CN115322949A

CN115322949A - Isolated culture method of human umbilical vein smooth muscle cells and application thereof

Info

Publication number: CN115322949A
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: 2022-11-11
Anticipated expiration: 2042-07-26
Also published as: CN115322949B

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 and washing umbilical cord, filling blood vessel with collagenase I solution, and incubating; cutting the vessel cavity into 1 to 2mm ³ After the tissue blocks are processed, the ratio of 3 to 5 blocks/cm is calculated ² The density of (b) is planted in a culture flask; upwards arranging the bottle bottom with the tissue block, adding complete culture solution, standing, turning over the culture bottle to immerse the tissue block in the complete culture solution, culturing, and periodically replacing the complete culture solution; the tissue mass was removed and serial subcultures were performed. The method optimizes the separation culture method of the human umbilical vein smooth muscle cells, improves the activity of the cells, has higher proliferation speed, high purity and good survival rate, can be continuously passed 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 diseases have become the first diseases that threaten human health. Vascular disease is often the starting disease and there is a lack of effective treatment. Abnormal growth of vascular smooth muscle cells is closely related to the development and progression of vascular disease and disease prognosis. Vascular smooth muscle cells are an important component of the vascular wall, and the occurrence of many cardiovascular diseases is closely related to the proliferation, migration and differentiation of the cells, such as vascular inflammation, plaque formation, atherosclerosis, restenosis, pulmonary hypertension and the like. Studies have shown that most of the pathogenic cells in atherosclerotic plaques (the leading cause of heart attack and stroke) are derived from vascular smooth muscle cells.

Therefore, it is a hot spot of current research to explore the pathogenesis related to cardiovascular diseases by taking human vascular smooth muscle cells as experimental research objects; the vascular smooth muscle cells cultured in vitro have important significance for researching the physiological function, the drug action and the pathophysiological change under the action of various pathogenic factors.

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

Human umbilical vein smooth muscle cells are isolated from human umbilical tissue, are one of important structural component cells of the umbilical vein, and play an important role in the normal physiological process of the organism. The umbilical cord isThe mammal has a tubular structure connecting fetus and placenta, and is in the shape of rope, smooth and transparent surface, and contains connective tissue, a umbilical vein and a pair of umbilical arteries. In the uterus, the uterine arteries are in close proximity to the capillaries in the maternal portion of the placenta, to the fetal capillaries in the daughter portion of the placenta, where CO is carried out between the maternal and fetal blood ₂ 、O ₂ Exchange of metabolic waste and nutrients. The umbilical artery carries waste products from the fetus to the placenta, and the umbilical vein carries O ₂ And nutrients are delivered from the placenta to the fetus. Umbilical cord is a waste product during childbirth, which makes umbilical vein smooth muscle cells cultured in vitro into model cells for studying blood vessels.

However, the existing umbilical vein smooth muscle cell isolation method is adopted, and the cells obtained by culture are often low in passage number, long in amplification time and poor in viability, so that how to provide a human umbilical vein smooth muscle cell isolation and culture method which is high in passage number, short in amplification time, good in cell viability and high in purity becomes a problem to be solved urgently.

Disclosure of Invention

In order to improve the activity and the passage times of the human umbilical vein smooth muscle cells obtained by separation culture, the application provides a separation culture method of the human umbilical vein smooth muscle cells and application thereof.

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 separating and culturing human umbilical vein smooth muscle cells comprises the following steps:

aseptically separating and washing umbilical cords, filling blood vessels with a collagenase I solution, and incubating;

cutting the vessel cavity into 1-2 mm ³ After the tissue blocks are processed, the ratio of 3 to 5 blocks/cm is calculated ² The density of (a) is planted in a culture flask;

upwards arranging the bottle bottom with the tissue block, adding complete culture solution, standing, turning over the culture bottle to immerse the tissue block in the complete culture solution, culturing, and periodically replacing the complete culture solution;

the tissue mass was removed and serial subcultures were performed.

In the application, the isolated culture method of the human umbilical vein smooth muscle cells is optimized, so that the activity of the cells is obviously improved, the proliferation speed is higher, the time required by increasing the number of the cells by 1 time is shortened, and the number of the cells can reach more than 1.5 times of that of the cells in the prior art within the same culture time; the obtained cells can be continuously passaged for more than 20 times and the stability of the genotype is ensured, and compared with the existing separation method, the continuous cell separation method has the advantages that the passage times are obviously improved, and the growth state of the cells is better; the purity of the cells after separation culture 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 cells can be used as model cells for related research 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 And the like.

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

In some embodiments, the density of the planting is 3 to 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 mass is controlled to be 1 to 2mm ³ Within the range of (2), the adherent and climbing efficiency of the cells can be improved, the number of the obtained umbilical vein smooth muscle cells is increased, and the utilization rate of raw materials is improved. If the volume of the tissue block is larger, the cell climbing is not facilitated, the number of finally obtained cells is less, and the waste of raw materials is caused; if the volume of the tissue block is small, the tissue block is greatly influenced by the external environment and is not easy to adhere to the wall, cells are easy to grow in a laminated manner and are not easy to obtainThe cell is a monolayer, and the physiological state of the cell is poor and the proliferation capacity is weak.

