Stem cell combined protection liquid as well as preparation method and application thereof

Abstract

The invention discloses a stem cell combined protection solution as well as a preparation method and application thereof, and particularly relates to the technical field of biological medicines. The protective liquid is prepared from 5.5 mg / ml to 6.5 mg / ml of sodium chloride, 0.2 mg / ml to 0.4 mg / ml of potassium chloride, 2.5 mg / ml to 3.5 mg / ml of sodium lactate, 0.1 mg / ml to 0.3 mg / ml of calcium chloride, 0.1 mu g / ml to 0.3 mu g / ml of glucose, 0.5 mu g / ml to 2 mu g / ml of neurotrophic factors, 0.3 mu g / ml to 2.1 mu g / ml of sodium ferulate and 0.5 mg / ml to 1.5 mg / ml of cerebroprotein hydrolysate. The stem cell combined protection solution developed by the invention is simple in component, is at an injection level, can effectively protect cells and is safe to use, each milliliter of the cell protection solution can effectively protect (1-5) * 10 < 7 > cells, the problems of small injection volume and insufficient cell quantity in the brain can be well solved, special treatment is not needed, and the cost is low. Direct injection can be realized, so that clinical application is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically relates to a stem cell combined protective solution, a preparation method thereof, and an application thereof. Background Art

[0002] Stereotactic intracerebral injection transplantation is currently the most widely used stem cell transplantation method in clinical treatment and research applications. Stereotactic intracerebral injection has the advantages of accurate positioning, small surgical trauma, short operation time, etc., so it is widely used and can be used to treat ischemic stroke, sequelae of cerebral hemorrhage, sequelae of brain injury, cerebral palsy in children, focal cerebral infarction, Parkinson's disease, Alzheimer's disease, etc. The stereotactic intracerebral injection transplantation method can concentrate all stem cells around the lesion to play a therapeutic role, and the improvement of nerve function is direct and rapid. However, the stereotactic intracerebral injection transplantation technology is highly difficult, including various aspects such as cell culture, preparation, and implantation. In addition, cell culture also requires GMP environmental requirements and investment in various instruments and equipment, resulting in high treatment costs, which greatly hinder the convenient use of stem cells for clinical treatment like drugs and increase the difficulty of promotion. Moreover, there is currently no suitable cell protection drug for intracerebral injection on the market. Most of the stem cell cryopreservation solutions contain DMSO, and even those without DMSO have a very complex composition and cannot be directly used for intracerebral injection; the time period for culturing new stem cells is relatively long, and safety detection cannot be carried out in time after processing, so they cannot be directly injected into the brain.

[0003] Most of the existing low-temperature cell drug protective solutions have a relatively complex composition, and the volume of the cell protective solution is too large and the cell concentration is insufficient, which is not suitable for intracerebral injection; therefore, how to prepare a stem cell combined protective solution is extremely important for the development of medicine. Summary of the Invention

[0004] To this end, the present invention provides a stem cell combined protective solution, a preparation method thereof, and an application thereof to solve the above problems.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A stem cell combined protective solution provided according to the first aspect of the present invention, the protective solution includes: sodium chloride 5.5 - 6.5 mg / ml, potassium chloride 0.2 - 0.4 mg / ml, sodium lactate 2.5 - 3.5 mg / ml, calcium chloride 0.1 - 0.3 mg / ml, glucose 0.1 - 0.3 μg / ml, nerve growth factor 0.5 - 2 μg / ml, sodium ferulate 0.3 - 2.1 μg / ml, and cerebroprotein hydrolysate 0.5 - 1.5 mg / ml.

[0007] Further, the protective solution comprises: sodium chloride 6 mg / ml, potassium chloride 0.3 mg / ml, sodium lactate 3.1 mg / ml, calcium chloride 0.2 mg / ml, glucose 0.21 μg / ml, neurotrophic factor 1 μg / ml, sodium ferulate 1.5 μg / ml, and cerebroprotein hydrolysate 1 mg / ml.

