Stem cell culture solution freeze-drying process

Abstract

The invention discloses a freeze-drying process of a stem cell culture solution. The freeze-drying process comprises the following steps: step 1, preparing stem cell factor freeze-dried powder; placing 35ml of the stem cell-rich factor solution in a 50ml centrifugal tube for centrifugation so as to remove cell debris and other large-particle impurities; step 2, adding sephadex into the centrifuged solution according to the concentration of 4g / 100ml, and soaking for 5.5 hours; 3, filtering by using a 100-mesh screen to remove gel swollen after adsorbing the inorganic salt solution to obtain a stem cell factor concentrated solution, and sub-packaging into penicillin bottles; step 4, finally putting into a freeze-drying machine for freeze-drying; the preparation method comprises the following steps: preparing a concentrated solution, and freeze-drying the concentrated solution, containing a plurality of cell factors secreted by mesenchymal stem cells, intervening an abnormal fat metabolism pathway of a body, reducing triglyceride synthesis and repairing damaged cells through the regulation effect of the cell factors, solving the problems of triglyceride synthesis and decomposition from a metabolism source, and improving the bioavailability of the stem cell factors.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, in particular to a freeze-drying process for stem cell culture fluid. Background Art

[0002] With the improvement of living standards, facial skin care and anti-aging have become an indispensable part of daily life. The root cause of human aging and wrinkles is the slow metabolism and reduced turnover of cells, and the aging and reduction of cells are closely related to stem cells.

[0003] Human stem cell culture supernatant refers to the liquid obtained by filtering the supernatant produced during the culture process of human mesenchymal stem cells and removing the stem cells. The remaining liquid is composed of three parts: culture medium, growth factors and exosomes. This part of the filter is rich in various nutrients, which have a nourishing effect on stem cells. It can keep stem cells alive and proliferate healthily. The human stem cell culture supernatant will be easier to store after freeze-drying.

[0004] However, the existing culture medium freeze-drying process cannot intervene in the body's abnormal fat metabolism pathway to restore fat metabolic balance through the regulation of cytokines, which reduces the bioavailability of stem cell factors. Therefore, we proposed a stem cell culture medium freeze-drying process to solve the above-mentioned problems. Summary of the invention

[0005] The purpose of the present invention is to provide a freeze-drying process for stem cell culture fluid to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A freeze-drying process for stem cell culture fluid comprises the following steps:

[0008] Step 1: Prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities;

[0009] Step 2: Add dextran gel at a concentration of 4 g / 100 ml to the solution obtained after centrifugation, and soak for 5.5 hours;

[0010] Step 3: Use a 100-mesh sieve to filter out the gel swollen by the adsorption of the inorganic salt solution to obtain the stem cell factor concentrate, which is then dispensed into vials;

[0011] Step 4: Finally, put it into the freeze dryer for freeze drying.

[0012] As a further scheme of the present invention: Step 1: prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities. The stem cell factor-rich solution is derived from the culture supernatant of mesenchymal stem cells, that is, the culture supernatant of mesenchymal stem cells is collected when the growth confluence of mesenchymal stem cells reaches 95%.

[0013] As a further solution of the present invention: Step 1: prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities, and the centrifugation time is 10 minutes.

[0014] As a further solution of the present invention: Step 2: Add dextran gel at a concentration of 4g / 100ml to the solution obtained after the above centrifugation, and soak for 5.5h. During the soaking, the inorganic salt molecules will enter the gel mesh, while the cytokine proteins and polypeptides will be excluded from the gel mesh due to their large molecular weight.

[0015] As a further solution of the present invention: Step 3: Use a 100-mesh sieve to filter and remove the gel swollen by adsorption of the inorganic salt solution to obtain a stem cell factor concentrate, which is dispensed into vials with a capacity of 2 ml to 5 ml.

[0016] As a further solution of the present invention: Step 4: finally put it into a freeze dryer for freeze drying, and after the freeze-dried powder is prepared, it is stored at a temperature of -20°C and sealed for storage.