In the application, the planting density is controlled to be 3-5 pieces/cm ² The range of (2) is favorable for obtaining monolayer cells, and can improve the vitality of the cells and increase the number of the obtained cells. If the planting density is high, the growing space of the cells climbed out of the tissue block is small, the phenomenon of multilayer cell stacking is easily generated, the continuous climbing out of the cells is influenced, and the number of the finally obtained cells is small; if the planting density is small, the material and information communication among the climbing-out cells is not facilitated, and the vitality of the cells is also influenced to a certain extent. In addition, if the tissue block is planted at too low a density, the cells which are not easy to climb out form a uniform single cell layer, and the cells are easy to gather around the tissue block to grow in a stacked manner, so that the phenomena of falling off and aging are easy to occur; the time required for the cells to reach the fusion degree corresponding to subculture is longer, so that the maintenance of the cell activity is not facilitated, and the cell proliferation efficiency is reduced.

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

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

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

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

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

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

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

In some specific embodiments, the collagenase type I solution has a collagenase type I concentration of 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, 2 to 2.5mg/mL, or the like.

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

Preferably, the method of incubation comprises:

the filled vessels were submerged in D-Hanks solution and incubated for 28-35 min.

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

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

Preferably, the incubation is carried out with CO transfer at 36.5-37.5 ℃ ₂ Incubations were performed in an incubator.

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

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

In the application, the umbilical vein blood vessels are filled and incubated by the method, and the vascular endothelial cells can be fully digested, so that the obtained umbilical vein smooth muscle cells have extremely high purity, and no other impurity cells are mixed. In addition, endothelial cells obtained by digestion can also 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 comprises the steps of firstly cutting off the blood vessel by using a cutter, and then scraping the endothelial cells by using a cotton swab or a blade, wherein the endothelial cells can not be completely removed, and other cells are mixed in the obtained umbilical vein smooth muscle cells, so that the purity is low; it may also result in a waste of material due to over-purging, resulting in a reduction in the total amount of cells obtained. In addition, there is a risk of injury to the operator when the knife is used to scrape off the endothelial cells, which has a certain safety risk.

In this application, the order of using type I collagenase to incubate earlier and digest endothelial cell and cut into the tissue piece is favorable to improving the purity of the umbilical vein smooth muscle cell that finally obtains, has also reduced the degree of difficulty of operation simultaneously. If the operation sequence of cutting into tissue blocks and then digesting is adopted, the degree of digestion is difficult to determine during digestion, and if the degree of digestion is lower, the purity of the finally obtained umbilical vein smooth muscle cells is lower due to incomplete digestion of endothelial cells; if the degree of digestion is too high, it will result in loss of smooth muscle cells, resulting in a smaller number of cells being finally obtained. In addition, the inner wall and the outer wall of the blood vessel are not easy to distinguish after being cut into tissue blocks, and difficulty is brought to subsequent planting operation.

Preferably, after the incubation, the method further comprises the step of washing the vein by using the PBS solution, and the washing times can be 1-5 times.

Preferably, the complete culture solution comprises fetal bovine serum, streptomycin qing mixed solution and DMEM/F12 basal medium.

Preferably, the volume percentage of the fetal calf 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% to 19%, 18% to 20%, 18% to 21%, 19% to 20%, 19% to 21%, 19% to 22%, 20% to 21%, 20% to 22%, 21% to 22%, or the like.

In a specific embodiment, the fetal bovine serum is 18%, 19%, 20%, 21%, 22%, etc. by volume in the complete culture medium.

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

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

In a specific embodiment, the volume percentage of the penicillin streptomycin mixed solution in the complete culture solution is 0.8%, 0.9%, 1%, 1.1%, 1.2% or the like.

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

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

Preferably, the amount of the complete culture solution added to the culture flask is 1.5 to 2mL.

In some embodiments, the total medium is added to the flask in an amount of 1.5 to 1.7mL, or 1.7 to 2mL, etc.

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

In this application, the addition through control complete culture solution is 1.5~ 2mL, has reduced the impact of complete culture solution to the tissue piece among the culture process to it is more firm to make the tissue piece adhere to the wall, and the quantity that the cell climbed out is more, and speed is faster.

Preferably, the standing time is 8-16 h.

In some specific embodiments, the time of standing 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 standing time is 8h, 10h, 12h, 14h or 16h, and the like.

In the application, the standing time is controlled to be 8-16 h, the tissue blocks adhere to the wall more firmly and are not easy to fall off, and the cell climbing amount and the cell climbing speed are closely related to each other in the subsequent process.

