阅读说明：本技术 干细胞培养方法 (Stem cell culture method ) 是由 胡赓熙 于 2020-07-17 设计创作，主要内容包括：在此公开了一种干细胞培养方法,其特征在于,包括在一个大气压之外加有额外压力的气体环境中培养干细胞的步骤,所述额外压力为60-140mmHg。(Disclosed herein is a stem cell culture method characterized by comprising the step of culturing stem cells in an atmosphere to which an additional pressure is applied in addition to atmospheric pressure, the additional pressure being 60-140 mmHg.)

1. A method for culturing stem cells, comprising the step of culturing stem cells in an atmosphere to which an additional pressure is applied in addition to atmospheric pressure, the additional pressure being 60 to 140mmHg, such as 70 to 120mmHg, 75 to 115mmHg, 80 to 110mmHg, 85 to 100mmHg, or 90 to 95 mmHg; wherein the stem cells do not include cells isolated or obtained from a human embryo that has been more than 14 days post-fertilization or that has undergone in vivo development.

2. The method according to claim 1, wherein the additional pressure is periodically fluctuated in the range of 60-140mmHg, such as 70-120mmHg or 75-115mmHg, preferably with a sinusoidal or sinusoidally varying periodicity.

3. A method according to claim 2, wherein the frequency of the periodic fluctuation is 12-20 times/min, such as 13-18 times/min, 14-17 times/min, 14-16 times/min or 13-15 times/min.

4. The method of claim 1, wherein the additional pressure is constant.

5. The method according to claim 1, wherein the oxygen concentration in the gaseous environment is 2-20%, such as 2-7%, 2-5%, 4-7%, 5-7%.

6. The method of claim 1, wherein the stem cells in said step are primary stem cells and/or passaged stem cells.

7. The method of claim 1, wherein the stem cell is a human stem cell.

8. The method of claim 1, wherein the stem cell is a mesenchymal stem cell, such as a human adipose mesenchymal stem cell, a human endometrial mesenchymal stem cell, a human hair follicle mesenchymal stem cell, a human umbilical cord mesenchymal stem cell.

9. The method according to claim 1, wherein the temperature is 36.5-37.5 ℃, preferably 37 ℃.

10. A stem cell obtained according to the method of any one of claims 1-9.

Technical Field

The invention relates to the field of stem cells, in particular to a culture method of stem cells.

Background

Stem cells are a type of pluripotent cells with self-replicating ability, which can differentiate into various functional cells under certain conditions. The embryonic stem cell is a totipotent stem cell with the strongest differentiation capability, but the clinical application prospect of the embryonic stem cell is not clear at present due to the influence of multiple factors such as the tumorigenicity, ethical problems and the like. Mesenchymal Stem Cells (MSCs) are members of the stem cell family, are present in a variety of tissues (e.g., bone marrow, umbilical cord blood and umbilical cord tissue, placental tissue, adipose tissue, etc.), and have multipotent differentiation potential. The stem cells have the potential of differentiating into various mesenchymal series cells (such as osteogenic cells, chondrogenic cells, adipogenic cells and the like) or non-mesenchymal series cells, have unique cytokine secretion function and immunoregulation and anti-inflammatory functions, and can be used for treating immune-related diseases such as systemic lupus erythematosus, Crohn's disease and graft-versus-host disease; there are also some basic studies and clinical trials that demonstrate that mesenchymal stem cells can reduce atherosclerosis.

Efficient culture of stem cells has been a problem that the art is expected to solve. In the field, the culture efficiency of the stem cells is generally improved from the perspective of medium screening, and the efficiency and activity of the stem cell culture are rarely considered from factors such as oxygen concentration, pressure and the like. The literature reports that the oxygen tension in normal human bone marrow is 3.591-6.517 kPa, which is equivalent to the oxygen volume fraction of 4-7%, namely that the mesenchymal stem cells live in the hypoxic environment under the normal state. In addition, it has also been reported that a better effect is obtained by culturing mesenchymal stem cells by additionally applying pressure. However, reports on the screening and optimization of gas condition parameters such as oxygen concentration, pressure parameters, etc. in stem cell culture conditions have not been found. At the same time, there is a continuing need for more optimal culture protocols in the field of stem cell research and production.

Disclosure of Invention

The invention provides a new optimized stem cell culture scheme through the research and optimization of the gas environment in the culture condition. The stem cells cultured by the method of the invention show better biological activity, including proliferation capacity, independent survival capacity, differentiation capacity, dryness maintenance, aging resistance and the like.

One aspect of the present invention is a stem cell culture method comprising the step of culturing stem cells in an atmosphere to which an additional pressure is added to the atmosphere, the additional pressure being 60 to 140 mmHg. The additional pressure may be static or dynamic, e.g. periodically fluctuating.

In one embodiment, the gaseous environment has an oxygen concentration of 2% to 20%, preferably a low oxygen environment, for example a low oxygen concentration of 2% to 7%.

Also provided herein are stem cells cultured by the methods of the invention.

As used herein, the term "stem cell" does not include cells isolated or obtained from human embryos that have been more than 14 days post-fertilization or that have undergone in vivo development. The stem cell is preferably a mesenchymal stem cell.

Drawings

Figure 1 shows the fold expansion of mesenchymal stem cells of hair follicles cultured under different conditions.

