CN112063579B - Serum-free bone marrow culture medium and preparation method and application thereof - Google Patents

Serum-free bone marrow culture medium and preparation method and application thereof Download PDF

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CN112063579B
CN112063579B CN202010976255.7A CN202010976255A CN112063579B CN 112063579 B CN112063579 B CN 112063579B CN 202010976255 A CN202010976255 A CN 202010976255A CN 112063579 B CN112063579 B CN 112063579B
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李晓辉
王亦平
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Shanghai Peihui Biotechnology Development Co ltd
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Abstract

The invention provides a serum-free bone marrow culture medium, a preparation method and application thereof. The culture medium comprises basal medium, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL-2, cpG oligonucleotide, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, and NaH 2 PO 4 、Na 2 HPO 4 Human lymphocyte culture supernatant from rhu-EPO culture. The invention is convenient for quality control between batches; the rhu-EPO cultured human lymphocyte culture supernatant is adopted to provide growth factors required by bone marrow growth, so that the production cost is reduced; sodium glycerophosphate and the like are used as a pH buffer system, so that the pH value is more stable, and the pH value is stable in the preservation process. The rhIL-2 and CpG oligonucleotide are adopted to stimulate the activity of B cells, and more karyotypes are obtained when the B cells are used for karyotype analysis.

Description

Serum-free bone marrow culture medium and preparation method and application thereof
Technical Field
The invention relates to the field of cell culture, in particular to a serum-free 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.
The traditional marrow culture medium consists of RPMI1640 basic culture medium, fetal bovine serum, antibiotics, sodium bicarbonate and the like; in order to enhance the effect of bone marrow culture media, in recent years, there have been some bone marrow culture 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 good effects are obtained. However, the above bone marrow culture medium contains fetal bovine serum, has complex components, and is easy to cause pollution of fungi, mycoplasma and the like; also, serum quality standards are different, which easily causes unstable quality of the medium for mass production and is expensive. In addition, the 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 meeting the production requirements, and are high in cost.
Therefore, in order to reduce the cost, facilitate mass production and have stable quality, the preparation and application of the serum-free bone marrow cell culture medium with good growth of bone marrow cells, multiple and clear division phases, good morphology and good dispersity are particularly important.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the serum-free marrow culture medium, so that when the cultured marrow cells are used for G-banding chromosome karyotype analysis, the background is clear, the chromosome splitting phases are multiple, the morphology and the dispersity are good, the observation is easy, the cost is low, and the quality is stable.
In addition, the invention also provides a preparation method and application of the serum-free bone marrow culture medium.
In order to solve the technical problems, the invention is realized by adopting the following technical scheme:
a serum-free bone marrow culture medium comprises basal medium, and L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL-2, cpG oligonucleotide, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, naH 2 PO 4 、Na 2 HPO 4 Human lymphocyte culture supernatant from rhu-EPO culture.
Preferably, the basal medium is RPMI1640 medium.
Preferably, the serum-free bone marrow medium comprises the following components in concentration:
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 in the basal medium is 80-120mg/L; the concentration of sodium selenite in the basal medium is 10-20nM; the concentration of glutamine in the basal medium is 10-30mM; the concentration of rhIL-2 in the basal medium is 0.005-0.05mg/L; the concentration of CpG oligonucleotide in the basal medium is 0.5-5uM; the concentration of linoleic acid in the basal medium is 0.1-0.5mg/L; the concentration of linolenic acid in the basal medium is 0.1-0.5mg/L; the concentration of stearic acid in basal medium is 5-15uM; the concentration of BSA in the basal medium is 2g-10g/L; the concentration of the sodium glycerophosphate in the basal medium is 5-15g/L; the concentration of HEPES in the basal medium is 3-6g/L; naH (NaH) 2 PO 4 The concentration in the basal medium is 1-5g/L; na (Na) 2 HPO 4 The concentration in the basal medium is 0.5-1.5g/L; the concentration of rhu-EPO cultured human lymphocyte culture supernatant in basal medium is 0.5-5mL/L.