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

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

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

Preferably, the standing is carried out under the conditions that the humidity is 95% -100% and the CO content is higher than the preset value ₂ CO with concentration of 4-6% ₂ And (5) standing in an incubator.

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

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

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

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

Preferably, the method of resting comprises:

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

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

half of the liquid is changed for 1 time every 3 to 5 days.

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

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

In the application, a half-amount liquid changing mode is adopted, so that the stress reaction of the cells caused by the abrupt change of the culture environment can be reduced, the physiological state of the cells is maintained in a relatively stable environment, the cell climbing speed is higher, and the state is better.

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

In some specific embodiments, the tissue mass is removed under conditions that result in a degree of cell confluence of 85% to 87%, 87% to 90%, or the like.

In a specific embodiment, the condition for removing the tissue mass is that the degree of cell fusion reaches 85%, 87%, 90%, or the like.

Preferably, the fusion degree of the cells is not less than 90% during the continuous subculture, and the subculture is carried out by means of TrypLE solution digestion.

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 particular embodiment, the degree of fusion of the cells is 90%, 92%, 94%, 96%, 98%, or the like.

In this application, use the trypLE solution to carry out subculture, compare with pancreatin solution, the trypLE solution is gentler to the digestion of cell, is difficult for causing cell apoptosis to make the cell keep higher activity, and the adherent proportion is higher again after the cell digestion, is favorable to subsequent division and cultivation, also can reduce the cell loss because of the passage operation arouses.

Preferably, the method for TrypLE solution digestion comprises:

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

gently blowing and beating the cells, collecting the cells into a centrifugal tube, centrifuging the cells for 3 to 5min at 850 to 1000rpm, and removing supernatant;

resuspend cells in 5-10 mL complete medium, count, according to cell density, according to 1.

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 method for isolating and culturing human umbilical vein smooth muscle cell according to the first aspect.

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

In some specific embodiments, the number of passages of the human umbilical vein smooth muscle cells is 20 to 23, 20 to 25, 20 to 28, 20 to 30, 23 to 25, 23 to 28, 23 to 30, 25 to 28, 25 to 30, or 28 to 30, etc.

In a specific embodiment, the number of passages of the human umbilical vein smooth muscle cells is 20, 23, 25, 28, or 30, etc.

Preferably, the human umbilical vein smooth muscle cells are preserved in China center for type culture Collection with the preservation number of CCTCC NO: c202270, preservation date of 2022, 4 months and 20 days.

In the present application, the human umbilical vein smooth muscle cell is named human umbilical vein smooth muscle cell line HUVSMC.

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

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

1. the method optimizes the isolated culture method of the human umbilical vein smooth muscle cells, and the vascular endothelial cells are fully digested by incubating with the collagenase I solution, so that the prepared human umbilical vein smooth muscle cells have extremely high purity, the umbilical vessels can be fully utilized, and the utilization rate of raw materials is improved; by controlling the size of the tissue block and the density of 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, and the proliferation state of cells is improved; by controlling the addition of the complete culture solution and the standing condition, the wall-adhering efficiency of the tissue blocks is improved, the cell climbing efficiency is higher, and the cell climbing speed is higher; improves the subculture mode and improves the activity of cells.

2. The performance of the human umbilical vein smooth muscle cells obtained by the separation and culture method is better: the proliferation speed is faster, the total number of the cells obtained by adopting the technical scheme of the application can reach more than 1.5 times of the number of the cells obtained by the traditional method in the same culture time, and 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 can be carried out for more than 20 times, the stability of the genotype can be maintained, and the method can be applied to the preparation of large-scale cells and has important research value.

Drawings

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

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

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

Fig. 4 is a morphological picture (magnification =40 times) of human umbilical vein smooth muscle cells re-attached after trypsinization in example 7 of the present application.

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

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

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

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

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

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

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

FIG. 12 is a photograph showing the statistical results of the positive rates of α -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 photograph showing the results of cell growth number 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 photograph showing the results of serial passage number 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 (magnification = 40-fold) of human umbilical vein smooth muscle cells subcultured 1 time in comparative example 5.

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

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

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

Detailed Description

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

1. washing the blood clot inside and outside umbilical vein blood vessel with D-Hanks solution containing 0.4-0.6% heparin sodium at 36.5-37.5 deg.c, clamping one end of the blood vessel with umbilical cord clamp, injecting I in 0.5-2.5 mg/mL from the other end of the vein with syringeThe collagenase solution is clamped and closed by an umbilical cord clamp until the blood vessel is full; the filled cord was immersed in D-Hanks solution and transferred to CO at 36.5-37.5 deg.C ₂ Incubating for 28-35 min in an incubator; the umbilical cord clamps are removed and the vein is rinsed 1-5 times with PBS solution.

2. Cutting the blood vessel cavity longitudinally into a volume of 1-2 mm ³ After the tissue blocks are processed, the ratio of 3 to 5 blocks/cm is calculated ² Is planted in a T25 flask.