[0008] In a normal temperature environment, various enzymes and other biological macromolecules in cells react and metabolize vigorously, consuming a large amount of oxygen and potentially producing a large amount of secretions. These physiological processes generate a large number of oxygen free radicals and lipid peroxides, which can cause changes in the environmental pH and osmotic pressure, resulting in cell swelling and death. At low temperatures, cell metabolism is lower, and these problems can be greatly alleviated. The cell protective solution of the present invention increases the oxygen content by increasing the cell and space ratio, and a low temperature of about 12°C can solve this problem.

[0009] When stem cells are preserved using the above-mentioned cell transport preservation solution, low temperature helps to reduce cell metabolism and extend the cell preservation time. However, although stem cells are in a low metabolic state, they still require certain nutrients for metabolism. In addition to adding glucose as an energy source to provide basic energy supply for cells, the storage and transport solution of the present invention also adds: sodium chloride, potassium chloride, sodium lactate, calcium chloride, neurotrophic factor, sodium ferulate, and cerebroprotein hydrolysate. Among them, sodium chloride, potassium chloride, sodium lactate, and calcium chloride are formulated into a balanced salt solution according to the component ratio. Its electrolyte concentration, pH, osmotic pressure, etc. are close to those of the extracellular fluid, and it can be used as a good cell solution. The combined use of several components can better ensure the normal activity and metabolism of cells, improve the adaptability to harsh environments, and the components are simple, clear, and the main components are of medical grade, which can be directly used in the human body without any risk.

[0010] Sodium chloride, potassium chloride, sodium lactate, and calcium chloride: These electrolytes and acid-base balance regulators help to maintain the osmotic pressure and acid-base balance of the extracellular fluid, providing a stable condition close to the physiological environment for stem cells, which is crucial for the survival and normal function of cells.

[0011] Glucose: As an energy source for cells, it provides necessary energy support for stem cells and helps to maintain their metabolic activities.

[0012] Neurotrophic factor: It has a protective and nutritional effect on nerve cells and may help to maintain the health and function of stem cells, especially neural stem cells.

[0013] Sodium ferulate: It has an antioxidant effect and can protect cells from oxidative stress damage, which is beneficial for cell survival in a non-ideal environment.

[0014] Cerebroprotein Hydrolysate: Provides nutrients required for stem cell growth and repair, and helps in the recovery and regeneration of stem cells.

[0015] A method for preparing a stem cell combined protective solution according to the second aspect of the present invention, the method comprising: uniformly mixing the raw materials with water to obtain the solution.

[0016] An application of a stem cell combined protective solution according to the third aspect of the present invention in the preparation of a stereotactic intracerebral injection transplantation protective solution.

[0017] Further, the stereotactic intracerebral injection transplantation protective solution comprises a bone marrow mesenchymal stem cell suspension and the protective solution as described above.

[0018] Further, the cell suspension is (1 - 5)×10 7 / ml.

[0019] Further, the stereotactic intracerebral injection transplantation protective solution is prepared by placing the cell suspension in a 5 ml cryopreservation tube with a breathable membrane, with 1 ml of cell drug protective solution in each tube, wherein the space ratio between the cell suspension and the cryopreservation tube is maintained within the range of 1:4.

[0020] The present invention has the following advantages:

[0021] The stem cell combined protective solution developed by the present invention has a simple composition and all are of injection grade. It can not only effectively protect cells and is safe to use, but also each milliliter of cell protective solution can effectively protect (1 - 5)×10 7 cells, which can well solve the problems of small intracerebral injection volume and insufficient cell quantity. Moreover, it can be injected without special treatment or centrifugation and then reselecting and treating with physiological saline according to the required concentration, thus facilitating clinical application.

