A culture medium for efficiently and rapidly culturing primary mouse pulmonary artery endothelial cells

Abstract

The present invention relates to a culture medium for efficiently and rapidly culturing primary mouse pulmonary artery endothelial cells, belonging to the technical field of culture medium preparation, and well solving the problems such as high price, slow cell crawling speed and poor cell state of the primary mouse pulmonary artery endothelial cell culture medium on the current market. The culture medium comprises the following components: glucose, L-glutamine, sodium pyruvate, sodium bicarbonate, fetal bovine serum, heparin, vascular endothelial growth factor, epidermal growth factor, vasopressin, endothelin, phenol red, Earle's balanced salt solution, and ultrapure water. This culture medium can culture primary mouse pulmonary artery endothelial cells economically, rapidly and efficiently, and has the characteristics of good cell growth, stable phenotype and meeting the requirements of subsequent experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of primary endothelial cell culture media, and specifically relates to the preparation and application of a culture medium for efficiently and rapidly culturing primary pulmonary artery endothelial cells of mice. Background Art

[0002] Pulmonary hypertension (PAH) is a clinical syndrome characterized by a continuous increase in pulmonary vascular resistance caused by different etiologies and pathogenesis. The main pathological mechanisms of PAH are the dysfunction of pulmonary artery endothelial cells and the proliferation of smooth muscle cells, etc. Vascular endothelial cell injury is the initiating factor for the occurrence of many cardiovascular diseases. The dysregulation of endothelial cell signaling functions will cause various effects such as abnormal vasoconstriction and relaxation functions and inflammation, and thus promote the occurrence and development of PAH. Therefore, culturing pulmonary artery endothelial cells is of great significance for studying the specific mechanisms of endothelial cell dysfunction in patients with pulmonary hypertension and providing new therapeutic targets.

[0003] Currently, the culture media used for culturing primary pulmonary artery endothelial cells of mice on the market have the following problems: First, during the culture of primary pulmonary artery endothelial cells of mice using commercially available culture media, it is found that the cells crawl out slowly, the culture time required is long, and the endothelial cells are extremely prone to differentiation, poor homogeneity, senescence or death, etc. Second, during the use of commercially available culture media, there are many heterologous cells such as pulmonary artery vascular smooth muscle cells and fibroblasts, which makes the survival space required for culturing primary pulmonary artery endothelial cells of mice smaller, and often makes it very difficult for the pulmonary artery endothelial cells to crawl out. Third, the price of commercially available endothelial cell culture media is relatively high, and for research groups that need to culture primary pulmonary artery endothelial cells of mice in large quantities for a long time, the cost of purchasing the required culture media is relatively large. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the existing endothelial cell culture media on the market, such as high price, difficult and slow cell crawling out, etc., and to provide a new economical, rapid and efficient culture medium for primary pulmonary artery endothelial cells of mice, which is suitable for the culture system of primary pulmonary artery endothelial cells of mice.

[0005] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0006] A medium for efficiently and rapidly culturing primary mouse pulmonary artery endothelial cells, the formula of which includes: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Endothelin (ET), Phenol red, Earle's equilibrium salt solution, etc.

[0007] Preferably, the concentration of Glucose used each time is 4000 mg / L, the concentration of L-glutamine is 4200 mg / L, the concentration of Sodium Pyruvate is 3000 mg / L, the concentration of Sodium bicarbonate is 4000 mg / L, the concentration of Fetal Bovine Serum (FBS) is 20%, the concentration of Heparin is 50 ng / mL, the concentration of Vascular Endothelial Growth Factor 164 (VEGF164) is 20 ng / mL, the concentration of Epidermal Growth Factor (EGF) is 30 pg / mL, the concentration of Vasopressin (AVP) is 0.001 mmol / L, the concentration of Endothelin (ET) is 0.001 mmol / L, the concentration of Phenol red is 20 mg / L, and the concentration of Earle's equilibrium salt solution is 0.5%.

[0008] Furthermore, another object of the present invention is to provide a preparation method of the above-mentioned medium for efficiently and rapidly culturing primary mouse pulmonary artery endothelial cells, including the following steps:

[0009] First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water. Then, sequentially add Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), and Endothelin (ET) to the basal culture medium. Next, mix it with Earle's equilibrium salt solution at a ratio of 1:1. Finally, add Phenol red for pH color indication.

[0010] Furthermore, another object of the present invention is to provide the application of the above-mentioned medium for efficiently and rapidly culturing primary mouse pulmonary artery endothelial cells, and the application is to use this medium to culture primary mouse pulmonary artery endothelial cells economically, rapidly, and efficiently to meet the research on the molecular mechanism of pulmonary artery diseases.

[0011] Furthermore, another object of the present invention is to provide a method for culturing primary mouse pulmonary artery endothelial cells, which includes the following steps:

[0012] 1) After anesthetizing the mouse, quickly remove the lung, isolate its pulmonary artery, and place it in a 35 mm cell culture dish after rinsing it three times with phosphate buffered saline (PBS).

