CN115322949B

CN115322949B - Isolated culture method of human umbilical vein smooth muscle cells and application thereof

Info

Publication number: CN115322949B
Application number: CN202210887570.1A
Authority: CN
Inventors: 郭瑞敏; 曹毓琳; 程世翔; 李伟
Original assignee: Tianjin Economic And Technological Development Zone Tangyi Cell Intelligent Manufacturing And Nerve Trauma Repair Research Institute; Tangyi Holdings Shenzhen Ltd
Current assignee: Tianjin Economic And Technological Development Zone Tangyi Cell Intelligent Manufacturing And Nerve Trauma Repair Research Institute; Tangyi Holdings Shenzhen Ltd
Priority date: 2022-07-26
Filing date: 2022-07-26
Publication date: 2024-03-15
Anticipated expiration: 2042-07-26
Also published as: CN115322949A

Abstract

The application relates to the technical field of cell separation culture, and particularly discloses a separation culture method of human umbilical vein smooth muscle cells and application thereof, wherein the separation culture method of the human umbilical vein smooth muscle cells comprises the following steps: aseptically separating umbilical cord, washing, filling blood vessel with type I collagenase solution, and incubating; cutting off a vascular cavity to obtain a volume of 1-2 mm ³ After the tissue blocks are arranged, the volume is 3-5 blocks/cm ² Is planted in a culture flask; the bottle bottom planted with the tissue blocks is upwards, the complete culture solution is added, the culture bottle is turned over after standing, the tissue blocks are immersed into the complete culture solution for culture, and the complete culture solution is replaced periodically; tissue pieces were removed and serial subcultured. The method optimizes the separation culture method of the human umbilical vein smooth muscle cells, improves the activity of the cells, has the advantages of higher proliferation speed, high purity and good survival rate, can continuously passage for more than 20 times, and has important research value.

Description

Isolated culture method of human umbilical vein smooth muscle cells and application thereof

Technical Field

The application relates to the technical field of cell separation culture, in particular to a separation culture method of human umbilical vein smooth muscle cells and application thereof.

Background

Cardiovascular disease has become a leading disease that threatens human health. Vascular disease is often the originating disease and lacks effective therapeutic means. Abnormal growth of vascular smooth muscle cells is closely related to the formation, development and prognosis of vascular disease. Vascular smooth muscle cells are important components of the vascular wall, and many cardiovascular diseases occur in close relationship with their proliferation, migration and differentiation, such as vascular inflammation, plaque formation, atherosclerosis, restenosis, pulmonary arterial hypertension, etc. Studies have shown that most of the pathogenic cells in atherosclerotic plaques (the major cause of heart attacks and strokes) are derived from vascular smooth muscle cells.

Therefore, the study of pathogenesis related to cardiovascular diseases by taking human vascular smooth muscle cells as an experimental study object is a hot spot of current study; the vascular smooth muscle cells cultured in vitro have important significance for researching physiological functions, drug actions and pathophysiological changes under the action of various pathogenic factors.

In addition, more and more studies are gradually beginning to replace experimental animals with organoids constructed in vitro, and blood vessels are essential as important structures of skin in the process of constructing complete skin organoids, and there have been some studies on the construction of skin organoids using vascular smooth muscle cells.

Human umbilical vein smooth muscle cells are isolated from human umbilical vein tissue, are one of the important structural constituent cells of umbilical veins, and play an important role in the normal physiological process of the body. The umbilical cord is a tubular structure of a mammal that connects the fetus to the placenta, and is shaped as a rope, with a smooth and transparent surface, and contains connective tissue, an umbilical vein, and a pair of umbilical arteries. In the uterus, the uterine arteries run in the capillaries of the maternal part of the placenta, close to the fetal capillaries of the daughter part of the placenta, where CO is carried out between the maternal and fetal blood ₂ 、O ₂ Exchange of metabolic waste and nutrients. Umbilical artery for fetal productionIs delivered to the placenta, umbilical vein carries O ₂ And nutrient transport from the placenta to the fetus. Umbilical cord is a waste material during childbirth, which makes umbilical vein smooth muscle cells cultured in vitro a model cell for studying blood vessels.

However, by adopting the existing separation method of umbilical vein smooth muscle cells, the cultured cells tend to have fewer passages, longer expansion time and poorer activity, so how to provide a separation culture method of human umbilical vein smooth muscle cells with more passages, short expansion time, good cell activity and higher purity becomes a problem to be solved urgently.

Disclosure of Invention

In order to improve the activity and the passage times of the isolated and cultured human umbilical vein smooth muscle cells, the application provides an isolated and cultured method for the human umbilical vein smooth muscle cells and application thereof, and the umbilical vein smooth muscle cells obtained by the isolated and cultured method have the advantages of good activity, high purity, short expansion time, capability of large-scale continuous passage culture for more than 20 generations and important application value in related theoretical research.

In a first aspect, the present application provides a method for isolated culture of human umbilical vein smooth muscle cells, which adopts the following technical scheme:

a method for isolated culture of human umbilical vein smooth muscle cells, the method comprising:

aseptically separating umbilical cord, washing, filling blood vessel with type I collagenase solution, and incubating;

cutting off the vascular cavity to obtain a volume of 1-2 mm ³ After the tissue block of (3) to (5) blocks/cm ² Is planted in a culture flask;

the bottle bottom planted with the tissue blocks is upwards, the complete culture solution is added, the culture bottle is turned over after standing, the tissue blocks are immersed into the complete culture solution for culture, and the complete culture solution is replaced periodically;

tissue pieces were removed and serial subcultured.

In the application, the activity of the cells is obviously improved, the proliferation speed is faster, the time required for increasing the number of the cells by 1 time is shortened by optimizing the separation culture method of the human umbilical vein smooth muscle cells, and the number of the cells can reach more than 1.5 times of that of the existing method in the same culture time; the obtained cells can be continuously passaged for more than 20 times and the stability of genotypes is ensured, and compared with the existing separation method, the cell growth state is better while the passaging times are obviously improved; the purity of the separated and cultured cells is high and can reach more than 98%, the survival rate of the cells is more than 99.5%, the activity is good, and the separated and cultured cells can be used as model cells for related researches and have important research significance.

In some specific embodiments, the tissue mass has a volume of 1 to 1.5mm ³ Or 1.5-2 mm ³ Etc.

In a specific embodiment, the volume of the tissue mass is 1mm ³ 、1.5mm ³ Or 2mm ³ Etc.