[0022] The protective solution of the present invention is a cell drug protective solution for intracerebral injection of which all the components can be directly used in the human body; the stem cell combined protective solution of the present invention contains a large number of cells per milliliter and has a high concentration, and can be centrifuged, solving the problem of the small amount of liquid required for intracerebral injection; the present invention uses a 5 ml cryopreservation tube with a breathable membrane cover, solving the problem of cell air replacement; the present invention uses a medical injection preparation with a simple composition and safe application; the protective solution of the present invention has a long storage time, saving valuable time for the traditional Chinese medicine company to detect and apply fresh preparations for medicine production; the cryopreservation tube of the present invention is portable, convenient for transportation, simple to operate, and can be injected without special treatment or centrifugation and then reselecting and treating with physiological saline according to the required concentration, thus facilitating clinical application and solving the limitations in the absence of corresponding environment and equipment during application, and therefore also greatly saving costs. Description of the Drawings

[0023] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only exemplary. For those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained based on the provided drawings.

[0024] The structures, ratios, sizes, etc. illustrated in this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they do not have substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the effects that the present invention can produce and the purposes that can be achieved, should still fall within the scope that can be covered by the technical content disclosed in the present invention.

[0025] Figure 1 It is a comparison graph of normal culture and culture after being protected by a cell protection drug provided in Experimental Example 1 of the present invention. Among them, A is the graph of normal culture, and B is the graph of culture after being protected by a cell protection drug;

[0026] Figure 2 It is a flow cytometry detection graph provided in Embodiment 1 of the present invention. Among them, A is the flow cytometry detection graph of normal culture; B is the flow cytometry detection graph of culture after being protected by a cell protection drug;

[0027] Figure 3 It is a CCK-8 cell proliferation viability detection graph provided in Embodiment 1 of the present invention;

[0028] Figure 4 It is a three-lineage differentiation graph provided in Embodiment 1 of the present invention. Among them, A is the graph of normal culture, and B is the graph of culture after being protected by a cell protection drug. Detailed implementation manners

[0029] The following specific embodiments illustrate the implementation manners of the present invention. Those familiar with this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope protected by the present invention.

[0030] Neurotrophic factor: Wuhan Heite Biopharmaceutical Co., Ltd., National Medicine Approval Number S20060051;

[0031] Cerebroprotein hydrolysate: Harbin Pharmaceutical Co., Ltd., National Medicine Approval Number H20051202.

[0032] Embodiment 1

[0033] This embodiment provides a stem cell combined protective solution:

[0034] Sodium chloride 6 mg / ml, potassium chloride 0.3 mg / ml, sodium lactate 3.1 mg / ml, calcium chloride 0.2 mg / ml, glucose 0.21 μg / ml, nerve growth factor 1 μg / ml, sodium ferulate 1.5 μg / ml, cerebroprotein hydrolysate 1 mg / ml.

[0035] Example 2

[0036] This embodiment provides a stem cell combined protective solution:

[0037] Sodium chloride 5.5 mg / ml, potassium chloride 0.2 mg / ml, sodium lactate 2.5 mg / ml, calcium chloride 0.1 mg / ml, glucose 0.1 μg / ml, nerve growth factor 0.5 μg / ml, sodium ferulate 0.3 μg / ml, cerebroprotein hydrolysate 0.5 mg / ml.

[0038] Example 3

[0039] This embodiment provides a stem cell combined protective solution:

[0040] Sodium chloride 6.5 mg / ml, potassium chloride 0.4 mg / ml, sodium lactate 3.5 mg / ml, calcium chloride 0.3 mg / ml, glucose 0.3 μg / ml, nerve growth factor 2 μg / ml, sodium ferulate 2.1 μg / ml, cerebroprotein hydrolysate 1.5 mg / ml.

[0041] Example 4

[0042] Preparation method of the stem cell combined protective solution of Examples 1-3:

[0043] Sodium chloride, potassium chloride, sodium lactate, calcium chloride, glucose, nerve growth factor, sodium ferulate, and cerebroprotein hydrolysate are added with water to form a mixture with a unit concentration and mixed evenly to obtain the solution.

[0044] Experimental Example 1

[0045] 1. Cell culture

[0046] 1) Take 45 ml of bone marrow blood donated by volunteers, add it to a centrifuge tube and centrifuge at 3000 rpm for 30 minutes. Use a 2ML pipette to aspirate the buffy coat layer, dilute it with PBS at a ratio of 1:1, and then add it to a 50 ml centrifuge tube containing the same volume of lymphocyte separation solution. The operation must be slow and the liquid level should not be broken. After it spreads evenly at the bottom, the speed can be increased uniformly until the scale reaches 45 ml.