FIG. 2 shows the generation numbers and the expansion multiples of the mesenchymal stem cells of hair follicles under different culture conditions, wherein the generation numbers are represented by Pn, and n is a natural number.

Fig. 3 shows CFU rates of umbilical cord and adipose mesenchymal stem cells cultured under different conditions.

Fig. 4 shows the osteogenesis rate (fig. 4A) and the adipogenesis rate (fig. 4B) of mesenchymal stem cells of hair follicles cultured under different conditions.

Figure 5 shows a sinusoidal or quasi-sinusoidal fluctuation of the additional pressure applied outside one atmosphere in the range of 75-115mmHg in one embodiment.

Above, the only difference between the "different (culture) conditions" is the gas environment difference characteristic represented in the figure as follows: "20%" means 20% oxygen concentration in the environment, no additional pressure is applied; "5%" means 5% oxygen concentration with no additional pressure applied; "5% + static" means an oxygen concentration of 5%, with an additional constant pressure of 95mmHg applied beyond 1 atmosphere; "5% + move" means that the oxygen concentration is 5%, and that an additional pressure is applied beyond 1 atmosphere, said additional pressure being a periodic fluctuation of sinusoidal or quasi-sinusoidal variation in the range 75-115mmHg with a frequency of 14 times/min.

FIG. 6 is a schematic diagram showing a hypoxic static pressure (5% + static) apparatus in an example in which cells are cultured in a pressure tank at an oxygen concentration of 5% and an additional pressure is applied at 1 atmosphere, the additional pressure being maintained at 95 mmHg.

FIG. 7 shows a schematic diagram of a hypoxic dynamic pressure (5% + dynamic) apparatus in an example in which cells are cultured in a pressure tank with 5% oxygen concentration, and an additional pressure is applied at 1 atmosphere with periodic fluctuations of sinusoidal or quasi-sinusoidal variation in the range of 75-115 mmHg. In the figure, the line from the control module to the pressure tank shown by the arrow represents a pressure increasing air path, and the line from the pressure tank to the control module shown by the arrow represents a pressure reducing air path; the control module comprises a software control and a pressurization air pump, a decompression valve and a pressurization valve on an air path.

Detailed description of the preferred embodiments

As used herein, unless otherwise specified, the singular forms "a", "an", and "the" preceding singular forms "a", "an", and "the" include plural referents.

Herein, unless otherwise specified, each technical feature mentioned herein, such as components, contents, steps, condition parameters, parameter values, components, connections, working relationships, and the like, is not limited to the respective embodiments and examples specifically described herein, and any other combination thereof is also within the scope of the present invention.

Herein, unless otherwise specified, numerical ranges expressed as two end values are to be considered as specifically disclosing all real numbers between the two end values and numerical ranges formed by combining two of them. Furthermore, unless specifically stated otherwise, when multiple alternative ranges or values are specifically described for the same parameter, these endpoints and values may be arbitrarily combined, and the ranges so derived are within the scope of the present invention provided they are within the broadest continuous range specifically described.

As used herein, unless otherwise specified, a stated numerical value, whether with the antecedent "about," encompasses the range of that numerical value by + -10%, and can also be within the range of + -5%, + -3%, + -2%, + -1%, or + -0.5%.

Herein, unless otherwise specified, all scientific and technical terms shall have the meaning according to what is known or known in the art, in particular in the field of stem cells, mesenchymal stem cells, for example as described in textbooks, experimental manuals or prior art documents.

Provided herein is a method for culturing stem cells, the method comprising the step of culturing stem cells in an atmosphere in which an additional pressure is applied in addition to atmospheric pressure, the additional pressure being about 60-140 mmHg.

Herein, "gaseous environment" refers to the gaseous conditions in the environment in which the cell culture is located, e.g., the gaseous environment in an incubator, and is characterized primarily by gas composition and gas pressure. Typically, the cell culture gas environment, including the stem cells, is at one atmosphere pressure, about 5% CO₂And about 95% relative humidity.

The process of the present invention applies an additional pressure of about 60-140mmHg, in addition to one atmosphere. It is known that the diastolic blood pressure of a normal person is 60 to 90mmHg and the systolic blood pressure is 140 to 90 mmHg. Therefore, the present invention well simulates the pressure environment of stem cells in the human body by applying additional pressure. In some embodiments, the additional pressure is about 70-120mmHg, about 75-115mmHg, about 80-110mmHg, about 85-100mmHg, or about 90-95 mmHg. For example, the pressurization and regulation may be accomplished using the gas delivery system and the pressure sensing device of the incubator.

In some embodiments, "one atmosphere" refers to ambient atmosphere without additional pressurization, i.e., atmospheric pressure. Preferably, "one atmosphere" is one standard atmosphere, i.e., 760 mmHg.

In the present invention, the additional pressure may be static, i.e. constant, or may be dynamic. In some embodiments, the additional pressure is static, i.e., constant, e.g., constant at any value in the range of 70 to 120mmHg, 75 to 115mmHg, 80 to 110mmHg, 85 to 100mmHg, 90 to 95mmHg, e.g., 95 mmHg. Herein, the "constant" pressure includes the case where the pressure is substantially constant, i.e. the fluctuation amplitude with respect to the nominal pressure value is not more than 10%, preferably not more than 5%, 3%, 2%, 1% or 0.5%, preferably infinitely close to 0% and includes the case of zero fluctuation. Means for maintaining a cell culture gaseous environment stable, including maintaining a constant gas pressure, are well known to those skilled in the art, such as conventional incubators and their gas delivery systems and monitoring systems.