In some specific embodiments, the density of the planting is 3-4 pieces/cm ² Or 4 to 5 pieces/cm ² 。

In a specific embodiment, the density of the planting is 3 pieces/cm ² 4 pieces/cm ² Or 5 pieces/cm ² 。

In the present application, the volume of the tissue block is controlled to be 1-2 mm ³ Can improve the adherence and climbing-out efficiency of cells, increase the number of obtained umbilical vein smooth muscle cells and improve the utilization rate of raw materials. If the volume of the tissue block is large, the climbing out of cells is not facilitated, the number of the finally obtained cells is small, and the waste of raw materials is caused; if the volume of the tissue block is smaller, the tissue block is greatly influenced by the external environment, the wall is not easy to adhere, the cells are easy to laminate and grow, single-layer cells are not easy to obtain, the physiological state of the cells is also poor, and the proliferation capacity is weak.

In the application, the planting density is controlled to be 3-5 pieces/cm ² Is beneficial to the acquisition of monolayer cells, can promote the activity of the cells and increase the number of the acquired cells. If the planting density is large, the growth space of the climbing-out cells in the tissue block is small, the phenomenon of multilayer cell lamination is easy to generate,and also affects the continued climbing out of cells, resulting in a smaller number of cells that are ultimately obtained; if the density of the planting is smaller, the material and information communication between the cells are not facilitated, and the activity of the cells is also affected to a certain extent. In addition, if the density of tissue mass planting is too small, the cells which are unfavorable for climbing out form a uniform single cell layer, and the cells are easy to gather around the tissue mass and are stacked and grown, so that the phenomena of falling and aging are easy to occur; the time required for the cells to reach the corresponding fusion degree of subculture is longer, which is not beneficial to the maintenance of the cell activity, and meanwhile, the proliferation efficiency of the cells is reduced.

Preferably, the method of washing comprises washing with a D-Hanks solution containing 0.4% to 0.6% heparin sodium at 36.5-37.5 ℃.

In some specific embodiments, the temperature of the D-Hanks solution is 36.5 to 37℃or 37 to 37.5℃or the like.

In a specific embodiment, the temperature of the D-Hanks solution is 36.5 ℃, 37 ℃ or 37.5 ℃ or the like.

In some specific embodiments, the concentration of heparin sodium in the D-Hanks solution is 0.4% to 0.5% or 0.5% to 0.6% or the like.

In a specific embodiment, the concentration of heparin sodium in the D-Hanks solution is 0.4%, 0.5% or 0.6%, etc.

In the application, the coagulation of blood can be delayed by adding heparin sodium into the D-Hanks solution, so that the residual blood in the umbilical cord is flushed more cleanly.

Preferably, the concentration of the type I collagenase in the type I collagenase solution is 0.5-2.5 mg/mL.

In some specific embodiments, the concentration of type I collagenase in the type I collagenase solution is 0.5 to 1mg/mL, 0.5 to 1.5mg/mL, 0.5 to 2mg/mL, 1 to 1.5mg/mL, 1 to 2mg/mL, 1 to 2.5mg/mL, 1.5 to 2mg/mL, 1.5 to 2.5mg/mL, or 2 to 2.5mg/mL, etc.

In a specific embodiment, the collagenase type I solution has a collagenase type I concentration of 0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL, or the like.

Preferably, the method of incubating comprises:

immersing the filled blood vessel by using a D-Hanks solution, and incubating for 28-35 min.

In some specific embodiments, the incubation time may be, for example, 28 to 30 minutes, 28 to 33 minutes, 30 to 35 minutes, 33 to 35 minutes, or the like.

In a specific embodiment, the incubation time may be, for example, 28min, 30min, 33min, 35min, or the like.

Preferably, the incubation is carried out with CO transferred to a temperature of 36.5-37.5 DEG C ₂ Incubation was performed in an incubator.

In some specific embodiments, the CO ₂ The temperature of the incubator is 36.5-37 ℃ or 37-37.5 ℃.

In a specific embodiment, the CO ₂ The temperature of the incubator is 36.5 ℃, 37 ℃ or 37.5 ℃ and the like.

According to the method, umbilical vein blood vessels are filled and incubated, and vascular endothelial cells can be fully digested, so that the obtained umbilical vein smooth muscle cells are guaranteed to have extremely high purity, and other impurity cells are not mixed. In addition, the endothelial cells obtained by digestion can be cultured for other purposes, so that the utilization rate of umbilical vessels is improved, and the method is more economical and efficient.

The traditional method for removing the vascular endothelial cells is to cut off the blood vessel by a cutter and then scrape the endothelial cells by a cotton swab or a blade, which can lead to incomplete removal of the endothelial cells, and other cells are mixed in the obtained umbilical vein smooth muscle cells, so that the purity is lower; the total amount of cells obtained may be reduced by wasting the raw material due to excessive removal. In addition, there is also a risk of injury to the operator when scraping endothelial cells using a knife, with a certain safety risk.

In the application, the sequence of firstly using the type I collagenase to incubate and digest endothelial cells and then shearing tissue blocks is adopted, so that the purity of the finally obtained umbilical vein smooth muscle cells is improved, and meanwhile, the operation difficulty is reduced. If the operation sequence of firstly cutting into tissue blocks and then digesting is adopted, the digestion degree is difficult to determine during digestion, and if the digestion degree is light, the purity of the finally obtained umbilical vein smooth muscle cells is low due to incomplete digestion of endothelial cells; if the degree of digestion is too high, this will result in the loss of smooth muscle cells, resulting in a smaller number of cells being obtained. In addition, after the tissue blocks are cut, the inner wall and the outer wall of the blood vessel are not easy to distinguish, and the difficulty is brought to the subsequent planting operation.

Preferably, after the incubation, the method further comprises a step of washing the venous blood vessel by using a PBS solution, wherein the washing time can be 1-5 times.

Preferably, the complete culture broth comprises fetal bovine serum, a mixture of green streptomycin and DMEM/F12 basal medium.

Preferably, the volume percentage of the fetal bovine serum in the complete culture solution is 18-22%.

In some specific embodiments, the volume percentage of the fetal bovine serum in the complete culture solution is 18% -19%, 18% -20%, 18% -21%, 19% -20%, 19% -21%, 19% -22%, 20% -21%, 20% -22%, 21% -22%, or the like.

In a specific embodiment, the volume percentage of the fetal bovine serum in the complete culture broth is 18%, 19%, 20%, 21% or 22%, etc.

Preferably, the volume percentage of the green streptomycin mixed solution in the complete culture solution is 0.8-1.2%.

In some specific embodiments, the volume percentage of the green streptomycin mixed solution in the complete culture solution is 0.8% -0.9%, 0.8% -1%, 0.8% -1.1%, 0.9% -1%, 0.9% -1.1%, 0.9% -1.2%, 1% -1.1%, 1% -1.2% or 1.1% -1.2% or the like.