[0017] As a further solution of the present invention: the lyophilized powder also includes a permeable cell cryoprotectant that can penetrate into cells.

[0018] As a further solution of the present invention: the osmotic cell cryoprotectant mainly includes the following components in parts by weight: 3-5 parts of glycerol, 3.5-12 parts of dimethyl sulfoxide, 0.5-5 parts of ethylene glycol, 3-7 parts of propylene glycol, 0.8-3 parts of acetamide, 4-8 parts of methanol, and 68-90 parts of ion buffer solvent.

[0019] As a further solution of the present invention: the permeable cell cryoprotectant mainly includes the following components in parts by weight: 4-6 parts of glycerol, 4-12 parts of dimethyl sulfoxide, 1-6 parts of ethylene glycol, 2-8 parts of propylene glycol, 0.8-5 parts of acetamide, 5-9 parts of methanol, and 65-92 parts of ion buffer solvent.

[0020] As a further solution of the present invention: the osmotic cell cryoprotectant mainly includes the following components in parts by weight: 3.5-7 parts of glycerol, 3-11 parts of dimethyl sulfoxide, 0.8-6.5 parts of ethylene glycol, 3-8 parts of propylene glycol, 0.6-4 parts of acetamide, 5-8 parts of methanol, and 67-87 parts of ion buffer solvent.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The stem cell culture medium is freeze-dried through a concentrated liquid freeze-drying process. It contains a variety of cytokines secreted by mesenchymal stem cells. Through the regulatory effect of cytokines, it intervenes in the body's abnormal fat metabolism pathways, reduces triglyceride synthesis, and repairs damaged cells. It solves the problem of triglyceride synthesis and decomposition from the metabolic source, thereby restoring fat metabolism balance and improving the bioavailability of stem cell factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a schematic diagram of the freeze-drying process of the stem cell culture medium of the present invention. DETAILED DESCRIPTION

[0024] Example 1

[0025] In one embodiment, Figure 1 As shown, a freeze-drying process for stem cell culture fluid comprises:

[0026] Step 1: Prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities;

[0027] Step 2: Add dextran gel at a concentration of 4 g / 100 ml to the solution obtained after centrifugation, and soak for 5.5 hours;

[0028] Step 3: Use a 100-mesh sieve to filter out the gel swollen by the adsorption of the inorganic salt solution to obtain the stem cell factor concentrate, which is then dispensed into vials;

[0029] Step 4: Finally, put it into the freeze dryer for freeze drying;

[0030] The preparation method of dextran gel comprises: weighing dextran, adding purified water, adding alkali, stirring to dissolve, adding a cross-linking agent, stirring and standing, and then performing a cross-linking reaction; adjusting the reaction solution to slightly acidic with an acid, swelling at room temperature, passing through a sieve, repeatedly washing with purified water three times, and then grinding and crushing to form gel particles; adding an osmotic pressure regulator, a pH regulator, and purified water to the gel particles, and stirring at a high speed to obtain a dextran gel;

[0031] The molecular structure of the dextran is composed of 90%-95% D-glucose connected by α-1,6-glycosidic bonds, and the remaining 5%-10% D-glucose connected by α-1,3-glycosidic bonds, and the molecular weight of the dextran ranges from 100 to 2000 KDa; the selected dextran is selected from dextran 20, dextran 40 or dextran 70; dextran 70 is further preferred; the mass concentration of the dextran in the aqueous solution is 10%-60%, preferably 20%-30%;

[0032] The catalyst is selected from alkaline compounds, more preferably, the catalyst is selected from alkali metal hydroxides and / or alkali metal carbonates; further preferably, the catalyst is selected from at least one of sodium hydroxide or potassium hydroxide; preferably, the mass concentration of the catalyst in the aqueous solution is 0.5% to 10%, preferably 0.5% to 1%;

[0033] Step 1: Prepare stem cell factor freeze-dried powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities. The stem cell factor-rich solution is derived from the culture supernatant of mesenchymal stem cells, that is, the culture supernatant of mesenchymal stem cells is collected when the growth confluence of mesenchymal stem cells reaches 95%;

