Ips cell culture medium and culture method

Abstract

The invention discloses an ips cell culture medium and a culture method, and aims to solve the technical problems that the current ips cell culture medium is slow in growth speed and relatively low in activity in the ips cell culture process, and the ips cells are easily damaged due to osmotic pressure change, shear force, oxidation and bubble action in the culture process. Comprising the following raw materials in percentage by weight: 90%-91% of water; the mass part of amino acid is 1%-2%; the mass part of vitamins is 1%-2%; 2%-5% of carbohydrate by mass; and the mass part of the inorganic salt is 1-3%. The ips cell culture medium provided by the invention can provide a necessary energy source, support cell energy metabolism, maintain osmotic pressure and pH value of cells, maintain the pH value of a culture solution in a range suitable for cell growth, supplement amino acids in the cell culture medium, promote cell growth and activity, and improve cell quality. And moreover, the phenomenon that the ips cells are damaged due to osmotic pressure change, shearing force, oxidation and bubble action in the culture process is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of IPS cell culture, in particular to an IPS cell culture medium and a culture method. Background Art

[0002] Stem cells (embryonic stem cells, artificial pluripotent stem cells (iPS cells), etc.) are cultured using culture media containing serum. For example, fetal bovine serum (FBS) is widely used as an important additive for cell proliferation. However, when cultured stem cells are used for medical purposes, components from foreign species may become a source of infection with blood-borne pathogens or foreign antigens. In addition, there is the possibility that differences between serum batches may cause deviations in culture results. Therefore, in recent years, the use of culture media with clear chemical compositions to culture stem cells has gradually become the mainstream.

[0003] In the process of realizing the invention, the inventors found that there are at least the following problems in the prior art that have not been solved: the traditional iPS cell culture medium has a slow growth rate and low activity during the iPS cell culture process, and iPS cells are easily damaged by osmotic pressure changes, shear force, oxidation and bubble effects during the culture process. Therefore, it is necessary to design a new technical solution to solve the problem. Summary of the invention

[0004] The purpose of the present invention is to provide an iPS cell culture medium and a culture method to solve the technical problems that the current iPS cell culture medium has a slow growth rate and relatively low activity during the iPS cell culture process, and the iPS cells are easily damaged by osmotic pressure changes, shear force, oxidation and bubble effects during the culture process.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: an IPS cell culture medium and a culture method, comprising the following raw materials and their weight fractions: 90%-91% by weight of water; 1%-2% by weight of amino acids; 1%-2% by weight of vitamins; 2%-5% by weight of carbohydrates; 1%-3% by weight of inorganic salts; 2%-4% by weight of optimized nutrients and 1%-3% by weight of protective agents.

[0006] As a preferred embodiment of the present invention, the IPS cell culture medium includes the following raw materials and their weight fractions: 91% by mass of water; 1% by mass of amino acids; 1% by mass of vitamins; 2% by mass of carbohydrates; 1% by mass of inorganic salts; 2% by mass of optimized nutrients and 2% by mass of protective agents.

[0007] As a preferred embodiment of the present invention, the IPS cell culture medium comprises the following raw materials and their weight fractions: 90% by mass of water; 1.5% by mass of amino acids; 1.5% by mass of vitamins; 3% by mass of carbohydrates; 1% by mass of inorganic salts; 3% by mass of optimized nutrients and 3% by mass of protective agents.

[0008] As a preferred embodiment of the present invention, the culturing method of the iPS cell culture medium comprises the following culturing steps:

[0009] S1. First, coat the culture dish with Matrix;

[0010] S2. After taking out the cryotube from the liquid nitrogen tank, quickly transfer it to the biosafety cabinet and thaw it with the heated complete medium. Then centrifuge the thawed medium using a centrifuge. After centrifugation, remove the supernatant and add an appropriate amount of iCell complete thawed medium. After gently blowing and aspirating, add it to the culture dish and place it in a 37°C incubator for 24 hours before replacing it with the complete medium.

