A cell drug protection solution for injection, a preparation method and application thereof

Abstract

The application discloses a cell medicine protection liquid for injection and a preparation method and application thereof, and particularly relates to the technical field of biological medicine. The protection liquid comprises the following components: 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 ug / ml, glucose 0.15-0.3 ug / ml, neurotrophic factor 0.5-2 ug / ml, and compound amino acid 4 ul / 100 ml. The protection liquid can be used for storing cell medicine for human lumbar puncture injection, and the medicine storage time is long, which helps to save valuable time for new fresh preparation detection and application of drug companies. The application uses a freeze storage tube, which is portable, convenient to transport and simple to operate, and does not need special treatment and direct application, thereby solving the limitation of no corresponding environment and equipment application, and greatly saving the cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a cell drug protection solution for injection and a preparation method and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) have the ability to secrete various neurotrophic factors and have the potential for self-renewal, multi-directional differentiation and immune regulation. Therefore, mesenchymal stem cell transplantation has great potential in the treatment of various nervous system diseases such as ischemic cerebral stroke, multiple sclerosis, Parkinson's disease and spinal cord injury. It is widely used in the fields of biological medicine such as nervous system, bone and joint system, genetic immune system, etc. Stem cell therapy is a new field, and the development of targeted stem cell drugs and targeted treatment programs is the direction of future development. Stem cell therapy brings new hope to patients. Stem cell therapy for nervous system injury often uses lumbar puncture stem cell transplantation, and the method of subarachnoid injection is used for transplantation. The injection amount is generally 2-3ml (cell amount is 20-30 million), which has the advantages that the transplanted cells can flow through the entire brain and spinal cord along the circulation pathway of cerebrospinal fluid, and the cells can reach anywhere with lesions; and the trauma is very small, only a needle eye of a syringe needle is left on the skin of the waist, and each operation only takes about 10 minutes, greatly reducing the pain of patients. In order to ensure the quality, effectiveness, safety, convenience and popularization of stem cell drugs in application, the development of stem cell drug protection solution is imminent.

[0003] The existing cell protection solution is basically divided into frozen cell protection solution and low-temperature cell protection solution.

[0004] 1. The frozen protection solution basically contains DMSO, which is toxic to cells and cannot be injected locally. If injection is needed, centrifugal cleaning treatment is required before injection, which will cause certain damage and loss to cells, and the cells need to be treated in a corresponding environment and equipment, which increases the difficulty of application.

[0005] 2. Most of the existing low-temperature protection solution has complex components, uses pH buffer, antioxidant, various osmotic pressure stabilizers and various oxygen free radical scavengers, of course, there are other components for cell metabolism in addition, only the protection of cells is considered, and the safety and convenience of direct application are ignored. The cell drug preservation solution developed by the present application has simple components, which are injection grade, can effectively protect cells, and is safe to use.

[0006] Most existing cell preservation solutions are not suitable for direct use in human lumbar puncture injections, limiting their role in the clinical development of cell therapy towards pharmaceutical applications. Furthermore, they protect a relatively small number of cells per milliliter, requiring large quantities of preservation solution for large cell applications, while lumbar puncture and injection volumes are relatively small. Especially for adherent cultured cells, future clinical applications will require specialized personnel in GMP-standard operating rooms, utilizing equipment such as centrifuges and biosafety cabinets, significantly hindering the convenient clinical use of cells like pharmaceuticals. In contrast, the preservation solution developed in this invention consists entirely of injectable-grade components that can be directly injected into the human body. It requires no special processing or only simple on-demand processing, similar to direct injection of pharmaceuticals, thus facilitating clinical application.

[0007] Existing cell protection solutions have complex compositions, so it is urgent to invent a cell drug protection solution specifically for lumbar puncture injection. Summary of the Invention

[0008] Therefore, the present invention provides an injectable cell drug preservative solution, its preparation method, and its application to solve the above-mentioned problems.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] According to a first aspect of the present invention, a cell drug preservative solution for injection is provided, the preservative solution comprising: 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, and compound amino acids 4 μl / 100ml.

