CN110669730B - Human peripheral blood lymphocyte culture medium - Google Patents
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Abstract
The invention belongs to the technical field of cell culture media, and discloses a human peripheral blood lymphocyte culture medium. The culture medium mainly comprises the following components: RPMI1640 medium powder, sodium bicarbonate, HEPES free acid, gentamicin sulfate, heparin sodium, mercaptoethanol, glutamine, calcium ions, iron ions, titanium ions and phytohemagglutinin. The culture medium of the invention can contain serum or protein components, and can also not contain serum or protein components, and the culture medium mainly utilizes certain specific metal ions to promote cell proliferation. The culture medium has the advantages of definite components, stable performance and low cost, can effectively promote the lymphoblast proliferation and division in human whole blood, and has higher lymphocyte transformation rate and lymphocyte division index.
Description
Technical Field
The invention belongs to the technical field of cell culture media, and particularly relates to a formula of a human peripheral blood lymphocyte culture medium and a preparation method thereof.
Background
Lymphocytes in human peripheral blood are almost in the GI phase (G0 phase) and generally do not divide any more, and when a mitogen such as concanavalin (ConA), Lipopolysaccharide (LPS), interleukin 2(IL-2), Phytohemagglutinin (PHA) or an antigenic substance is added to a medium, small lymphocytes are stimulated by an inducer to be converted into lymphoblasts, and then enter mitosis. After short-term cell culture, treatment with colchicine solution, hypotonic reaction and fixation, partial mitotic metaphase cell can be obtained. The method is widely applied in clinical medicine, virology, pharmacology, genotoxicology and the like.
Known causes affecting human peripheral blood lymphocyte growth are:
1) viral or microbial infection: when cells are placed in culture in vitro, the virus or microorganism has a competitive effect on the cells, leading to cell death once the cells are contaminated. Therefore, the living environment of the cells is ensured to be free from pollution during the culture process.
2) Nutrient deficiency: the important prerequisite for the normal growth and metabolism of cells is sufficient nutrients, including various proteins, amino acids, vitamins, trace elements, carbohydrates, etc. required for maintaining the normal metabolism of cells, and substances (growth factors) for promoting cell growth and division, such as growth hormone, nitrogen-containing hormone such as insulin, etc., steroid hormone such as testosterone, progesterone, etc. During the cell culture process, nutrients are gradually consumed, and if the supply of substances in the culture medium is insufficient, the cells grow slowly, and the cell culture result is influenced. Therefore, the culture medium should contain enough nutrients to satisfy the requirements for cell growth and proliferation. Besides conventional vitamins, amino acids, inorganic salts and trace elements, a certain amount of protein, serum, growth factors and the like are usually added into a culture medium to meet the requirements of cell growth and proliferation.
3) The culture environment is unstable: the culture environment includes culture temperature, whether carbon dioxide is required, stable pH value, etc. The constant and proper temperature can maintain the vigorous growth of cells, and if the temperature deviates from the optimal temperature, the normal metabolism of the cells is affected, and the cells die in severe cases. Oxygen and carbon dioxide are one of the essential conditions for maintaining the cell survival, and the oxygen functions to participate in the tricarboxylic acid cycle, generate energy for the growth and proliferation of the cell and synthesize various components required by the cell growth; the main function of carbon dioxide is to maintain the pH of the cell culture system. Proper pH is also an important prerequisite for cell growth and proliferation, and metabolites produced during cell culture affect the change of pH value and thus cell growth.
Of the above three factors, the second is the most difficult to achieve. Therefore, the key point for solving the above problems is to develop a culture medium suitable for the growth of human peripheral blood lymphocytes, and the conventional culture medium is generally to add sufficient serum (5-10% by volume) to a synthetic medium, because the serum contains various growth factors, trace elements and the like in addition to various proteins and polypeptides to support the growth of cells and promote the proliferation of the cells. However, because the serum is derived from animal blood, the age, diet difference, health condition and the like of the animals have great influence on serum components, and the quality of the serum of different batches has great fluctuation; furthermore, due to the influence of factors such as individual differences of animals, breeding environment, and serum extraction and processing conditions, the serum may contain components harmful to cell growth, such as viruses and heat sources. And the harmful factors can not be removed in the culture medium processing link or the culture process. Although the current large-scale serum processing factory strictly controls the quality of animals in the links of collection, processing, storage and the like, the inter-batch difference of serum is still obvious and is an important factor causing the inter-batch difference of the culture effect of the cell culture medium.