In some embodiments, the additional pressure is dynamic, hereinafter also referred to as "dynamic pressure". In some embodiments, the additional pressure is periodically varied in the range of 60-140 mmHg. The periodic fluctuation can be fixed frequency or variable frequency, and the amplitude of each period can be the same or different. Thus, taking the 60-140mmHg range as an example, the extremes of the range represent the pressure values of the lowest valley and the highest peak, respectively. Preferably, the additional pressure fluctuates in a range, i.e., amplitude, from about 70 to 120mmHg, more preferably from about 75 to 115 mmHg. Wherein 75mmHg is the median of the diastolic pressure of normal people (60-90mmHg), and 115mmHg is the median of the systolic pressure (140-90 mmHg). Thus, the additional dynamic pressure applied by the present invention mimics well in amplitude the periodically changing pressure environment in which stem cells are located in vivo. Preferably, the fluctuations are periodic fluctuations of sinusoidal or sinusoidal-like variations, such as shown in fig. 5.

For example, the means for applying dynamic pressure may be as follows: and (3) communicating the chamber in which the culture is positioned with a pressure changing device, and applying air pressure to the chamber by the pressure changing device. In one embodiment, the pressure changing device comprises a pressurizing air path and a pressure reducing air path, the chamber where the culture is located is communicated with the pressurizing air path and the pressure reducing air path, and the pressurizing air path is communicated with the air source. The chamber may be an inner chamber in which a culture of a culture device, such as an incubator, pressure tank or pressure capsule, culture tank, etc., is located. The gas source may be, for example, a gas tank, or may be the ambient gas in which the culture chamber is located. The gas provided by the gas source corresponds to the gas composition required by the invention. The pressurization gas path comprises a pressurization gas pump and an optional valve such as a pressurization valve. The pressure reducing gas circuit includes a valve, such as a pressure reducing valve, and an optional pressure reducing gas pump. In one embodiment, the pressurizing air pump applies the air from the air source to the chamber where the culture is located through the pressurizing air path under the control of software, when the detected air pressure reaches the set upper limit, the pressurizing air path is closed, the pressure reducing valve is opened at the same time, the air pressure of the chamber where the culture is located is reduced through the pressure reducing air path, when the air pressure is reduced to the set lower limit, the pressure reduction is stopped, the pressure reducing air path is closed, the pressurizing air path is re-opened, and the steps are repeated in such a circulating way to form air pressure fluctuation. The air pump and the valve can be connected with a software control, and the operation control of pressure increasing and reducing is carried out through a program in the software control, so that the pressure in the pressure tank periodically fluctuates along with time, such as sinusoidal or quasi-sinusoidal fluctuation. In one embodiment, the pressure reducing gas circuit comprises a pressure reducing pump, and the pressure of the chamber in which the culture is located is reduced by pumping gas from the pressure reducing pump. In another embodiment, the pressure reducing air circuit can omit a pressure reducing air pump for pumping air from the culture chamber, and the pressure inside the chamber is released by utilizing the pressure difference between the inside and the outside of the chamber through a pressure reducing valve so as to realize pressure reduction.

In another embodiment, the pressure changing device comprises a cylinder. The chamber where the culture is located is communicated with the air cylinder, the air pressure of the chamber is changed through the movement of the piston of the air cylinder in the cylinder body, the reciprocating motion of the piston in the cylinder body enables the air volume of the chamber to be periodically changed, the air pressure is periodically pulsed along with the periodic change, and the pressure change can be set and controlled according to needs, for example, the pressure change is enabled to form sine or similar sine change.

In some embodiments, the frequency of the periodic fluctuations of the additional dynamic pressure is about 12 to 20 times/minute, preferably about 13 to 18 times/minute, about 14 to 17 times/minute, about 14 to 16 times/minute, or about 13 to 15 times/minute, such as about 12, 13, 14, 15, 16, or 17 times/minute. In some embodiments, the frequency of the periodic fluctuations of the additional dynamic pressure is constant. It is known that the respiratory rate of normal persons is between 12 and 20 times per minute. Therefore, the dynamic pressure applied additionally by the present invention also well simulates the periodically changing pressure environment of the stem cells in vivo in frequency.

In the present invention, the gas composition in the gas atmosphere can be referred to or matched with the conventional setting of stem cell culture, such as CO, except for the oxygen concentration₂Concentration about 5%, relative humidity about 95%. In some embodiments, the gaseous environment comprises about 2% to about 20% oxygen, and the remaining gas other than oxygen is selected from any other component of the air other than oxygen, other gases that are not harmful to stem cells, or combinations thereof, such as nitrogen (N) gas₂) Carbon dioxide (CO)₂) Or a combination thereof. In some embodiments, the concentration of oxygen in the gaseous environment is between about 2% and 7%, between about 2% and 5%, between about 4% and 7%, between about 5% and 7%. Means for maintaining a stable cell culture gaseous environment, including maintaining a stable gas composition, are well known to those skilled in the art, such as a common incubator and its carbon dioxide gas control system and humidity control system. For example, a three-gas incubator can be used to produce low oxygen concentrations.