3. The bottle bottom with the tissue block is upward, 1.5 to 2mL of complete culture solution containing 18 to 22 volume percent of fetal calf serum, 0.8 to 1.2 volume percent of streptomycin mixed solution and the balance of DMEM/F12 basal medium is added, and the mixture is heated at the temperature of 36.5 to 37.5 ℃, the humidity is 95 to 100 percent and CO ₂ CO with concentration of 4-6% ₂ Standing in an incubator for 8-16 h, slowly turning over the culture bottle to slowly immerse the tissue blocks in the complete culture solution, culturing, and changing the culture solution for 1 time every 3-5 days.

4. Removing the tissue block when the fusion degree of the cells creeping out from the periphery of the tissue block reaches 85-90 percent, adding 1-2 mL of TrypLE solution, digesting for 1-2 min at 36.5-37.5 ℃, and adding the PBS solution with the same volume to stop the digestion reaction; gently blowing and beating the cells, collecting the cells into a centrifuge tube, centrifuging the cells at 850-1000 rpm for 3-5 min, discarding the supernatant, resuspending the cells by using 5-10 mL of complete culture solution, counting the cells, inoculating the cells into a culture bottle according to the cell density according to the ratio of 1 to 1.

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

The instrument information used in the above procedure 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 cell obtained by the separation culture method, the purity of the human umbilical vein smooth muscle cell can reach more than 98%, the survival rate can reach more than 99.5%, and the cell can be continuously passed for more than 20 times and maintain the stability of the genotype, so that the cell can be used as a cell model to be applied to related researches.

The human umbilical vein smooth muscle cell is named as human umbilical vein smooth muscle cell line HUVSMC, is preserved in China center for type culture Collection with the preservation number of CCTCC NO: c202270, preservation date of 2022, 4 months and 20 days.

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

Examples

Example 1

In this example, human umbilical vein smooth muscle cells were isolated from human umbilical vein and cultured, the method steps were as follows:

1. washing coagulated blood mass inside and outside the umbilical vein blood vessel with D-Hanks solution containing 0.5% heparin sodium at 37 deg.C, clamping one end of the blood vessel with an umbilical cord clamp, injecting 1.5mg/mL type I collagenase solution from the other end of the vein with an injector until the blood vessel is full, and clamping with the umbilical cord clamp; the filled cord was immersed in D-Hanks solution and transferred to CO at 37 deg.C ₂ Incubating for 30min in a incubator; the umbilical cord clamps were removed and the vein was rinsed 3 times with PBS solution.

2. Cutting the vessel cavity longitudinally into 1.5mm ³ After the tissue blocks are obtained, the ratio of 4 blocks/cm is calculated ² Is planted in a T25 flask.

3. The bottom of the bottle with the tissue block is upward, and 1.8mL of a solution containing 20% (volume percentage) fetal calf serum and 1% (volume percentage) fetal calf serum are added) The streptomycin mixture and DMEM/F12 basic culture medium in balance are mixed at 37 deg.c and 98% humidity, and are completely cultured in CO ₂ CO concentration of 5% ₂ Standing in an incubator for 12h, slowly turning over the culture bottle to slowly immerse the tissue blocks in the complete culture solution, culturing, and changing the culture solution for 1 time every 3-5 days.

4. When the fusion degree of the cells climbing out of the periphery of the tissue block reaches 85-90 percent, removing the tissue block, and adding 1.5mL of trypLE ^TM Digesting the Select solution for 1-2 min at 37 ℃, and adding an equal volume of PBS solution to terminate the digestion reaction; gently blow the cells down, collect the cells into a centrifuge tube, centrifuge the cells for 4min at 900rpm by using a low-speed centrifuge, discard the supernatant, resuspend the cells by using 8mL of complete culture solution, count the cells, insert the cells into a culture flask according to cell density according to 1 or 1.

5. The cultured cells were observed under a microscope.

Example 2

In this example, human umbilical vein smooth muscle cells were isolated and cultured from human umbilical veins, which are different from example 1 only in that DMEM basal medium was used instead of DMEM/F12 basal medium in step 3, and the rest of the procedure and steps were the same as those in example 1.

Example 3

This example, which was conducted in the same manner as example 1 except that Ham's F-12 basal medium was used instead of DMEM/F12 basal medium in step 3, was used to isolate and culture human umbilical vein smooth muscle cells from human umbilical veins.

Example 4

This example was conducted in the same manner as example 1 except that the amount of the complete culture medium added in step 3 was 5mL, and the other operations and steps were the same as in example 1.

Example 5

This example was separated and cultured from human umbilical vein to obtain human umbilical vein smooth muscle cells, and only differs from example 1 in that in step 3, the standing time was 6 hours, and the rest of the operation and steps were the same as those in example 1.

Example 6

This example was conducted in the same manner as example 1 except that the human umbilical vein smooth muscle cells were isolated and cultured from the human umbilical vein and the only difference between this example and example 1 was that the time for standing was 20 hours in step 3.