[0047] 2) After the separation liquid is completely added, gently place it in a centrifuge. Centrifuge at 400 g for 30 min (note to adjust the deceleration and acceleration). After centrifugation, extract the white layer separated by gradient density centrifugation. Slowly suck it out with a straight tube (the white layer), then make up to 45 ml with PBS and place it in the centrifuge for centrifugation at 2000 rpm for 5 min.

[0048] 3) After centrifugation, pour out the supernatant and leave the required cells. Take an appropriate amount of red blood cell lysate with a straight tube to disperse the cells to achieve the purpose of lysing red blood cells (control the time at about 3 min). After the lysis time, add PBS to 45 ml and place it in the centrifuge for centrifugation at 2000 rpm for 5 min. When the centrifugation time is up, pour out the waste liquid, add PBS to 45 ml and place it in the centrifuge for centrifugation at 2000 rpm for 5 minutes.

[0049] 4) After centrifugation, disperse the cells with imported complete medium (α - mem). After counting, add them to a 150 cm² culture flask, and add 25 ml of medium to each flask. Then place it in a carbon dioxide constant temperature incubator.

[0050] 5) After static culture for 24 h, pour out the supernatant, then wash the bottom of the flask with saline. After washing, add imported medium for liquid change. Observe the cell growth status every day after liquid change, and give liquid change every 3 d until the cells grow to about 80% in the flask. Generally, it can be passaged and amplified to P1 generation in about 10 days. In the later stage, passage once every 5 days. After reaching the eighth generation and the cell confluence is above 80%, start the cell preservation solution experiment. After taking cell photos and retaining them, carry out digestion treatment. Discard the medium in the culture flask, add an appropriate amount of normal saline to wash the cell surface, pour out the washing solution after washing, suck out the residual liquid with a 10 ml pipette, then add 3 ml of trypsin for digestion at room temperature for 3 - 5 min, and then use an appropriate amount of termination solution to terminate digestion. Transfer the cell suspension to a 50 ml centrifuge tube, and use normal saline to rinse the culture flask to collect the residual cells. After rinsing, transfer them to the 50 ml centrifuge tube in the same way. Centrifuge at 500 g for 5 min to collect the cells. After centrifugation, use normal saline washing solution, centrifuge at 500 g for 5 min and wash twice, then resuspend, filter through a 100 μm mesh sieve, take samples for counting, and adjust the cell density to (1 - 5)×10 7 / ml to make a cell suspension.

[0051] 2. Preservation

[0052] Finally, place the cell suspension in a 5 ml cryopreservation tube with a breathable membrane, and add 1 ml of the cell drug protection solution prepared in Example 1 to each tube, where the space ratio range of the cell suspension to the cryopreservation tube is kept at 1:4.

[0053] 3. Store or transport using a biological vaccine refrigerator, set the temperature to 12°C, conduct safety tests for sterility, endotoxin, microorganisms, mycoplasma, etc. No sterility, endotoxin, microorganisms, mycoplasma, etc. were detected. After passing the safety test, perform CT / MRI scan and localization, input the images into a computer, use the computer to plan the transplantation target point and surgical path, after local anesthesia, drill a hole in the skull, insert a probe, directly or after centrifuging according to the required cell quantity and injection volume and resuspending the cells with normal saline for adjustment, pump in the stem cell drug suspension with a microinfusion pump.

[0054] 4. Back-cultivation experiment

[0055] The cells stored in the cell drug protective solution for 24 hours and the cells cultured in normal medium were re-attached and cultured. As Figure 1 shown, when the cell confluence reached 80%, CCK-8 cell proliferation viability detection and three-lineage differentiation detection were performed, and there were no significant differences.

[0056] 5. Flow cytometry was performed on the cells before and after the stem cell experiment, and there were no significant differences in the expression of specific markers.

[0057] According to the conventional flow cytometry results as Figure 2 and Table 1 shown, after normal culture and culture with the cell protection drug, the culture with the cell protection drug was slightly better than normal culture.

