Preparation method and application of repair and regeneration type stem cells for renal injury

Abstract

The invention relates to the technical field of stem cell preparation. The invention provides a preparation method and application of repair and regeneration type stem cells for renal injury, and the preparation method comprises the following steps: (1) inoculating stem cells into a first culture medium, and carrying out primary culture to obtain a primary cell population; and (2) transferring the primary cell population into a second culture medium, and continuously culturing to obtain the repaired and regenerated stem cells. According to the preparation method, the problems existing in an existing culture medium and an existing culture method are solved, the used culture medium does not need to be frequently replaced, and the GMP clinical-grade production standard is met; and the dynamic environment parameter design in the preparation process is adaptive to automatic equipment, so that the manual operation cost is remarkably reduced. Metabolic regulation and control of the culture medium are dynamically and deeply combined with environmental parameters, on the premise that complex equipment does not need to be transformed, the problems that in a traditional culture method, dryness is difficult to maintain, cost is high, and operation is tedious are solved, and the method has extremely high industrialization landing potential.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell preparation, and in particular to a preparation method and application of repair and regeneration stem cells for kidney damage. Background Art

[0002] In existing stem cell culture processes, traditional serum-free culture media rely on high concentrations of exogenous growth factors, which are costly and can easily trigger the activation of abnormal signaling pathways (such as tumorigenicity). Existing metabolic additives, however, have fixed concentrations and cannot dynamically adapt to the metabolic needs of stem cells at different stages of proliferation. Commercially available, ready-made serum-free culture media require frequent replacement to maintain nutrients, which is cumbersome and prone to contamination.

[0003] During the culture process, a static atmosphere can easily lead to the accumulation of oxidative stress, accelerating stem cell aging. Constant temperature and humidity conditions, on the other hand, fail to mimic physiological fluctuations in the body, reducing the stability of stemness-related genes. While hypoxic culture can delay differentiation, prolonged hypoxia inhibits mitochondrial function, leading to decreased proliferation efficiency.

[0004] It can be seen that the existing culture media and culture methods have some problems that restrict the development of stem cells. Therefore, the development of new culture methods is particularly important. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of repair and regenerative stem cells for kidney damage, improve the problems existing in existing culture media and culture methods, and cultivate high-quality repair and regenerative stem cells.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing repair and regenerative stem cells for kidney damage, comprising the following steps:

[0008] (1) inoculating stem cells into a first culture medium and performing preliminary culture to obtain a primary cell population;

[0009] (2) The primary cell population is transferred to a second culture medium and cultured to obtain repair and regeneration stem cells.

[0010] Preferably, the type of stem cells in step (1) is mesenchymal stem cells, which are derived from human fat, bone marrow, dental pulp, umbilical cord, placenta or umbilical cord blood.

[0011] Preferably, in step (1), the first culture medium is AIM-V culture medium.

[0012] Preferably, the temperature of the preliminary culture in step (1) is 36-39° C., the oxygen concentration is 10-13%, and the carbon dioxide concentration is 2.5-3.5%.

[0013] Preferably, the initial culture time in step (1) is 2 to 4 days.

[0014] Preferably, the second culture medium in step (2) is based on AIM-V culture medium, with additional additions of 1-3 mM pyruvate, 1.5-2.5 mM taurine, 0.5-1 μM CHIR9902, 8-12 nM trichostatin A, and 0.5-0.8 g / L glucose.

[0015] Preferably, the temperature for continuing the culture in step (2) is 36-39° C., and the carbon dioxide concentration is 2.5-3.5%;

[0016] During the continued cultivation, low temperature treatment is performed every 10 to 12 hours, the temperature of the low temperature treatment is 30 to 33° C., and the time of the low temperature treatment is 20 to 30 minutes;

[0017] The oxygen concentration during the continued culture process changes once every 8 hours. In each cycle, the oxygen concentration of 10-13% is maintained for 4-6 hours, and the oxygen concentration of 4-6% is maintained for 2-4 hours.

[0018] Preferably, the time for continuing the culture in step (1) is 3 to 7 days.

[0019] The present invention also provides repair and regeneration stem cells prepared by the preparation method.

