CN115181725A

CN115181725A - Cell culture method using music, cell, preparation and application

Info

Publication number: CN115181725A
Application number: CN202210917169.8A
Authority: CN
Inventors: 高东; 尹淞炼; 卢瑞珊
Original assignee: Guangzhou Xiankangda Biotechnology Co ltd
Current assignee: Guangzhou Xiankangda Biotechnology Co ltd
Priority date: 2022-08-01
Filing date: 2022-08-01
Publication date: 2022-10-14

Abstract

The application provides a cell culture method using music, cells, a preparation and application, and relates to the field of cell culture. The cell culture method using music performs fumigation on cells by playing music in the environment for culturing the umbilical cord stem cells, the obtained umbilical cord stem cells have stable quality and meet clinical requirements, and the potential therapeutic effect of the obtained cells is more obvious than that of the conventional culture method. Compared with the prior art, the method adopts a music fumigation method to culture the umbilical cord stem cells, and the obtained umbilical cord stem cells are superior to cells obtained by the conventional culture method in terms of cell particle size, cell viability and cell purity.

Description

Cell culture method using music, cell, preparation and application

Technical Field

The application relates to the field of cell culture, in particular to a cell culture method, cells, a preparation and application by using music.

Background

Clinical experiments in the medical field prove that the music can cause the changes of respiration, blood pressure, heart beating and blood flow in physiology, and has the effective effects of relaxing the body and mind, refreshing the mind, inducing deep sleep and the like. The endocrine system and digestive system of people who frequently listen to music are adjusted, and the normal listening to music can also relieve skin aging and prolong the service life. The music therapy is not inferior to the efficacy of drug therapy in the field of spirit, has no adverse reaction of drugs, and can resist pressure, relieve headache and migraine, improve insomnia, relieve depression, regulate hormone in vivo, relieve pruritus, relieve pain, and inspire infant intelligence.

At present, the main parameters of the traditional culture method of umbilical cord mesenchymal stem cells are as follows: 5% of CO ₂ 95% humidity, 37 ℃; the culture system is generally: basic culture medium, 5-10% of nutrient components, factor additives and the like. The growth rate of the umbilical cord stem cells is related to the adding ratio of nutrient components and factor additives, and the proliferation rate of the umbilical cord stem cells is directly influenced. However, the nutrient-rich culture environment inhibits the secretory function of the umbilical cord stem cells, and causes a situation of impaired efficacy of the umbilical cord stem cells in clinical use. Therefore, it is highly desirable to develop a novel culture method, which can control the addition ratio of nutrients and factor additives in the umbilical cord stem cells to 5-8% (the general addition ratio is 10%), increase various cell parameters of the umbilical cord stem cells, and enhance the paracrine function of the umbilical cord stem cells.

Disclosure of Invention

The application aims to provide a cell culture method using music, and aims to solve the problem that the proliferation rate and the secretion function of umbilical cord mesenchymal stem cells obtained by the conventional cell culture method cannot be achieved at the same time.

To achieve the above object, the present application provides a cell culture method using music, which plays music in a culture environment of the cells.

Preferably, the cell is an umbilical cord stem cell.

Preferably, the music comprises any one or more of light music, classical music, orchestral music;

preferably, the light music comprises music played by any one or more musical instruments of pianos, whistles, sourdles, zithers, lutes and erhu.

Preferably, the cells are cultured using a basal medium + a complete medium with 5% to 10% nutrients;

preferably, said cells are cultured using DMEM +10% FBS complete medium.

Preferably, the culture environment of the cells is 5% ₂ 37 ℃ and 95% humidity.

Preferably, the volume of the music is 20-40 db.

Preferably, the playing time of the music is 8-12 hours/day.

The application also provides an umbilical cord stem cell which is obtained by culturing the cell culture method using the music;

preferably, the particle size of the umbilical cord stem cells within the P5 generation is 11-14 μm.

The present application also provides a formulation comprising the umbilical cord stem cells described above.

The application also provides application of the umbilical cord stem cells or the preparation in preparing medicaments for treating depression and mental diseases.

Compared with the prior art, the beneficial effect of this application includes:

according to the cell culture method using music, the cells are fumigated by playing the music in the environment for culturing the umbilical cord stem cells, the obtained umbilical cord stem cells are stable in quality and meet clinical requirements, and the potential treatment effect of the obtained cells is more obvious than that of the conventional culture method.

Compared with the prior art, the method for culturing the umbilical cord stem cells by adopting the music fumigation method is pioneering in the scheme, the obtained umbilical cord stem cells do not have abnormal conditions in particle size, cell viability and cell purity, but the umbilical cord stem cells obtained by the phonological therapy have stronger proliferation capacity and more excellent immunoregulation effect than the cells obtained by the conventional culture method.