[0011] Furthermore, the protective solution comprises 6 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, 3.1 mg / ml sodium lactate, 0.2 mg / ml calcium chloride, 0.21 μg / ml glucose, 1 μg / ml neurotrophic factor, and 4 μl / 100ml compound amino acids.

[0012] According to a second aspect of the present invention, a method for preparing an injectable cell drug preservative solution is provided, the method comprising mixing the raw materials of the injectable cell drug preservative solution evenly to obtain the solution.

[0013] The application of an injectable cell drug preservative solution provided by a third aspect of the present invention in the preparation of a cell drug preservative solution for direct human lumbar puncture injection.

[0014] Furthermore, the cell drug preservative solution for direct human lumbar puncture injection comprises bone marrow mesenchymal stem cell suspension and the cell drug preservative solution for injection as described above.

[0015] Further, the bone marrow mesenchymal stem cell suspension cell density is (1-2) x 10 7 / ml.

[0016] Further, the cell drug protection liquid directly used for human lumbar puncture injection is mixed by finally placing the cell suspension in a 5ml cryopreservation tube with a breathable film, and adding 1ml of the cell drug protection liquid into each tube, wherein the space ratio of the cell suspension to the cryopreservation tube is kept at 1:4.

[0017] Further, the cell drug directly used for human lumbar puncture injection is transported by using a biological vaccine refrigeration box during transportation, and the temperature is set to 16℃.

[0018] Since in a normal temperature environment, various enzymes and other biological macromolecules in the cell react and metabolize vigorously, consume a large amount of oxygen, and may secrete a large amount of substances. These physiological processes can generate a large amount of oxygen free radicals and lipid peroxides, cause changes in environmental pH and osmotic pressure, and cause cell swelling and death. At low temperature, cell metabolism is low, and these problems can be greatly alleviated. The cell protection liquid of the present application increases the oxygen content by increasing the cell and space ratio, and the problem can be solved by controlling the temperature at about 16℃.

[0019] When stem cells are stored in the above-mentioned cell transportation and storage liquid, low temperature helps to reduce cell metabolism and prolong cell storage time. However, although stem cells are in a low metabolic state, they still need certain nutrients for metabolism. In addition to adding glucose as an energy substance to provide basic energy supply for cells, the storage and transportation liquid of the present application also adds: sodium chloride, potassium chloride, sodium lactate, calcium chloride, neurotrophic factors, and compound amino acids. Among them, sodium chloride, potassium chloride, sodium lactate, calcium chloride, neurotrophic factors, and compound amino acids are formulated into a balanced salt solution according to the component ratio, and the electrolyte concentration, pH, and osmotic pressure thereof are close to that of extracellular fluid, which can be used as a good cell solution. The use of several components can better ensure the normal metabolic activity of cells and improve their adaptability to harsh environments. Moreover, the components are simple, clear, and mainly medical grade, and can be directly used in the human body without any risk.

[0020] The various components in the formula of the present application are combined together to provide a stable environment for cell drugs to protect the activity and function of cells during storage and transportation. Sodium chloride and potassium chloride help maintain the osmotic pressure and electrolyte balance of extracellular fluid, which is essential for the survival and normal function of cells. Sodium lactate can regulate acid-base balance and help maintain the physiological environment of cells. Calcium chloride helps maintain the stability of the cell membrane. Glucose as an energy source for cells can provide necessary energy support for cells. Neurotrophic factors have protective and nutritional effects on nerve cells and help maintain the health and function of nerve cells. Compound amino acids provide nutrients required for cell growth and repair.

[0021] The present application has the following advantages:

[0022] The protective solution of the present application is a protective solution for cell drugs that can be directly used in the human body; the protective solution of the present application contains a large number of cells per milliliter, with high concentration, solving the problem of a small number of cells in lumbar puncture injection liquid; the present application uses a 5-milliliter gas-permeable film-covered cryopreservation tube, solving the problem of air replacement required by cells; the present application uses a medical injection preparation, with simple components and safe application.

[0023] The protective solution of the present application can preserve cell drugs for direct lumbar puncture injection in the human body, allowing long-term drug preservation, and gaining valuable time for new drug companies to detect and apply fresh preparations; the present application uses a cryopreservation tube, which is portable, convenient for transportation, and easy to operate, without the need for special treatment or simple treatment, and can be directly applied, solving the limitations of application due to the lack of corresponding environment and equipment, thus greatly saving costs. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only exemplary, and for those skilled in the art, other drawings can be obtained from the provided drawings without creative labor.