Example 7

This example was carried out in the same manner as example 1 except that in step 4, pancreatin was used in an amount of 0.25% by mass as the volume, and the rest of the procedure and the steps were the same as in example 1.

Example 8

In this example, human umbilical vein smooth muscle cells were isolated from human umbilical vein by culture, the method steps were as follows:

1. washing coagulated blood mass inside and outside umbilical vein with sterile separated umbilical cord with D-Hanks solution containing 0.5% heparin sodium at 37 deg.C, cutting blood vessel cavity longitudinally, and cutting into volume of 1.5mm ³ The tissue mass of (a); the tissue mass was immersed in 1.5mg/mL collagenase type I solution and transferred to CO at 37 ℃ ₂ Incubating for 30min in a box; the tissue mass was washed 3 times with PBS solution.

2. The tissue blocks after washing are divided into 4 blocks/cm ² Is planted in a T25 flask.

3-5. Same as example 1.

Comparative example 1

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

Comparative example 2

This comparative example, in which human umbilical vein smooth muscle cells were isolated from human umbilical vein and cultured, differs from example 1 only in that 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, in which human umbilical vein smooth muscle cells were obtained by isolation culture from human umbilical vein, differs from example 1 only in that the tissue mass was seeded at a density of 1 mass/cm in step 2 ² The rest of the operation and the steps were the same as in example 1.

Comparative example 4

This comparative example, in which human umbilical vein smooth muscle cells were obtained by isolated culture from human umbilical vein, differs from example 1 only in that the tissue piece was seeded at a density of 7 pieces/cm in step 2 ² The rest of the operation and the steps were the same as in example 1.

Comparative example 5

This comparative example provides a commercially available human umbilical vein smooth muscle cell, purchased from GmbH, having a product number CP-H084.

Comparative example 6

This comparative example provides a commercially available human umbilical vein smooth muscle cell from Johan new boat Biotech, inc. in Shanghai under the trade designation DFSC-EC-01.

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 morphology observation, purity identification, viability determination, growth number determination, and serial passage number determination.

Cell morphology observation

The cells were seeded into a culture dish, and the morphology of the cells was observed periodically under a microscope.

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

FIG. 3 shows TrypLE of human umbilical vein smooth muscle cells in example 1 of the present application ^TM Morphological pictures of re-adherence after digestion of Select solution (magnification)Number =40 times).

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

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

After 3 days of primary cell isolation culture in example 1, it was seen that cells in the shape of spindle, triangle or sector were crawled out of the tissue mass, with the nucleus oval and centered; after 2 weeks, the cells are confluent, most of the cells are stretched into long spindle shapes, rich cytoplasm and branched bulges, and the cells are arranged in parallel to form a single layer or a part of areas with multiple layers and overlapped growth with fluctuating heights; when the cell density is low, the cells are often interwoven into a net shape; at high density, the fibers are arranged in a vortex or fence pattern (see FIG. 1). After passage, the cells grow faster, can be confluent within 2 to 3 days, and maintain the morphological characteristics and growth characteristics (see figure 2).

Compared with the example 1, the DMEM basal medium used in the example 2 replaces the DMEM/F12 basal medium, the Ham's F-12 basal medium is used in the example 3 replaces the DMEM/F12 basal medium, the morphology of the umbilical vein smooth muscle cells obtained by separation has no obvious change compared with the example 1, but the proliferation speed is slightly reduced, which shows that the DMEM/F12 basal medium is beneficial to improving the physiological state of the cells and promoting the proliferation and division of the cells.

The volume of the complete culture solution added in example 4 is large, and the impact of the culture solution on the tissue blocks in the standing and culturing processes is large, so that the tissue blocks are easy to fall off and not easy to adhere to the wall, the number of the climbed out cells is small, and the cell morphology has no obvious change compared with that of example 1; in example 5, the standing time is short, the tissue blocks are not adhered tightly, the subsequent cell climbing is influenced, the number of the climbed cells is small, and the cell morphology is not obviously different from that of example 1; in example 6, the time of standing was long, the surface of the tissue mass was dried, the tissue mass was completely exfoliated, and umbilical vein smooth muscle cells were not obtained.

In addition, subculturing with TrypLE solution is also beneficial to maintaining the viability of the cells. In example 1, the TrypLE solution is used for digesting the cells, most of the cells are attached again quickly after digestion treatment, the morphology of the cells is not changed, and the physiological state is better (see figure 3). Compared with example 1, in example 7, when digestion is performed by using pancreatin, most cells cannot be attached again after digestion treatment, cell morphology begins to become round, and apoptosis occurs, indicating that the state of the cells is poor and the activity is weak (see fig. 4). The above results indicate that digestion with TrypLE solution is important for maintaining the viability of human umbilical vein smooth muscle cells.