[0034] Step 1: Prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge for 10 minutes to remove cell debris and other large particles;

[0035] Step 2: Add dextran gel at a concentration of 4 g / 100 ml to the solution obtained after the above centrifugation, and soak for 5.5 hours. During the soaking, inorganic salt molecules will enter the gel mesh, while cytokine proteins and polypeptides will be excluded from the gel mesh due to their large molecular weight;

[0036] Step 3: Use a 100-mesh sieve to filter out the gel swollen by the adsorption of the inorganic salt solution to obtain the stem cell factor concentrate, which is dispensed into vials with a capacity of 2 ml to 5 ml.

[0037] Step 4: Finally, freeze-dry the freeze-dried powder in a freeze dryer. After the freeze-dried powder is prepared, it is stored at -20°C and sealed. The freeze-dried powder also includes a permeable cell cryoprotectant that can penetrate into the cells.

[0038] The permeable cell cryoprotectant mainly includes the following components in parts by weight: 4 parts of glycerol, 8 parts of dimethyl sulfoxide, 3.5 parts of ethylene glycol, 5 parts of propylene glycol, 2.1 parts of acetamide, 6 parts of methanol, and 85 parts of ion buffer solvent;

[0039] Example 2

[0040] In one embodiment, Figure 1 As shown, a freeze-drying process for stem cell culture fluid comprises:

[0041] Step 1: Prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities;

[0042] Step 2: Add dextran gel at a concentration of 4 g / 100 ml to the solution obtained after centrifugation, and soak for 5.5 hours;

[0043] Step 3: Use a 100-mesh sieve to filter out the gel swollen by the adsorption of the inorganic salt solution to obtain the stem cell factor concentrate, which is then dispensed into vials;

[0044] Step 4: Finally, put it into the freeze dryer for freeze drying;

[0045] The preparation method of dextran gel comprises: weighing dextran, adding purified water, adding alkali, stirring to dissolve, adding a cross-linking agent, stirring and standing, and then performing a cross-linking reaction; adjusting the reaction solution to slightly acidic with an acid, swelling at room temperature, passing through a sieve, repeatedly washing with purified water three times, and then grinding and crushing to form gel particles; adding an osmotic pressure regulator, a pH regulator, and purified water to the gel particles, and stirring at a high speed to obtain a dextran gel;

[0046] The molecular structure of the dextran is composed of 90%-95% D-glucose connected by α-1,6-glycosidic bonds, and the remaining 5%-10% D-glucose connected by α-1,3-glycosidic bonds, and the molecular weight of the dextran ranges from 100 to 2000 KDa; the selected dextran is selected from dextran 20, dextran 40 or dextran 70; dextran 70 is further preferred; the mass concentration of the dextran in the aqueous solution is 10%-60%, preferably 20%-30%;

[0047] The catalyst is selected from alkaline compounds, more preferably, the catalyst is selected from alkali metal hydroxides and / or alkali metal carbonates; further preferably, the catalyst is selected from at least one of sodium hydroxide or potassium hydroxide; preferably, the mass concentration of the catalyst in the aqueous solution is 0.5% to 10%, preferably 0.5% to 1%;

[0048] Step 1: Prepare stem cell factor freeze-dried powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities. The stem cell factor-rich solution is derived from the culture supernatant of mesenchymal stem cells, that is, the culture supernatant of mesenchymal stem cells is collected when the growth confluence of mesenchymal stem cells reaches 95%;

[0049] Step 1: Prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge for 10 minutes to remove cell debris and other large particles;

[0050] Step 2: Add dextran gel at a concentration of 4 g / 100 ml to the solution obtained after the above centrifugation, and soak for 5.5 hours. During the soaking, inorganic salt molecules will enter the gel mesh, while cytokine proteins and polypeptides will be excluded from the gel mesh due to their large molecular weight;

[0051] Step 3: Use a 100-mesh sieve to filter out the gel swollen by the adsorption of the inorganic salt solution to obtain the stem cell factor concentrate, which is dispensed into vials with a capacity of 2 ml to 5 ml.