[0011] S3. Culture the iPS cells using iPS cell culture medium;

[0012] S4. Discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium and magnesium ions;

[0013] S5. Add digestion solution to the culture bottle and place it in a 37°C incubator for 1-2 minutes. Observe the cell digestion and terminate the digestion appropriately.

[0014] S6. Gently blow the cells again, aspirate the cell suspension, remove the supernatant after centrifugation, add culture medium and divide into new culture dishes according to proportion;

[0015] S7. Coat the culture dish again, then aspirate the culture medium, wash with PBS and add freezing solution, finally divide the cell suspension into cryopreservation tubes and cryopreserve according to the standard freezing procedure.

[0016] As a preferred embodiment of the present invention, the Matrix is ​​stored at -20°C and thawed in a 4°C refrigerator or on ice before use. After the Matrix is ​​thawed, it is rapidly diluted with PBS liquid in proportion, added to a culture dish, and placed in a 37°C incubator for 4 hours. The coating liquid is removed before use.

[0017] As a preferred embodiment of the present invention, the main components of the inorganic salt are Na+, K+, Ca2+, Mg2+, Cl-, PO43-, SO42- and HCO3-.

[0018] As a preferred embodiment of the present invention, the main components of the optimized nutrients are L-glutamine, sodium pyruvate, Hank's balanced salt solution, NaHCO3-CO2 buffer system and MEM non-essential amino acids.

[0019] As a preferred embodiment of the present invention, the protective agent is serum, albumin, polyethylene glycol (PEG), non-ionic surfactant Pluronic F68 or one of other high molecular polymers.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The iPS cell culture medium of the present invention can provide necessary energy sources, support cell energy metabolism, maintain the osmotic pressure and pH value of cells, keep the pH value of the culture solution within a range suitable for cell growth, and supplement the amino acids in the cell culture medium, thereby promoting cell growth and activity, and avoids the phenomenon that iPS cells are damaged by osmotic pressure changes, shear force, oxidation and bubble effects during the culture process, thereby improving the survival rate of iPS cells during the culture process. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0023] Embodiment 1: An iPS cell culture medium and a culture method, comprising the following raw materials and their weight fractions: 90%-91% by weight of water; 1%-2% by weight of amino acids; 1%-2% by weight of vitamins; 2%-5% by weight of carbohydrates; 1%-3% by weight of inorganic salts; 2%-4% by weight of optimized nutrients and 1%-3% by weight of protective agents.

[0024] Specifically, the IPS cell culture medium includes the following raw materials and their weight fractions: 91% by weight of water; 1% by weight of amino acids; 1% by weight of vitamins; 2% by weight of carbohydrates; 1% by weight of inorganic salts; 2% by weight of optimized nutrients and 2% by weight of protective agents.

[0025] Furthermore, the IPS cell culture medium includes the following raw materials and their weight fractions: 90% by weight of water; 1.5% by weight of amino acids; 1.5% by weight of vitamins; 3% by weight of carbohydrates; 1% by weight of inorganic salts; 3% by weight of optimized nutrients and 3% by weight of protective agents.

[0026] Furthermore, the culturing method of the iPS cell culture medium comprises the following culturing steps:

[0027] S1. First, coat the culture dish with Matrix;

[0028] S2. After taking out the cryotube from the liquid nitrogen tank, quickly transfer it to the biosafety cabinet and thaw it with the heated complete medium. Then centrifuge the thawed medium using a centrifuge. After centrifugation, remove the supernatant and add an appropriate amount of iCell complete thawed medium. After gently blowing and aspirating, add it to the culture dish and place it in a 37°C incubator for 24 hours before replacing it with the complete medium.

[0029] S3. Culture the iPS cells using iPS cell culture medium;

[0030] S4. Discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium and magnesium ions;

[0031] S5. Add digestion solution to the culture bottle and place it in a 37°C incubator for 1-2 minutes. Observe the cell digestion and terminate the digestion appropriately.

[0032] S6. Gently blow the cells again, aspirate the cell suspension, remove the supernatant after centrifugation, add culture medium and divide into new culture dishes according to proportion;

[0033] S7. Coat the culture dish again, then aspirate the culture medium, wash with PBS and add freezing solution, finally divide the cell suspension into cryopreservation tubes and cryopreserve according to the standard freezing procedure.