In a specific embodiment, the volume percentage of the green streptomycin mixture in the complete culture solution is 0.8%, 0.9%, 1%, 1.1% or 1.2%, etc.

As the preferable technical scheme, the complete culture solution contains 18-22% of fetal calf serum and 0.8-1.2% of green streptomycin mixed solution by volume percent, and the balance is DMEM/F12 basal medium.

In the application, the components of the complete culture solution are optimized, the DMEM/F12 basic culture medium is selected, the addition amount of the fetal calf serum and the green streptomycin mixed solution is optimized, the proliferation state of cells is improved, the division speed is faster, the number of cells is more in the same culture time, and the physiological state is better.

Preferably, the addition amount of the complete culture solution in the culture flask is 1.5-2 mL.

In some specific embodiments, the amount of the complete culture solution added to the flask is 1.5 to 1.7mL, 1.7 to 2mL, or the like.

In a specific embodiment, the amount of the complete culture solution added to the flask is 1.5mL, 1.7mL, 2mL, or the like.

In this application, through controlling the addition of complete culture solution to be 1.5 ~ 2mL, reduced the impact of complete culture solution to the tissue piece in the culture process to make the tissue piece adherence more firm, the cell climbs out more, and the speed is faster.

Preferably, the time of the standing is 8-16 hours.

In some specific embodiments, the time of the resting is 8 to 10 hours, 8 to 12 hours, 8 to 14 hours, 10 to 12 hours, 10 to 14 hours, 10 to 16 hours, 12 to 14 hours, 12 to 16 hours, 14 to 16 hours, or the like.

In a specific embodiment, the time of rest is 8h, 10h, 12h, 14h, 16h, or the like.

In the application, the time of standing is controlled to be 8-16 h, the tissue block is more firmly attached, and the tissue block is not easy to fall off, and is closely related to the climbing-out quantity and the climbing-out speed of cells in the subsequent process.

Preferably, the temperature of the rest is 36.5-37.5 ℃.

In some specific embodiments, the temperature of the resting is 36.5 to 37 ℃, or 37 to 37.5 ℃, or the like.

In a specific embodiment, the resting temperature is 36.5 ℃, 37 ℃, 37.5 ℃, or the like.

Preferably, the said standing is wetThe degree is 95-100%, and CO ₂ CO with concentration of 4-6% ₂ Standing in an incubator.

In some specific embodiments, the CO ₂ The humidity of the incubator is 95-98% or 98-100%.

In a specific embodiment, the CO ₂ The humidity of the incubator is 95%, 98% or 100%, etc.

In some specific embodiments, the CO ₂ CO of incubator ₂ The concentration is 4% -5% or 5% -6%.

In a specific embodiment, the CO ₂ CO of incubator ₂ The concentration is 4%, 5% or 6%.

Preferably, the method of standing includes:

at the temperature of 36.5-37.5 ℃ and the humidity of 95-100 percent, CO ₂ CO with concentration of 4-6% ₂ And standing for 8-16 h in an incubator.

Preferably, the method for periodically replacing the complete culture solution comprises the following steps:

half liquid is changed for 1 time every 3-5 days.

In some specific embodiments, the half-change interval is 3 to 4 days or 4 to 5 days, etc.

In a specific embodiment, the half-change interval is 3 days, 4 days, 5 days, etc.

In the application, the mode of half liquid exchange is adopted, so that stress response generated by cells due to sudden change of a culture environment can be reduced, the physiological state of the cells is maintained in a relatively stable environment, the climbing-out speed of the cells is higher, and the state is better.

Preferably, the condition for removing the tissue blocks is that the cell fusion degree reaches 85% -90%.

In some specific embodiments, the conditions for removing tissue mass are such that the degree of cell fusion reaches 85% to 87% or 87% to 90%, etc.

In a specific embodiment, the conditions for removing tissue mass are such that cell fusion levels of 85%, 87%, 90%, etc. are achieved.

Preferably, in the continuous subculture, the degree of fusion of the cells is not less than 90%, and the subculture is performed by means of digestion with a TrypLE solution.

In some specific embodiments, the degree of fusion of the cells is 90% to 92%, 90% to 94%, 90% to 96%, 90% to 98%, 92% to 94%, 92% to 96%, 92% to 98%, 94% to 96%, 94% to 98%, 96% to 98%, or the like.

In a specific embodiment, the degree of fusion of the cells is 90%, 92%, 94%, 96% or 98%, etc.

In this application, use the TrypLE solution to carry out subculture, compare with pancreatin solution, the TrypLE solution is milder to the digestion of cell, is difficult for causing apoptosis to make the cell keep higher vigor, and the reattachment proportion after the cell digestion is higher, is favorable to subsequent division and culture, also can reduce the cell loss because of the operation of passaging causes.

Preferably, the method of TrypLE solution digestion comprises:

adding 1-2 mL of TrypLE solution, digesting for 1-2 min at 36.5-37.5 ℃, and then adding an equal volume of PBS solution to terminate the digestion reaction;

gently blowing off cells, collecting the cells into a centrifuge tube, centrifuging at 850-1000 rpm for 3-5 min, and discarding the supernatant;

the cells are resuspended in 5-10 mL of complete culture solution, counted, and inoculated into culture flasks at 1:1 or 1:2 according to the cell density, and the solution is changed 1 time every 2-3 days.

Preferably, the isolated culture method of human umbilical vein smooth muscle cells further comprises the step of identifying the cultured cells.

In a second aspect, the present application provides a human umbilical vein smooth muscle cell isolated and cultured by the isolated and cultured method of human umbilical vein smooth muscle cells of the first aspect.

Preferably, the number of passages of human umbilical vein smooth muscle cells is 20 or more.

In some specific embodiments, the human umbilical vein smooth muscle cells are passaged 20-23 times, 20-25 times, 20-28 times, 20-30 times, 23-25 times, 23-28 times, 23-30 times, 25-28 times, 25-30 times, 28-30 times, or the like.

In a specific embodiment, the human umbilical vein smooth muscle cells are passaged 20 times, 23 times, 25 times, 28 times, 30 times, or the like.

Preferably, the human umbilical vein smooth muscle cells are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: c202270, 4 months and 20 days of 2022.

In the present application, the above human umbilical vein smooth muscle cells are named human umbilical vein smooth muscle cell line HUVSMC.

In the application, the human umbilical vein smooth muscle cells obtained by the separation culture method of the human umbilical vein smooth muscle cells have good activity, the cell proliferation and division speed is higher, the survival rate is high, the purity is good, and the method has important research value.

