Cell low-temperature transfer preserving fluid as well as preparation method and use method thereof
By using multi-component cell low-temperature transport preservation solution, including glucose, human albumin and chloride, the problem of the decline in cell viability rate during long-term and long-distance transport in the prior art is solved, and efficient and safe cell preservation and transport are achieved.
Patent Information
- Application Number
- CN202510086219.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-04-29
AI Technical Summary
Existing cell low-temperature transport and preservation fluid cannot effectively maintain the high cell viability rate during long-term and long-distance transport, and there are problems of safety and cost.
The low-temperature transport and preservation solution of cells composed of sterilized injection water, glucose, human albumin, sodium chloride, potassium chloride and calcium chloride is used to provide nutritional support and stable osmotic pressure to prevent cell aggregation and death through the combination of multiple components.
This storage solution can maintain cell viability above 85% within 72 hours, ensuring high survival rate of cells during transport, suitable for large-scale production applications, and reducing transportation costs.
Smart Images
Figure CN119896212A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell cryogenic transport and preservation, and in particular to a cell cryogenic transport and preservation solution and a preparation method and a use method thereof.
[0002] Cell therapy refers to the bioengineering of autologous or allogeneic somatic cells or stem cells to form cell products with specific functions to treat diseases. Since the 1990s, cell therapy has gradually developed into an important treatment method, especially in the treatment of cancer, genetic diseases, injury repair, degenerative diseases and autoimmune diseases. It has shown great potential. After entering the 21st century, the successful application of CAR-T cell therapy marked a new stage in cell therapy, especially in the treatment of hematological malignancies. At present, the global cell therapy R&D pipeline continues to increase, and new technologies involving a variety of immune cells and stromal cells are also under continuous research. With the vigorous development of regenerative medicine and cell therapy, the low-temperature storage and transportation of cell products have become a key link.
[0003] The low-temperature storage and transportation of cell products is a key technical issue involving the quality of cell products. The cell survival rate and vitality caused by the storage and transportation of cells in vitro will limit the therapeutic effect of cells in vivo. As the active ingredient of cell therapy products, living cells have high requirements for the medium in which cells survive during storage and transportation. How to maintain high cell viability during transportation time and ensure the clinical safety of the product is the main difficulty in the current clinical application of cells. Therefore, finding a cell low-temperature transport preservation solution to ensure the safety of cell storage and transportation is of great significance for the clinical application of cells.
[0004] At present, there are three cell transportation methods shown in relevant literature: 1) The frozen transportation method using liquid nitrogen or dry ice is cumbersome and has high transportation costs. The repeated freezing and thawing of cells causes a decrease in cell survival rate, which affects the treatment effect. In addition, most cryoprotectants contain dimethyl sulfoxide (DMSO), which is toxic and can cause nausea, vomiting, rash, abdominal pain, hypotension, severe neurological diseases, renal failure, cardiovascular diseases and other adverse reactions after injection into the human body. 2) The normal temperature transportation method is only suitable for short-distance transportation, and the intracellular biochemical reactions are active. The changes in pH and osmotic pressure can cause cell swelling and death. Dead cells will condense into insoluble flocs, affecting the safety of cell product transfusion. 3) The low-temperature transportation method can not only slow down the metabolic rate of cells, but also avoid the safety risks caused by resuscitation and reoperation. It is a more ideal transportation method than the first two. However, there is no low-temperature cell transportation preservation solution that can maintain cell activity for a long time and can be directly transfused.
[0005] Therefore, a cell cryogenic transport preservation solution with clear ingredients, high safety and easy preparation is sought to solve the problems of existing cell preservation solutions, such as short cell preservation time, inability to transport cells over long distances, rapid decline in cell activity and inability to be conveniently used in clinical practice. Summary of the invention
[0006] Based on the problems existing in the background technology, the present invention proposes a cell low-temperature transport preservation solution and a preparation method and a use method thereof.
[0007] A cell low-temperature transport preservation solution comprises the following components: 100 g of sterile injection water, 5 g of glucose, 5 g of human serum albumin (protein), 0.85 g of sodium chloride, 0.03 g of potassium chloride and 0.028 g of calcium chloride.
[0008] The method for preparing the cell low-temperature transport preservation solution comprises the following steps: mixing the above components evenly, filtering after dissolution, and aseptically packaging.