[0034] It is worth noting that the Matrix is ​​stored at -20°C and thawed in a 4°C refrigerator or on ice before use. After the Matrix is ​​thawed, it is quickly diluted with PBS liquid in proportion, added to a culture dish, and placed in a 37°C incubator for 4 hours. The coating liquid is removed before use.

[0035] It is worth noting that the main components of the inorganic salt are Na+, K+, Ca2+, Mg2+, Cl-, PO43-, SO42- and HCO3-.

[0036] It is worth mentioning that the main components of the optimized nutrients are L-glutamine, sodium pyruvate, Hank's balanced salt solution, NaHCO3-CO2 buffer system and MEM non-essential amino acids.

[0037] It is worth emphasizing that the protective agent is serum, albumin, polyethylene glycol (PEG), non-ionic surfactant Pluronic F68 or one of other high molecular polymers.

[0038] Embodiment 1

[0039] The IPS cell culture medium comprises the following raw materials and their weight fractions: 91% by weight of water; 1% by weight of amino acid; 1% by weight of vitamin; 2% by weight of carbohydrate; 1% by weight of inorganic salt; 2% by weight of optimized nutrients and 2% by weight of protective agent.

[0040] The cultivation steps are as follows:

[0041] S1. First, coat the culture dish with Matrix;

[0042] S2. After taking out the cryotube from the liquid nitrogen tank, quickly transfer it to the biosafety cabinet and thaw it with the heated complete medium. Then centrifuge the thawed medium using a centrifuge. After centrifugation, remove the supernatant and add an appropriate amount of iCell complete thawed medium. After gently blowing and aspirating, add it to the culture dish and place it in a 37°C incubator for 24 hours before replacing it with the complete medium.

[0043] S3. Culture the iPS cells using iPS cell culture medium;

[0044] S4. Discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium and magnesium ions;

[0045] S5. Add digestion solution to the culture bottle and place it in a 37°C incubator for 1-2 minutes. Observe the cell digestion and terminate the digestion appropriately.

[0046] S6. Gently blow the cells again, aspirate the cell suspension, remove the supernatant after centrifugation, add culture medium and divide into new culture dishes according to proportion;

[0047] S7. Coat the culture dish again, then aspirate the culture medium, wash with PBS and add freezing solution, finally divide the cell suspension into cryopreservation tubes and cryopreserve according to the standard freezing procedure.

[0048] Embodiment 2

[0049] The IPS cell culture medium comprises the following raw materials and their weight fractions: 90% by weight of water; 1.5% by weight of amino acids; 1.5% by weight of vitamins; 3% by weight of carbohydrates; 1% by weight of inorganic salts; 3% by weight of optimized nutrients and 3% by weight of protective agents.

[0050] The cultivation steps are as follows:

[0051] S1. First, coat the culture dish with Matrix;

[0052] S2. After taking out the cryotube from the liquid nitrogen tank, quickly transfer it to the biosafety cabinet and thaw it with the heated complete medium. Then centrifuge the thawed medium using a centrifuge. After centrifugation, remove the supernatant and add an appropriate amount of iCell complete thawed medium. After gently blowing and aspirating, add it to the culture dish and place it in a 37°C incubator for 24 hours before replacing it with the complete medium.

[0053] S3. Culture the iPS cells using iPS cell culture medium;

[0054] S4. Discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium and magnesium ions;

[0055] S5. Add digestion solution to the culture bottle and place it in a 37°C incubator for 1-2 minutes. Observe the cell digestion and terminate the digestion appropriately.

[0056] S6. Gently blow the cells again, aspirate the cell suspension, remove the supernatant after centrifugation, add culture medium and divide into new culture dishes according to proportion;

[0057] S7. Coat the culture dish again, then aspirate the culture medium, wash with PBS and add freezing solution, finally divide the cell suspension into cryopreservation tubes and cryopreserve according to the standard freezing procedure.