[0025] The structures, proportions, sizes, etc. shown in the specification are only used to cooperate with the content disclosed in the specification, to be understood and read by those skilled in the art, and do not define the limiting conditions for the implementation of the present application, so they do not have technical significance. Any modification of structure, change of proportion relationship or adjustment of size, without affecting the effects and purposes that the present application can produce, should still fall within the scope of the technical content disclosed by the present application.

[0026] Figure 1Normal culture and cell drug protection liquid protection after culture comparison chart provided for the experimental example 1 of the present application, wherein A is a normal culture chart, B is a cell drug protection liquid protection after culture chart;

[0027] Figure 2 Flow detection chart provided for the example 1 of the present application, wherein A is a normal culture flow detection chart; B is a cell drug protection liquid protection after culture flow detection chart;

[0028] Figure 3 CCK-8 cell proliferation activity detection chart provided for the example 1 of the present application;

[0029] Figure 4 Three differentiation charts provided for the example 1 of the present application. DETAILED DESCRIPTION

[0030] The embodiments of the present application will be described in detail by specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the specification. Obviously, the described embodiments are part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0031] Neurotrophic factor: Wuhan Haitian Biopharmaceutical Co., Ltd. National Drug Code S20060051;

[0032] Compound amino acid: Shijiazhuang Four Pharmaceutical Co., Ltd. National Drug Code H9993803.

[0033] Example 1

[0034] The present embodiment provides a cell drug protection liquid for injection:

[0035] The protection liquid comprises sodium chloride 6mg / ml, potassium chloride 0.3mg / ml, sodium lactate 3.1mg / ml, calcium chloride 0.2mg / ml, glucose 0.21μg / ml, neurotrophic factor 1μg / ml, compound amino acid 4μl / 100ml.

[0036] Example 2

[0037] The present embodiment provides a cell drug protection liquid for injection:

[0038] The protection liquid comprises: sodium chloride 5.5mg / ml, potassium chloride 0.2mg / ml, sodium lactate 2.5mg / ml, calcium chloride 0.1mg / ml, glucose 0.1μg / ml, neurotrophic factor 0.5μg / ml, compound amino acid 4μl / 100ml.

[0039] Example 3

[0040] The present embodiment provides a cell drug protective solution for injection:

[0041] The protective solution comprises: 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, neurotrophic factor 2 μg / ml, compound amino acid 4 μl / 100 ml.

[0042] Example 4

[0043] The present embodiment provides a preparation method of the cell drug protective solution for injection of Examples 1-3:

[0044] Sodium chloride, potassium chloride, sodium lactate, calcium chloride, glucose, neurotrophic factor, compound amino acid are added to sterile water to prepare a unit concentration mixture, which is uniformly mixed to obtain the protective solution.

[0045] Example 5

[0046] The present embodiment provides a cell drug protective solution for direct injection into the human lumbar puncture:

[0047] 1. Cell culture

[0048] Take 45 ml of bone marrow blood donated by a volunteer, add it to a centrifuge tube, and centrifuge at 3000 rpm for 30 minutes. Use a 2 mL pipette to suck the white membrane 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 lymph separation solution. The operation must be slow and the liquid level should not be broken. After the bottom is leveled, the speed can be increased. Stop when the scale reaches 45 ml.

[0049] After the separation solution is completely added, gently place it in the centrifuge. Centrifuge at 400g for 30 minutes (note to adjust the speed down and up). After centrifugation, extract the white layer separated by gradient density centrifugation. Slowly suck out the white layer with a straight tube, then add PBS to 45 ml and put it into the centrifuge to centrifuge at 2000 rpm for 5 minutes.

[0050] After centrifugation, discard the supernatant and leave the desired cells. Use a straight tube to take an appropriate amount of red blood cell lysis solution to disperse the cells and achieve the purpose of lysing red blood cells (the time is controlled at about 3 minutes). After the lysis time, add PBS to 45 ml and put it into the centrifuge to centrifuge at 2000 rpm for 5 minutes. After the centrifugation time, discard the waste liquid, add PBS to 45 ml and put it into the centrifuge to centrifuge at 2000 rpm for 5 minutes.