[0013] 2) Use a sterile surgical blade to cut the pulmonary artery into pieces, digest it with Collagenase, centrifuge it, and place it in the above-mentioned medium for culture.

[0014] 3) Replace the fresh medium at 3 days of culture. When the primary mouse pulmonary artery endothelial cells grow to 80% - 90% confluence, passage the cells. After passage 2 - 3 times, use the cells for subsequent experiments.

[0015] Preferably, in step 1), when isolating the pulmonary artery, immerse the whole lung tissue in PBS solution pre-cooled at 4°C in advance. Under a dissecting microscope, use ophthalmic scissors to quickly remove the tissue around the pulmonary artery and isolate it to the third branch of the pulmonary artery. Cut the isolated pulmonary artery along the longitudinal axis, transfer it into a laminar flow hood, and then wash the pulmonary artery 3 times with sterile PBS solution. Aspirate the remaining PBS solution and transfer the pulmonary artery into a new 35 mm diameter cell culture dish.

[0016] Preferably, in step 2), use a sterile surgical blade to chop the pulmonary artery tissue into 1 mm 3 tissue blocks; digest with Collagenase for 20 min; the rotation speed during centrifugation is 1700 g and the time is 3 min.

[0017] Preferably, in step 3), culture in an incubator containing 3-7% CO2 by volume, the temperature of the incubator is 30-40 °C, and pay attention to retaining 1 / 5 of the original culture medium when changing the liquid.

[0018] The culture medium and culture method provided by the present invention can establish an economical, rapid and efficient preparation process for primary mouse pulmonary artery endothelial cells, can maintain and promote the proliferation of primary mouse pulmonary artery endothelial cells, are rich in various growth factors required for endothelial cell growth, and the concentration and ratio of various growth factors meet the physiological requirements, and can significantly promote the growth of primary mouse pulmonary artery endothelial cells.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1) The present invention provides a culture medium for efficiently and rapidly culturing primary mouse pulmonary artery endothelial cells. Using this culture medium, primary mouse pulmonary artery endothelial cells can be prepared quickly and efficiently. This method has the advantages of fast cell proliferation speed, large quantity, good morphology, etc., and overcomes the disadvantages of difficult and slow outgrowth of primary mouse pulmonary artery endothelial cells in the case of using commercially available culture media at present.

[0021] 2) By scientifically analyzing the nutritional components required for the growth and development of vascular endothelial cells and adjusting the components and concentrations of the culture medium, the present invention improves the culture effect while reducing the culture cost, effectively overcomes the problem of high price of commercially available endothelial cell culture media at present, and greatly reduces the expenses for long-term experimental research related to the culture of primary mouse pulmonary artery endothelial cells.

[0022] 3) The culture medium provided by the present invention uses Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), and Endothelin (ET) as the main components, and then is mixed with 0.5% Earle's equilibrium salt solution in a 1:1 ratio, and Phenol red is added for pH color indication, thus meeting the requirements for continuous subculture of primary mouse pulmonary artery endothelial cells in vitro; the test results show that the primary mouse pulmonary artery endothelial cells cultured with the culture medium provided by the present invention have a uniform morphology, meet the experimental standards, have a high proliferation efficiency, and the culture effect is significantly higher than that of other commercially available culture media.

[0023] 4) In the culture medium provided by the present invention, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), and Endothelin (ET) are used in combination. While promoting the proliferation of primary mouse pulmonary artery endothelial cells, they can also inhibit the growth of pulmonary artery smooth muscle cells and fibroblasts, thereby greatly improving the purity and proliferation efficiency of primary mouse pulmonary artery endothelial cells. Brief Description of the Drawings

[0024] Figure 1 It is a bright-field image (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Example 1 of the present invention;

[0025] Figure 2 It is a bright-field image (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Example 2 of the present invention;

[0026] Figure 3 It is a bright-field image (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Comparative Example 1;

[0027] Figure 4Bright-field images (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Comparative Example 2;

[0028] Figure 5 Bright-field images (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Comparative Example 3;

[0029] Figure 6 Bright-field images (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Comparative Example 4;

[0030] Figure 7 Bright-field images (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Comparative Example 5;

[0031] Figure 8 Bright-field images (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Comparative Example 6;

[0032] Figure 9 Bright-field images (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Comparative Example 7;

[0033] Figure 10 Bright-field images (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Comparative Example 8; Detailed implementation manners

[0034] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

[0035] I. Preparation of primary mouse pulmonary artery endothelial cell culture medium

[0036] Example 1

[0037] An efficient and rapid culture medium for primary mouse pulmonary artery endothelial cells, comprising the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Endothelin (ET), Phenol red, Earle's equilibrium salt solution (EBSS), and Ultrapure water.