[0020] The present invention also provides the use of the repair and regeneration stem cells in preparing products for treating kidney damage.

[0021] The present invention provides a preparation method and application of repair and regenerative stem cells for kidney injury, comprising the following steps: (1) inoculating stem cells into a first culture medium, and preliminarily culturing to obtain a primary cell group; (2) transferring the primary cell group to a second culture medium, and continuing to culture to obtain repair and regenerative stem cells. The preparation method of the present invention improves the problems existing in existing culture media and culture methods. The culture medium used does not require frequent liquid changes and meets GMP clinical-grade production standards; the dynamic environmental parameter design in the preparation process is adapted to automated equipment, which significantly reduces manual operation costs. By deeply combining the metabolic regulation of the culture medium with the dynamicization of environmental parameters, the present invention solves the pain points of traditional culture methods such as difficulty in maintaining stemness, high cost, and cumbersome operation without the need for complex equipment modification, and has extremely strong potential for industrialization. DETAILED DESCRIPTION

[0022] The present invention provides a method for preparing repair and regenerative stem cells for kidney damage, comprising the following steps:

[0023] (1) inoculating stem cells into a first culture medium and performing preliminary culture to obtain a primary cell population;

[0024] (2) The primary cell population is transferred to a second culture medium and cultured to obtain repair and regeneration stem cells.

[0025] Preferably, the type of stem cells in step (1) is mesenchymal stem cells, and the mesenchymal stem cells are preferably derived from human fat, bone marrow, dental pulp, umbilical cord, placenta or umbilical cord blood.

[0026] Preferably, the first culture medium in step (1) is AIM-V culture medium.

[0027] Preferably, the temperature of the preliminary culture in step (1) is preferably 36-39° C., more preferably 37-38° C., the oxygen concentration is preferably 10-13%, more preferably 11-12%, and the carbon dioxide concentration is preferably 2.5-3.5%, more preferably 3%.

[0028] Preferably, the initial culture time in step (1) is preferably 2 to 4 days, more preferably 3 days.

[0029] Preferably, the second culture medium in step (2) is preferably based on AIM-V culture medium, with additional additions of 1-3 mM pyruvate, 1.5-2.5 mM taurine, 0.5-1 μM CHIR99021, 8-12 nM trichostatin A, and 0.5-0.8 g / L glucose, and more preferably 2 mM pyruvate, 2 mM taurine, 0.7-0.8 μM CHIR99021, 10 nM trichostatin A, and 0.6-0.7 g / L glucose.

[0030] Preferably, the temperature for continued cultivation in step (2) is preferably 36-39°C, more preferably 37-38°C, and the carbon dioxide concentration is preferably 2.5-3.5%, more preferably 3%;

[0031] During the continued cultivation, the low-temperature treatment is preferably performed every 10 to 12 hours. The temperature of the low-temperature treatment is preferably 30 to 33°C, more preferably 31 to 32°C, and the time of the low-temperature treatment is preferably 20 to 30 minutes, more preferably 25 minutes.

[0032] The oxygen concentration in the continued culture process is preferably changed once in a cycle of 8 hours, and in each cycle the oxygen concentration condition of 10-13% is maintained for 4-6 hours, and the oxygen concentration condition of 4-6% is maintained for 2-4 hours. It is further preferably changed once in a cycle of 8 hours, and in each cycle the oxygen concentration condition of 11-12% is maintained for 5 hours, and the oxygen concentration condition of 5% is maintained for 3 hours.

[0033] Preferably, the continued culturing time in step (1) is preferably 3 to 7 days, more preferably 5 days.

[0034] The present invention also provides repair and regeneration stem cells prepared by the preparation method.

[0035] The present invention also provides the use of the repair and regeneration stem cells in preparing products for treating kidney damage.

[0036] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] A method for preparing repair and regenerative stem cells for kidney damage comprises the following steps:

[0039] (1) Mesenchymal stem cells (derived from bone marrow) were inoculated into AIM-V medium (Thermo Fisher Scientific) and cultured at 36°C, 10% oxygen concentration, and 3.5% carbon dioxide concentration. Primary cell populations were obtained after 4 days of initial culture.