[0058] The data parameters of traditional skin repair fluid are shown in Table 1:

[0059]

[0060] Example 1 Skin repair liquid data parameter table 2 is as follows:

[0061]

[0062] Example 2 Skin repair liquid data parameter table 3 is as follows:

[0063]

[0064] In summary, referring to the data comparison of Table 1, Table 2 and Table 3, it can be concluded that the ips cell culture medium of the present invention can provide the necessary energy source, support cell energy metabolism, maintain the osmotic pressure and pH value of the cells, keep the pH value of the culture solution within a range suitable for cell growth, and supplement the amino acids in the cell culture medium, thereby promoting cell growth and activity, and avoiding the phenomenon that ips cells are damaged by osmotic pressure changes, shear force, oxidation and bubble effects during the culture process, thereby improving the survival rate of ips cells during the culture process.

[0065] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0066] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An iPS cell culture medium, characterized in that: The invention comprises the following raw materials and their weight fractions: 90%-91% by weight of water; 1%-2% by weight of amino acid; 1%-2% by weight of vitamin; 2%-5% by weight of carbohydrate; 1%-3% by weight of inorganic salt; 2%-4% by weight of optimized nutrient substance and 1%-3% by weight of protective agent.

2. An iPS cell culture medium according to claim 1, characterized in that: The invention comprises the following raw materials and their weight fractions: 91% by weight of water; 1% by weight of amino acid; 1% by weight of vitamin; 2% by weight of carbohydrate; 1% by weight of inorganic salt; 2% by weight of optimized nutrient substance and 2% by weight of protective agent.

3. An iPS cell culture medium according to claim 1, characterized in that: The invention comprises the following raw materials and their weight fractions: 90% by weight of water; 1.5% by weight of amino acid; 1.5% by weight of vitamin; 3% by weight of carbohydrate; 1% by weight of inorganic salt; 3% by weight of optimized nutrient substance and 3% by weight of protective agent.

4. The method for culturing an iPS cell culture medium according to claim 1, characterized in that: The cultivation steps are as follows: S1. First, coat the culture dish with Matrix; S2. After taking out the cryotube from the liquid nitrogen tank, quickly transfer it to the biosafety cabinet and thaw it with the heated complete medium. Then centrifuge the thawed medium using a centrifuge. After centrifugation, remove the supernatant and add an appropriate amount of iCell complete thawed medium. After gently blowing and aspirating, add it to the culture dish and place it in a 37°C incubator for 24 hours before replacing it with the complete medium. S3. Culture the iPS cells using iPS cell culture medium; S4. Discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium and magnesium ions; S5. Add digestion solution to the culture bottle and place it in a 37°C incubator for 1-2 minutes. Observe the cell digestion and terminate the digestion appropriately. S6. Gently blow the cells again, aspirate the cell suspension, remove the supernatant after centrifugation, add culture medium and divide into new culture dishes according to proportion; S7. Coat the culture dish again, then aspirate the culture medium, wash with PBS and add freezing solution, finally divide the cell suspension into cryopreservation tubes and cryopreserve according to the standard freezing procedure.

5. The method for culturing an iPS cell culture medium according to claim 4, characterized in that: The matrix is ​​stored at -20°C and thawed in a 4°C refrigerator or on ice before use. After the matrix is ​​thawed, it is quickly diluted with PBS liquid in proportion, added to a culture dish, and placed in a 37°C incubator for 4 hours. The coating liquid is removed before use.

6. The method for culturing an iPS cell culture medium according to claim 4, characterized in that: The main components of the inorganic salt are Na+, K+, Ca2+, Mg2+, Cl-, PO43-, SO42- and HCO3-.

7. The method for culturing an iPS cell culture medium according to claim 1, characterized in that: The main components of the optimized nutrients are L-glutamine, sodium pyruvate, Hank's balanced salt solution, NaHCO3-CO2 buffer system and MEM non-essential amino acids.

8. The method for culturing an iPS cell culture medium according to claim 1, characterized in that: The protective agent is one of serum, albumin, polyethylene glycol (PEG), non-ionic surfactant Pluronic F68 or other high molecular polymers.

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