[0038] The concentration of Glucose in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of L-glutamine in the primary mouse pulmonary artery endothelial cell medium is 4200 mg / L. The concentration of Sodium Pyruvate in the primary mouse pulmonary artery endothelial cell medium is 3000 mg / L. The concentration of Sodium bicarbonate in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of Fetal Bovine Serum (FBS) in the primary mouse pulmonary artery endothelial cell medium is 20%, and the brand used is Gbico (Gibco is a world-famous animal serum brand, produced in South America, with high quality and high stability, meeting the FDA quality system requirements of many countries such as the United States). The concentration of Heparin in the primary mouse pulmonary artery endothelial cell medium is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the primary mouse pulmonary artery endothelial cell medium is 20 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the primary mouse pulmonary artery endothelial cell medium is 30 pg / mL. The concentration of Vasopressin (AVP) in the primary mouse pulmonary artery endothelial cell medium is 0.001 mmol / L. The concentration of Endothelin (ET) in the primary mouse pulmonary artery endothelial cell medium is 0.001 mmol / L. The concentration of Phenol red in the primary mouse pulmonary artery endothelial cell medium is 20 mg / L. The concentration of Earle's equilibrium salt solution (EBSS) in the primary mouse pulmonary artery endothelial cell medium is 0.5%.

[0039] Further, the preparation method of the above-mentioned mouse primary pulmonary artery endothelial cell culture medium comprises the following steps: First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water; then sequentially add the Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), and Endothelin (ET) at the above concentrations to the basal culture medium; then mix it with Earle's Balanced Salt Solution (EBSS) at a ratio of 1:1, and add Phenol red for pH color indication.

[0040] Table 1 Mouse primary pulmonary artery endothelial Cell culture conditions

[0041]

[0042] Example 2

[0043] A highly efficient and rapid culture medium for mouse primary pulmonary artery endothelial cells, comprising the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Endothelin (ET), Earle's Balanced Salt Solution (EBSS), and Phenol red.

[0044] The concentration of Glucose in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of L-glutamine in the primary mouse pulmonary artery endothelial cell medium is 4200 mg / L. The concentration of Sodium Pyruvate in the primary mouse pulmonary artery endothelial cell medium is 3000 mg / L. The concentration of Sodium bicarbonate in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of Fetal Bovine Serum (FBS) in the primary mouse pulmonary artery endothelial cell medium is 20%, and the brand used is Gbico. The concentration of Heparin in the primary mouse pulmonary artery endothelial cell medium is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the primary mouse pulmonary artery endothelial cell medium is 15 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the primary mouse pulmonary artery endothelial cell medium is 20 pg / mL. The concentration of Vasopressin (AVP) in the primary mouse pulmonary artery endothelial cell medium is 0.001 mmol / L. The concentration of Endothelin (ET) in the primary mouse pulmonary artery endothelial cell medium is 0.002 mmol / L. The concentration of Phenol red in the primary mouse pulmonary artery endothelial cell medium is 20 mg / L. The concentration of Earle's equilibrium salt solution (EBSS) in the primary mouse pulmonary artery endothelial cell medium is 0.5%.

[0045] Further, the preparation method of the above-mentioned mouse primary pulmonary artery endothelial cell culture medium includes the following steps: First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water; sequentially add a certain concentration of Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), and Endothelin (ET) to the basal culture medium. Then, mix it with Earle's Balanced Salt Solution (EBSS) at a ratio of 1:1, and add Phenol red for pH colorimetric indication.

[0046] Table 2 Mouse primary pulmonary artery endothelial Cell culture conditions

[0047]

[0048] Comparative Example 1

[0049] Different from Example 1, the concentration of FBS is 10%, and ET is not contained.

[0050] An efficient and rapid culture medium for primary mouse pulmonary artery endothelial cells, comprising the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Phenol red, Earle's Balanced Salt Solution (EBSS), and Ultrapure water, etc.

[0051] The concentration of Fetal Bovine Serum (FBS) in the primary mouse pulmonary artery endothelial cell medium is 10%, and the brand used is Gbico. The concentration of Glucose in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of L-glutamine in the primary mouse pulmonary artery endothelial cell medium is 4200 mg / L. The concentration of Sodium Pyruvate in the primary mouse pulmonary artery endothelial cell medium is 3000 mg / L. The concentration of Sodium bicarbonate in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of Heparin in the primary mouse pulmonary artery endothelial cell medium is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the primary mouse pulmonary artery endothelial cell medium is 20 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the primary mouse pulmonary artery endothelial cell medium is 30 pg / mL. The concentration of Vasopressin (AVP) in the primary mouse pulmonary artery endothelial cell medium is 0.001 mmol / L. The concentration of Phenol red in the primary mouse pulmonary artery endothelial cell medium is 20 mg / L. The concentration of Earle's equilibrium salt solution in the primary mouse pulmonary artery endothelial cell medium is 0.5%.

[0052] Further, the method for preparing the above-mentioned primary mouse pulmonary artery endothelial cell culture medium includes the following steps: First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water; sequentially add a certain concentration of Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), and Vasopressin (AVP) to the basal culture medium. Then, mix it with 0.5% Earle's equilibrium salt solution at a ratio of 1:1, and add Phenol red for pH color indication.