The method of the present invention can be used for culturing various stem cells, including embryonic stem cells, adult stem cells, induced pluripotent stem cells. The adult stem cells may be, for example, adipose, uterine membrane, hair follicle, or umbilical cord stem cells, among others. Human stem cells are preferred. It is to be noted that the term "stem cell" as used herein does not include cells isolated or obtained from human embryos that have been fertilized for more than 14 days or have undergone in vivo development. In some embodiments, the methods of the invention are used to culture mesenchymal stem cells, such as (human) adipose, uterine membrane, hair follicle or umbilical cord mesenchymal stem cells.

In some embodiments, the step of culturing the stem cells in the additional pressurized environment is or includes expansion, maintenance and/or passaging of the primary cells. In some embodiments, the step of culturing the stem cells in the additional pressurized environment is or includes maintenance, expansion and/or passage of progeny cells. In some embodiments, the expansion, maintenance, passage are cultured from the primary cell, i.e., in an additional pressurized environment.

In the present invention, other culture conditions such as culture medium and temperature, besides the gas atmosphere, are referred to or meet the conventional setting of stem cell culture. For example, any medium used to culture stem cells, such as mesenchymal stem cells, may be employed in the methods of the invention. For example, the method of the present invention may employ a suitable culture temperature of any stem cell, such as mesenchymal stem cells, generally 36.5-37.5 ℃, preferably 37 ℃.

Also provided herein are stem cells obtained by culturing in a method of the invention. The stem cells cultured by the method of the invention show better biological activity, including proliferation capacity, independent survival capacity, differentiation capacity, dryness maintenance, aging resistance and the like.

Examples

The present invention will be described in further detail with reference to examples. It should be understood that these examples are given for illustrative purposes only, and are not intended to limit the scope of the present invention.

In the following examples, "different conditions" or "different culture conditions" means that the cell culture conditions involved in the comparison differ from each other only by the unique gas environment characteristic indicated below, such a set of different culture conditions for comparison is referred to simply as "4 conditions" in which the pressure values are both gauge pressures, i.e., pressures applied in addition to one atmosphere:

normal oxygen (20%): oxygen concentration is 20%, and no additional pressure is applied;

hypoxia (5%): the oxygen concentration is 5%, and no additional pressure is applied;

hypoxic static pressure (5% + resting): oxygen concentration is 5%, a constant pressure of 95mmHg is additionally applied under 1 atmosphere, a pressure tank is placed in a hypoxia culture box (ESCO company), cell culture is carried out in the pressure tank, the pressure tank is inflated and pressurized by an air pump, when the pressure reaches 95mmHg, a slow release valve is slowly opened on the premise of not stopping inflation and pressurization, so that air inlet and outlet are dynamically balanced, and the gas in the tank is maintained at 95mmHg, which is shown in figure 6 in detail;

hypoxic dynamic pressure (5% + kinetic): oxygen concentration 5%, an additional pressure was applied at 1 atmosphere, with a periodic fluctuation of sinusoidal or quasi-sinusoidal variation in the range 75-115mmHg at a frequency of 14 times/min, see FIG. 5. As shown in FIG. 7, a pressure tank was placed in a hypoxic incubator (ESCO Co.) and cell culture was performed in the pressure tank. The pressure tank is connected with a pressurization gas circuit and a decompression gas circuit. In the figure, the line from the control module to the pressure tank shown by the arrow represents a pressure increasing air path, and the line from the pressure tank to the control module shown by the arrow represents a pressure reducing air path; the control module comprises a pressure increasing air pump, a pressure reducing air pump, a pressure increasing valve and a pressure reducing valve which are controlled by software and arranged on an air circuit. The method comprises the steps of opening a pressure increasing valve of a pressure increasing gas circuit under the control of software control, starting a pressure increasing gas pump, pumping gas in an incubator to charge and pressurize the pressure tank by the pressure increasing gas pump, closing the pressure increasing gas pump and the pressure increasing valve of the pressure increasing gas circuit when the pressure reaches 115mmHg, stopping pressurizing, simultaneously opening a pressure reducing valve of the pressure reducing gas circuit, starting the pressure reducing gas pump, reducing the pressure by the gas in the pressure reducing gas pump pumping tank, closing the pressure reducing valve and the pressure reducing gas pump of the pressure reducing gas circuit when the pressure is reduced to 75mmHg, stopping reducing the pressure, re-opening the pressure increasing gas circuit, namely opening the pressure increasing valve and the pressure increasing gas pump, and repeating the steps in the same way, wherein the alternating frequency is controlled to be 14 times/minute by program software.

Example 1: comparison of expansion fold of mesenchymal stem cells of hair follicle cultured under different conditions

1) Intact human hair follicle tissue was obtained and carefully placed on the bottom of a 1.5mL EP tube using microscopic forceps. Adding enzymatic hydrolysate TripLE (Gibco-12604021) to 5 μ L per hair follicle, and adding 5% CO at 37 deg.C₂The incubator was allowed to stand for 3 hours, the bottom of the tube was gently flicked every 1 hour, and gently mixed.