[0040] (2) AIM-V medium (Thermo Fisher Scientific) was used as the base, and pyruvate 3 mM, taurine 1.5 mM, CHIR9902 10.5 μM, trichostatin A 12 nM, and glucose 0.8 g / L were added to obtain the second medium;

[0041] (3) The primary cell population was transferred to a second culture medium and continued to be cultured at 36°C and a carbon dioxide concentration of 3.5%. During the continued culture, the oxygen concentration was changed once every 8 hours. In each cycle, the oxygen concentration was maintained at 13% for 4 hours and at 6% for 4 hours. At the same time, a low-temperature treatment was performed every 12 hours, and the temperature was lowered to 30°C during the low-temperature treatment and maintained for 30 minutes each time.

[0042] (4) The culture process is continued for 7 days to obtain repair and regeneration stem cells.

[0043] Example 2

[0044] A method for preparing repair and regenerative stem cells for kidney damage comprises the following steps:

[0045] (1) Mesenchymal stem cells (derived from placenta) were inoculated into AIM-V medium (Thermo Fisher Scientific) and cultured at 39°C, with an oxygen concentration of 13% and a carbon dioxide concentration of 2.5%. Primary cell populations were obtained after 2 days of initial culture.

[0046] (2) AIM-V medium (Thermo Fisher Scientific) was used as the base, and 1 mM pyruvate, 2.5 mM taurine, 11 μM CHIR9902, 8 nM trichostatin A, and 0.5 g / L glucose were added to obtain the second medium;

[0047] (3) The primary cell population was transferred to a second culture medium and continued to be cultured at 39°C and a carbon dioxide concentration of 2.5%. During the continued culture, the oxygen concentration was changed once every 8 hours. In each cycle, the oxygen concentration was maintained at 10% for 6 hours and at 4% for 2 hours. At the same time, a low-temperature treatment was performed every 10 hours, and the temperature was lowered to 33°C during the low-temperature treatment and maintained for 20 minutes each time.

[0048] (4) The culture process is continued for 3 days to obtain repair and regeneration stem cells.

[0049] Example 3

[0050] A method for preparing repair and regenerative stem cells for kidney damage comprises the following steps:

[0051] (1) Mesenchymal stem cells (derived from umbilical cord blood) were inoculated into AIM-V medium (Thermo Fisher Scientific) and cultured at 37°C, with an oxygen concentration of 12% and a carbon dioxide concentration of 3%. Primary cell populations were obtained after 3 days of initial culture.

[0052] (2) AIM-V medium (Thermo Fisher Scientific) was used as the base, and 2 mM pyruvate, 2 mM taurine, 10.8 μM CHIR9902, 10 nM trichostatin A, and 0.6 g / L glucose were added to obtain the second medium;

[0053] (3) The primary cell population was transferred to a second culture medium and cultured at 37°C under conditions of 3% carbon dioxide concentration. During the continued culture, the oxygen concentration was changed once every 8 hours. In each cycle, the oxygen concentration was maintained at 12% for 5 hours and at 5% for 3 hours. At the same time, a low-temperature treatment was performed every 11 hours, and the temperature was lowered to 32°C during the low-temperature treatment and maintained for 25 minutes each time.

[0054] (4) The culture process is continued for 5 days to obtain repair and regeneration stem cells.

[0055] Test Example 1: Kidney Injury Treatment Test

[0056] 1. Forty SD male rats weighing 200 ± 10 g were purchased from Shanghai Slake Laboratory Animal Co., Ltd. Before the experiment, the rats were randomly divided into four groups of 10 rats each, numbered as experimental groups 1 to 3 and a control group.

[0057] 2. A rat renal injury model was established using oleic acid in rats from experimental groups 1-3. The specific method was as follows: After anesthetizing the rats, a midline incision was made in the abdomen, the left renal artery was isolated, and oleic acid was injected via the left renal artery at a standard dose of 0.15 mL / kg. Rats in the control group were injected with normal saline using the same method and standard dose.