Drawings

To more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments are briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope of the present application.

FIG. 1 is the result of isotype control assay of P3 generation cells of umbilical cord stem cells in logarithmic growth phase in example 2;

FIG. 2 shows the results of detecting the markers of the P3 passage cells of umbilical cord stem cells in logarithmic growth phase in example 2;

FIG. 3 is the result of isotype control assay of umbilical cord stem cell P3 generation cells in logarithmic growth phase of comparative example 1;

FIG. 4 is a graph showing the results of detecting markers of umbilical cord stem cells P3 passage cells in the logarithmic growth phase of comparative example 1;

FIG. 5 is a graph showing cell proliferation profiles of example 1, example 2 and comparative example 1.

Detailed Description

The terms as used herein:

"prepared from … …" is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of … …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the subject of the claims rather than immediately after the subject matter, it defines only the elements described in that clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4," "1 to 3," "1 to 2 and 4 to 5," "1 to 3 and 5," and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

In these examples, the parts and percentages are by mass unless otherwise indicated.

"parts by mass" means the basic unit of measure indicating the mass ratio of the plurality of components, and 1 part may represent any unit mass, for example, 1g, 2.689g, and the like. If we say that the part by mass of the component A is a part by mass and the part by mass of the component B is B part by mass, the ratio of the part by mass of the component A to the part by mass of the component B is a: b. alternatively, the mass of the A component is aK and the mass of the B component is bK (K is an arbitrary number, and represents a multiple factor). It is unmistakable that, unlike the parts by mass, the sum of the parts by mass of all the components is not limited to 100 parts.

"and/or" is used to indicate that one or both of the illustrated conditions may occur, e.g., a and/or B includes (a and B) and (a or B).

Embodiments of the present application will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

1. Primary culture of umbilical cord stem cells

Taking umbilical cord of healthy infant delivered by caesarean section at term, aseptically cleaning in super clean bench, peeling off umbilical cord amnion and arteriovenous, taking out Wharton jelly, shearing with surgical scissors, and cutting with DMEM +10%Resuspension of FBS complete medium, plating into 15cm dishes, transfer to 5% CO ₂ And culturing at 37 deg.C and 95% humidity.

And supplementing the solution after 5 days until the cells climb out, removing the tissue blocks, supplementing a new complete culture medium for continuous culture, and obtaining P0 generation cells after the cells form larger clones, so as to carry out passage.

2. Subculturing umbilical cord stem cells

(1) Taking passable P0 generation cells, discarding original culture solution in a dish, adding 10mLPBS buffer solution to gently wash the growth surface of the cells, and discarding washing solution.

(2) Adding 2mL0.25% trypsin-0.02% EDTA, and shaking the plate to cover the pancreatin uniformly on the bottom of the dish; the cell space is enlarged and the cytoplasm is retracted when observed under a microscope; when the long fusiform cells become round and bright, digestion is immediately terminated by the addition of 10 volumes of complete medium.

(3) Repeatedly pumping until all cells are dropped into single cell suspension, transferring the suspension into a centrifuge tube, and centrifuging at room temperature for l500rpm/5min.

(4) Discarding the supernatant, adding DMEM +10% FBS complete medium to resuspend the cells at a density of 1X 10 ⁵ cells were subcultured. Placing the flask at 37 deg.C and 5% CO ₂ And culturing in a cell culture box with the saturated humidity of 95 percent.

(5) And obtaining P1 generation cells when the cells grow to be fused, and performing cell amplification culture. Subculturing according to the operations of the steps (1) to (4) to obtain the cells of the P2 generation to the P5 generation of the umbilical cord stem cells.

Wherein, the whole-period culture environment of the umbilical cord stem cells in the embodiment 1 plays piano soft music, the volume of the piano soft music is 30 decibels, and the playing time is 8 h/day.

Example 2

Unlike example 1, the whole-cycle culture environment of umbilical cord stem cells of example 2 plays classical music.

Comparative example 1

Unlike example 1, the whole-cycle culture environment of the umbilical cord stem cells of comparative example 1 has no music, but is a normal medium.

Test example 1 Activity measurement

The P3 generation cells of the umbilical cord stem cells cultured in examples 1 to 2 and comparative example 1 were selected and adjusted to have a cell density of 3 to 4X 10 ⁶ cell/mL. According to the cell suspension: 0.4% trypan blue =3:1 (v: v) was mixed well, 20. Mu.L of the cell mixture was added to a cell counting plate, and cell viability was measured by a Countstar cell counter. The number of the umbilical cord stem cell P3 generation cells is extracted and the activity is detected, as shown in the table 1.