[0009] The method for using the above-mentioned cell cryogenic transport preservation solution comprises the following steps: uniformly mixing cells (stem cells and immune cells) with the cell cryogenic transport preservation solution described in any one of claims 1-2 to obtain a cell suspension, transferring the suspension into a sterile blood transfer bag and then placing the suspension into a vaccine box, and placing the vaccine box partition into an ice box.
[0010] Preferably, the cell concentration in the cell suspension is 1×10 6 -1×10 8 Pieces / mL.
[0011] Preferably, the stem cells are selected from umbilical cord mesenchymal stem cells, which are prepared by the following specific operation: take an umbilical cord, remove Wharton's jelly and cut into pieces of 1 to 3 mm 3 The cells were cultured and passaged in complete stem cell culture medium, and the digested cells were centrifuged, washed with D-PBS after centrifugation, filtered with a 100 μm cell strainer, and centrifuged to obtain umbilical cord mesenchymal stem cells.
[0012] Preferably, the immune cells are selected from umbilical cord blood NK cells, which are prepared by the following specific operation: taking a portion of umbilical cord blood and using the Ficoll separation method to separate the umbilical cord blood mononuclear cells, then using trophoblast cells and Corning 581 culture medium to expand and culture the NK cells for 16 days, harvesting the cells by centrifugation, washing with PBS after centrifugation, and filtering the cells with a 100 μm cell filter, and centrifuging to obtain umbilical cord blood NK cells.
[0013] The technical effects of the present invention are as follows:
[0014] (1) The present invention uses human serum albumin, which is highly safe, does not contain animal serum, and has no possibility of infection by animal-borne pathogens.
[0015] (2) The invention has the advantages of simple ingredients, convenient preparation, low cost, and the preparation can be directly reinfused, and is suitable for large-scale production and application.
[0016] (3) The present invention can be used for long-distance and long-term transportation of cell preparations to preserve cell viability and proliferation capacity.
[0017] (4) The present invention prolongs the time for maintaining stem cell activity, ensuring that the cell viability remains above 85% and the total number of cells remains above 90% after 72 hours of transportation, which can still meet the requirements for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Cell viability after cell harvesting and before preparation.
[0019] Figure 2 Effects of various cell transport and preservation solutions on the cell viability of umbilical cord mesenchymal stem cells within 72 hours.
[0020] Figure 3 Effects of various cell transport and preservation solutions on the cell viability of umbilical cord blood NK cells within 72 hours.
[0020] Figure 4 Cell viability before cell preparation. DETAILED DESCRIPTION
[0022] The commonly used low-temperature cell transport fluid is 0.9% sodium chloride injection, but due to its single component, it can only keep cells alive for a short period of time and cannot meet the needs of long-term and long-distance transportation. The present invention can ensure that the cells still have a cell viability of more than 85% after 72 hours.
[0023] In terms of stability, conventional 0.9% sodium chloride injection has low stability, and cells are prone to agglomeration and death, thus affecting the safety of clinical application. The present invention can make cells evenly dispersed and not prone to agglomeration by combining multiple components.
[0024] When preserving cells, 0.9% sodium chloride injection can only be stored at low temperatures (e.g., 2-8°C), but low temperature storage cannot be preserved for a long time, which may be due to the inability to meet the long-term nutritional and osmotic pressure requirements of the cells, resulting in cell death and aggregation. The cell cryotransport preservation solution of the present invention can keep the cell viability above 85% after 72 hours of low temperature storage (e.g., 2-8°C).
[0025] Based on this, the present invention provides a method with high safety, low cost, and the ability to stably maintain the viability of cells during transport, which is composed of sterile water for injection, glucose, human albumin (protein), sodium chloride, potassium chloride, and calcium chloride. Glucose and human albumin are used as nutritional protective agents to provide nutritional support to cells. Sodium chloride, potassium chloride, and calcium chloride are used as osmotic pressure stabilizers to provide stable osmotic pressure to cells and reduce the damage caused by osmotic pressure to cells.
[0026] The cell transport preservation solution prepared by the invention through the coordination of sterile injection water, glucose, human serum albumin (protein), sodium chloride, potassium chloride and calcium chloride has less damage to cells and a higher cell survival rate during the transport process.
[0027] The cell low-temperature transport preservation solution comprises the following components: 100 g of sterile injection water, 5 g of glucose, 5 g of human serum albumin (protein), 0.85 g of sodium chloride, 0.03 g of potassium chloride, and 0.028 g of calcium chloride.