In summary, the present application has the following beneficial effects:

1. the separation culture method of the human umbilical vein smooth muscle cells is optimized, the I-type collagenase solution is used for incubation, and vascular endothelial cells are fully digested, so that the prepared human umbilical vein smooth muscle cells are guaranteed to have extremely high purity, umbilical vessels can be fully utilized, and the utilization rate of raw materials is improved; by controlling the size of the tissue mass and the density of the inoculation, the number of the obtained cells is increased, and the activity of the cells is improved; the components of the complete culture solution are optimized, so that the proliferation state of cells is improved; by controlling the addition amount of the complete culture solution and the standing condition, the adherence efficiency of the tissue block is improved, the climbing-out efficiency of the cells is higher, and the speed is higher; the subculture mode is improved, and the activity of cells is improved.

2. The human umbilical vein smooth muscle cells obtained by the separation culture method have better performance: the proliferation speed is faster, and the total number of cells obtained by adopting the technical scheme of the application can be more than 1.5 times of the number of cells obtained by the traditional method in the same culture time, so that the time required by proliferation is obviously shortened; the purity is high and can reach more than 98%, the survival rate is high and can reach more than 99.5%, the cell activity is better, the continuous passage is carried out for more than 20 times, the stability of the genotype is maintained, the method can be applied to the preparation of large-scale cells, and the method has important research value.

Drawings

Fig. 1 is a morphological picture (magnification = 40 times) of human umbilical vein smooth muscle cells originally cultured in example 1 of the present application.

Fig. 2 is a morphological picture of human umbilical vein smooth muscle cells subcultured 1 time in example 1 of the present application (magnification = 40 times).

FIG. 3 shows the expression of human umbilical vein smooth muscle cells in TrypLE in example 1 of the present application ^TM Morphology pictures of the reattachment after Select solution digestion (magnification = 40 fold).

Fig. 4 is a morphological image of human umbilical vein smooth muscle cells in example 7 of the present application re-adherent after pancreatin digestion (magnification = 40 x).

Fig. 5 is a morphological picture of human umbilical vein smooth muscle cells subcultured 1 time in example 8 of the present application (magnification = 40 times).

Fig. 6 is a morphological picture (magnification = 40 times) of human umbilical vein smooth muscle cells originally cultured in comparative example 1 of the present application.

Fig. 7 is a morphological picture (magnification = 40 times) of human umbilical vein smooth muscle cells originally cultured in comparative example 2 of the present application.

Fig. 8 is a morphological picture (magnification = 40 times) of human umbilical vein smooth muscle cells originally cultured in comparative example 3 of the present application.

Fig. 9 is a morphological picture (magnification = 40 times) of human umbilical vein smooth muscle cells originally cultured in comparative example 4 of the present application.

Fig. 10 is a graph (magnification = 20 times) of the results of α -SMA staining of human umbilical vein smooth muscle cells prepared in example 1 of the present application.

Fig. 11 is a picture (magnification=20×) of the result of Colponin1 staining of human umbilical vein smooth muscle cells prepared in example 1 of the present application.

FIG. 12 is a graph of the positive rate statistics of alpha-SMA and Colponin1 staining of human umbilical vein smooth muscle cells prepared in example 1 of the present application.

FIG. 13 is a photograph showing the results of cell viability assay of human umbilical vein smooth muscle cells prepared in example 1, comparative example 5 and comparative example 6 of the present application.

FIG. 14 is a graph showing the results of cell growth count measurement of human umbilical vein smooth muscle cells prepared in example 1, comparative example 5 and comparative example 6 of the present application.

FIG. 15 is a graph showing the results of continuous passage count measurement of human umbilical vein smooth muscle cells prepared in example 1, comparative example 5 and comparative example 6 of the present application.

Fig. 16 is a morphological picture of human umbilical vein smooth muscle cells subcultured 1 time in comparative example 5 (magnification=40).

Fig. 17 is a morphological picture of human umbilical vein smooth muscle cells subcultured 10 times in comparative example 5 (magnification=40).

Fig. 18 is a morphological picture of human umbilical vein smooth muscle cells subcultured 1 time in example 1 (magnification = 40 times).

Fig. 19 is a morphological picture of human umbilical vein smooth muscle cells subcultured 20 times in example 1 (magnification = 40 times).

Detailed Description

The application provides a separation culture method of human umbilical vein smooth muscle cells, which comprises the following steps:

1. flushing the blood coagulation blocks inside and outside the umbilical vein by using a D-Hanks solution containing 0.4 to 0.6 percent of heparin sodium at the temperature of between 36.5 and 37.5 ℃ through the asepsis separated umbilical cord, clamping one end of the vein by using an umbilical cord clamp, injecting a type I collagenase solution of between 0.5 and 2.5mg/mL from the other end of the vein to filling the vein by using a syringe, and clamping by using the umbilical cord clamp; immersing the filled umbilical cord with D-Hanks solution, transferring to CO at 36.5-37.5 DEG C ₂ Incubating for 28-35 min in an incubator; the umbilical cord clamp was removed and the vein was flushed 1-5 times with PBS solution.

2. Longitudinally cutting off the vascular cavity to obtain a volume of 1-2 mm ³ After the tissue block of (3) to (5) blocks/cm ² Is planted in T25 flasks.

3. 1.5-2 mL of complete culture solution containing 18-22% (volume percentage) fetal bovine serum, 0.8-1.2% (volume percentage) green streptomycin mixed solution and the balance DMEM/F12 basic culture medium are added upwards at the temperature of 36.5-37.5 ℃ and the humidity of 95-100%, CO ₂ CO with concentration of 4-6% ₂ Standing for 8-16 h in an incubator, slowly turning over the culture flask to enable the tissue blocks to slowly submerge into the complete culture solution, and culturing, wherein the half amount of the culture solution is replaced for 1 time every 3-5 days later.

4. Removing tissue blocks when the fusion degree of cells climbing out around the tissue blocks reaches 85% -90%, adding 1-2 mL of TrypLE solution, digesting for 1-2 min at 36.5-37.5 ℃, and then adding an equal volume of PBS solution to terminate the digestion reaction; gently blowing off the cells, collecting the cells into a centrifuge tube, centrifuging at 850-1000 rpm for 3-5 min, discarding the supernatant, re-suspending the cells with 5-10 mL of complete culture solution, counting, inoculating the cells into a culture flask according to the cell density at 1:1 or 1:2, changing the solution 1 time every 2-3 days, and carrying out passaging again until the fusion degree of the cells is not lower than 90%.

5. The cultured cells were observed under a microscope and the cultured cells were identified.

In the above steps, the instrument information used is shown in table 1.