Patent CN106520694A discloses a human peripheral blood lymphocyte culture medium, which comprises the following main components in concentration: 55g/L of RPMI1640 culture medium, 1.5g/L of calf serum, 4.76g/L of collagen, 3.75g/L of sodium hyaluronate, 1.43g/L of glucagon, 6.8 mu g/L of thyroid-stimulating hormone, 22.56mg/L of total glucosides of paeony, 2.75g/L of linoleic acid, 0.32g/L of butanediamine, 3.5mg/L of water and ferric gluconate, 4.25 mu g/L of glutathione and 3.15mg/L of polylysine. Patent CN106754694A discloses a human body peripheral blood lymphocyte culture medium, which comprises the following main components and concentrations: 55g/L of RPMI1640 medium, 27g/L of Ham's 12 medium, 1.68g/L of calf serum, 3.75g/L of hyaluronic acid, 4.12g/L of casein phosphopeptide, 5.32g/L of phytohemagglutinin, 89.3 mu g/mL of lycium barbarum polysaccharide, 4.2g/L of baicalin and 0.58g/L of nisin. The culture media have the advantages of high lymphocyte transformation rate and high division index, but bovine serum, various proteins, polypeptides and other biological extracts (such as total glucosides of paeony, lycium barbarum polysaccharides and baicalin) are added into the culture media, because most of the components are extracted through blood, tissues and the like, the animal sources and feeding conditions are different, the animal individual difference, the age, the health condition and other factors of the animals can influence the content and the proportion of various effective components in serum, such as proteins, growth factors, polypeptides and the like; the purity and the biological activity of the extract fluctuate due to factors such as an extraction process, an extraction reagent and the like; the risk of pollution of harmful factors such as viruses and heat sources, protein denaturation, heavy metal pollution and the like can be brought if the processing link is controlled improperly; the above influencing factors are difficult to control accurately, so that the culture effect is unstable, and large batch-to-batch difference exists. In addition, because serum and protein contain more nutrients, are suitable for the growth of microorganisms, are easy to cause the growth and propagation of bacteria, fungi and the like, and cause the failure of the culture medium, the culture medium usually needs to be added with antibiotics at higher concentration and needs to be stored under a freezing condition. In addition, the prices of serum, proteins and other biological extracts are generally high, and the cost of the culture medium is high.
Therefore, if a human peripheral blood lymphocyte culture medium which does not contain serum, protein or other food extracts, has simple and definite components, good effect, stable performance and low cost can be developed, the culture medium has higher economic prospect and social benefit.
Disclosure of Invention
Aiming at the problem that the components are complex and difficult to control because the existing cell culture medium contains serum, protein and other biological extracts, the invention provides a human peripheral blood lymphocyte culture medium and a preparation method thereof. The culture medium has simple components, easily obtained raw materials, good reagent stability, high conversion rate and division index of cultured lymphocytes and low cost.
The invention is realized by the following technical scheme:
a culture medium for human peripheral blood lymphocytes mainly comprises the following components and concentrations thereof: 8-12g/L of RPMI1640 medium, 1.0-3.0g/L of sodium bicarbonate, 2.0-3.0g/L of HEPES free acid, 0-10% (V/V) of bovine serum, 0-0.01g/mL of bovine serum albumin, 25-75 mu g/mL of gentamicin sulfate, 6-8U/mL of heparin sodium, 5-10 mu mol/L of mercaptoethanol, 0.1-0.5mmol/L of glutamine, 0.6-1.0mmol/L of calcium ions, 1-3 mu mol/L of iron ions, 10-30 mu mol/L of phytohemagglutinin, and the solvent is ultrapure water.
Preferably, the human peripheral blood lymphocyte culture medium consists of the following components and the concentrations thereof: 10.4g/L of RPMI1640 medium, 2.0g/L of sodium bicarbonate, 2.38g/L of HEPES free acid, 0% or 5% of bovine serum, 50 mu g/mL of gentamicin sulfate, 6.9U/mL of heparin sodium, 7.15 mu mol/L of mercaptoethanol, 0.25mmol/L of glutamine, 0.8mmol/L of calcium ions, 1.86 mu mol/L of iron ions, 20.8 mu mol/L of titanium ions and 54mg/L of phytohemagglutinin.