[0028] The present invention also provides a method for using the cell transport preservation solution, which comprises:
[0029] Umbilical cord mesenchymal stem cells were selected as stem cells and prepared by the following specific operation: an umbilical cord was taken to remove Wharton's jelly and cut into pieces of 1-3 mm 3 The cells were centrifuged, washed with D-PBS, filtered with a 100 μm cell strainer, and centrifuged to obtain umbilical cord mesenchymal stem cells. The cells obtained by centrifugation were divided into 1×10 5 ~1×10 7 / mL, and put it into a blood transfer bag and transfer it to the vaccine box for storage.
[0030] Umbilical cord blood NK cells were selected as immune cells and prepared by the following specific operation: a portion of umbilical cord blood was taken and the umbilical cord blood mononuclear cells were separated by Ficoll separation method, and then the NK cells were expanded and cultured with trophoblast cells and Corning 581 medium for 16 days, the cells were harvested and centrifuged, washed with PBS after centrifugation, and filtered with a 100 μm cell filter, and the umbilical cord blood NK cells were obtained by centrifugation. The cells obtained by centrifugation were 1×10 6 ~1×10 8 / mL, and put it into a blood transfer bag and transfer it to the vaccine box for storage.
[0031] The following embodiments and drawings are used to describe the implementation methods of the present invention in detail, so that the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.
[0032] Three different cell transport storage solutions were selected for experimental comparison:
[0033] Cell transport preservation solution A: 0.9% sodium chloride injection
[0034] Cell transport preservation solution B: Cell preservation solution (commercially purchased product, brand: Haoyang)
[0035] Cell transport preservation solution C: sterile water for injection 100g, glucose 5g, human serum albumin (protein) 5g, sodium chloride 0.85g, potassium chloride 0.03g, calcium chloride 0.028g.
[0036] Experimental procedures
[0037] Umbilical cord mesenchymal stem cells were selected as stem cells and prepared by the following specific operation: an umbilical cord was taken to remove Wharton's jelly and cut into pieces of 1-3 mm 3 The cells were cultured and passaged in complete stem cell culture medium, and the digested cells were centrifuged, washed with D-PBS after centrifugation, filtered with a 100 μm cell strainer, and centrifuged to obtain umbilical cord mesenchymal stem cells.
[0038] Umbilical cord blood NK cells were selected as immune cells and prepared by the following specific operation: a portion of umbilical cord blood was taken and the Ficoll separation method was used to separate the umbilical cord blood mononuclear cells, and then the NK cells were expanded and cultured with trophoblast cells and Corning 581 culture medium for 16 days, the cells were harvested and centrifuged, washed with PBS after centrifugation, and the cells were filtered with a 100 μm cell filter and centrifuged to obtain the umbilical cord blood NK cells.
[0039] After cell culture is completed, cell viability needs to be tested. Use CountStar cell counter to detect cell quantity and viability.
[0040] Through the analysis of test results, Figure 1 The viability of both cells exceeded 90%, meeting the preparation requirements.
[0041] According to the aforementioned cell transport preservation solutions A, B, and C, the two cells (umbilical cord mesenchymal stem cells and umbilical cord blood NK cells) were resuspended in three cell preservation solutions to prepare preparations. The concentration of the two cell preparations was 1×10 6 Pieces / mL.
[0042] The umbilical cord mesenchymal stem cells were divided into six parts (each group had its own control group), and the cells were resuspended in cell transport preservation solution A, B, and C respectively, put into blood transfer bags, and stored in a 4°C refrigerator.
[0043] The umbilical cord blood NK cells were divided into six parts (each group had one group of self-control), and the cells were resuspended in cell transport preservation solution A, B, and C respectively, put into blood transfer bags, and stored in a 4°C refrigerator.
[0044] Cell detection: samples were taken at 0h, 2h, 4h, 6h, 12h, 24h, 36h, and 72h to test cell viability.
[0045] The experimental results are shown in Table 1 below:
[0046] Table 1
[0047] It can be seen from Table 1 that the cell viability of the cell transport preservation solution used decreased with the extension of time, and the maximum decrease from 0h to 72h was 64.28%; Figure 2 , Figure 3 It can be seen that the cell viability of cell transport solution C is significantly lower than that of cell transport solution A and cell transport solution B. Through mutual comparison, it is found that the results of cell viability of the three cell transport solutions are relatively stable and the differences are small. Therefore, cell transport solution C has the best performance for cell transport.