In example 8, the cells were in various forms by cutting into a tissue mass and then digesting with collagenase type I solution (see FIG. 5). Presumably, the reason for this was that the endothelial cells were not completely digested, resulting in a mixture of endothelial cells and umbilical vein smooth muscle cells. In addition, the umbilical vein is cut into small pieces and then inoculated, and the inner wall and the outer wall of the umbilical vein are difficult to distinguish in the process of inoculation. All of the above factors result in the obtaining of umbilical vein smooth muscle cells with low purity.

Compared with example 1, the tissue mass in comparative example 1 has a larger volume, the amount of the cells crawled out is smaller, and the cell fusion degree corresponding to passage is difficult to achieve and subsequent passage culture is performed (see fig. 6); in comparative example 2, the volume of the tissue block is small, it can be seen that the tissue block has fallen off, the cells crawled out are in a laminated shape, subculture is not easy to perform, the activity of the cells is also influenced to a certain extent, and the morphology of the cells is also changed to a certain extent (see fig. 7); in comparative example 3, the tissue mass was seeded at a lower density, the number of cells climbed out was lower, no information or material communication was possible between cells, and the influence on the viability of cells was greater (see fig. 8); in comparative example 4, the tissue block is seeded at a higher density, cells are aggregated together to form a macroscopic "cell mass", the cells are not uniformly distributed in the culture flask, the morphology of the cells is changed to a certain extent, and the subsequent subculture operation is influenced to a certain extent (see fig. 9). The results show that the size of the tissue block and the inoculation density have certain influence on the number, the shape and the activity of the obtained human umbilical vein smooth muscle cells.

Comparative examples 5 and 6 are commercial products, and 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 and inoculated into a petri dish in which a treated cover glass was placed in advance, and after the cells grew into a monolayer, the cover glass was taken out and washed 2 times with PBS.

The cells were fixed for 20min using 4% tissue cell fixative and washed 3 times with PBS. Triton X-100 was permeabilized at room temperature for 5-10 min, and then washed with PBS 3 times for 5min each time.

Cells were blocked using 1% BSA for 30-60 min at room temperature.

Primary antibodies were incubated overnight with Anti- α -SMA antibody and Anti-collagen 1 antibody at a dilution ratio of 1. The Secondary Antibody was 1. Mu.g/mL of Goat anti-Rabbit IgG (H + L) high hly Cross-Adsorbed Secondary Antibody, alexa Fluor ^TM 546 and Goat anti-Rabbit IgG (H + L) high hly Cross-Adsorbed Secondary Antibody, alexa Fluor ^TM Plus488, incubation at room temperature in the dark for 1h, rinsing with PBS 3 times for 5min each. Finally, the plate was rinsed 1 more time with distilled water, mounted with DAPI-containing anti-fluorescence decay mounting agent, observed under a fluorescence inverted microscope and photographed, and the positive rate was calculated with 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 1-5, 7 and comparative examples 1-4 is higher, and is more than 95%, which indicates that the purity of the obtained human umbilical vein smooth muscle cells can be ensured to a great extent by using a mode of firstly digesting with collagenase I solution and then shearing into tissue blocks for inoculation, and conditions are created for ensuring the accuracy of related research results.

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

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

As can be seen from FIGS. 10 to 12, the alpha-SMA positivity of the umbilical vein smooth muscle cells prepared in example 1 was > 99%, the Colponin1 positivity was > 98%, indicating that the cell purity was at least 98% or more.

In comparison with examples 1 to 5, example 7 and comparative examples 1 to 4, in example 6, the tissue mass was dried and shrunk due to an excessively long standing time, and the purity could not be identified because the corresponding cells were not obtained. In example 8, the method of cutting into tissue blocks and then digesting with collagenase type i solution resulted in the mixture of multiple cells obtained due to incomplete digestion of endothelial cells and the inability to distinguish between the inner wall and the outer wall of the umbilical vein blood vessels, and thus the cell purity was low, only 75%.

Comparative example 5 and comparative example 6 are commercially available products, and the cell purity after purity identification by the above method is 93% and 92%, respectively, which shows that the purity of the human umbilical vein smooth muscle cells obtained by the technical scheme of the application is higher than that of the commercially available products.

Cell viability assay

Cells in logarithmic growth phase are taken, stained with PI for 5min, 20 mu L of the cells are dropped on a cell counting plate, and the viable cell rate is counted on a fluorescent cell analyzer. The mean was calculated in 3 replicates.

The statistics of the viable cell rate of umbilical vein smooth muscle cells in examples 1 to 8 and comparative examples 1 to 6 are shown in table 4.

TABLE 4 statistics 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 human umbilical vein smooth muscle cells obtained in examples 1, 4 to 5 and 8 have very high viability, all of which is above 99.0%, and even can be as high as 99.5%. In example 4, the addition amount of the complete culture solution was large, the tissue mass was likely to fall off, and in example 5, the time for the standing was short, and the tissue mass was not firmly attached to the wall. The cells prepared in example 8, although of lower purity, still have good viability.