[0058] Table 1

[0059]

[0060] 6. CCK-8 detection

[0061] As Figure 3 shown, the cells stored in the cell drug protective solution for 24 hours and the cells cultured in normal medium were re-attached and cultured. When the cell confluence reached 80%, subsequent CCK-8 cell proliferation viability detection was performed, and it was found that there were no significant differences between the two.

[0062] 7. Three-lineage differentiation detection

[0063] As Figure 4 shown, the cells stored in the cell drug protective solution for 24 hours and the cells cultured in normal medium were re-attached and cultured. When the cell confluence reached 80%, subsequent three-lineage differentiation detection was performed, and it was found that there were no significant differences between the two.

[0064] 8. Cell quantity and viability detection

[0065] The survival rates of different cell suspensions are shown in Table 2 - Table 6, and there were no significant differences in cell survival rates.

[0066] Table 2

[0067] Time 0h 6h 12h 18h 24h 26h Quantity <![CDATA[1.1x10 7 / ml]]> <![CDATA[1.03x10 7 / ml]]> <![CDATA[1.02x10 7 / ml]]> <![CDATA[1.01x10 7 / ml]]> <![CDATA[1.04x10 7 / ml]]> <![CDATA[1.06x10 7 / ml]]> Survival Rate 98.99％ 98.95％ 99.01％ 98.91％ 98.97％ 98.62％

[0068] Table 3

[0069] Time 0h 6h 12h 18h 24h 26h Quantity <![CDATA[2.07x10 7 / ml]]> <![CDATA[2.05x10 7 / ml]]> <![CDATA[2.01x10 7 / ml]]> <![CDATA[2.10x10 7 / ml]]> <![CDATA[2.06x10 7 / ml]]> <![CDATA[2.11x10 7 / ml]]> Survival Rate 98.65％ 98.59％ 98.69％ 98.63％ 98.37％ 98.25％

[0070] Table 4

[0071] Time 0h 6h 12h 18h 24h 26h Quantity <![CDATA[3x10 7 / ml]]> <![CDATA[3.03x10 7 / ml]]> <![CDATA[3.10x10 7 / ml]]> <![CDATA[3.08x10 7 / ml]]> <![CDATA[3.09x10 7 / ml]]> <![CDATA[3.07x10 7 / ml]]> Survival Rate 98.59％ 98.13％ 98.11％ 97.98％ 98.78％ 98.15％

[0072] Table 5

[0073] Time 0h 6h 12h 18h 24h 26h Quantity <![CDATA[4x10 7 / ml]]> <![CDATA[4.01x10 7 / ml]]> <![CDATA[4.06x10 7 / ml]]> <![CDATA[4.01x10 7 / ml]]> <![CDATA[4.05x10 7 / ml]]> <![CDATA[4.07x10 7 / ml]]> Survival Rate 97.97％ 98.12％ 97.91％ 98.18％ 97.98％ 97.85％

[0074] Table 6

[0075] Time 0h 6h 12h 18h 24h 26h Quantity <![CDATA[5x10 7 / ml]]> <![CDATA[5.06x10 7 / ml]]> <![CDATA[5.09x10 7 / ml]]> <![CDATA[5.02x10 7 / ml]]> <![CDATA[5.05x10 7 / ml]]> <![CDATA[2.07x10 7 / ml]]> Survival Rate 98.36％ 98.17％ 98.13％ 97.96％ 97.95％ 97.85％

[0076] Experimental Example 2

[0077] 1. The safety detection data is shown in Table 7 as follows.

[0078] Table 7

[0079]

[0080] 2. Animal experiment:

[0081] To provide a scientific basis for the safety evaluation of the cell protection drug. Under strictly controlled conditions in this example, 120 healthy SD rats (60 males and 60 females) with a body weight of about 200 g and aged 6 - 9 months were selected to be intracerebrally injected with the cell protection drug of Example 2, and 40 rats (20 males and 20 females) in 2 batches were injected with normal saline as a control. After injection, the rats were observed continuously for at least 14 days. The observation indexes included clinical symptoms (such as animal appearance, behavior, diet, response to stimuli, secretions, excretions), death conditions (death time, reactions before death, etc.), changes in body weight (before injection and at the end of the observation period), and the blood routine, blood biochemical indexes, and organ - body weight ratios were measured and the normal reference value ranges were determined. After the observation period ended, the animals were euthanized and dissected.