TABLE 1 determination of the viability of the umbilical cord stem cells at the P3 generation in examples 1 to 2 and comparative example 1

Grouping	Number of viable cells (cells/mL)	Total cell number (cells/mL)	Rate of cell viability
				Example 1	(3.54±0.24)×10 ⁶	(3.78±0.41)×10 ⁶	93.65±1.24％
Example 2	(3.48±0.31)×10 ⁶	(3.62±0.38)×10 ⁶	96.13±2.08％
				Comparative example 1	(3.42±0.26)×10 ⁶	(3.73±0.28)×10 ⁶	91.68±1.58％

* Indicating significant difference compared to the comparative example, P <0.05.

As can be seen from the above table, examples 1 and 2 are not significantly different from comparative example 1. The viability rate of the umbilical cord stem cells obtained by the music fumigation method is not different from that of the cells obtained by the normal culture method; the cells obtained by the sonic therapy culture have no abnormal change in morphology.

Test example 2 adherence rate measurement

The umbilical cord mesenchymal stem cell P3 generation cells obtained by culturing in examples 1 to 2 and comparative example 1 are separated and cultured, and then the density is adjusted to be 2 x 10 ⁵ cells/mL, seeded 8mL into 9cm diameter dishes (T = 1.6X 10) ⁶ cells) after the cells naturally grow adherent for 2 hours, removing nonadherent cells, performing enzymolysis by using 0.25% trypsin, and calculating the number of adherent cells to obtain the adherence rate, wherein the results are shown in table 2.

TABLE 2 results of measurement of adherence rate of umbilical cord stem cells obtained in examples 1 to 2 and comparative example 1

Grouping	Number of adherent cells 2h (× 10) ⁶ )	Wall sticking rate
			Example 1	1.48±0.13	92.50％
Example 2	1.46±0.18	91.25％
			Comparative example 1	1.27±0.34*	79.38％*

* Representing significant difference compared with comparative example 1, P <0.05.

As can be seen from table 2, examples 1 and 2 are significantly different from comparative example 1. The adherence rate of the umbilical cord stem cell obtained by the music fumigation method at 2h is demonstrated, and compared with the adherence rate of the umbilical cord stem cell obtained by the culture without the music therapy, the activity of the umbilical cord stem cell is stronger.

Test example 3 flow assay

The umbilical cord stem cell P3 generation cells in the logarithmic growth phase of example 2 and comparative example 1 were taken, and the cell density was adjusted to 1X 10 ⁶ The cell suspension of cells is prepared by respectively taking 2.5 μ L of monoclonal antibodies of anti-human CD105, CD90, CD73, CD14, CD45, CD19, CD34 and HLA-DR, adding 500 μ L of cell suspension, incubating at room temperature in dark for 20min, simultaneously establishing isotype control, centrifuging at 1500r/min for 5min, discarding supernatant, washing with 10 FBS-containing PBS for 2 times, resuspending with 500 μ L1640, and detecting on a machine. The results of isotype control detection of P3 generation cells of umbilical cord stem cells in logarithmic growth phase in example 2 are shown in FIG. 1, and the results of marker detection are shown in FIG. 2; the results of isotype control detection of P3 generation cells of umbilical cord stem cells in logarithmic growth phase of comparative example 1 are shown in FIG. 3, and the results of marker detection are shown in FIG. 4.

From the flow results of example 2, the umbilical cord stem cells highly express CD44, CD73, CD90 and CD105, and the expression rates are 100.0%, 100.00%, 99.98% and 99.83% respectively; the expression rates of low-expression HLA-DR, CD14, CD34, CD19 and CD45 are 0.07 percent respectively.

From the flow results of comparative example 1, the umbilical cord stem cells highly express CD73, CD90 and CD105, and the expression rates are 99.6%, 99.92% and 97.19%, respectively; the expression rates of low-expression HLA-DR, CD45, CD34 and CD14 are respectively 0.03%.

The results meet the surface antigen characteristics of the mesenchymal stem cells (the identification standard of the mesenchymal stem cells issued by the International Stem cell society ISCT, namely positive expression of CD73, CD90 and CD105 is more than 95.0 percent, negative expression of HLA-DR and CD45 is less than 2.0 percent), which indicates that the umbilical cord stem cells do not have the phenomenon of differentiation and still maintain the characteristics of the stem cells.

Test example 4 cell proliferation test

Taking the umbilical cord mesenchymal stem cells of examples 1 and 2 and comparative example 1, culturing for 12h, 24h, 48h and 72h, digesting the cells with 0.25% trypsin +0.02% EDTA to form cell suspension, mixing the cell suspension with 0.2% trypan blue, and mixing the mixture according to the weight ratio of the cell suspension: trypan blue (v: v) =1:1, the number and viability of cells were calculated using countstar, and the cell growth tendency was obtained, and the results are shown in fig. 5.