[0053] Table 3 Mouse primary pulmonary artery endothelial Cell culture conditions

[0054] Factor Original condition Existing condition Glucose 4000 mg / L / L-glutamine 4200 mg / L / Sodium Pyruvate 3000 mg / L / Sodium bicarbonate 4000 mg / L / FBS concentration / 10%, Gbico Heparin concentration 50 ng / mL / VEGF164 concentration 20 ng / mL / EGF concentration 30 pg / mL / AVP concentration 0.001 mmol / L / EBSS 0.5% / Phenol red 20 mg / L /

[0055] Comparative Example 2

[0056] Different from Example 1, the concentration of FBS was changed to 15%, and ET was not included.

[0057] An efficient and rapid culture medium for primary mouse pulmonary artery endothelial cells, comprising the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Earle's Balanced Salt Solution (EBSS), and Phenol red.

[0058] The concentration of Fetal Bovine Serum (FBS) in the primary mouse pulmonary artery endothelial cell medium is 15%, and the brand used is Gbico. The concentration of Glucose in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of L-glutamine in the primary mouse pulmonary artery endothelial cell medium is 4200 mg / L. The concentration of Sodium Pyruvate in the primary mouse pulmonary artery endothelial cell medium is 3000 mg / L. The concentration of Sodium bicarbonate in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of Heparin in the primary mouse pulmonary artery endothelial cell medium is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the primary mouse pulmonary artery endothelial cell medium is 20 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the primary mouse pulmonary artery endothelial cell medium is 30 pg / mL. The concentration of Vasopressin (AVP) in the primary mouse pulmonary artery endothelial cell medium is 0.001 mmol / L. The concentration of Phenol red in the primary mouse pulmonary artery endothelial cell medium is 20 mg / L. The concentration of Earle's equilibrium salt solution in the primary mouse pulmonary artery endothelial cell medium is 0.5%.

[0059] Further, the preparation method of the above-mentioned mouse primary pulmonary artery endothelial cell culture medium includes the following steps: First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water; sequentially add a certain concentration of Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), and Vasopressin (AVP) to the basal culture medium. Then, mix it with Earle's Balanced Salt Solution (EBSS) at a ratio of 1:1, and add Phenol red for pH color indication.

[0060] Table 4 Mouse primary pulmonary artery endothelial Cell culture conditions

[0061] Factor Original condition Existing condition Glucose 4000 mg / L / L-glutamine 4200 mg / L / Sodium Pyruvate 3000 mg / L / Sodium bicarbonate 4000 mg / L / FBS concentration / 15%, Gbico Heparin concentration 50 ng / mL / VEGF164 concentration 20 ng / mL / EGF concentration 30 pg / mL / AVP concentration 0.001 mmol / L / EBSS 0.5% / Phenol red 20 mg / L /

[0062] Comparative Example 3

[0063] Different from Example 1, the brand of FBS was changed to Evergreen and it does not contain ET.

[0064] An efficient and rapid culture medium for mouse primary pulmonary artery endothelial cells, comprising the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Earle's Balanced Salt Solution (EBSS), and Phenol red.

[0065] The concentration of FBS in the primary mouse pulmonary artery endothelial cell medium is 20%, and the brand of FBS is Evergreen (Evergreen is a kind of FBS produced by Hangzhou Sijiqing Bioengineering Materials Co., Ltd., which is a series of FBS without mycoplasma and phage and with low endotoxin). The concentration of Glucose in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of L-glutamine in the primary mouse pulmonary artery endothelial cell medium is 4200 mg / L. The concentration of Sodium Pyruvate in the primary mouse pulmonary artery endothelial cell medium is 3000 mg / L. The concentration of Sodium bicarbonate in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of Heparin in the primary mouse pulmonary artery endothelial cell medium is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the primary mouse pulmonary artery endothelial cell medium is 20 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the primary mouse pulmonary artery endothelial cell medium is 30 pg / mL. The concentration of Vasopressin (AVP) in the primary mouse pulmonary artery endothelial cell medium is 0.001 mmol / L. The concentration of Phenol red in the primary mouse pulmonary artery endothelial cell medium is 20 mg / L. The concentration of Earle's equilibrium salt solution (EBSS) in the primary mouse pulmonary artery endothelial cell medium is 0.5%.

[0066] Furthermore, the method for preparing the above-mentioned primary mouse pulmonary artery endothelial cell culture medium includes the following steps: First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water. Then, sequentially add a certain concentration of Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), and Vasopressin (AVP) to the basal culture medium. Then, mix it with Earle's Balanced Salt Solution (EBSS) at a ratio of 1:1, and add Phenol red for pH color indication.

[0067] Table 5 Mouse primary pulmonary artery endothelial Cell culture conditions

[0068] Factor Original condition Existing condition Glucose 4000 mg / L / L-glutamine 4200 mg / L / Sodium Pyruvate 3000 mg / L / Sodium bicarbonate 4000 mg / L / FBS concentration / 20%, Evergreen Heparin concentration 50 ng / mL / VEGF164 concentration 20 ng / mL / EGF concentration 30 pg / mL / AVP concentration 0.001 mmol / L / EBSS 0.5% / Phenol red 20 mg / L /

[0069] Comparative Example 4

[0070] Different from Example 1, the brand of FBS is Excell and it does not contain ET.