Table 1 instrument information

In the present application, information on the reagents used is shown in table 2.

TABLE 2 Experimental reagent information

The application also provides the human umbilical vein smooth muscle cells obtained by the separation culture method, the purity of the human umbilical vein smooth muscle cells can reach more than 98%, the survival rate can reach more than 99.5%, and the human umbilical vein smooth muscle cells can be continuously passaged for more than 20 times and maintain the stability of genotypes, so that the human umbilical vein smooth muscle cells can be used as a cell model for related researches.

The human umbilical vein smooth muscle cells are named as human umbilical vein smooth muscle cell line HUVSMC, and are preserved in China Center for Type Culture Collection (CCTCC) NO: c202270, 4 months and 20 days of 2022.

The present application will be described in further detail with reference to fig. 1 to 19, examples 1 to 9, and comparative examples 1 to 6.

Examples

Example 1

The method for obtaining the human umbilical vein smooth muscle cells by separating and culturing from the human umbilical vein comprises the following steps:

1. flushing the blood coagulation blocks inside and outside the umbilical vein by using a D-Hanks solution containing 0.5% heparin sodium at 37 ℃ through the asepsis separated umbilical cord, clamping one end of the vein by using an umbilical cord clamp, injecting 1.5mg/mL of type I collagenase solution from the other end of the vein by using a syringe until the vein is full, and clamping by using the umbilical cord clamp; immersing the filled umbilical cord with D-Hanks solution, transferring it to CO at 37 DEG C ₂ Incubating for 30min in a incubator; the umbilical cord clamps were removed and the veins were flushed 3 times with PBS solution.

2. Longitudinally cutting off the vascular cavity to obtain a volume of 1.5mm ³ After the tissue block of (C), the volume of the tissue block is 4 blocks/cm ² Is planted in T25 flasks.

3. Adding 1.8mL of a complete culture solution containing 20% (volume percent) fetal bovine serum, 1% (volume percent) green streptomycin mixed solution and the balance DMEM/F12 basal medium into the bottle bottom with the tissue blocks planted upwards, and heating at 37deg.C with humidity of 98% and CO ₂ CO at a concentration of 5% ₂ Standing for 12h in an incubator, slowly turning over the culture flask to enable the tissue blocks to slowly submerge into the complete culture solution, and culturing, wherein the half amount of the culture solution is replaced for 1 time every 3-5 days later.

4. The fusion degree of cells climbing out around the tissue block reaches 85% -90%, and the group is removedWeaving blocks, adding 1.5mL TrypLE ^TM Select solution, digesting for 1-2 min at 37 ℃, and then adding an equal volume of PBS solution to terminate the digestion reaction; gently blowing off the cells, collecting the cells into a centrifuge tube, centrifuging at 900rpm for 4min by using a low-speed centrifuge, discarding the supernatant, re-suspending the cells by using 8mL of complete culture solution, counting, inoculating the cells into a culture flask according to the cell density at a ratio of 1:1 or 1:2, changing the solution 1 time every 2-3 days, and carrying out passaging again until the fusion degree of the cells is not lower than 90%.

5. The cultured cells were observed under a microscope.

Example 2

The present example was isolated from human umbilical vein and cultured to obtain human umbilical vein smooth muscle cells, and the difference from example 1 was only that in this example, the DMEM basal medium was used instead of DMEM/F12 basal medium in step 3, and the rest of the operations and steps were the same as in example 1.

Example 3

The present example was isolated from human umbilical vein and cultured to obtain human umbilical vein smooth muscle cells, and the difference from example 1 was that in this example, in step 3, ham's F-12 basal medium was used instead of DMEM/F12 basal medium, and the rest of the operations and steps were the same as in example 1.

Example 4

The present example was conducted to obtain human umbilical vein smooth muscle cells from human umbilical veins by isolation, and the difference from example 1 was that in the present example, the total amount of the culture medium added in step 3 was 5mL, and the other operations and steps were the same as in example 1.

Example 5

The present example was isolated from human umbilical vein and cultured to obtain human umbilical vein smooth muscle cells, and was different from example 1 only in that the time of standing in step 3 was 6 hours, and the rest of the operations and steps were the same as in example 1.

Example 6

The present example was isolated from human umbilical vein and cultured to obtain human umbilical vein smooth muscle cells, and was different from example 1 only in that the time of standing in step 3 was 20 hours, and the rest of the operations and steps were the same as in example 1.

Example 7

The present example was isolated from human umbilical vein and cultured to obtain human umbilical vein smooth muscle cells, and the difference from example 1 was that in this example, digestion was performed using pancreatin having an equivalent volume mass fraction of 0.25% in step 4, and the rest of the operations and steps were the same as in example 1.

Example 8

1. flushing the umbilical cord with D-Hanks solution containing 0.5% heparin sodium at 37deg.C to flush blood coagulation blocks inside and outside umbilical vein, cutting off vascular cavity longitudinally, and cutting into 1.5mm volume ³ Is a block of tissue; the tissue mass was immersed in 1.5mg/mL type I collagenase solution and transferred to CO at 37 ℃ ₂ Incubating for 30min in a incubator; tissue pieces were washed 3 times with PBS solution.

2. The washed tissue blocks are pressed to 4 blocks/cm ² Is planted in T25 flasks.

3 to 5. The same as in example 1.

Comparative example 1

This comparative example was isolated from human umbilical vein and cultured to obtain human umbilical vein smooth muscle cells, which was different from example 1 only in that in this comparative example, blood vessels were cut to a volume of 5mm in step 2 ³ The rest of the operations and steps are the same as in example 1.

Comparative example 2

This comparative example was isolated from human umbilical vein and cultured to obtain human umbilical vein smooth muscle cells, and was different from example 1 only in that in this comparative example, blood vessels were cut to a volume of 0.5mm in step 2 ³ The rest of the operations and steps are the same as in example 1.

Comparative example 3

This comparative example was isolated from human umbilical vein and cultured to obtain human umbilical vein smooth muscle cells, which was different from example 1 only in that in this comparative example, the inoculation density of the tissue mass was 1 mass/cm in step 2 ² The rest of the operations and steps are the same as in example 1.

Comparative example 4

This comparative example was isolated from human umbilical vein and cultured to obtain human umbilical vein smooth muscle cells, which was different from example 1 only in that in this comparative example, the inoculation density of the tissue mass was 7 pieces/cm in step 2 ² The rest of the operations and steps are the same as in example 1.

Comparative example 5

This comparative example provides a commercially available human umbilical vein smooth muscle cell, available from the marsupium life technologies, inc., under the designation CP-H084.

