Wherein the basal medium is RPMI1640 medium, and the dosage of each additive component is as follows:
penicillin/streptomycin 8ul/ml;
96ul/ml of fetal bovine serum;
96ul/ml of human lymphoma cell culture;
wherein penicillin is 10000U/ml, and streptomycin is 10000 μg/ml. In other embodiments, other concentrations of penicillin and streptomycin may be selected for use in formulating the culture medium.
Wherein, the human lymphoma cell culture is prepared by the following method: cells are recovered and passaged, when the color of the passaged cell culture medium changes to yellow, the cells are collected and centrifuged, and after centrifugation, the supernatant is collected and filtered to obtain a cell culture.
More specifically, the method for preparing the human lymphoma cell culture comprises the following steps:
a. the super clean bench was wiped clean with benzalkonium bromide and irradiated with ultraviolet light for 30 minutes.
b. The frozen tube was removed from the liquid nitrogen tank and immediately placed in a 37 ℃ water bath to shake and promote rapid thawing of its contents within 2 minutes.
c. Transferring the cell suspension into 10ml of RPMI-1640 medium containing 9.6% fetal bovine serum, 37 ℃ and 5% CO 2 Culturing.
d. When the cells reached about 50% confluency, 4ml of trypsin (EDTA-containing) 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 for 5min incubation, microscopic examination, and if all cells had fallen off, 10ml of medium was added and the cells were washed away with a pipette.
f. Then, 30ml of fresh medium was added, and the cell suspension one by one was transferred to a cell culture flask containing 10ml of medium, and the cells were gently mixed.
g. The flask was placed in an incubator (unscrewed), and when the cell density reached about 50% confluency, the medium was changed to a final concentration of about 40 ml.
h. The medium was ready to collect when it turned yellow in color. The cultured cells were transferred to a 50ml round bottom polypropylene centrifuge tube (labeled accordingly) with a serum pipette and centrifuged at 2000rpm for 10min.
i. The supernatant was collected and filtered through a 0.22 μm sterile filter (no cell pellet was encountered during collection), i.e., human lymphoma cell cultures. The filtered cultures may be stored in aliquots at-20℃if not used.
The components are added into a basic culture medium according to the respective concentration, fully dissolved and uniformly mixed, and then filtered and sterilized by a 0.22uM filter membrane and packaged into 5 ml/bottle.
In other embodiments, cell cultures from other concentrations of penicillin/streptomycin, fetal bovine serum, and human lymphomas may be used to formulate the medium.
Example 5
Preparing control culture medium
Control medium was prepared by the method of reference examples 1 to 4. The control culture medium only contains basic culture medium, fetal bovine serum, penicillin and streptomycin; the basic culture medium of the control culture medium is RPMI1640, the concentration of fetal bovine serum in the basic culture medium is 100mL/L, and the concentration of penicillin in the basic culture medium is 1 multiplied by 10 5 U/L; the concentration of streptomycin in the basal medium is 50mg/L, naHCO 3 The concentration in the basal medium was 2g/L.
The components are added into a basic culture medium according to the respective concentration, fully dissolved and uniformly mixed, and then filtered, sterilized and packaged into 5 ml/bottle by a 0.22uM filter membrane;
example 6
Application effect examples
Bone marrow cells of the same sample were cultured using the bone marrow medium of example 1, the bone marrow medium of example 2 without rhu-EPO culture human lymphocyte culture supernatant, the bone marrow medium of example 3 without human lymphocyte culture supernatant added, the bone marrow control medium of example 4 and the control medium of example 5, respectively, and used for G-banding pattern analysis after harvesting, as follows:
(1) Preparing a culture medium: the culture medium was prepared according to the methods of example 1, example 2, example 3, example 4, and example 5.
(2) Inoculating: the bone marrow cells are processed into a mixture of 1.5 to 2 multiplied by 10 7 Each inoculated into each culture medium in the step (1).
(3) Culturing: 37 ℃ and 5% CO 2 Is cultured for 24 hours or 48 hours in an incubator.
(4) Termination of the culture: colchicine was added about 2 hours before termination of the culture.
(5) Harvesting the cells: the flask was gently shaken and the culture was transferred to a 15mL centrifuge tube, centrifuged at 1500rpm for 10min. The supernatant was aspirated, leaving about 0.5 to 1.0mL of the mixture.
(6) Hypotonic: slowly adding 8-10 ml of 0.075mol/L KCL solution with the pre-temperature of 37 ℃ along the pipe wall, blowing and uniformly mixing, and then placing the mixture in a water bath box with the temperature of 37 ℃ for 25-30 minutes, and shaking the centrifuge pipe for three times left and right during the period to ensure that the cells are hypotonic uniformly.
(7) Pre-fixing: 1ml of a freshly prepared fixing solution of 3:1 methanol and glacial acetic acid is added, and the mixture is blown and mixed uniformly, and the mixture is subjected to water bath at 37 ℃ for 10 minutes. Centrifugation at 1500rpm for 5 minutes. The supernatant was aspirated, leaving about 0.5 to 1.0mL of mix.
(8) Fixing: adding 6-8 ml of freshly prepared 3:1 methanol and glacial acetic acid fixing solution, blowing and mixing uniformly, 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 of mix.
(9) Step 8 is repeated, the supernatant is sucked, after about 0.5 to 1.0mL of the supernatant is left to be uniformly mixed, the supernatant is sucked off by centrifugation at 1500rpm for 10 minutes, a proper amount of fixing solution is reserved, and the cell suspension with proper concentration is prepared by adding the fixing solution.