[0051] After centrifugation, disperse the cells with imported complete medium (α-mem), count and add 150 cm 2The flask was placed in a carbon dioxide incubator.

[0052] After 24h of static culture, the supernatant was discarded, and the bottom of the flask was washed with saline. Then, the flask was filled with fresh medium. The cell growth was observed every day after the medium was changed. The medium was changed every 3 days until the cells grew to about 80% of the flask. The cells were usually subcultured to the P1 generation after about 10 days. The cells were subcultured every 5 days in the later stage. The eighth generation of bone marrow mesenchymal stem cells were collected after being digested with 0.05% trypsin (centrifugation at 2000 rpm for 5 minutes). The cells were blown apart using a stem cell transport solution, and the cell density was adjusted to (1-2)

[0053] ×10 7 / ml to prepare a cell suspension.

[0054] 2. Preservation

[0055] The cell suspension was finally placed in a 5ml cryogenic tube with a gas-permeable membrane. 1ml of the cell drug protection solution was added to each tube. The ratio of the cell suspension to the space in the cryogenic tube was maintained at 1:4. The cell drug directly used for human lumbar puncture injection was obtained. The cell drug was transported using a biological vaccine refrigerator, and the temperature was set to 16°C. The cell drug directly used for human lumbar puncture injection was used for animal experiments and toxicological tests in subsequent experiments.

[0056] Experimental Example 1

[0057] The cell drug protection solution directly used for human lumbar puncture injection of Example 5 was subjected to flow cytometry, CCK-8 cell proliferation activity detection, three differentiation detection, viability detection, quantity detection, and back-culture experiments.

[0058] 1. Back-culture experiment

[0059] Cells stored for 24h using the cell drug protection solution were reattached and cultured in normal medium, as shown in Figure 1 . When the cells were fused to 80%, subsequent CCK-8 cell proliferation activity detection and three differentiation detection were performed.

[0060] 2. Flow cytometry

[0061] Flow cytometry was performed by a conventional method. The results are shown in Figure 2 and Table 1.

[0062] The flow cytometry data are shown in Table 1.

[0063] Table 1

[0064]

[0065] 3. CCK-8 cell proliferation activity detection

[0066] As shown in Figure 3 , the cells stored for 24 h in the cell drug protection solution were re-plated and cultured with normal culture medium. When the cells were fused to 80%, the subsequent CCK-8 cell proliferation activity detection was performed. It was found that there was no significant difference between the two.

[0067] 4. Three differentiation detections

[0068] As shown in Figure 4 , the cells stored for 24 h in the cell drug protection solution were re-plated and cultured with normal culture medium. When the cells were fused to 80%, the subsequent CCK-8 cell proliferation activity detection was performed. It was found that there was no significant difference between the two.

[0069] 5. Cell number and viability detection

[0070] The survival rates of different cell suspensions are shown in Tables 2 and 3.

[0071] Table 2

[0072] Time 0h 6h 12h 18h 24h 26h Number 1 x 10 7 / ml]] 1.05 x 10 7 / ml 1.08 x 10 7 / ml 1.06 x 10 7 / ml 1.02 x 10 7 / ml 1.08 x 10 7 / ml Viability 98.95％ 98.91％ 98.89％ 99.01％ 98.61％ 98.52％

[0073] Table 3

[0074] Time 0h 6h 12h 18h 24h 26h Number 2 x 10 7 / ml 2.03 x 10 7 / ml 1.97 x 10 7 / ml 2.01 x 10 7 / ml 2.05 x 10 7 / ml 2.07 x 10 7 / ml Viability 97.95％ 98.13％ 97.91％ 97.65％ 97.35％ 97.85％

[0075] As shown in Tables 2 and 3, there is no difference in cell survival rate.