As can be seen from FIG. 5, in examples 1 and 2, compared with comparative example 1, the cord stem cells cultured by the phonotherapy exhibit faster and stronger proliferation capacity at 24h and 48 h; culturing for 72h; the cells of example 1 and example 2 reached the plateau phase in a shorter time. The cell obtained by the scheme of the application has shorter proliferation cycle and stronger proliferation capacity.

Test example 5 comparison of the number of human peripheral leukocytes, the proportion of lymphocytes and the proportion of monocytes

Co-culturing the umbilical cord mesenchymal stem cells of examples 1 and 2 and the umbilical cord mesenchymal stem cells of the comparative example 1 with the human peripheral blood mononuclear cells for 1 day, 5 days and 10 days; the results of comparison of the number of human peripheral leukocytes, the lymphocyte ratio and the neutrophil ratio are shown in Table 3.

TABLE 3 comparison of the number of leukocytes, the proportion of lymphocytes and the proportion of neutrophils in human peripheral blood mononuclear cells (x. + -.s)

As is clear from table 3, in examples 1 and 2, the proliferation and the increase in the ratio of leukocytes, lymphocytes, and neutrophils were observed in examples 1 and 2 on the 5 th and 10 th days of co-culture, as compared with comparative example 1. The umbilical cord stem cells obtained by the music fumigation method can promote the proliferation of the number of leucocytes in the human peripheral blood mononuclear cells; meanwhile, the proportion of lymphocytes and neutrophils is up-regulated, which shows that the stem cells obtained by the phonological culture have the function of up-regulating the human immunity compared with the stem cells obtained by the normal culture method.

Test example 6 Depression rat treatment test

Modeling a depressed rat: inclining the mouse cage (45 ℃,2 h); plantar electrical stimulation (40v, 50 times); fasting and water deprivation and day and night reversal (24 h alternation); suspending tail (1 min/time); startle stimulation (150db, 25hz, 10s/time), etc., rats were stimulated alternately with the 2 stress methods described above daily for a total of 28 days. The control rats did not receive any stimulation.

After the molding is finished, the rat tail of the model rat is intravenously injected with the cells of the example 1, the cell of the example 2 and the cell of the comparative example 1 to be treated as a treatment group, the rat of the model olanzapine as an antidepressant is injected as a positive group, the treatment is continuously carried out for 15 days, and the rat of the control group is administered with the same amount of physiological saline. After the treatment is finished, the expression change of the molecules related to the tissue level of each group of depressed rats is detected, and the result is shown in table 4.

TABLE 4 comparison of the content of monoamine neurotransmitter 5-HT, DA in rat brain tissue (mean. + -. Variance)

* Representing significant differences compared with the model group, P <0.05.

# indicates a significant difference compared to the control group, P <0.05.

The color of the product is marked by a difference in significance compared with that of comparative example 1, wherein P is less than 0.05.

As can be seen from table 4, the control group, the positive group, the treatment group of example 1, the treatment group of example 2, and the treatment group of comparative example 1 were significantly different from the model group, and the modeling was successful.

Example 1, example 2 and comparative example 1, the content of 5-HT and DA detected in the three groups is compared with that of a control group, and no significant difference exists, which indicates that the umbilical cord stem cells can improve the secretion of monoamine neurotransmitters in rat brain tissues; meanwhile, the 5-HT and DA content of rats in the treatment groups of example 1 and example 2 is higher than that of rats in the treatment group of comparative example 1, which shows that the stem cells obtained by the phonological culture can remarkably up-regulate the secretion of monoamine neurotransmitters by the nerve cells compared with the stem cells obtained by the normal culture method.

Finally, it should be noted that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.

Moreover, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the application and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Claims

1. A cell culture method using music, characterized in that music is played in a culture environment of the cells.

2. The cell culture method according to claim 1, wherein the cell is an umbilical cord stem cell.

3. The cell culture method according to claim 1 or 2, wherein the music comprises any one or more of light music, classical music, and orchestral music;

4. The cell culture method according to claim 3, wherein the cells are cultured using a basal medium +5% to 10% nutrient complete medium;

preferably, the cells are cultured using DMEM +10% FBS complete medium.

5. The cell culture method according to claim 4, wherein the culture environment of the cells is 5% CO ₂ 37 ℃ and 95% humidity.

6. The cell culture method according to claim 1 or 2, wherein the volume of the music is 20 to 40 decibels.

7. The cell culture method according to claim 1 or 2, wherein the music is played for a period of 8-12 h/day.

8. An umbilical cord stem cell cultured by the cell culture method using music according to any one of claims 1 to 7;

9. A preparation comprising the umbilical cord stem cells of claim 8.

10. Use of the umbilical cord stem cells of claim 8 or the formulation of claim 9 for the manufacture of a medicament for the treatment of depression, psychiatric disorders.