[0071] A highly efficient and rapid culture medium for primary mouse pulmonary artery endothelial cells, comprising the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Earle's Balanced Salt Solution (EBSS), and Phenol red.

[0072] The concentration of FBS in the primary mouse pulmonary artery endothelial cell medium is 20%, and the brand of FBS is Excell (Excell is produced in South America and its products meet the internationally renowned FBS detection standards and are suitable for the culture of conventional cell lines). The concentration of Glucose in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of L-glutamine in the primary mouse pulmonary artery endothelial cell medium is 4200 mg / L. The concentration of Sodium Pyruvate in the primary mouse pulmonary artery endothelial cell medium is 3000 mg / L. The concentration of Sodium bicarbonate in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of Heparin in the primary mouse pulmonary artery endothelial cell medium is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the primary mouse pulmonary artery endothelial cell medium is 20 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the primary mouse pulmonary artery endothelial cell medium is 30 pg / mL. The concentration of Vasopressin (AVP) in the primary mouse pulmonary artery endothelial cell medium is 0.001 mmol / L. The concentration of Phenol red in the primary mouse pulmonary artery endothelial cell medium is 20 mg / L. The concentration of Earle's equilibrium salt solution (EBSS) in the primary mouse pulmonary artery endothelial cell medium is 0.5%.

[0073] Further, the method for preparing the above-mentioned primary mouse pulmonary artery endothelial cell culture medium comprises the following steps: First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water; sequentially add a certain concentration of Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), and Vasopressin (AVP) to the basal culture medium. Then, mix it with Earle's Balanced Salt Solution (EBSS) at a ratio of 1:1, and add Phenol red for pH color indication.

[0074] Table 6 Mouse primary pulmonary artery endothelial Cell culture conditions

[0075] Factor Original condition Existing condition Glucose 4000 mg / L / L-glutamine 4200 mg / L / Sodium Pyruvate 3000 mg / L / Sodium bicarbonate 4000 mg / L / FBS concentration / 20%, Excell Heparin concentration 50 ng / mL / VEGF164 concentration 20 ng / mL / EGF concentration 30 pg / mL / AVP concentration 0.001 mmol / L / EBSS 0.5% / Phenol red 20 mg / L /

[0076] Comparative Example 5

[0077] Different from Example 2, the concentration of VEGF164 is 10 ng / mL, and the concentration of EGF is 10 pg / mL.

[0078] A culture medium for efficiently and rapidly culturing primary mouse pulmonary artery endothelial cells, comprising the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Endothelin (ET), Earle's Balanced Salt Solution (EBSS), and Phenol red.

[0079] The concentration of Fetal Bovine Serum (FBS) in the primary mouse pulmonary artery endothelial cell medium is 20%, and the brand used is Gbico. The concentration of Glucose in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of L-glutamine in the primary mouse pulmonary artery endothelial cell medium is 4200 mg / L. The concentration of Sodium Pyruvate in the primary mouse pulmonary artery endothelial cell medium is 3000 mg / L. The concentration of Sodium bicarbonate in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of Heparin in the primary mouse pulmonary artery endothelial cell medium is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the primary mouse pulmonary artery endothelial cell medium is 10 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the primary mouse pulmonary artery endothelial cell medium is 10 pg / mL. The concentration of Vasopressin (AVP) in the primary mouse pulmonary artery endothelial cell medium is 0.001 mmol / L. The concentration of Endothelin (ET) in the primary mouse pulmonary artery endothelial cell medium is 0.002 mmol / L. The concentration of Phenol red in the primary mouse pulmonary artery endothelial cell medium is 20 mg / L. The concentration of Earle's equilibrium salt solution in the primary mouse pulmonary artery endothelial cell medium is 0.5%.

[0080] Further, the method for preparing the above-mentioned primary mouse pulmonary artery endothelial cell culture medium comprises the following steps: First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water; sequentially add a certain concentration of Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), and Endothelin (ET) to the basal culture medium. Then, mix it with Earle's Balanced Salt Solution (EBSS) at a ratio of 1:1, and add Phenol red for pH colorimetric indication.

[0081] Table 7 Mouse primary pulmonary artery endothelial Cell culture conditions

[0082] Factor Original condition Existing condition Glucose 4000 mg / L / L-glutamine 4200 mg / L / Sodium Pyruvate 3000 mg / L / Sodium bicarbonate 4000 mg / L / FBS concentration 20%, Gbico / Heparin concentration 50 ng / mL / VEGF164 concentration / 10 ng / mL EGF concentration / 10 pg / mL AVP concentration 0.001 mmol / L / ET concentration 0.002 mmol / L / EBSS 0.5% / Phenol red 20 mg / L /

[0083] Comparative Example 6

[0084] Differing from Example 2, the concentration of VEGF164 is 10 ng / mL, the concentration of EGF is 10 pg / mL, and ET is not contained.