Preferably, the preparation method of the rhu-EPO-cultured human lymphocyte culture supernatant in the serum-free 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.
Preferably, the CpG oligonucleotide in the serum-free bone marrow culture medium is DSP-30 and is modified by phosphorothioate.
The preparation method of the serum-free bone marrow culture medium comprises the following steps:
and (3) taking all the components of the serum-free bone marrow culture medium, adding the components into a basic culture medium according to the respective concentration, fully dissolving and uniformly mixing, and then filtering, sterilizing and packaging the mixture into 5 ml/bottle by using a 0.22uM filter membrane.
The invention also provides the application of the serum-free marrow culture medium in the analysis of marrow chromosome karyotype, and the marrow cells are mixed with the serum-free marrow culture medium according to the ratio of 1.5-2 multiplied by 10 7 Inoculating into the serum-free bone marrow cell culture medium, and placing into 5% CO at 37deg.C 2 Culturing for 24 hours or 48 hours in an incubator without colchicine added 2 hours before the termination of the culture; 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 serum-free marrow culture medium provided by the invention does not contain fetal bovine serum, so that the quality control among batches is facilitated; the rhu-EPO cultured human lymphocyte culture supernatant is adopted to provide growth factors required by bone marrow growth, so that the production cost is reduced; sodium glycerophosphate, HEPES and NaH are adopted 2 PO 4 、Na 2 HPO 4 As a pH buffer system, the pH value is more stable, and the pH value is stable in the preservation process. The rhIL-2 and CpG oligonucleotide are adopted to stimulate the activity of B cells, and more karyotypes are obtained when the B cells are used for karyotype analysis. The marrow cells cultured by the serum-free 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, good dispersity and more stable test results. The serum-free bone marrow culture medium has low cost and is convenient to popularize and use.
Detailed Description
The present invention will be further described below for the sake of clarity and intuition to those skilled in the art.
The serum-free marrow culture medium comprises a basal culture medium RPMI1640, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL-2, cpG oligonucleotide, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES and NaH 2 PO 4 、Na 2 HPO 4 Human lymphocyte culture supernatant of rhu-EPO culture; wherein:
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 inThe concentration in the basal medium was 1X 10 5 U/L; the concentration of streptomycin in the basal medium is 50mg/L; the concentration of sodium pyruvate in the basal medium is 80-120mg/L; the concentration of sodium selenite in the basal medium is 10-20nM; the concentration of glutamine in the basal medium is 10-30mM; the concentration of rhIL-2 in the basal medium is 0.005-0.05mg/L; the concentration of CpG oligonucleotide in the basal medium is 0.5-5uM; the concentration of linoleic acid in the basal medium is 0.1-0.5mg/L; the concentration of linolenic acid in the basal medium is 0.1-0.5mg/L; the concentration of stearic acid in basal medium is 5-15uM; the concentration of BSA in the basal medium is 2g-10g/L; the concentration of the sodium glycerophosphate in the basal medium is 5-15g/L; the concentration of HEPES in the basal medium is 3-6g/L; naH (NaH) 2 PO 4 The concentration in the basal medium is 1-5g/L; na (Na) 2 HPO 4 The concentration in the basal medium is 0.5-1.5g/L; the concentration of rhu-EPO cultured human lymphocyte culture supernatant in basal medium is 0.5-5mL/L.
The preparation method of 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 10min, collecting supernatant, centrifuging at 4500rpm for 20 min, and collecting supernatant.
The CpG oligonucleotide in serum-free marrow culture medium is DSP-30 and is modified with thiophosphoric acid.
The following examples are given with a preferred choice of amounts.