2) After 3 hours of enzymolysis, the external hair root sheath of the hair follicle can be seen to be completely enzymolyzed under the microscope, and the hair shaft part can not be completely enzymolyzed. The part without enzymolysis needs to be taken out, and is lightly blown and beaten for 10 times by using a 100-plus 1000 mu L liquid transferring gun to completely mix the enzymolysis liquid. Standing for 1 minute until the hair shafts which are not subjected to enzymolysis sink to the bottom of the EP tube, and absorbing the upper layer enzymolysis suspension to obtain the primary mesenchymal stem cells.

To one well of a six-well plate, 25 μ L of the primary mesenchymal stem cell suspension obtained by enzymatic hydrolysis pooled from 5 hair follicles was added, and then 2mL of amniotic fluid medium (purchased from baiyunshan beidy biopharmaceutical, inc., guangzhou) was added for resuspension. The six-hole plate is respectively put into 4 conditions of normal oxygen (20%), hypoxia (5%), hypoxia static pressure (5% + static) and hypoxia dynamic pressure (5% + dynamic) for culture at 37 ℃, and the culture solution is changed every 3 days.

3) Culturing for 10-12 days, observing that the cell density in the six-hole plate reaches more than 80%, discarding the culture medium, adding 0.5mL of tryptlE digestive juice at the bottom of the six-hole plate, placing the six-hole plate in an incubator at 37 ℃ for digesting for 3 minutes, then adding 1mL of amniotic fluid culture medium into the six-hole plate to terminate the digestion, taking out the supernatant, placing the supernatant into a 15mL centrifuge tube, rinsing the hole plate 1 time with 1mL of the amniotic fluid culture medium, and adding the rinsing solution into the centrifuge tube. Centrifuging for 5 minutes in a centrifuge at 1500r.p.m., discarding the supernatant, adding 1mL of amniotic fluid culture medium for resuspension, counting, inoculating to a T25 culture flask, and culturing to obtain the P1 generation mesenchymal stem cells.

4) The cells were continuously subcultured to P12 passages, and the cells under 4 conditions were maintained under the respective culture conditions. Fold expansion-number of cells harvested per passage/number of seeded cells. In this example, the strict 5000 cells/cm was adopted²Is passaged, and the area of one T25 flask is 25cm²That is, one T25 culture bottle is inoculated with 1.25X 10 cells each time⁵For each cell, the fold expansion equation was expressed as "fold expansion ═ number of cells harvested per time/(1.25X 10)⁵)”。

The results are shown in FIGS. 1 and 2. The result shows that the expansion multiple of the mesenchymal stem cells cultured in the hypoxia state is higher than that of the normal oxygen state, the expansion multiple is further improved by pressurization, and the expansion multiple of the mesenchymal stem cells is the highest under the condition of the hypoxia dynamic pressure.

Example 2: comparison of clone formation abilities of mesenchymal stem cells cultured under different conditions

Colony formation is one of the effective methods for determining the proliferative capacity of cells. When a single cell is cultured in vitro, the progeny of the cell form a population of cells called clones. At this time, each clone contains more than 50 cells with the size of 0.3-1.0mm, and the proliferation potential of the cells can be quantitatively analyzed by counting the clone formation rate, so that the proliferation capacity and the independent survival capacity of the cells can be known.

Human umbilical cord mesenchymal stem cell test:

1) obtaining complete human umbilical cord tissue, using large surgical scissors to cut off two ends of the umbilical cord by 1cm respectively, then rinsing for many times to remove blood, using small scissors and tissue forceps to completely tear open a amniotic membrane layer along a position close to an umbilical vein, flattening, and then using the tissue forceps to remove two umbilical arteries and one umbilical vein in the tissue.

2) The vessel-removed tissue was transferred to a 50ml centrifuge tube containing 10ml of TripLE (Gibco-12604021) as a digestive enzyme solution, and the tissue was minced with a large surgical scissors and the tissue pieces were cut to 1mm³And (4) sealing with a sealing film, transferring to a shaking table at 37 ℃, rotating at 60-80 times/min, and digesting for 3 hours by shaking.

3) After digestion, 30ml Hank balanced salt solution is added to dilute and mix evenly, liquid in the tube is not layered after mixing evenly and is light yellow, the liquid is colloidal, and the slow filtration is carried out by using a 100 mu m cell filter screen.

4) And subpackaging the filtrate into 13 15ml centrifuge tubes, centrifuging for 6 minutes at the normal temperature by 400g, slowly removing the supernatant after the centrifugation is finished, adding 1ml of DMEM/F12 (purchased from Gibco company) for resuspension of the precipitated cells, mixing to one tube, centrifuging for 6 minutes at the normal temperature by 400g, removing the supernatant, and resuspending by 1ml of DMEM/F12 to obtain the primary mesenchymal stem cells. The cells were counted and inoculated into a T25 flask using Edes medium (purchased from Edes Biotech, Inc., Shanghai).