[0085] An efficient and rapid culture medium for primary mouse pulmonary artery endothelial cells, comprising the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Earle's Balanced Salt Solution (EBSS), and Phenol red.

[0086] In the culture medium for primary mouse pulmonary artery endothelial cells, the concentration of Fetal Bovine Serum (FBS) is 20%, and its brand is Gbico. The concentration of Glucose in the culture medium for primary mouse pulmonary artery endothelial cells is 4000 mg / L. The concentration of L-glutamine in the culture medium for primary mouse pulmonary artery endothelial cells is 4200 mg / L. The concentration of Sodium Pyruvate in the culture medium for primary mouse pulmonary artery endothelial cells is 3000 mg / L. The concentration of Sodium bicarbonate in the culture medium for primary mouse pulmonary artery endothelial cells is 4000 mg / L. The concentration of Heparin in the culture medium for primary mouse pulmonary artery endothelial cells is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the culture medium for primary mouse pulmonary artery endothelial cells is 10 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the culture medium for primary mouse pulmonary artery endothelial cells is 10 pg / mL. The concentration of Vasopressin (AVP) in the culture medium for primary mouse pulmonary artery endothelial cells is 0.001 mmol / L. The concentration of Phenol red in the culture medium for primary mouse pulmonary artery endothelial cells is 20 mg / L. The concentration of Earle's equilibrium salt solution (EBSS) in the culture medium for primary mouse pulmonary artery endothelial cells is 0.5%.

[0087] Further, the method for preparing the above-mentioned primary mouse pulmonary artery endothelial cell culture medium includes the following steps: First, prepare a basic culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water; sequentially add a certain concentration of Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), and Vasopressin (AVP) to the basic culture medium. Then, mix it with Earle's Balanced Salt Solution (EBSS) at a ratio of 1:1, and add Phenol red for pH color indication.

[0088] Table 8 Mouse primary pulmonary artery endothelial Cell culture conditions

[0089] Factor Original condition Existing condition Glucose 4000 mg / L / L-glutamine 4200 mg / L / Sodium Pyruvate 3000 mg / L / Sodium bicarbonate 4000 mg / L / FBS concentration 20%, Gbico / Heparin concentration 50 ng / mL / VEGF164 concentration / 10 ng / mL EGF concentration / 10 pg / mL AVP concentration 0.001 mmol / L / EBSS 0.5% / Phenol red 20 mg / L /

[0090] Comparative Example 7

[0091] Different from Example 2, other components remain unchanged, and ET is not included.

[0092] An efficient and rapid culture medium for primary mouse pulmonary artery endothelial cells, comprising the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Vasopressin (AVP), Earle's Balanced Salt Solution (EBSS), and Phenol red.

[0093] The concentration of Fetal Bovine Serum (FBS) in the primary mouse pulmonary artery endothelial cell medium is 20%, and the brand used is Gbico. The concentration of Glucose in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of L-glutamine in the primary mouse pulmonary artery endothelial cell medium is 4200 mg / L. The concentration of Sodium Pyruvate in the primary mouse pulmonary artery endothelial cell medium is 3000 mg / L. The concentration of Sodium bicarbonate in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of Heparin in the primary mouse pulmonary artery endothelial cell medium is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the primary mouse pulmonary artery endothelial cell medium is 20 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the primary mouse pulmonary artery endothelial cell medium is 30 pg / mL. The concentration of Vasopressin (AVP) in the primary mouse pulmonary artery endothelial cell medium is 0.001 mmol / L. The concentration of Phenol red in the primary mouse pulmonary artery endothelial cell medium is 20 mg / L. The concentration of Earle's equilibrium salt solution (EBSS) in the primary mouse pulmonary artery endothelial cell medium is 0.5%.

[0094] Furthermore, the method for preparing the above-mentioned primary mouse pulmonary artery endothelial cell culture medium comprises the following steps: First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water; then sequentially add a certain concentration of Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), and Vasopressin (AVP) to the basal culture medium. Then, mix it with Earle's Balanced Salt Solution (EBSS) at a ratio of 1:1, and add Phenol red for pH color indication.

[0095] Table 9 Mouse primary pulmonary artery endothelial Cell culture conditions

[0096] Factor Original condition Existing condition Glucose 4000 mg / L / L-glutamine 4200 mg / L / Sodium Pyruvate 3000 mg / L / Sodium bicarbonate 4000 mg / L / FBS concentration 20%, Gbico / Heparin concentration 50 ng / mL / VEGF164 concentration 20 ng / mL / EGF concentration 30 pg / mL / AVP concentration 0.001 mmol / L / EBSS 0.5% / Phenol red 20 mg / L /

[0097] Comparative Example 8

[0098] Different from Example 2, it does not contain AVP and ET.

[0099] An efficient and rapid culture medium for primary mouse pulmonary artery endothelial cells comprises the following components: Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), Epidermal Growth Factor (EGF), Earle's Balanced Salt Solution (EBSS), and Phenol red.