Preferably, the preparation method of the human peripheral blood lymphocyte culture medium comprises the following steps:
1) 10.4g of RPMI1640 medium powder was dissolved in 1L of ultrapure water, and 2.0g of sodium bicarbonate and 2.38g of HEPES free acid were added thereto and mixed well.
2) Adding 5% of total volume of bovine serum or adding no bovine serum, and mixing.
3) The following were added to achieve the following final concentrations: 50 mu g/mL of gentamicin sulfate, 6.9U/mL of heparin sodium, 7.15 mu mol/L of mercaptoethanol, 0.25mmol/L of glutamine, 0.8mmol/L of calcium ions, 1.86 mu mol/L of iron ions and 20.8 mu mol/L of the sodium sulfate, and the components are mixed uniformly.
4) Mixing the above solutions, filtering with 0.22 μm filter membrane, adding phytohemagglutinin under aseptic condition to reach final concentration of 54mg/L, and mixing.
Compared with the existing culture medium, the culture medium of the invention has the following advantages:
1) has definite components, easy and accurate control of the addition amount and stable culture effect. The culture medium of the invention can be free of bovine serum or protein components, and even in the scheme of adding bovine serum or protein components, the addition amount is obviously lower than the addition amount level in the prior art. The invention mainly depends on specific metal ions (calcium ions, iron ions and titanium ions) to promote the proliferation of lymphocytes besides basic vitamins, amino acids, inorganic salts, carbohydrates and other components contained in RPMI 1640. Compared with serum, protein or other biological extracts, the inorganic salt metal ions have definite components, and the addition amount can be accurately controlled to control the culture effect of the culture medium. In the traditional culture medium, biological extracts such as serum and protein are added to promote cell growth and proliferation, the substances come from biological tissues and contain complex components and undefined effective components, components or microorganisms which are not beneficial to cell growth can be introduced in the extraction process, and the components of serum and protein in different batches are greatly different, so that the culture effect of the culture medium is unstable.
2) The culture medium has low cost. The method uses specific inorganic salts of calcium, iron, titanium and the like to partially or completely replace serum, protein and various growth factors in the conventional culture medium. Compared with inorganic salts such as calcium, iron, titanium and the like in unit mass, the prices of fetal calf serum, protein and the like for cell culture are higher by more than 10 times, and the prices of various growth factors extracted from biological tissues are higher; therefore, the culture medium prepared by the method has obvious advantages in cost compared with the traditional culture medium.
3) The stability of the reagent is good. A large amount of serum, protein and the like are added into a conventional culture medium, wherein the effective components in the serum mainly comprise various proteins and some growth factors. Proteins are high molecular compounds formed by connecting various amino acids by peptide bonds, and exist in the form of colloid in aqueous solution. When the pH value of the solution is changed, freeze thawing, microorganism growth and the like, the charge and the space structure of protein molecules are changed, so that the biological activity of the protein is changed; the change in charge on the protein surface causes the colloid to break down, gradually causing the molecules to aggregate with each other and precipitate. Therefore, serum or protein containing media usually need to be frozen below-18 ℃ to delay the change of proteins, so as to be preserved for a long time and maintain the culture effect. The culture medium does not contain protein macromolecular compounds, various effective components stably exist in a solution form in water, and can be stored for a long time at 4 ℃, and the culture effect is continuous and stable.
4) The culture medium has good culture effect. Compared with the conventional lymphocyte culture medium, the culture medium can realize good culture effect only by a plurality of specific metal ions under the condition of not adding serum, protein, growth factors and other food extracts, and the lymphocyte transformation rate and the lymphocyte division index are higher than those of the conventional lymphocyte culture medium.
Detailed description of the preferred embodiments
The present invention is further described in detail with reference to the following embodiments, which are not intended to limit the invention, and those skilled in the art can make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.
Example 1 culture Medium for human peripheral blood lymphocytes
10.4g/L of RPMI1640 medium, 2.0g/L of sodium bicarbonate, 2.38g/L of HEPES free acid, 5% of bovine serum, 50 mu g/mL of gentamicin sulfate, 6.9U/mL of heparin sodium, 7.15 mu mol/L of mercaptoethanol, 0.25mmol/L of glutamine, 0.8mmol/L of calcium ions, 1.86 mu mol/L of iron ions, 15 mu mol/L of titanium ions and 54mg/L of phytohemagglutinin.