Compared with the examples 1, 4-5 and 8, the change of the basic culture medium in the examples 2 and 3 has certain influence on the viability of the cells, which shows that the selection of proper culture medium is important for maintaining the physiological state of the obtained human umbilical vein smooth muscle cells. Example 6 no umbilical vein smooth muscle cells were obtained, and therefore no cell viability assay could be performed. In example 7, when digestion was performed using pancreatin, the damage to the cells was large and the decrease in cell viability was significant.

The tissue mass in comparative example 1 has a large volume, which is not beneficial to the climbing out and growth of cells; in comparative example 2, the volume of the tissue mass is small, cells grow in a laminated manner, and the physiological state is poor; in comparative example 3, the tissue block has low inoculation density, which is not beneficial to the exchange of materials and information among cells; the tissue mass in comparative example 4 was seeded at a higher density and had a smaller cell growth space, thus affecting the normal growth of cells. The factors all have certain influence on the vitality of the cells, which shows that the physiological state of the cells can be improved and the vitality of the cells can be improved only by controlling the size of the tissue block within a proper range and matching with proper inoculation density.

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

In addition, the cell viability of the human umbilical vein smooth muscle cells of example 1, comparative example 5 and comparative example 6 were compared, 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 viability 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 has statistical significance.

Cell growth quantification assay

Taking cells in logarithmic growth phase, adjusting cell density to 1 × 10 ⁵ mL, cells were seeded into 96-well plates at 100 μ L per well, with 8 duplicate wells per set. After the cells adhere to the wall, at 72h, a CCK8 experiment is carried out by using a CCK8 kit, and OD is measured by using an enzyme-labeling instrument _450nm The value is obtained. And calculating the cell growth number at different time points according to the cell growth curve.

The results of measuring 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 measurement of cell growth

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 2.9X 10 after 72h culture ⁵ Per well; in example 4, the addition amount of the complete culture solution is too large, the tissue block is easy to fall off, in example 5, the standing time is short, the tissue block is not firmly attached to the wall, although the number of the cells obtained initially is small, the raw materials are not fully utilized, and the raw materials are wasted, the proliferation efficiency of the cells is not influenced, and when the initial number of the inoculated cells is controlled to be the same, the total number of the cells obtained in the same time of culture is not obviously different; in example 8, the method of cutting tissue into pieces and then digesting the pieces with collagenase type I solution had no significant effect on the proliferation rate of cells.

Compared with the examples 1, 4 to 5 and 8, the DMEM/F12 or Ham's F12 basal medium used in the examples 2 and 3 has a remarkable influence on the growth rate of the cells, and the number of the cells is obviously reduced; in example 6, the tissue blocks are dried and shrunk due to too long standing time, cells cannot grow, and the number of the grown cells cannot be identified because corresponding cells are not obtained; in example 7, the cells are digested by pancreatin, so that the damage to the cells is large, the growth rate of the cells is obviously influenced, and the growth rate is obviously reduced.

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) and the seeding density is smaller (comparative example 3) or larger (comparative example 4), the exchange of materials and information between cells or between cells and the culture environment is affected, so that the physiological state of the cells is affected, and the proliferation rate of the obtained human umbilical vein smooth muscle cells is slower. It is shown that the size of the tissue block and the planting density are important factors for the viability maintenance and the proliferation rate of the cells.

Comparative example 5 and comparative example 6, which are commercially available products, showed a proliferation rate slightly lower than that of example 1 according to the test results, indicating that the umbilical vein smooth muscle cells obtained by the method of the present application had a faster proliferation rate.

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

FIG. 14 is a photograph showing the results of the cell growth number measurement of the 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, which took a shorter time; the number of cells was greater in the same culture time, about 1.5 times that of the other two cells, and the difference was statistically significant when compared with the growth rates of the other two groups of cells, P < 0.05.

Cell serial passage number determination

And (4) continuously subculturing the cells in sequence, carrying out STR detection and analysis, and observing the maximum passable times of the cells.

All experimental data were statistically analyzed using statistical software SPSS24.0, and comparisons between groups were statistically significant using the F-test for differences of α =0.05 and p < 0.05.

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

TABLE 6 determination of the number of successive passages of cells

As can be seen from Table 6, the human umbilical vein smooth muscle cells prepared in example 1 had good physiological status and many passage times, up to 23; in example 4, the amount of the complete culture medium added was large, and in example 5, the time for the standing was short, and although the number of cells obtained initially was small due to the loose attachment of the tissue mass, the physiological state of the cells was not significantly affected, and the number of passages of the cells was not significantly affected.