Example 1
The serum-free bone marrow medium of this example comprises: basic culture medium RPMI1640 and L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL-2, cpG oligonucleotide, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, naH added thereto 2 PO 4 、Na 2 HPO 4 Human lymphocyte culture supernatant of rhu-EPO culture; wherein:
l-ascorbic acidThe concentration in the basal medium is 5mg/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 in the basal medium is 80mg/L; the concentration of sodium selenite in basal medium was 20nM; glutamine was at a concentration of 30mM in basal medium; the concentration of rhIL-2 in the basal medium is 0.01mg/L; the concentration of CpG oligonucleotide in the basal medium was 2uM; the concentration of linoleic acid in the basal medium is 0.3mg/L; the concentration of linolenic acid in the basal medium is 0.3mg/L; the concentration of stearic acid in the basal medium was 15uM; BSA concentration in basal medium was 2g/L; the concentration of sodium glycerophosphate in the basal medium is 10g/L; HEPES concentration in basal medium was 6g/L; naH (NaH) 2 PO 4 The concentration in the basal medium is 2g/L; na (Na) 2 HPO 4 The concentration in the basal medium is 1g/L; the concentration of rhu-EPO cultured human lymphocyte culture supernatant in the basal medium is 5mL/L; the CpG oligonucleotide is DSP-30 and is modified by phosphorothioate;
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 10min, collecting supernatant, centrifuging at 4500rpm for 20 min, 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 cells/mL into RPMI1640 medium containing 10IU/L rhu-EPO, and transferring the cells/mL into 5% CO at 37 DEG C 2 Culturing. (in other embodiments, rhu-EPO from other concentrations can be used to prepare human lymphocyte cultures of the corresponding examplesCulture supernatant).
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 lymphocyte culture supernatant was used immediately or was filtered with a 0.22uM filter and stored at-20℃or below for further use.
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
Serum-free bone marrow cell culture medium without human lymphocyte culture supernatant
Serum-free bone marrow cell culture medium containing no human lymphocyte culture supernatant was prepared as in example 1, and the serum-free bone marrow cell culture medium of this example differs from that of example 1 only in that it contains no human lymphocyte culture supernatant.
Specifically, the serum-free bone marrow cell culture medium without human lymphocyte culture supernatant comprises a basal medium RPMI1640, and L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL-2, cpG oligonucleotide, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, naH added into the basal medium 2 PO 4 、Na 2 HPO 4 The method comprises the steps of carrying out a first treatment on the surface of the Wherein:
the concentration of the L-ascorbic acid in the basal medium is 5mg/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 in the basal medium is 80mg/L; the concentration of sodium selenite in basal medium was 20nM; glutamine was at a concentration of 30mM in basal medium; the concentration of rhIL-2 in the basal medium is 0.01mg/L; the concentration of CpG oligonucleotide in the basal medium was 2uM; linoleic acid in basal mediumThe concentration of (3) is 0.3mg/L; the concentration of linolenic acid in the basal medium is 0.3mg/L; the concentration of stearic acid in the basal medium was 15uM; BSA concentration in basal medium was 2g/L; the concentration of sodium glycerophosphate in the basal medium is 10g/L; HEPES concentration in basal medium was 6g/L; naH (NaH) 2 PO 4 The concentration in the basal medium is 2g/L; na (Na) 2 HPO 4 The concentration in the basal medium is 1g/L; the CpG oligonucleotide is DSP-30 and is modified by phosphorothioate.
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
Serum-free bone marrow medium without rhu-EPO cultured human lymphocyte culture supernatant
Serum-free bone marrow medium was prepared as in example 1 without human lymphocyte culture supernatant of rhu-EPO culture, and not without human lymphocyte culture supernatant.