The preparation method comprises the following steps:
1) 10.4g of RPMI1640 medium powder was dissolved in 1L of ultrapure water, and 2.0g of sodium bicarbonate and 2.38g of HEPES free acid were added thereto and mixed well.
2) Add 5% bovine serum of the total volume and mix well.
3) The following substances are added to make the final concentration meet the formula requirement: gentamicin sulfate, heparin sodium, mercaptoethanol, glutamine, calcium ions, iron ions and titanium ions, and mixing uniformly.
4) Mixing the above solutions, filtering with 0.22 μm filter membrane, adding phytohemagglutinin according to formula under aseptic condition, and mixing.
Example 2 culture Medium for human peripheral blood lymphocytes
10.4g/L of RPMI1640 medium, 2.0g/L of sodium bicarbonate, 2.38g/L of HEPES free acid, 0.5% of bovine serum albumin, 50 mu g/mL of gentamicin sulfate, 7.2U/mL of heparin sodium, 7.5 mu mol/L of mercaptoethanol, 0.2mmol/L of glutamine, 0.9mmol/L of calcium ions, 1.9 mu mol/L of iron ions, 15 mu mol/L of titanium ions and 72mg/L of phytohemagglutinin.
The formulation method was similar to example 1.
Example 3 culture Medium for human peripheral blood lymphocytes
10.4g/L of RPMI1640 medium, 2.0g/L of sodium bicarbonate, 2.38g/L of HEPES free acid, 50 mu g/mL of gentamicin sulfate, 6.9U/mL of heparin sodium, 7.15 mu mol/L of mercaptoethanol, 0.25mmol/L of glutamine, 0.8mmol/L of calcium ions, 1.86 mu mol/L of iron ions, 20.8 mu mol/L of titanium ions and 54mg/L of phytohemagglutinin.
The formulation method was similar to example 1.
Example 4 use of human peripheral blood lymphocyte culture Medium
Peripheral blood was drawn from human with heparin sodium anticoagulation tube. A certain brand of culture medium on the market is taken as a control, and the three culture media are tested together. The medium was first rewarmed at 37 ℃. Mixing human anticoagulation liquid by turning upside down at ultra clean bench or alcohol lamp, inoculating 0.5ml blood into culture bottle containing 5ml culture medium (28-35 drops of No. 7 needle seed blood), and mixing. The flask was allowed to stand in an incubator at 37 ℃ for cultivation.
After culturing for 70-72 hours, 50. mu.l of colchicine with a concentration of 10. mu.g/ml is added to the culture flask and the culture is continued for 1.5 hours. The flask was opened, the medium was transferred to a 15ml centrifuge tube after a slight blow with a pipette, centrifuged at 1500rpm for 10 minutes, the supernatant was aspirated off, and about 0.5ml of the supernatant and the pellet were left. 5ml of 0.075M potassium chloride solution preheated to 37 ℃ is added, the mixture is stirred evenly by a suction pipe and hypotonic for 30 minutes in a water bath at 37 ℃. 1ml of the fixation solution (methanol: glacial acetic acid ═ 3:1) prepared in situ was added thereto, the mixture was gently mixed, the mixture was left at room temperature for 10 minutes, centrifuged at 1500rpm for 10 minutes, and the supernatant was aspirated off to leave about 0.5ml of the supernatant and the precipitate. 5ml of fresh fixative was slowly added along the tube wall, gently beaten and mixed well, left at room temperature for 30 minutes, centrifuged at 1500rpm for 10 minutes, and the supernatant was aspirated off. The fixation was repeated once and the supernatant was centrifuged off. Adding a proper amount of fresh fixing solution to adjust to a proper cell concentration, and dripping tablets in a chromosome dispersion instrument. Dispersing conditions are as follows: the temperature is 25 ℃, the humidity is 50 percent, and the slide is taken out after being completely dried. The slices were oven-baked at 80 ℃ for 3 hours, followed by chromosome banding staining.
The total cell number and the number of dividing phase cells in 4 fields which are uniformly distributed are observed under a microscope, and the division index is calculated. Division index is 100% of the number of cells in the division phase/total number of cells. The results are as follows:
the peripheral blood lymphocyte culture medium prepared according to the three cases of the invention can culture peripheral blood lymphocytes, the division index reaches more than 3.5-4.5, and the division index of a certain brand culture medium used as a contrast is only 2.8, which shows that the culture effect of the culture medium of the invention is obviously better than that of the conventional culture medium.