Comparative example 6

This comparative example provides a commercially available human umbilical vein smooth muscle cell, available from Shanghai under the designation DFSC-EC-01, qiao Xin boat biotechnology limited.

Performance detection

The human umbilical vein smooth muscle cells prepared in examples 1 to 8 and comparative examples 1 to 6 were identified and tested, specifically including morphological observation, purity identification, viability assay, growth number assay, and serial passage number assay.

Cell morphology observations

Cells were seeded into petri dishes and the morphology of the cells was observed under a microscope at regular intervals.

After 3 days of primary isolation and culture of the cells of example 1, it was seen that the cells, which were spindle, triangle or sector shaped, were climbing out of the tissue mass, nuclear oval, centered; after 2 weeks, the cells are converged, most of the cells are stretched to be long fusiform, the cytoplasm is rich and has branched protrusions, and the cells are arranged in parallel to form a single layer or partial area for multi-layer overlapping growth, and the height is fluctuant; when the cell density is low, the cells are often interwoven into a net shape; at high densities, the arrangement is in a swirl or a grating (see FIG. 1). Cells grew faster after passage, confluent for 2-3 days, and maintained the morphological and growth characteristics described above (see FIG. 2).

Compared with example 1, the DMEM basal medium used in example 2 is used for replacing the DMEM/F12 basal medium, the Ham's F-12 basal medium is used for replacing the DMEM/F12 basal medium in example 3, and the morphology of the isolated umbilical vein smooth muscle cells is not obviously changed as in example 1, but the proliferation speed is slightly slowed, which indicates that the use of the DMEM/F12 basal medium is helpful for improving the physiological state of the cells and promoting the proliferation and division of the cells.

The total culture solution added in the example 4 has more volume, and the impact of the culture solution on the tissue block is more in the standing and culturing process, so that the tissue block is easy to fall off and is not easy to adhere to the wall, the number of the crawled cells is less, but the cell morphology is not obviously changed compared with the example 1; in the embodiment 5, the standing time is shorter, the tissue blocks are not tightly adhered, the follow-up climbing of cells is influenced, the number of the climbing cells is smaller, and the cell morphology is not obviously different from that of the embodiment 1; in example 6, the time for standing was long, the surface of the tissue mass was dried, and the tissue mass was completely exfoliated, and umbilical vein smooth muscle cells were not obtained.

In addition, subculturing with the TrypLE solution is also advantageous in maintaining the viability of the cells. In example 1, cells were digested with TrypLE solution, most cells were reattached soon after digestion treatment, the morphology of the cells was unchanged, and the physiological state was also good (see FIG. 3). In comparison with example 1, in example 7, the cells were digested with pancreatin, most of the cells were not adhered again after the digestion treatment, the cell morphology began to become round, apoptosis occurred, and the cell state was poor, and the viability was also weak (see fig. 4). The above results demonstrate that digestion with a TrypLE solution is important for maintaining the viability of human umbilical vein smooth muscle cells.

In example 8, cells were used in various forms by cutting into tissue pieces and then digesting with a type I collagenase solution (see FIG. 5). It is speculated that the possible cause is incomplete digestion of endothelial cells, resulting in the obtained cells being a mixture of endothelial cells and umbilical vein smooth muscle cells. In addition, the umbilical vein blood vessel is cut into small blocks and then inoculated, and the inner wall and the outer wall of the umbilical vein blood vessel are difficult to distinguish in the inoculation process. All of the above factors result in lower purity of the obtained umbilical vein smooth muscle cells.

Compared with example 1, the tissue block in comparative example 1 has larger volume, the number of cells climbing out is smaller, and the cell fusion degree corresponding to passage is difficult to achieve and the subsequent subculture is carried out (see FIG. 6); in the comparative example 2, the volume of the tissue block is smaller, the tissue block is seen to fall off, the climbing cells are in a layered shape, subculture is not easy to carry out, the activity of the cells is also affected to a certain extent, and the morphology of the cells is also changed to a certain extent (see fig. 7); the inoculation of the tissue mass in comparative example 3 has a smaller density, the number of the crawled cells is smaller, and the information and substances cannot be exchanged between the cells, so that the influence on the activity of the cells is larger (see fig. 8); the inoculation density of the tissue mass in comparative example 4 was high, the cells were aggregated together to form macroscopic "cell clusters", the distribution of cells in the flask was also not uniform, the morphology of the cells was also changed to some extent, and the subsequent subculture operation was also affected to some extent (see fig. 9). The results show that the size of the tissue mass and the density of the inoculation have a certain effect on the number, morphology and viability of the obtained human umbilical vein smooth muscle cells.

Comparative example 5 and comparative example 6 are commercial products, the physiological state of the cells after subculture is good, the cell morphology is uniform, and the proliferation speed is high.

Cell purity identification

Cells in the logarithmic growth phase were taken, inoculated into a petri dish in which a treated coverslip was previously placed, and after the cells grew into a monolayer, the coverslip was taken out and washed 2 times with PBS.

Fixed with 4% tissue cell fixative for 20min and washed 3 more times with PBS. Triton X-100 was allowed to pass through at room temperature for 5-10 min, and washed with PBS 3 times for 5min each.

The cells were blocked with 1% BSA at room temperature for 30-60 min.

The antibodies were incubated overnight at 1:200 and 1:100 dilution ratio, respectively, at 4℃with Anti- α -SMA Anti-ibody and Anti-Colponin1 Anti-ibody, and rinsed 3 times with PBS for 5min each. The secondary antibodies were each 1. Mu.g/mL of Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, alexa Fluor ^TM 546 and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, alexa Fluor ^TM Plus488, incubated at room temperature for 1h in the dark, rinsed 3 times with pbs for 5min each. Finally, rinsing again with distilled water for 1 time, sealing with anti-fluorescence decay sealing tablet containing DAPI, observing and photographing under a fluorescence inversion microscope, and calculating the positive rate by using Image Pro Plus.

The purity statistics of umbilical vein smooth muscle cells in examples 1 to 8 and comparative examples 1 to 6 are shown in Table 3.

TABLE 3 purity statistics

Group of	Purity (%)
		Example 1	98
Example 2	97
		Example 3	98
Example 4	97
		Example 5	96
Example 6	0
		Example 7	95
Example 8	75
		Comparative example 1	96
Comparative example 2	96
		Comparative example 3	96
Comparative example 4	95
		Comparative example 5	93
Comparative example 6	92

As can be seen from Table 3, the purity of the human umbilical vein smooth muscle cells prepared in examples 1 to 5, example 7 and comparative examples 1 to 4 is high and is above 95%, which means that the purity of the obtained human umbilical vein smooth muscle cells can be ensured to a great extent by adopting a mode of firstly using the I-type collagenase solution for digestion and then shearing the obtained human umbilical vein smooth muscle cells into tissue blocks for inoculation, and conditions are created for ensuring the accuracy of related research results.