Specifically, the serum-free bone marrow culture medium without rhu-EPO cultured human lymphocyte culture supernatant comprises a basal medium RPMI1640, and L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL-2, cpG oligonucleotide, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, naH added into the basal medium 2 PO 4 、Na 2 HPO 4 Human lymphocyte culture supernatant; wherein:
the concentration of the L-ascorbic acid in the basal medium is 5mg/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 in the basal medium is 80mg/L; the concentration of sodium selenite in basal medium was 20nM; glutamine was at a concentration of 30mM in basal medium; the concentration of rhIL-2 in the basal medium is 0.01mg/L; the concentration of CpG oligonucleotide in the basal medium was 2uM; linoleic acid on the basis ofThe concentration in the culture medium is 0.3mg/L; the concentration of linolenic acid in the basal medium is 0.3mg/L; the concentration of stearic acid in the basal medium was 15uM; BSA concentration in basal medium was 2g/L; the concentration of sodium glycerophosphate in the basal medium is 10g/L; HEPES concentration in basal medium was 6g/L; naH (NaH) 2 PO 4 The concentration in the basal medium is 2g/L; na (Na) 2 HPO 4 The concentration in the basal medium is 1g/L; the concentration of the human lymphocyte culture supernatant in the basal medium is 5mL/L; the CpG oligonucleotide is DSP-30 and is modified by phosphorothioate;
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 lymphocyte culture supernatant was used immediately or was filtered with a 0.22uM filter and stored at-20℃or below for further use.
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
Serum-free bone marrow cell culture medium without rhIL-2 and CpG oligonucleotide
Serum-free bone marrow cell culture medium without rhIL-2 and CpG oligonucleotides was prepared as in example 1. The serum-free bone marrow cell culture medium without rhIL-2 and CpG oligonucleotide comprises basic culture medium RPMI1640, and L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL-2, cpG oligonucleotide, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, naH added into the basic culture medium 2 PO 4 、Na 2 HPO 4 Human lymphocyte culture supernatant of rhu-EPO culture; wherein:
the concentration of the L-ascorbic acid in the basal medium is 5mg/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 in the basal medium is 80mg/L; the concentration of sodium selenite in basal medium was 20nM; glutamine was at a concentration of 30mM in basal medium; the concentration of linoleic acid in the basal medium is 0.3mg/L; the concentration of linolenic acid in the basal medium is 0.3mg/L; the concentration of stearic acid in the basal medium was 15uM; BSA concentration in basal medium was 2g/L; the concentration of sodium glycerophosphate in the basal medium is 10g/L; HEPES concentration in basal medium was 6g/L; naH (NaH) 2 PO 4 The concentration in the basal medium is 2g/L; na (Na) 2 HPO 4 The concentration in the basal medium is 1g/L; the concentration of rhu-EPO cultured human lymphocyte culture supernatant in the basal medium is 5mL/L;
the preparation method of the rhu-EPO cultured human lymphocyte culture 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 10min, collecting supernatant, centrifuging at 4500rpm for 20 min, and collecting supernatant. Preparation of human lymphocyte culture supernatant in more detailThe following are provided:
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 cells/mL into RPMI1640 medium containing 10IU/L rhu-EPO, and transferring the cells/mL into 5% CO at 37 DEG C 2 Culturing. (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 touched during collection), centrifuging at 4500rpm for 10 minutes, and collecting supernatant (no sediment is touched during collection) to obtain lymphocyte culture supernatant. The obtained lymphocyte culture supernatant can be used immediately or can be stored below-20deg.C for use after filtration with 0.22uM filter membrane.
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 5
Control medium for serum-free bone marrow medium
A control medium containing only basal medium, L-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, naH was prepared according to the method of example 1, example 2, example 3 2 PO 4 、Na 2 HPO 4 The method comprises the steps of carrying out a first treatment on the surface of the Wherein:
the concentration of the L-ascorbic acid in the basal medium is 5mg/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 in the basal medium is 80mg/L; selenious acidSodium concentration in basal medium was 20nM; glutamine was at a concentration of 30mM in basal medium; the concentration of linoleic acid in the basal medium is 0.3mg/L; the concentration of linolenic acid in the basal medium is 0.3mg/L; the concentration of stearic acid in the basal medium was 15uM; BSA concentration in basal medium was 2g/L; the concentration of sodium glycerophosphate in the basal medium is 10g/L; HEPES concentration in basal medium was 6g/L; naH (NaH) 2 PO 4 The concentration in the basal medium is 2g/L; na (Na) 2 HPO 4 The concentration in the basal medium was 1g/L.