5) Changing the culture solution every 3 days, culturing for 9 days, removing the culture medium when the cell density in the T25 culture bottle reaches more than 80%, adding 1mL of TryplE digestive juice at the bottom of the culture bottle, then placing the culture bottle in an incubator at 37 ℃ for digestion for 3 minutes, then adding 3mL of Edes culture medium into the culture bottle by using a 10mL pipette to terminate digestion, taking out the supernatant, placing the supernatant in a 15mL centrifuge tube, adding 1mL of Edes culture medium to rinse the bottom of the bottle for 1 time, and adding the rinse solution into the centrifuge tube. Centrifuging for 5 minutes at 1500r.p.m. in a centrifuge, discarding the supernatant, adding 1mL Edes culture medium for resuspension, counting and inoculating to a T25 culture flask, and transferring to P1 generation umbilical cord mesenchymal stem cells.

6) Continuously subculturing the cells, and culturing the P6 generation cells at the ratio of 5000 cells/cm²The density of (2) was inoculated into 4T 25 flasks and cultured at 37 ℃ under the 4 conditions described above. After 4 days, collect the fine powderCells were resuspended in Edes medium, made into cell suspensions and counted.

7) Colony Forming Units (CFU) were calculated:

inoculating the cells cultured under different conditions to 10cm²Petri dish, 2200 cells/dish. After being plated, the mixed solution is shaken up and respectively put into an incubator for cultivation, and the cultivation conditions are respectively the same as the cultivation conditions in the step 6). The culture period is 14 days, and the liquid is changed every 3 days in the culture process and the growth condition of cell communities is observed.

When the number of most of the individual clone cells in each dish was greater than 50, 2mL of 4% paraformaldehyde was added to each dish, the cells were fixed at 4 ℃ for 60 minutes, and the cells were washed 1 time with PBS. 2mL of clean and impurity-free crystal violet staining solution is added into each culture dish to stain cells for 30 minutes.

Cells were washed, air dried, photographed, and clone counted. The number of Colony Forming Units (CFU) in each dish was counted, and then the CFU rate was calculated: CFU rate is CFU number/inoculum, which in this example is 2200.

The results are shown in FIG. 3. Under the condition of hypoxia dynamic pressure, the CFU rate of the umbilical cord mesenchymal stem cells is the highest and reaches 3.55 percent.

Human adipose-derived mesenchymal stem cell assay:

1) obtaining a complete human adipose surgery sample, washing solid adipose tissues after surgery by using a cell washing solution, removing blood vessels and connective tissues visible to naked eyes by using medical straight-pointed-end ophthalmic scissors and medical bent toothed ophthalmic forceps, and taking 5mL of fat in a 50mL centrifuge tube.

2) Adding twice volume of enzymolysis working solution (10mg/mL collagenase I, solvent DMEM/F12) (10mL) at volume ratio of 1:2, mixing, and shearing adipose tissue to 1mm with sterile medical narrow-head fine scissors³The method comprises the following steps of forming muddy flesh, screwing a tube cover, sealing a sealing film, obliquely placing the muddy flesh into a constant-temperature shaking table at 37 ℃ for digesting for 1 hour at a shaking speed of 80r.p.m. until no obvious fat particles exist, repeatedly and lightly blowing digested fat tissues for 4-5 times by using a 10mL pipette, adding 15mL of washing liquor, turning upside down, uniformly mixing, terminating digestion, centrifuging at the room temperature of 1500r.p.m. for 5 minutes, and removing a supernatant.

3) The pellet was resuspended in 20mL of cell wash, and all of the resuspension was passed through a 100 μm cell screen, followed by 5mL of cell wash to wash the cell screen. Centrifuge at 1500r.p.m. for 5min at room temperature and discard the supernatant. Cells were suspended in 5mL of DMEM-F12, counted, and inoculated into a T25 flask (amniotic fluid medium, purchased from Baiyunshan Bydi Bio-pharmaceuticals, Guangzhou). After culturing the cells at 37 ℃ for 48 hours, changing the liquid to wash off the nonadherent erythrocytes, and continuing culturing, wherein the liquid is changed once every four days, so that the primary mesenchymal stem cells are obtained. Culturing for 7-9 days, and subculturing when the fusion degree reaches 80% -90%. And (3) digesting the trypLE for 2 minutes at room temperature, adding 2 times of volume of amniotic fluid culture medium to terminate digestion, centrifuging, removing a supernatant, adding 1mL of amniotic fluid culture medium to resuspend, counting, and inoculating to a T25 culture flask to obtain the P1 generation adipose-derived mesenchymal stem cells.

4) Continuously subculturing the cells, and culturing the P4 generation cells at the ratio of 5000 cells/cm²The density of (2) was inoculated into 4T 25 flasks and cultured at 37 ℃ under the 4 conditions described above. After 4 days, the cells were collected, resuspended in amniotic fluid medium, made into a cell suspension and counted.

5) Colony Forming Units (CFU) were calculated as described previously.

The results are shown in FIG. 3. The results show that the CFU rate of the adipose-derived mesenchymal stem cells cultured under the hypoxia condition is obviously higher than that of the normal oxygen, and the CFU rate of the adipose-derived mesenchymal stem cells under the hypoxia dynamic pressure condition is the highest and reaches 8.18%.

Example 3: comparison of osteogenic differentiation and adipogenic differentiation of hair follicle mesenchymal stem cells cultured under different conditions

Mesenchymal stem cells of hair follicle were prepared and subcultured as described in example 1, and cultured and subcultured under 4 conditions, respectively, from the primary cells. Harvesting the P5 generation hair follicle mesenchymal stem cells to perform osteogenic differentiation and adipogenic differentiation induction.