[0052] Step 4: Finally, freeze-dry the freeze-dried powder in a freeze dryer. After the freeze-dried powder is prepared, it is stored at -20°C and sealed. The freeze-dried powder also includes a permeable cell cryoprotectant that can penetrate into the cells.

[0053] The permeable cell cryoprotectant mainly includes the following components in parts by weight: 5 parts of glycerol, 8 parts of dimethyl sulfoxide, 3 parts of ethylene glycol, 5 parts of propylene glycol, 3 parts of acetamide, 7 parts of methanol, and 86 parts of ion buffer solvent;

[0054] Example 3

[0055] In one embodiment, Figure 1 As shown, a freeze-drying process for stem cell culture fluid comprises:

[0056] Step 1: Prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities;

[0057] Step 2: Add dextran gel at a concentration of 4 g / 100 ml to the solution obtained after centrifugation, and soak for 5.5 hours;

[0058] Step 3: Use a 100-mesh sieve to filter out the gel swollen by the adsorption of the inorganic salt solution to obtain the stem cell factor concentrate, which is then dispensed into vials;

[0059] Step 4: Finally, put it into the freeze dryer for freeze drying;

[0060] The preparation method of dextran gel comprises: weighing dextran, adding purified water, adding alkali, stirring to dissolve, adding a cross-linking agent, stirring and standing, and then performing a cross-linking reaction; adjusting the reaction solution to slightly acidic with an acid, swelling at room temperature, passing through a sieve, repeatedly washing with purified water three times, and then grinding and crushing to form gel particles; adding an osmotic pressure regulator, a pH regulator, and purified water to the gel particles, and stirring at a high speed to obtain a dextran gel;

[0061] The molecular structure of the dextran is composed of 90%-95% D-glucose connected by α-1,6-glycosidic bonds, and the remaining 5%-10% D-glucose connected by α-1,3-glycosidic bonds, and the molecular weight of the dextran ranges from 100 to 2000 KDa; the selected dextran is selected from dextran 20, dextran 40 or dextran 70; dextran 70 is further preferred; the mass concentration of the dextran in the aqueous solution is 10%-60%, preferably 20%-30%;

[0062] The catalyst is selected from alkaline compounds, more preferably, the catalyst is selected from alkali metal hydroxides and / or alkali metal carbonates; further preferably, the catalyst is selected from at least one of sodium hydroxide or potassium hydroxide; preferably, the mass concentration of the catalyst in the aqueous solution is 0.5% to 10%, preferably 0.5% to 1%;

[0063] Step 1: Prepare stem cell factor freeze-dried powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities. The stem cell factor-rich solution is derived from the culture supernatant of mesenchymal stem cells, that is, the culture supernatant of mesenchymal stem cells is collected when the growth confluence of mesenchymal stem cells reaches 95%;

[0064] Step 1: Prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge for 10 minutes to remove cell debris and other large particles;

[0065] Step 2: Add dextran gel at a concentration of 4 g / 100 ml to the solution obtained after the above centrifugation, and soak for 5.5 hours. During the soaking, inorganic salt molecules will enter the gel mesh, while cytokine proteins and polypeptides will be excluded from the gel mesh due to their large molecular weight;

[0066] Step 3: Use a 100-mesh sieve to filter out the gel swollen by the adsorption of the inorganic salt solution to obtain the stem cell factor concentrate, which is dispensed into vials with a capacity of 2 ml to 5 ml.

[0067] Step 4: Finally, freeze-dry the freeze-dried powder in a freeze dryer. After the freeze-dried powder is prepared, it is stored at -20°C and sealed. The freeze-dried powder also includes a permeable cell cryoprotectant that can penetrate into the cells.

[0068] The permeable cell cryoprotectant mainly includes the following components in parts by weight: 5 parts of glycerol, 6.5 parts of dimethyl sulfoxide, 4.2 parts of ethylene glycol, 5 parts of propylene glycol, 2.2 parts of acetamide, 6.5 parts of methanol, and 88 parts of ion buffer solvent;

[0069] The present invention is freeze-dried from a concentrated solution, which contains cytokines secreted by a variety of mesenchymal stem cells. The cytokines can regulate the body's abnormal fat metabolism pathways, reduce triglyceride synthesis, and repair damaged cells, thereby resolving triglyceride synthesis and decomposition from the source of metabolism, thereby restoring fat metabolism balance and improving the bioavailability of stem cell factors.