[0100] The concentration of Fetal Bovine Serum (FBS) in the primary mouse pulmonary artery endothelial cell medium is 20%, and the brand used is Gbico. The concentration of Glucose in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of L-glutamine in the primary mouse pulmonary artery endothelial cell medium is 4200 mg / L. The concentration of Sodium Pyruvate in the primary mouse pulmonary artery endothelial cell medium is 3000 mg / L. The concentration of Sodium bicarbonate in the primary mouse pulmonary artery endothelial cell medium is 4000 mg / L. The concentration of Heparin in the primary mouse pulmonary artery endothelial cell medium is 50 ng / mL. The concentration of Vascular Endothelial Growth Factor 164 (VEGF164) in the primary mouse pulmonary artery endothelial cell medium is 15 ng / mL. The concentration of Epidermal Growth Factor (EGF) in the primary mouse pulmonary artery endothelial cell medium is 20 pg / mL. The concentration of Phenol red in the primary mouse pulmonary artery endothelial cell medium is 20 mg / L. The concentration of Earle's equilibrium salt solution (EBSS) in the primary mouse pulmonary artery endothelial cell medium is 0.5%.

[0101] Further, the preparation method of the above primary mouse pulmonary artery endothelial cell medium includes the following steps: First, prepare a basal culture medium containing Glucose, L-glutamine, Sodium Pyruvate, Sodium bicarbonate, and Ultrapure water; sequentially add a certain concentration of Fetal Bovine Serum (FBS), Heparin, Vascular Endothelial Growth Factor 164 (VEGF164), and Epidermal Growth Factor (EGF) to the basal culture medium. Then, mix it with Earle's Balanced Salt Solution (EBSS) at a ratio of 1:1, and add Phenol red for pH color indication.

[0102] Table 10 Mouse primary pulmonary artery endothelial Cell culture conditions

[0103] Factor Original condition Existing condition Glucose 4000 mg / L / L-glutamine 4200 mg / L / Sodium Pyruvate 3000 mg / L / Sodium bicarbonate 4000 mg / L / FBS concentration 20%, Gbico / Heparin concentration 50 ng / mL / VEGF164 concentration 15 ng / mL / EGF concentration 20 pg / mL / EBSS 0.5% / Phenol red 20 mg / L /

[0104] II. Isolation and culture of mouse primary pulmonary artery endothelial cells

[0105] 1) Acute isolation of mouse pulmonary artery tissue

[0106] Anesthetize the mouse with isoflurane and disinfect the body surface with iodophor cotton balls. Open the mouse's abdominal cavity and cut off one of its kidneys. Cut open the thoracic cavity along the sternum to fully expose the mouse's heart and lung tissues. Take out the lung and place it in a Petri dish containing 4°C PBS. Fix the lung tissue with a pin. Carefully remove the tissue around the pulmonary artery under a dissecting microscope. During the separation of the pulmonary artery, avoid pulling the blood vessel forcefully to prevent affecting the vascular activity. Longitudinally cut the isolated mouse pulmonary artery and then place it in a 35 mm Petri dish containing 4°C sterile PBS solution.

[0107] 2) Culture of primary mouse pulmonary artery endothelial cells

[0108] Transfer the isolated mouse pulmonary artery obtained above into a laminar flow hood, wash the blood vessel 3 times with sterile PBS solution, aspirate the residual PBS solution, transfer it into a new 35 mm Petri dish, and quickly cut the pulmonary artery into tissue pieces about 1 mm 3 in size with a sterile scalpel. Transfer the pulmonary artery tissue pieces into a new 35 mm Petri dish again, digest with collagenase for 20 min, then transfer the digestion solution into a 15 mL centrifuge tube and centrifuge at 1700 rpm for 3 min. Aspirate and discard the supernatant, add 0.7 mL of medium, and place it in a CO2 incubator (37°C, 5% CO2) for adherent culture. Take out the Petri dish after 3 h, aspirate and discard the non-adherent tissue, slowly add 3 mL of medium dropwise, and place it in a CO2 incubator for culture. Change the medium after 3 d. After adherent culture for 6 - 7 d, remove the redundant tissue, and then change the medium once every 2 - 3 d. When changing the medium, retain a part (about 1 / 5 volume) of the old medium. Passage the cells when the endothelial cells grow to 80% - 90% confluence.

[0109] 3) Passage of mouse pulmonary artery endothelial cells

[0110] Aspirate and discard the old culture medium, gently rinse the cells three times with PBS buffer, then add 0.25% Trypsin-EDTA (trypsin), place the culture dish in a CO2 incubator, after about 3 min, add culture medium to terminate digestion, gently pipette to suspend the cells, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1700 rpm for 3 min. Aspirate and discard the supernatant after centrifugation, resuspend the cells with complete culture medium, transfer the cell suspension into two new 60 mm culture dishes for culture until the endothelial cells grow to 80 - 90% confluence, and use the cells for subsequent experiments after subculturing 2 - 3 times.