[0082] There were no abnormal clinical symptoms before and after injection, no deaths occurred, the body weights of all rats increased, the blood routine, blood biochemical indexes, and organs were all within the normal ranges, and after dissection, the organ volumes, colors, and textures were normal without any abnormalities, as specifically shown in Table 8.

[0083] Table 8

[0084] Test Items Number of Animals Clinical Symptoms Body Weight Before Injection Body Weight 14 Days After Injection Number of Deaths Organs After Dissection Drug Administration Group 120 No Abnormality Around 200g Around 250g None No Abnormality Control Group 40 No Abnormality Around 200g Around 250g None No Abnormality

[0085] Experimental Example 3

[0086] Control group 1: Use the product imported from Biolifesolutions: HypoThermosol. This preservation solution is a commonly used scientific research grade preservation solution.

[0087] Control group 2: Normal saline.

[0088] Experimental group: The stem cell combined preservation solution of Experimental Example 1 of the present invention (the components of the preservation solution are the components of Example 1).

[0089] After Experimental Example 1 (cell culture), the cell suspension was finally placed in a 5 ml cryotube with a breathable membrane, and 1 ml of the cell drug preservation solutions of Control group 1, Control group 2 and the experimental group was added to each tube, and the space ratio range of the cell suspension to the cryotube was maintained at 1:4.

[0090] Refrigeration of the present invention and normal saline: Place the cryotube in a refrigerator at 12 °C for 5 days.

[0091] Control group 1: Refrigerate for 5 days according to the instructions.

[0092] Verification: Take out the cryotube, dilute it with 5 times the volume of normal saline, and calculate the cell survival rate, as shown in Table 9.

[0093] Table 9

[0094]

[0095] As can be seen from Table 9, the preservation solution of the present invention has a better effect than the commercially available drug preservation solution.

[0096] Although the present invention has been described in detail above with general descriptions and specific embodiments, on the basis of the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A stem cell joint protective solution, characterized in that: The protective solution comprises: sodium chloride 5.5-6.5 mg / ml, potassium chloride 0.2-0.4 mg / ml, sodium lactate 2.5-3.5 mg / ml, calcium chloride 0.1-0.3 mg / ml, glucose 0.1-0.3 μg / ml, neurotrophic factor 0.5-2 μg / ml, sodium ferulate 0.3-2.1 μg / ml, and brain protein hydrolyzate 0.5-1.5 mg / ml.

2. A stem cell joint protective solution according to claim 1, characterized in that: The protective solution includes: sodium chloride 6 mg / ml, potassium chloride 0.3 mg / ml, sodium lactate 3.1 mg / ml, calcium chloride 0.2 mg / ml, glucose 0.21 μg / ml, neurotrophic factor 1 μg / ml, sodium ferulate 1.5 μg / ml, and brain protein hydrolyzate 1 mg / ml.

3. A method for preparing a stem cell combined protective solution, characterized in that: The method comprises: adding water to the raw materials and mixing them evenly to obtain the product.

4. Application of a stem cell combined protective solution in the preparation of stereotactic intracerebral injection transplant protective solution.

5. The use according to claim 4, characterized in that: The stereotactic brain injection transplantation protective solution comprises a bone marrow mesenchymal stem cell suspension and the protective solution according to claim 1.

6. The use according to claim 5, characterized in that: The cell suspension is (1-5)×10 7 / ml.

7. The use according to claim 5, characterized in that: The stereotactic brain injection of transplant protection solution is to place the cell suspension in a 5 ml cryopreservation tube with a gas permeable membrane, each tube has 1 ml of cell drug protection solution, wherein the spatial ratio range of the cell suspension and the cryopreservation tube is maintained at 1:4.

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