Among them, the results of immunofluorescent staining of umbilical vein smooth muscle cells prepared in example 1 using α -SMA and Colponin1 and positive rate statistics are shown in fig. 10, 11 and 12.

Fig. 10 is a graph (magnification = 20 times) of the results of α -SMA staining of human umbilical vein smooth muscle cells prepared in example 1 of the present application. Fig. 11 is a picture (magnification=20×) of the result of Colponin1 staining of human umbilical vein smooth muscle cells prepared in example 1 of the present application. FIG. 12 is a graph of the positive rate statistics of alpha-SMA and Colponin1 staining of human umbilical vein smooth muscle cells prepared in example 1 of the present application.

As can be seen from FIGS. 10 to 12, the umbilical vein smooth muscle cells prepared in example 1 have an alpha-SMA positive rate of > 99% and a Colponin1 positive rate of > 98%, which means that the cell purity can reach at least 98%.

In contrast to examples 1 to 5, example 7 and comparative examples 1 to 4, in example 6, the tissue mass was dried and shrunken due to the long standing time, and the corresponding cells were not obtained, so that purity could not be identified. In example 8, the method of cutting into tissue blocks and then digesting with the type I collagenase solution is adopted, and the obtained cells are a mixture of various cells due to incomplete digestion of endothelial cells and incapability of distinguishing the inner wall and the outer wall of umbilical vein blood vessels, so that the purity of the cells is low, namely 75%.

The purity of the cell obtained by the technical scheme of the application is higher than that of the commercial product, namely the cell purity is 93% and 92% after the purity identification is carried out by adopting the method, which is shown in the comparative example 5 and the comparative example 6.

Cell viability assay

Taking cells in logarithmic growth phase, staining for 5min with PI, dripping 20 mu L of cells onto a cell counting plate, and counting the living cell rate on a fluorescent cell analyzer. The average value was calculated in 3 replicates.

The statistical results of the viable cell rates of umbilical vein smooth muscle cells in examples 1 to 8 and comparative examples 1 to 6 are shown in Table 4.

TABLE 4 statistical results of viable cell rates

Group of	Viable cell Rate (%)
		Example 1	99.5
Example 2	89.2
		Example 3	85.3
Example 4	99.5
		Example 5	99.0
Example 6	0
		Example 7	80.3
Example 8	99.5
		Comparative example 1	82.5
Comparative example 2	83.4
		Comparative example 3	78.2
Comparative example 4	80.6
		Comparative example 5	98.5
Comparative example 6	98.7

As can be seen from Table 4, the viability of the human umbilical vein smooth muscle cells obtained in example 1, examples 4 to 5 and example 8 was extremely high, both above 99.0% and even as high as 99.5%. The total culture solution in example 4 is more in addition amount, the tissue blocks are easy to fall off, the standing time in example 5 is shorter, the tissue blocks are not firmly adhered, and although the number of human umbilical vein smooth muscle cells prepared by the two methods is smaller, the physiological state of the cells is good and the activity is extremely high. The cells prepared in example 8, although of lower purity, still had good viability.

In comparison with examples 1, 4 to 5 and 8, the replacement of the basal medium in examples 2 and 3 has a certain influence on the viability of the cells, indicating that the selection of an appropriate medium is important for maintaining the physiological state of the obtained human umbilical vein smooth muscle cells. Example 6 did not obtain umbilical vein smooth muscle cells, and therefore, cell viability was not measured. In example 7, the cells were significantly damaged by digestion with pancreatin, and the cell viability was significantly reduced.

The tissue mass of comparative example 1 is large in volume and is unfavorable for the climbing out and growth of cells; the tissue mass in comparative example 2 has smaller volume, cells grow in layers, and the physiological state is poor; the inoculation density of the tissue block in comparative example 3 is small, which is not beneficial to the material and information exchange between cells; the inoculation density of the tissue mass in comparative example 4 was large and the cell growth space was small, thereby affecting the normal growth of cells. The factors have certain influence on the cell viability, which means that the physiological state of the cells can be improved and the cell viability can be improved only by controlling the size of the tissue mass within a proper range and matching with proper inoculation density.

Comparative example 5 and comparative example 6 are commercial products with slightly lower cell viability than example 1, indicating that the physiological status of umbilical vein smooth muscle cells obtained using the isolated culture method of the present application is superior to other products on the market.

In addition, the human umbilical vein smooth muscle cells of example 1, comparative example 5 and comparative example 6 were subjected to cell viability comparison, and the results are shown in fig. 13.

Fig. 13 is a picture of the results of cell viability assays of human umbilical vein smooth muscle cells of example 1, comparative example 5, and comparative example 6 of the present application.

As can be seen from FIG. 13, under the same treatment conditions, the human umbilical vein smooth muscle cells prepared in example 1 have higher cell viability, the average activation rate can reach more than 99.5%, and compared with the other two groups of cells, P is less than 0.05, and the difference is statistically significant.

Cell growth quantitative determination

Taking cells in logarithmic growth phase, regulatingWhole cell density of 1X 10 ⁵ Per mL, cells were seeded into 96-well plates, 100 μl per well, and 8 multiplex wells were placed per group. After cell attachment, at 72h, CCK8 experiments were performed using CCK8 kit and OD was measured using an enzyme-labeled instrument _450nm Values. And calculating the cell growth quantity at different time points according to the cell growth curve.

The measurement results of the cell growth numbers of umbilical vein smooth muscle cells in examples 1 to 8 and comparative examples 1 to 6 are shown in Table 5.

TABLE 5 results of cell growth number measurement

As can be seen from Table 5, the number of human umbilical vein smooth muscle cells isolated in example 1 was the largest and reached 2.9X10 when cultured for 72 hours ⁵ A/hole; in example 4, the total culture solution was added in a larger amount, the tissue mass was easily dropped off, the time for standing was short in example 5, the tissue mass was not firmly adhered, although the number of cells originally obtained was small, the raw material was not sufficiently utilized, and the waste of the raw material was caused, but the proliferation efficiency of the cells was not affected, and when the initial number of inoculated cells was controlled to be the same, the total number of cells obtained by culturing for the same time was not significantly different; in example 8, the cells were digested with collagenase type I solution after they were cut into tissue pieces, and the proliferation rate of the cells was not significantly affected.