[0111] III. Culture Results

[0112] Figure 1 Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured in Example 1 for 7 d; using the culture medium of this example, the amount of cells crawling out is relatively large. Figure 2 Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured in Example 2 for 7 d; using the culture medium of this example, the amount of cells crawling out is large. Figure 3 Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured in Comparative Example 1 for 7 d; using the culture medium of this example, the amount of cells crawling out is very small, and there are contaminating cells. Figure 4 Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured in Comparative Example 2 for 7 d; using the culture medium of this example, the amount of cells crawling out is relatively small, and there are contaminating cells. Figure 5 Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured in Comparative Example 3 for 7 d; using the culture medium of this example, the amount of cells crawling out is small, and there are contaminating cells. Figure 6 Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured in Comparative Example 4 for 7 d; using the culture medium of this example, the amount of cells crawling out is relatively large, but the growth is slow. Figure 7 Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured in Comparative Example 5 for 7 d; using the culture medium of this example, the amount of cells crawling out is relatively small. Figure 8 Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured in Comparative Example 6 for 7 d; using the culture medium of this example, the amount of cells crawling out is relatively small. Figure 9 Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured in Comparative Example 7 for 7 d; using the culture medium of this example, the amount of cells crawling out is small, and the adherent tissue is less. Figure 10Bright-field cell images (200X) of primary mouse pulmonary artery endothelial cells cultured for 7 days in Comparative Example 8; using the medium of this example, cell outgrowth was slow.

Claims

1. A culture medium for efficiently and rapidly culturing primary mouse pulmonary artery endothelial cells, characterized in that, It comprises the following components: glucose, L-glutamine, sodium pyruvate, sodium bicarbonate, fetal bovine serum, heparin, vascular endothelial growth factor, epidermal growth factor, vasopressin, endothelin, phenol red, Earle's balanced salt solution and ultrapure water; the concentration of glucose is 4000 mg / L, the concentration of L-glutamine is 4200 mg / L, the concentration of sodium pyruvate is 3000 mg / L, the concentration of sodium bicarbonate is 4000 mg / L, the concentration of fetal bovine serum is 20%, the concentration of heparin is 50 ng / mL, the concentration of vascular endothelial growth factor is 20 ng / mL, the concentration of epidermal growth factor is 30 pg / mL, the concentration of vasopressin is 0.001 mmol / L, the concentration of endothelin is 0.001 mmol / L, and the concentration of phenol red is 20 mg / L, and the concentration of Earle's balanced salt solution is 0.5%.

2. The preparation method of a culture medium for efficiently and rapidly culturing primary pulmonary artery endothelial cells of mice according to claim 1, wherein, It comprises the following steps: firstly, add glucose, L-glutamine, sodium pyruvate, sodium bicarbonate, fetal bovine serum, heparin, vascular endothelial growth factor, epidermal growth factor, vasopressin and endothelin into ultrapure water in sequence, then mix it with Earle's balanced salt solution at a ratio of 1:1, and finally add phenol red for pH color development indication.

3. Use of a culture medium for efficiently and rapidly culturing primary pulmonary artery endothelial cells of mice according to claim 1, characterized in that: This culture medium is applied to the culture of primary mouse pulmonary artery endothelial cells.

4. A method for culturing primary pulmonary artery endothelial cells of mice, characterized in that, It comprises the following steps: 1) Take the lung of a mouse, isolate its pulmonary artery under a microscope, repeatedly rinse the pulmonary artery three times with phosphate buffered saline solution and then place it in a 35 mm cell culture dish; 2) Use a sterile surgical blade to chop the pulmonary artery into pieces, digest it with collagenase, centrifuge it, and place it in the culture medium described in Claim 1 for the culture of primary mouse pulmonary artery endothelial cells; 3) Replace the fresh culture medium at 3 d of culture. When the pulmonary artery endothelial cells grow to 80% - 90% confluence, subculture them. After the cells are subcultured 2 - 3 times, they are used for subsequent experiments.

5. The culture method of primary pulmonary artery endothelial cells of mice according to claim 4, characterized in that: In step 1), when isolating the pulmonary artery, immerse the whole lung tissue in PBS solution pre-cooled at 4°C in advance. Under a dissecting microscope, use an ophthalmic scissors to quickly remove the tissue around the pulmonary artery, isolate it to the third branch of the pulmonary artery, longitudinally cut open the pulmonary artery, transfer it into a laminar flow hood, then wash the pulmonary artery 3 times with sterile PBS solution, suck out the remaining PBS solution, and transfer the pulmonary artery into a new 35 mm diameter cell culture dish.

6. The culture method of primary pulmonary artery endothelial cells of mice according to claim 4, characterized in that: In step 2), the pulmonary artery tissue was minced into 1 mm 3 tissue pieces and digested with collagenase for 20 min. The centrifugation was carried out at 1700 rpm for 3 min.

7. The culture method of primary pulmonary artery endothelial cells of mice according to claim 4, characterized in that: In step 3), culture it in an incubator containing 3 - 7% CO2 by volume, the temperature of the incubator is 30 - 40°C, and 1 / 5 of the original culture medium is retained during medium change.

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