[0070] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A freeze-drying process for stem cell culture fluid, characterized in that: The following steps are involved: Step 1: Prepare stem cell factor lyophilized powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities; Step 2: Add dextran gel at a concentration of 4 g / 100 ml to the solution obtained after centrifugation, and soak for 5.5 hours; Step 3: Use a 100-mesh sieve to filter out the gel swollen by the adsorption of the inorganic salt solution to obtain the stem cell factor concentrate, which is then dispensed into vials; Step 4: Finally, put it into the freeze dryer for freeze drying.

2. A freeze-drying process for stem cell culture fluid according to claim 1, characterized in that: The step 1 is to prepare the stem cell factor freeze-dried powder; place 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuge to remove cell debris and other large particle impurities. The stem cell factor-rich solution is derived from the culture supernatant of mesenchymal stem cells, that is, the culture supernatant of mesenchymal stem cells is collected when the growth confluence of mesenchymal stem cells reaches 95%.

3. A freeze-drying process for stem cell culture fluid according to claim 1, characterized in that: The step 1: preparing stem cell factor lyophilized powder; placing 35 ml of stem cell factor-rich solution in a 50 ml centrifuge tube and centrifuging to remove cell debris and other large particle impurities, and the centrifugation time is 10 minutes.

4. A freeze-drying process for stem cell culture fluid according to claim 1, characterized in that: The second step is to add dextran gel at a concentration of 4 g / 100 ml to the solution obtained after the centrifugation, and soak for 5.5 hours. During the soaking, inorganic salt molecules will enter the gel mesh, while cytokine proteins and polypeptides will be excluded from the gel mesh due to their large molecular weight.

5. A freeze-drying process for stem cell culture fluid according to claim 1, characterized in that: Step three: filtering with a 100-mesh screen to remove the gel swollen by the adsorption of the inorganic salt solution, obtaining a stem cell factor concentrate, and dispensing the concentrate into vials with a capacity of 2 ml to 5 ml.

6. A freeze-drying process for stem cell culture fluid according to claim 1, characterized in that: Step 4: finally putting the freeze-dried powder into a freeze dryer for freeze drying. After the freeze-dried powder is prepared, it is stored at a temperature of -20°C and sealed.

7. A freeze-drying process for stem cell culture fluid according to claim 1, characterized in that: The freeze-dried powder also includes a permeable cell cryoprotectant that can penetrate into cells.

8. A freeze-drying process for stem cell culture fluid according to claim 7, characterized in that: The permeable cell cryoprotectant mainly includes the following components in parts by weight: 3-5 parts of glycerol, 3.5-12 parts of dimethyl sulfoxide, 0.5-5 parts of ethylene glycol, 3-7 parts of propylene glycol, 0.8-3 parts of acetamide, 4-8 parts of methanol, and 68-90 parts of ion buffer solvent.

9. A freeze-drying process for stem cell culture fluid according to claim 8, characterized in that: The permeable cell cryoprotectant mainly includes the following components in parts by weight: 4-6 parts of glycerol, 4-12 parts of dimethyl sulfoxide, 1-6 parts of ethylene glycol, 2-8 parts of propylene glycol, 0.8-5 parts of acetamide, 5-9 parts of methanol, and 65-92 parts of ion buffer solvent.

10. A freeze-drying process for stem cell culture fluid according to claim 9, characterized in that: The permeable cell cryoprotectant mainly includes the following components in parts by weight: 3.5-7 parts of glycerol, 3-11 parts of dimethyl sulfoxide, 0.8-6.5 parts of ethylene glycol, 3-8 parts of propylene glycol, 0.6-4 parts of acetamide, 5-8 parts of methanol, and 67-87 parts of ion buffer solvent.

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