[0076] Experimental Example 2

[0077] To provide a scientific basis for the safety evaluation of the cell protection drug protection solution. In this experimental example, under strict control conditions, 9 batches of 180 healthy SD rats (half male and half female) weighing about 200 g and 6-9 months old were selected to perform lumbar puncture injection of the cell protection drug of Example 5, and 2 batches of 40 (half male and half female) were injected with normal saline as a control. After injection, at least 14 days of continuous observation was performed, and the observation indexes included clinical symptoms (such as animal appearance, behavior, diet, response to stimulation, excretion, and excretion), death (time of death, pre-death reaction, etc.), changes in body weight (before injection and at the end of the observation period), and determination of blood routine, blood biochemical indexes, and viscera-to-body ratio to determine the normal reference value range. After the observation period, the animals were euthanized for dissection, and the results are shown in Table 4.

[0078] Table 4

[0079]

[0080] From Table 4, it can be seen that there is no abnormality in clinical symptoms before and after injection, no death, weight increase, blood routine, blood biochemical indexes, and viscera are all in normal range, and no abnormality is found in organ volume, color, and texture after dissection.

[0081] Experimental Example 3

[0082] Control group 1: using the product of Biolife Solutions Company: HypoThermosol. The preservative solution is a commonly used scientific grade preservative solution.

[0083] Control group 2: normal saline.

[0084] Experimental group: the cell drug protective solution (the protective solution composition is the composition of Example 2) of the present application is directly used for human lumbar puncture injection.

[0085] After Example 5 (cell culture), the cell suspension is finally placed in a 5ml cryopreservation tube with a breathable film, and 1ml of the cell drug protective solution of control group 1, control group 2 and experimental group is added to each tube.

[0086] Refrigeration of the present application and control group 2: the cryopreservation tube is placed in a refrigerator at 16℃ for 5 days.

[0087] Control group 1: according to the instructions, cold storage for 5 days.

[0088] Verification: the cryopreservation tube is taken out, diluted with 5 times the volume of normal saline, and the cell survival rate is calculated, as shown in Table 5.

[0089] Table 5

[0090]

[0091] From Table 5, it can be seen that the protective solution of the present application is better than the commercially available drug preservative solution.

[0092] Although the present application has been described in detail in the foregoing with general description and specific examples, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection required by the present application.

Claims

1. A cell drug protection solution for direct use in human lumbar puncture injection, characterized by, The cell drug protection solution directly used for human lumbar puncture injection comprises a bone marrow mesenchymal stem cell suspension and a cell drug protection solution for injection; the cell density of the bone marrow mesenchymal stem cell suspension is (1-2)×10 7 / ml; The cell medicine injection protective solution comprises 5.5-6.5 mg / ml of sodium chloride, 0.2-0.4 mg / ml of potassium chloride, 2.5-3.5 mg / ml of sodium lactate, 0.1-0.3 mg / ml of calcium chloride, 0.1-0.3 μg / ml of glucose, 0.5-2 μg / ml of neurotrophic factor and 4 μl / 100 ml of compound amino acid.

2. The cell drug protection solution for direct lumbar puncture injection into human body according to claim 1, characterized in that, The cell medicine injection protective solution comprises 6 mg / ml of sodium chloride, 0.3 mg / ml of potassium chloride, 3.1 mg / ml of sodium lactate, 0.2 mg / ml of calcium chloride, 0.21 μg / ml of glucose, 1 μg / ml of neurotrophic factor and 4 μl / 100 ml of compound amino acid.

3. The cell drug protection solution for direct lumbar puncture injection into human body according to claim 1, characterized in that, The preparation method of the cell medicine injection protective solution comprises uniformly mixing cell medicine injection protective solution raw materials to obtain the cell medicine injection protective solution.

4. The cell drug protection solution for direct lumbar puncture injection into human body according to claim 1, characterized in that, The cell medicine injection protective solution directly used for human lumbar puncture injection is prepared by mixing a cell suspension with 1 ml of the cell medicine injection protective solution in a 5 ml cryopreservation tube with a gas permeable membrane, wherein the ratio of the cell suspension to the space of the cryopreservation tube is 1:

4.

5. The cell drug protection solution for direct lumbar puncture injection into human body according to claim 1, characterized in that, The cell medicine directly used for human lumbar puncture injection is transported by using a biological vaccine refrigeration box, and the temperature is set to 16℃.

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