In comparison with examples 1, 4 to 5 and 8, examples 2 and 3 use DMEM/F12 or Ham's F basal medium, which significantly affects the growth rate of cells and significantly decreases the number of cells; in example 6, the tissue mass was dried up and shrank due to the long standing time, the cells could not grow, and the corresponding cells were not obtained, so that the identification of the number of cell growth could not be performed; in example 7, cells were digested with pancreatin, which resulted in greater damage to the cells, a significant impact on the growth rate of the cells, and a significant decrease in the growth rate.

Comparing the results of comparative examples 1 to 4, it can be seen that when the volume of the tissue mass is larger (comparative example 1) or smaller (comparative example 2), the inoculation density is smaller (comparative example 3) or larger (comparative example 4), both the mass and information exchange between cells or between cells and the culture environment is affected, thereby affecting the physiological state of the cells, and the proliferation rate of the obtained human umbilical vein smooth muscle cells is slower. Indicating that the size of the tissue mass and the planting density are important influencing factors of the cell maintenance activity and proliferation rate.

Comparative example 5 and comparative example 6 are commercial products, and according to the test results, the proliferation rates are slightly lower than those of example 1, indicating that umbilical vein smooth muscle cells obtained by the method in the present application have faster proliferation rates.

In addition, the growth numbers of human umbilical vein smooth muscle cells of example 1, comparative example 5 and comparative example 6 at 0, 12, 24, 36, 48, 60 and 72 hours were also compared, and the results are shown in fig. 14.

FIG. 14 is a photograph showing the results of cell growth count measurement of human umbilical vein smooth muscle cells of example 1, comparative example 5 and comparative example 6 of the present application.

As can be seen from fig. 14, under the same treatment conditions, the human umbilical vein smooth muscle cells prepared in example 1 grew to the same cell number, requiring a shorter time period; the number of cells is more in the same culture time, about 1.5 times of the other two cells, and compared with the growth speed of the other two groups of cells, P is less than 0.05, and the difference has statistical significance.

Cell serial passage number determination

The cells were serially subcultured in sequence and subjected to STR detection analysis, and the maximum passable times of the cells were observed.

All experimental data were statistically analyzed by the statistical software SPSS24.0, the comparison between groups was tested by F, α=0.05, p < 0.05 being statistically significant.

The results of measuring the number of cell serial passages of umbilical vein smooth muscle cells in examples 1 to 8 and comparative examples 1 to 6 are shown in Table 6.

TABLE 6 results of determination of the number of continuous passages of cells

As can be seen from Table 6, the human umbilical vein smooth muscle cells prepared in example 1 have better physiological state and more passages, which can reach 23 times; the total culture solution in example 4 was added in a larger amount, and the standing time in example 5 was shorter, and although the number of cells obtained initially was smaller due to the weak adhesion of the tissue mass, the physiological state of the cells was not significantly affected, and the passage number of the cells was not significantly affected.

Comparing examples 1 and 4-5, it can be seen that the use of DMEM/F12 basal medium (example 2) or Ham's F basal medium (example 3) also affects the metabolic level of cells and thus the cell growth algebra; in example 6, the cells were left to stand for 20 hours, the tissue mass was dried up and atrophic, and the cells were not grown nor subcultured; in example 7, digestion with pancreatin has a significant effect on the metabolic level of cells, and the number of growth algebra is significantly reduced; in example 8, the method of cutting into tissue blocks and then digesting with the type I collagenase solution was adopted, and although the results of the previous cell viability and proliferation capacity measurement showed no significant change compared with example 1, the number of passaging was significantly reduced, namely, only 15 times, presumably because the obtained cells had lower purity, and other types of cells were mixed, and the interaction between cells resulted in a reduction in the number of passaging.

The volume of the tissue block in the comparative example 1 is larger, the volume of the tissue block in the comparative example 2 is smaller, the inoculation density of the tissue block in the comparative example 3 is smaller, the inoculation density of the tissue block in the comparative example 4 is larger, the physiological states of cells are poorer, and the passage times are very limited, which means that the influence of the size of the tissue block and the inoculation density on the physiological states of the cells is very obvious, and only in a proper range, the cell viability can be improved and the passage times are increased.

Comparative example 5 and comparative example 6 are commercial products, and the number of passages is slightly lower than that of example 1, only 15 and 18, indicating that human umbilical vein smooth muscle cells isolated and cultured by the method of the present application were passaged more than those purchased from other companies.

The number of serial passages of human umbilical vein smooth muscle cells of example 1, comparative example 5 and comparative example 6 was compared, and the results are shown in fig. 15.

FIG. 15 is a graph showing the results of continuous passage count measurement of human umbilical vein smooth muscle cells of example 1, comparative example 5 and comparative example 6 of the present application.

As can be seen from FIG. 15, the human umbilical vein smooth muscle cells cultured in example 1 can be passaged to 23 passages, the genotypes are kept unchanged, while the cells of comparative examples 5 and 6 are passaged to 15 passages and 18 passages respectively, the cells begin to age, proliferate slowly, etc., and it is difficult to continue the passaging.

Fig. 16 is a morphological picture of human umbilical vein smooth muscle cells subcultured 1 time in comparative example 5 (magnification=40). Fig. 17 is a morphological picture of human umbilical vein smooth muscle cells subcultured 10 times in comparative example 5 (magnification=40).

Fig. 18 is a morphological picture of human umbilical vein smooth muscle cells subcultured 1 time in example 1 (magnification = 40 times). Fig. 19 is a morphological picture of human umbilical vein smooth muscle cells subcultured 20 times in example 1 (magnification = 40 times).

Comparing fig. 16 and 17, it can be seen that the morphology of the human umbilical vein smooth muscle cells in comparative example 5 was significantly changed after 10 times of subculture, indicating that the cells were aged and apoptotic, and the viability was weakened; in contrast, the morphology of the human umbilical vein smooth muscle cells prepared in example 1 after passage for 20 times has not been significantly changed (compare fig. 18 and 19), which proves that the physiological state of the cells is good and the division and proliferation can be continued. In addition, after STR detection, the human umbilical vein smooth muscle cells in comparative example 5 were passaged for 10 passages, most cells had developed gene mutation, while the cells in example 1 were still less mutated after 20 passages, so the isolated and cultured cells of example 1 were more suitable for large-scale subculture.

Example 9

The human umbilical vein smooth muscle cells obtained by the isolated culture in example 1 are preserved in this example and named HUVSMC, which is preserved in China center for type culture Collection with the preservation address of China, university of Wuhan, and post code of 430072 with the preservation number of CCTCC NO: c202270, 4 months and 20 days of 2022.

The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.

Claims

1. The human umbilical vein smooth muscle cells are characterized in that the human umbilical vein smooth muscle cells are preserved in China Center for Type Culture Collection (CCTCC) NO: c202270, 4 months and 20 days of 2022.