(10) Preparation and preservation of cell suspensions. The cell suspension is gently stirred by a suction tube and then is sucked a small amount, and is dropped onto a clean lipid-free glass slide which is inclined at one end by 15 ℃ and is soaked by ice water or 20% ethanol, wherein each piece of the glass slide is dropped by 2 to 3 drops, and then the glass slide is placed in a drying air box at 75 ℃ for 2 to 3 hours.
The slide is soaked in 1% HCl overnight in advance, washed by a large amount of clean water and then soaked in 95% ethanol for standby. Before use, the soaked slide is taken out, washed by a large amount of clean water and placed in a refrigerator with the temperature of 2-8 ℃ for standby.
(11) And (3) band display: the slide was first digested in 0.025% pancreatin solution (ph=7.2-7.4) pre-warmed to 37 ℃ for about 1 minute and 30 seconds, each time with non-identical time. One piece can be tested first, then the action time of pancreatin can be regulated according to the banding effect, then the piece is rinsed twice in 0.85% NaCl solution which is preheated to 37 ℃, the piece is dyed in Giemsa solution (Giemsa stock solution and phosphate buffer solution with PH=6.8 are mixed according to the ratio of 1:10-20) which is preheated to 37 ℃ for 10 minutes, tap water is washed clean, after the piece is dried by a blower, the form is observed under a microscope, the splitting phase is counted, 5 vision statistical cells and the splitting phase are selected, the splitting phase index is calculated, and the statistical result is shown in table 1.
TABLE 1
Remarks: 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 in Table 1 show that: after the addition of the human lymphocyte culture supernatant, the bone marrow cell culture effect is better, and the division phase is more, but rhu-EPO is required to be added in the culture of human lymphocytes in the process of obtaining the human lymphocyte culture supernatant. If the bone 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 bone marrow culture medium without adding the human lymphocyte supernatant, and no obvious difference exists. Compared with the bone marrow control medium, the cultured bone marrow cells of the bone marrow cell culture medium of the invention are G-flaked, have high division index, and have low variation coefficient although there is no statistical difference. The method is used for diagnosis by bone marrow cell chromosome karyotype analysis, saves time and labor, and has more accurate diagnosis result. 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 morphology is good and the dispersion is good.
In addition, the preparation method of the bone marrow culture medium is simple, the cell culture supernatant is low in use amount compared with a bone marrow control culture medium, the human lymphoma cell culture supernatant meeting the production requirement is easy to obtain, the mass 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 RPMI1640 3 The invention, which can be used for the preparation of the marrow culture medium according to the corresponding examples, is not shown or described one by one, since the technical effect achieved by the culture medium depends on the component species in the culture mediumThe main effect of the variation of the amount of each component is the quality of the technical effect, so those skilled in the art should understand that the modification or equivalent substitution of the present invention is within the protection scope of the present invention without departing from the spirit and scope of the technical scheme of the present invention.
Claims (5)
1. A bone marrow culture medium is characterized in that,
the bone marrow culture medium comprises basal culture medium, and human lymphocyte culture supernatant, naHCO, added with fetal calf serum, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, rhu-EPO 3 ；
The basic culture medium is RPMI1640 culture medium;
the concentration of the fetal bovine serum in the basal medium is 80-150mL/L;
the concentration of the L-ascorbic acid in the basal medium is 5-20mg/L;
the concentration of vitamin E in the basal medium is 5-15mg/L;
penicillin concentration in basal medium is 1×10 5 U/L；
The concentration of streptomycin in the basal medium is 50mg/L;
the concentration of sodium pyruvate is 80-120mg/L;
the concentration of sodium selenite in the basal medium is 10-20nM;
the concentration of rhu-EPO cultured human lymphocyte culture supernatant in the basal medium is 0.5-5mL/L;
NaHCO 3 the concentration in the basal medium is 1.5-2g/L; the preparation process of the rhu-EPO cultured human lymphocyte culture supernatant is as follows:
human lymphocytes 1×10 6 Inoculating 1-10 IU/L rhu-EPO in RPMI1640 culture medium, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10 minutes, collecting supernatant, centrifuging at 4500rpm for 20 minutes, and collecting supernatant.
2. The method for preparing a bone marrow medium according to claim 1, comprising the steps of:
and (3) taking all the components of the bone marrow culture medium, adding the components into a basic culture medium according to the use amount of the respective concentration, fully dissolving and uniformly mixing, and then filtering and sterilizing by a 0.22uM filter membrane, and sub-packaging into 5 ml/bottle to obtain the bone marrow culture medium.
3. The method of claim 2, wherein the rhu-EPO-cultured human lymphocyte culture supernatant is prepared by the following steps:
human lymphocytes 1×10 6 Inoculating 1-10 IU/L rhu-EPO in RPMI1640 culture medium, culturing for 3 days, collecting cells, centrifuging at 1500rpm for 10 minutes, collecting supernatant, centrifuging at 4500rpm for 20 minutes, and collecting supernatant.
4. Use of the bone marrow medium of claim 1 for the preparation of a bone marrow chromosome karyotype analysis reagent.
5. The application according to claim 4, wherein the step of applying comprises:
the bone marrow cells are processed into a mixture of 1.5 to 2 multiplied by 10 7 Inoculating into the above bone marrow cell culture medium, and placing into 5% CO at 37deg.C 2 Culturing in an incubator for 24 hours or 48 hours, and adding colchicine 2 hours before stopping culturing; cells were collected, hypotonic, pre-fixed, drip-plated, roast-plated, pancreatin digested, giemsa stained, and then subjected to chromosome karyotyping.
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| WO2020044538A1 (en) * | 2018-08-31 | 2020-03-05 | 株式会社Ｇｃリンフォテック | Serum-free medium for culturing human lymphocytes |
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