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 6
Bone marrow medium containing serum
The serum-containing bone marrow medium of the invention of Cheng Jianbing, xia Chengqing, chen Gongmei, etc. was used as a control for serum-free bone marrow medium, and test effect comparison was performed. The serum-containing bone marrow 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 media of the corresponding examples.
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, penicillin/streptomycin, fetal bovine serum, and human lymphoma cell cultures from other concentrations can be used to formulate the media of the corresponding examples.
Example 7
Control medium for bone marrow medium with serum
The procedure is as in example 5Serum bone marrow medium control medium was configured. 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 8
Application effect examples
Bone marrow cells of the same sample were cultured using the serum-free bone marrow medium of example 1, the serum-free bone marrow cell medium of example 2 without the human lymphocyte culture supernatant, the serum-free bone marrow medium of example 3 without the rhu-EPO culture, the serum-free bone marrow cell medium of example 4 without rhIL-2 and CpG oligonucleotides, the control medium of example 5, the serum-containing bone marrow medium of example 6 and the serum-containing bone marrow medium of example 7, respectively, as follows, and used for G-banding pattern analysis after harvesting, the specific procedures are as follows:
preparing a culture medium: the culture medium was prepared in the same manner as in examples 1 to 7.
Inoculating: the bone marrow cells are processed into a mixture of 1.5 to 2 multiplied by 10 7 And (3) inoculating the culture medium in the step (1).
Culturing: 37 ℃ and 5% CO 2 Is cultured for 24 hours or 48 hours in an incubator.
Termination of the culture: colchicine was added about 2 hours before termination of the culture.
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.
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.
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.
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.
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.
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.
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. Serum-free bone marrow cell culture medium without rhIL-2 and CpG oligonucleotide has poor culture effect. When the serum-free 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. Serum-free bone marrow medium has a higher split index and a lower coefficient of variation than bone marrow medium containing fetal bovine serum. Therefore, the bone marrow cells cultured by the serum-free bone marrow culture medium are used for G preparation, have more cell division phases, clear chromosomes, good morphology, better dispersion and more stable test results.
Example 9
Determination of pH stability
The pH change of each medium was measured by leaving the serum-free bone marrow medium of example 1, the serum-free bone marrow cell medium of example 2 without human lymphocyte culture supernatant, the serum-free bone marrow medium of example 3 without rhu-EPO culture supernatant, the serum-free bone marrow cell medium of example 4 without rhIL-2 and CpG oligonucleotide, the control medium of example 5, the serum-containing bone marrow medium of example 6 and the serum-containing bone marrow medium of example 7 at room temperature for one month at 4 ℃. The pH change results are shown in Table 2.
TABLE 2
The results in Table 2 show that: the invention adopts sodium glycerophosphate, HEPES and NaH 2 PO 4 、Na 2 HPO 4 As a pH buffer system, the pH is stable and unchanged during storage at room temperature and 4 ℃. Whereas bone marrow medium with serum adopts traditional NaHCO 3 As a buffer system, the pH of the solution is stable and greatly changed during the storage at room temperature and 4 ℃.
Therefore, the pH value of the serum-free bone marrow culture medium is more stable in the production and preservation processes.