Osteogenic differentiation and staining:

taking 2.5X 10 according to the viable cell concentration⁵Cells were placed in a 15mL centrifuge tube and complete growth medium was added to a total volume of 2.5mL, i.e., a final concentration of 1.0X 10⁵cells/mL. At 1 mL/well, i.e., 1.0X 10⁵Cell concentration of cells/well culture under 4 conditionsCulturing in a box for 3-4 days until the cell fusion rate is 90% -100%. The in-well medium was aspirated off in a clean bench and 1mL of osteogenic induction differentiation complete medium (purchased from Spanish Biotech Co., Ltd.) was added to each well. The medium was changed every 3 days, and when osteogenic differentiation was induced for 14 days, osteoblastic differentiated cells were stained with alizarin red S. The results of bone formation rate (%) are shown in FIG. 4A.

Adipogenic differentiation and staining:

taking 2.5X 10 according to the viable cell concentration⁵Cells were placed in a 15mL centrifuge tube and complete growth medium was added to a total volume of 2.5mL, i.e., a final concentration of 1.0X 10⁵cells/mL. At 1 mL/well, i.e., 1.0X 10⁵The cell concentration of the cells/hole is cultured in an incubator under 4 conditions for 3-4 days until the cell fusion rate is 90% -100%. The medium was aspirated off the wells in a clean bench and 1mL of adipogenic differentiation medium (purchased from STEMCELL) was added to each well. The culture medium is replaced every 3 days, and when adipogenic induction differentiation is carried out for 14 days, adipogenic differentiated cells are stained by oil red O staining solution. The results of the fat formation (%) are shown in FIG. 4B.

The results show that the osteogenic and adipogenic differentiation capacity of the mesenchymal stem cells cultured by hypoxia is stronger than that of normal oxygen, and the differentiation capacity is further enhanced by pressurization. Particularly, the osteogenic and adipogenic differentiation capacity of the mesenchymal cells under the condition of the hypoxic dynamic pressure culture is obviously enhanced.

Example 4: comparison of expression levels of sternness and senescence genes of mesenchymal stem cells cultured under different conditions

Preparing and culturing human hair follicle mesenchymal stem cells:

mesenchymal stem cells of hair follicle were prepared and subcultured as described in example 1, and cultured and subcultured under 4 conditions, respectively, from the primary cells. Except that the amniotic fluid medium was replaced by a saybolt medium (purchased from saybolt biotechnology limited). And reserving harvested P3 and P5 generation mesenchymal stem cells for detecting the expression level of the sternness gene.

Preparing and culturing human mesentery mesenchymal stem cells:

1) tissue isolation: the menstrual blood sample in a 50mL centrifuge tube was sequentially passed through an 18 mesh stainless steel cell sieve, a 36 mesh stainless steel cell sieve, an 80 mesh stainless steel cell sieve and a 100 μm filter membrane.

2) Monocyte isolation method:

subjecting the menstrual blood sample filtered in the step 1) to 400g, pre-centrifuging at 20 ℃ for 10 minutes, removing most of supernatant, leaving 2mL of supernatant and cell precipitate at the bottom, diluting the precipitate with a cleaning solution, wherein the total volume of the diluted sample is 2 times of the amount of pure blood, and uniformly mixing. 16mL of the room temperature lymphocyte separation solution was aspirated by a syringe, added to the bottom of a 50mL separation tube, the bottom was completely filled, and then 20mL of blood was directly added along the tube wall, 800g, and centrifuged at 20 ℃ for 15 minutes. The centrifugation is finished, and the sample is divided into four layers from top to bottom, the upper layer of the clapboard: a plasma layer, a leukocyte layer, and a lymphocyte separation liquid layer and a erythrocyte layer under the clapboard. Most of the upper plasma layer (about 15 mL) was aspirated with a 3mL bus pipette. The remaining 2mL of plasma and tunica albuginea layers were carefully aspirated with a 3mL Pasteur pipette and added to a new 50mL centrifuge tube.

3) Tissue culture

Washing once with a washing solution with twice the sample volume of the white membrane layer, adding 10mL of the washing solution into each tube to resuspend the precipitate, combining the precipitates in each tube, centrifuging each tube by 40mL, centrifuging each tube for a suspension solution with the volume exceeding 40mL, and discarding the supernatant. Then washed once more to remove the supernatant. 2ml of medium (7501 medium, purchased from ScienCell) was added and all cell pellets, including primary mesenchymal stem cells, were resuspended, counted, and 3X 10 cells were added⁵Per cm²The density of (2) was inoculated into a six-well plate, shaken well, and cultured at 37 ℃ under the 4 conditions described above. After 48 hours, the liquid was changed to wash off the nonadherent erythrocytes, and then the liquid was changed every 3 days. Culturing to 7 days, observing that the cell density in the 6-well plate reaches more than 80%, digesting for 3 minutes at 37 ℃ by using pancreatin, adding 7501 culture medium with 2 times volume to terminate digestion, centrifuging, discarding supernatant, adding 1mL7501 culture medium for re-suspension, counting and inoculating to a T25 culture flask, namely P1 generation.