Example 5 storage stability of human peripheral blood lymphocyte culture Medium
The serum-free and protein-free peripheral blood lymphocyte culture medium prepared in example 3 was stored at 4 ℃ for 12 months, and then taken out, and an experiment was performed in accordance with the method of example 4 using a commercially available brand of medium (-18 ℃ storage, in term of validity) as a control. The total cell number and the number of dividing phase cells in 4 fields which are uniformly distributed are observed under a microscope, and the division index is calculated. Division index is 100% of the number of cells in the division phase/total number of cells.
The results are as follows:
from the above data, it can be seen that the culture medium of example 3 still has better culture effect than the conventional culture medium after being stored at 4 ℃ for 12 months, which indicates that the culture medium of the present invention can be stably stored at 4 ℃ for a long time.
Claims (2)
1. A culture medium for human peripheral blood lymphocytes is characterized by comprising the following components in concentration: 10.4g/L of RPMI1640 medium, 2.0g/L of sodium bicarbonate, 2.38g/L of HEPES free acid, 50 mu g/mL of gentamicin sulfate, 6.9U/mL of heparin sodium, 7.15 mu mol/L of mercaptoethanol, 0.25mmol/L of glutamine, 0.8mmol/L of calcium ions, 1.86 mu mol/L of iron ions, 20.8 mu mol/L of titanium ions, 54mg/L of phytohemagglutinin and ultrapure water as a solvent; the human peripheral blood lymphocyte culture medium does not contain serum or protein components.
2. The culture medium for human peripheral blood lymphocytes according to claim 1, wherein the culture medium for human peripheral blood lymphocytes is prepared by the steps of:
1) weighing RPMI1640 culture medium powder, dissolving in 1L of ultrapure water, adding sodium bicarbonate and HEPES free acid according to the formula amount, and uniformly mixing;
2) adding gentamicin sulfate, heparin sodium, mercaptoethanol, glutamine, calcium ions, iron ions and titanium ions, enabling the final concentration to reach the formula requirement, and uniformly mixing;
3) mixing the above solutions, filtering with 0.22 μm filter membrane, and adding phytohemagglutinin under aseptic condition.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997048821A1 (en) * | 1996-06-19 | 1997-12-24 | Research Development Foundation | Biochemical analysis of antioxidant function |
| CN1275618A (en) * | 2000-06-21 | 2000-12-06 | 张国庆 | Method for cultivating T-lymphocytes |
| CN1280194A (en) * | 2000-06-21 | 2001-01-17 | 张国庆 | Culture fluid for T-lymphocytes |
| CN102352343A (en) * | 2011-09-28 | 2012-02-15 | 上海柯莱逊生物技术有限公司 | Lymphocyte culture medium and use method thereof |
| CN109055309A (en) * | 2018-08-02 | 2018-12-21 | 广州白云山拜迪生物医药有限公司 | A kind of serum-free cell culture medium and application thereof |
| CN109294986A (en) * | 2018-10-26 | 2019-02-01 | 深圳市润科生物科技有限公司 | A kind of T lymphocyte serum free medium and preparation method thereof |
-
2019
- 2019-10-23 CN CN201911011883.5A patent/CN110669730B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997048821A1 (en) * | 1996-06-19 | 1997-12-24 | Research Development Foundation | Biochemical analysis of antioxidant function |
| CN1275618A (en) * | 2000-06-21 | 2000-12-06 | 张国庆 | Method for cultivating T-lymphocytes |
| CN1280194A (en) * | 2000-06-21 | 2001-01-17 | 张国庆 | Culture fluid for T-lymphocytes |
| CN102352343A (en) * | 2011-09-28 | 2012-02-15 | 上海柯莱逊生物技术有限公司 | Lymphocyte culture medium and use method thereof |
| CN109055309A (en) * | 2018-08-02 | 2018-12-21 | 广州白云山拜迪生物医药有限公司 | A kind of serum-free cell culture medium and application thereof |
| CN109294986A (en) * | 2018-10-26 | 2019-02-01 | 深圳市润科生物科技有限公司 | A kind of T lymphocyte serum free medium and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 淋巴细胞经TCR-CD3活化增殖作用的分析;李鸣等;《免疫学杂志》;19951231;第11卷(第2期);91-94 * |
| 钛离子对T淋巴细胞体外增殖及活化的影响;陈富军等;《中国组织工程研究》;20161216;第20卷(第52期);7781-7787 * |
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