In conclusion, the serum-free marrow culture medium provided by the invention does not contain fetal bovine serum, so that the quality control among batches is facilitated; the human lymphocyte culture supernatant is adopted to provide the growth factors required by bone marrow growth, and the cell supernatant is low in use amount, easy to prepare, easy to obtain the human lymphocyte culture supernatant required by mass production and low in cost; sodium glycerophosphate, HEPES and NaH are adopted 2 PO 4 、Na 2 HPO 4 As a pH buffer system, the pH value is more stable, and the pH value is stable in the preservation process. The marrow cells cultured by the serum-free 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, good dispersity and more stable test results. The serum-free bone marrow culture medium has low cost and is convenient to popularize and use.
In other embodiments, from other concentrations of L-ascorbic acid, vitamin E, penicillin, streptavidinSodium pyruvate, sodium selenite, glutamine, rhIL-2, cpG oligonucleotides, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, naH 2 PO 4 、Na 2 HPO 4 The culture supernatant of rhu-EPO cultured human lymphocyte can also be used for preparing serum-free marrow culture medium according to the corresponding embodiment of the invention, the invention is not shown and described one by one, and the technical effect obtained by the culture medium depends on the types of components in the culture medium, and the change of the dosage of each component mainly has influence on the strength of the technical effect, so the invention has the advantages of simple and convenient operation, low cost, convenient operation, low cost and the like. It will be understood by those skilled in the art that various modifications and equivalent substitutions may be made thereto without departing from the spirit and scope of the present invention.

Claims (5)

1. A serum-free marrow culture medium is characterized by comprising a basal culture medium,
l-ascorbic acid, vitamin E, penicillin, streptomycin, sodium pyruvate, sodium selenite, glutamine, rhIL-2, cpG oligonucleotide, linoleic acid, linolenic acid, stearic acid, BSA, sodium glycerophosphate, HEPES, naH 2 PO 4 、Na 2 HPO 4 Human lymphocyte culture supernatant of rhu-EPO culture;
the basic culture medium is RPMI1640 culture medium;
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 in the basal medium is 80-120mg/L;
the concentration of sodium selenite in the basal medium is 10-20nM;
the concentration of glutamine in the basal medium is 10-30mM;
the concentration of rhIL-2 in the basal medium is 0.005-0.05mg/L;
the concentration of CpG oligonucleotide in the basal medium is 0.5-5uM;
the concentration of linoleic acid in the basal medium is 0.1-0.5mg/L;
the concentration of linolenic acid in the basal medium is 0.1-0.5mg/L;
the concentration of stearic acid in basal medium is 5-15uM;
the concentration of BSA in the basal medium is 2g-10g/L;
the concentration of the sodium glycerophosphate in the basal medium is 5-15g/L;
the concentration of HEPES in the basal medium is 3-6g/L;
NaH 2 PO 4 the concentration in the basal medium is 1-5g/L;
Na 2 HPO 4 the concentration in the basal medium is 0.5-1.5g/L;
the concentration of rhu-EPO cultured human lymphocyte culture supernatant in the basal medium is 0.5-5mL/L;
the preparation method of 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 10min, collecting supernatant, centrifuging at 4500rpm for 20 min, and collecting supernatant;
the CpG oligonucleotide in the serum-free marrow culture medium is DSP-30 and is modified by thiophosphoric acid.
2. The method for preparing serum-free bone marrow medium according to claim 1, comprising the steps of:
and (3) taking all the components of the serum-free bone marrow culture medium, adding the components into a basic culture medium according to the respective concentration, fully dissolving and uniformly mixing, and then filtering, sterilizing and packaging the mixture into 5 ml/bottle by using a 0.22uM filter membrane.
3. The method for preparing serum-free bone marrow medium according to claim 2, wherein,
the preparation method of 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.
4. Use of the serum-free bone marrow medium according to 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 serum-free bone marrow cell culture medium, and placing into 5% CO at 37deg.C 2 Culturing for 24 hours or 48 hours in an incubator without colchicine added 2 hours before the termination of the culture; cells were collected, hypotonic, pre-fixed, drip-plated, roast-plated, pancreatin digested, giemsa stained, and then subjected to chromosome karyotyping.
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