[0058] 3. 24 hours after the injection, the rats in experimental groups 1 to 3 were tested and found to have obvious renal ischemia, vascular endothelial damage, elevated plasma BUN and SCr, and fat embolism, indicating successful modeling. The rats in the control group had no obvious abnormalities.

[0059] 4. After the model was successfully established, the repair and regeneration stem cells prepared in Example 1 were injected into the tail vein of the rats in the experimental group 1. Each rat was injected with 1.5×10 6 Rats in test group 2 were treated with the repair and regeneration stem cells prepared in Example 2, and rats in test group 3 were treated with the repair and regeneration stem cells prepared in Example 3, according to the same methods and standards. An equal volume of normal saline was injected into the tail vein of the control group rats. Fifteen days after the first injection, a second injection was performed according to the same methods and standards.

[0060] Test results:

[0061] Fifteen days after the injection of the regenerative stem cells, rats in experimental groups 1-3 were retested and found to have significantly improved renal ischemic symptoms, with endothelial damage and tubular necrosis halted and improved. Plasma BUN and SCr levels recovered, and fat embolism was alleviated. Forty days after the injection, no significant differences were observed between the rats in experimental groups 1-3 and the control group. Furthermore, none of the rats in these groups showed any damage to other organs, which is common during oleic acid modeling.

[0062] As can be seen from the above embodiments, the present invention provides a preparation method and application of repair and regenerative stem cells for kidney injury, comprising the following steps: (1) inoculating stem cells into a first culture medium and performing preliminary culture to obtain a primary cell group; (2) transferring the primary cell group to a second culture medium and continuing to culture to obtain repair and regenerative stem cells. The preparation method of the present invention improves the problems existing in existing culture media and culture methods. The culture medium used does not require frequent liquid changes and meets GMP clinical-grade production standards. The dynamic environmental parameter design in the preparation process is adapted to automated equipment, which significantly reduces manual operation costs. By deeply combining the metabolic regulation of the culture medium with the dynamicization of environmental parameters, the present invention solves the pain points of traditional culture methods such as difficulty in maintaining stemness, high cost, and cumbersome operation without the need for complex equipment modification, and has extremely strong potential for industrialization.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing repair and regenerative stem cells for kidney damage, characterized in that: The steps include: (1) inoculating stem cells into a first culture medium and performing preliminary culture to obtain a primary cell population; (2) The primary cell population is transferred to a second culture medium and cultured to obtain repair and regeneration stem cells.

2. The method according to claim 1, characterized in that The type of stem cells in step (1) is mesenchymal stem cells, which are derived from human fat, bone marrow, dental pulp, umbilical cord, placenta or umbilical cord blood.

3. The method according to claim 2, characterized in that In step (1), the first culture medium is AIM-V culture medium.

4. The method according to claim 3, characterized in that The temperature of the initial culture in step (1) is 36-39° C., the oxygen concentration is 10-13%, and the carbon dioxide concentration is 2.5-3.5%.

5. The method according to claim 4, characterized in that The initial culture time in step (1) is 2 to 4 days.

6. The method according to claim 5, characterized in that In step (2), the second culture medium is based on AIM-V culture medium, and is additionally supplemented with 1-3 mM pyruvate, 1.5-2.5 mM taurine, 0.5-1 μM CHIR9902, 8-12 nM trichostatin A, and 0.5-0.8 g / L glucose.

7. The method according to claim 6, characterized in that The temperature for continuing the culture in step (2) is 36-39° C. and the carbon dioxide concentration is 2.5-3.5%; During the continued cultivation, low temperature treatment is performed every 10 to 12 hours, the temperature of the low temperature treatment is 30 to 33° C., and the time of the low temperature treatment is 20 to 30 minutes; The oxygen concentration during the continued culture process changes once every 8 hours. In each cycle, the oxygen concentration of 10-13% is maintained for 4-6 hours, and the oxygen concentration of 4-6% is maintained for 2-4 hours.

8. The method according to any one of claims 1 to 7, characterized in that The time for continuing the culture in step (1) is 3 to 7 days.

9. Repair and regeneration stem cells obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the repair and regeneration stem cells according to claim 9 in the preparation of products for treating kidney damage.