The cells are continuously subcultured to P8 generations, the cells under 4 conditions always maintain respective culture conditions, and harvested mesenchymal stem cells of P6 and P8 generations are reserved for detecting the expression level of senescence genes.

Detecting the expression level of the xerosis genes and the senescence genes:

extracting total RNA: collecting mesenchymal stem cells in a 1.5mL centrifuge tube, washing with PBS for 2 times, adding 1mL RNAlso Plus lysate (purchased from Takara corporation), standing on ice, adding 200 μ L chloroform, mixing thoroughly, after 5min, centrifuging at 4 ℃ for 12000r.p.m., 15 min, gently sucking 500 μ L of upper transparent water phase into a new 1.5mL centrifuge tube, adding isopyknol, gently reversing for 8 times, mixing, standing for 15 min, centrifuging at 4 ℃ for 12000r.p.m., 15 min, removing white precipitate at the bottom of the tube, discarding supernatant, washing the precipitate with 75% ethanol, centrifuging again, removing supernatant, uncovering, air drying the precipitate at room temperature, adding 15-20 μ L DNase/RNase Free H₂O(Invitrogen^TM) And fully mixing the mixture until the precipitate is completely dissolved.

Reverse transcription of total RNA into cDNA: reverse transcription was carried out using a reverse transcription kit "TransScript One-Step gDNA Removal and cDNA Synthesis Super Mix" (AT311-03, Beijing Quanyujin Biotechnology Ltd.). Specifically, 1. mu.g of total RNA was taken and Dnase/RNase Free H was added thereto according to the instructions₂Adding 1 mu L of random primer into the mixture until the volume is 7 mu L, uniformly mixing the mixture, and preparing RNA Mix at 65 ℃ for 5 minutes; then, other components were added to prepare a reverse transcription system according to the instructions. Reverse transcription in gradient PCR instrument, procedure: 10 minutes at 25 ℃, 30 minutes at 42 ℃ and 5 seconds at 85 ℃.

Reverse transcription system:

components	Volume of
		RNA Mix	8μL
2×TS Reaction Mix	10μL
		TransScript RT/RI Enzyme Mix	1μL
gDNA Remover	1μL
		Total volume	20μL

qPCR detection of gene expression: qPRCR was performed using the kit PerfectStart Green qPCR Super Mix (AQ601-04, Beijing Quanjin Biotechnology Co., Ltd.). Specifically, 180. mu.L of DNase/RNase Free Water (Invitrogen) was added to 20. mu.L of the cDNA-containing reverse transcription system^TM) Thereby obtaining a cDNA template solution. A fluorescence quantitative 96-well plate was used, and the composition shown in the following table was added to each well according to the instructions to form a 20. mu.L reaction system. After the sample is added into the 96-well plate, the plate is sealed by using a sealing film, and then the plate is put into a centrifuge at 4000r.p.m., and is separated for 1 minute at 4 ℃. The PCR amplification parameters were: denaturation at 94 ℃ for 30 seconds; then, the cycle was repeated 40 times at 94 ℃ for 5 seconds, 60 ℃ for 15 seconds and 72 ℃ for 15 seconds. Ct values were obtained.

Reaction system:

components	Volume of
		cDNA template	3μL
Upstream primer (10. mu.M)	0.5μL
		Downstream primer (10. mu.M)	0.5μL
2×TransScript Top/Tip Green qPCR SurperMix	10μL
		DNase/RNase-Free distilled water	6μL
Total volume	20μL

PCR primers:

and (3) data calculation and processing:

the Ct value represents the number of cycles that the fluorescence signal of the amplification product in the PCR amplification process passes when reaching a set threshold, and the number of the templates at the time is as follows: mx 2^Ct(M represents the initial number of templates).

Suppose, M₁、Ct₁Represents a gene of interest (i.e., senescence gene or sternness gene), M₂、Ct₂Representing an internal reference gene (generally considered to be a gene constantly expressed in mesenchymal stem cells, the GAPDH gene is used in this example), then:

M₁×2^Ct1＝M₂×2^Ct2

M₁/M₂＝2^Ct2/2^Ct1＝2^-(Ct1-Ct2)

then, M₁/M₂I.e. 2^-(Ct1-Ct2)Can remove cell number differenceThe different influences accurately reflect the number of the target gene initial templates under the same cell number.

The results of the detection of the expression level of the stem gene of human mesenchymal stem cells are shown in Table 1. As can be seen from the table, the expression levels of the 4 sternness genes of the mesenchymal stem cells of hair follicle under the hypoxic dynamic pressure culture condition were the highest regardless of the P3 generation or the P5 generation.

TABLE 1

The detection results of the human mesenchyma stem cell senescence gene expression level are shown in table 2. As can be seen from the table, the expression level of the 3 senescence genes of the mesenchymal stem cells under the hypoxic dynamic pressure culture condition was the lowest in both P6 generation and P8.

TABLE 2

In conclusion, the hypoxic and dynamic pressure culture conditions are favorable for up-regulating the dry gene expression and down-regulating the senescence gene of the mesenchymal stem cells.

While specific examples of the invention have been described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention. It is, therefore, intended that the appended claims cover all such modifications that are within the scope of this present invention.

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