[0048] Experiments were conducted by optimizing the cell storage time and the formula of cell transport solution C.
[0049] The design optimized the effects of the increase or decrease of formula components (sterile water for injection and sodium chloride remained unchanged) and the length of cell storage time. The specific changes in components are shown in Table 2.
[0050] Table 2
[0051]
[0052] Umbilical cord mesenchymal stem cells were selected as stem cells and prepared by the following specific operation: an umbilical cord was taken to remove Wharton's jelly and cut into pieces of 1-3 mm 3 The cells were cultured and passaged in complete stem cell culture medium, and the digested cells were centrifuged, washed with D-PBS after centrifugation, filtered with a 100 μm cell strainer, and centrifuged to obtain umbilical cord mesenchymal stem cells.
[0053] Umbilical cord blood NK cells were selected as immune cells and prepared by the following specific operation: a portion of umbilical cord blood was taken and the Ficoll separation method was used to separate the umbilical cord blood mononuclear cells, and then the NK cells were expanded and cultured with trophoblast cells and Corning 581 culture medium for 16 days, the cells were harvested and centrifuged, washed with PBS after centrifugation, and the cells were filtered with a 100 μm cell filter and centrifuged to obtain the umbilical cord blood NK cells.
[0054] After cell culture is completed, cell viability needs to be tested. Use CountStar cell counter to detect cell quantity and viability.
[0055] Through the analysis of test results, Figure 4 The viability of both cells exceeded 90%, meeting the preparation requirements.
[0056] According to the aforementioned cell transport storage solutions a, b, c, d, and e, the two types of cells (umbilical cord mesenchymal stem cells and umbilical cord blood NK cells) were resuspended to prepare preparations. The concentration of the two cell preparations was 1×10 6 Pieces / mL.
[0057] The umbilical cord mesenchymal stem cells were divided into five parts, and the cells were resuspended in cell transport preservation solutions a, b, c, d, and e respectively, put into blood transfer bags, and stored in a 4°C refrigerator.
[0058] The umbilical cord blood NK cells were divided into five parts, and the cells were resuspended in cell transport preservation solution a, b, c, d, and e respectively, and placed in blood transfer bags and stored in a 4°C refrigerator.
[0059] Cell detection: samples were taken at 0h, 2h, 4h, 6h, 12h, 24h, 36h, and 72h to test cell viability.
[0060] The experimental results are shown in Table 2 below:
[0061]
[0062] As can be seen from Table 2, among the five ratios, the cell viability of the cell transport preservation solution of the present invention after 6h, 12h, 24h, 36h and 72h was significantly higher than that of other groups, and was better than the other ratios. Compared with group a, the preservation effects of group b, d and e were poor, and the preservation effect of group c was the worst, which was significantly different from the other groups.
[0063] The results show that the cell transport preservation solution of the present invention has a significantly better preservation effect on umbilical cord mesenchymal stem cells and umbilical cord blood NK cells; from the perspective of preservation time, within 72 hours, the downward trend of cell viability is relatively gentle and stable as time goes by.
[0064] All of the above are primary implementations of this intellectual property and do not set limitations on other forms of implementation of this new product and / or new method. Those skilled in the art will use this important information and modify the above content to achieve similar implementations. However, all modifications or transformations based on the new product of this invention belong to the reserved rights.
[0065] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.
Claims
1. A cell cryogenic transport storage solution, characterized in that: Its components include:
2. The mesenchymal stem cell cryoprotectant according to claim 1, characterized in that: The mass ratio of sterile water for injection: glucose: human serum albumin (protein): sodium chloride: potassium chloride: calcium chloride is 100:5:5:0.85:0.03:0.
028.
3. The method for preparing the cell cryotransport preservation solution according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: mixing sterile injection water, glucose, human serum albumin, sodium chloride, potassium chloride and calcium chloride, filtering and packaging under nitrogen protection.
4. The method for using the cell cryotransport storage solution according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: uniformly mixing cells (stem cells and immune cells) with the cell low-temperature transport preservation solution described in any one of claims 1 to 2 to obtain a cell suspension, transferring the suspension into a sterile blood transfer bag and then placing the suspension into a vaccine box, and placing the partition of the vaccine box into an ice box.
5. The method for using the cell cryotransport storage solution according to claim 4, characterized in that: The cell concentration in the cell suspension was 1×10 6 -1×10 8 Pieces / mL.