Compared with examples 1 and 4-5, it can be seen that the use of DMEM/F12 basal medium (example 2) or Ham's F12 basal medium (example 3) also affects the metabolic level of cells and thus the generation of cell growth; in example 6, after standing for 20 hours, the tissue blocks are dried and shrunk, and cells can not grow and can not be subjected to subculture; in example 7, the digestion with pancreatin has obvious influence on the metabolic level of cells, and the growth algebra is obviously reduced; in example 8, the previous cell viability and proliferation potency measurements performed by cutting into tissue blocks and then digesting with collagenase type i solution showed no significant change compared to example 1, but the number of passages was significantly reduced, only 15, presumably due to the low purity of the cells obtained and the interaction between cells mixed with other types of cells, resulting in a reduction in the number of passages.

The tissue mass in the comparative example 1 has a large volume, the tissue mass in the comparative example 2 has a small volume, the tissue mass in the comparative example 3 has a small inoculation density, the tissue mass in the comparative example 4 has a large inoculation density, the physiological state of cells is poor, and the number of passages is very limited, which shows that the influence of the size of the tissue mass and the inoculation density on the physiological state of cells is very obvious, and the cell activity can be improved and the number of passages can be increased only if the size of the tissue mass and the inoculation density are controlled within a proper range.

Comparative example 5 and comparative example 6, both commercially available products, were also passaged slightly less than example 1, only 15 and 18 times, 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 results of comparing the number of serial passages of the human umbilical vein smooth muscle cells of example 1, comparative example 5 and comparative example 6 are shown in fig. 15.

FIG. 15 is a photograph showing the results of serial passage number 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 generations, and the genotype is kept unchanged, while the cells in comparative examples 5 and 6 are passaged to 15 generations and 18 generations respectively, and the aging, slow proliferation and other phenomena begin to appear, and the continuous passaging is difficult.

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

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

Comparing fig. 16 and fig. 17, it can be seen that the morphology of the human umbilical vein smooth muscle cells in comparative example 5 is significantly changed after subculture for 10 times, which indicates that the cells are aged and apoptotic, and the activity of the cells is weakened; in contrast, the morphology of the human umbilical vein smooth muscle cells prepared in example 1 after 20 passages did not change significantly (compare fig. 18 and 19), demonstrating that the cells have a good physiological status and can continue to divide and proliferate. In addition, according to STR detection, most of the cells in comparative example 5 have gene mutation after the human umbilical vein smooth muscle cells are passaged to 10 generations, and the cells in example 1 have less mutation after 20 passages, so that the cells separated and cultured in example 1 are more suitable for large-scale subculture.

Example 9

In this example, the human umbilical vein smooth muscle cells obtained by isolated culture in example 1 were deposited and named human umbilical vein smooth muscle cell line HUVSMC, which was deposited in the chinese type culture collection center at the deposit address of wuhan university, zip code 430072, and the collection number of CCTCC NO: c202270, preservation date of 2022, 4 months and 20 days.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims

1. A method for separating and culturing human umbilical vein smooth muscle cells is characterized by comprising the following steps:

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

cutting the vessel cavity into 1 to 2mm ³ After the tissue blocks are processed, the ratio of 3 to 5 blocks/cm is calculated ² The density of (a) is planted in a culture flask;

adding complete culture solution into the bottle with the bottom of the bottle planted with the tissue block upwards, standing, turning over the culture bottle to immerse the tissue block in the complete culture solution, culturing, and periodically replacing the complete culture solution;

the tissue mass was removed and serial subculture was performed.

2. The isolated culture method of human umbilical vein smooth muscle cells according to claim 1, wherein the collagenase type I is contained in the collagenase type I solution at a concentration of 0.5 to 2.5mg/mL.

3. The isolated culture method of human umbilical vein smooth muscle cells as claimed in claim 1, wherein the incubation method comprises:

and D-Hanks solution is used for immersing the filled blood vessel, and the incubation is carried out for 28 to 35min.

4. The isolated culture method of human umbilical vein smooth muscle cells as claimed in claim 1, wherein the complete culture solution comprises fetal bovine serum, streptomycin mixture and DMEM/F12 basal medium.

5. The isolated culture method of human umbilical vein smooth muscle cells as claimed in claim 1, wherein the amount of the complete culture medium added in the culture flask is 1.5 to 2mL.

6. The isolated culture method of the human umbilical vein smooth muscle cells as claimed in claim 1, wherein the standing time is 8 to 16 hours.

7. The isolated culture method of human umbilical vein smooth muscle cells according to claim 1, wherein the confluency of the cells is not less than 90% during the continuous subculture, and the subculture is performed by digestion with TrypLE solution.

8. Human umbilical vein smooth muscle cells isolated and cultured by the isolation and culture method of human umbilical vein smooth muscle cells according to any one of claims 1 to 7.

9. The human umbilical vein smooth muscle cell according to claim 8, wherein the number of passages of the human umbilical vein smooth muscle cell is 20 or more.

10. The human umbilical vein smooth muscle cell according to claim 8 or 9, wherein the human umbilical vein smooth muscle cell is preserved in China center for type culture Collection with the preservation number of CCTCC NO: c202270, preservation date of 2022, 4 months and 20 days.