Allograft colloid set and use method thereof
By using colloid sets of components such as sodium alginate, gelatin and sodium heparin, the quality and operability of gel matrix in animal experiments were solved, and efficient and safe cell culture and allograft were achieved at room temperature to form stable cell clumps improve the repeatability and safety of the experiment.
Patent Information
- Application Number
- CN202310702331.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-14
- Publication Date
- 2024-12-17
AI Technical Summary
The existing gel-like culture medium has animal quality differences and risk of pathogenic bacteria or endotoxins in animal experiments, and the operating temperature requirements are strict, which limits the flexibility and safety of cell culture, especially in the growth of tumor-related cells. Not effective.
Colloidal solution containing sodium alginate, gelatin, HEPES buffer and sodium heparin and divalent cationic salts is formed to form an allograft colloid set, which can be operated at room temperature, add growth factors to improve cell culture effect, and Suitable cell culture colloids are formed by mixing in a specific proportion.
It realizes cell culture colloids that are free of animal materials, biotoxicity and easy to operate. They are suitable for a variety of cell lines and form stable cell clumps in the experimental animals, reducing experimental errors and residual risks, and improving the ability of experiments. Repeatability.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to an allogeneic transplantation colloid kit and a method of using the same. Background Art
[0002] In the process of biomedical research, isolated cell lines are usually used as research models to study various phenomena and biological mechanisms in organisms. However, in real organisms, various cells have complex interactions, so the observed phenomena and biological mechanisms are often different from the results obtained from cell models. Therefore, animal experiments are a very important part of biomedical research.
[0003] However, when conducting animal experiments, such as animal experiments related to tumors, human tumor cells are not easy to grow in experimental animals. Therefore, a culture method has been developed to culture cells in a gel culture medium and then transplant them into experimental animals. However, the raw materials used in the gel culture medium currently used include animal raw materials, but the quality of each batch of animal raw materials may vary, and may also contain pathogens or endotoxins. In addition, the existing gel culture medium needs to be operated at a temperature within a specific range, which is not convenient to use. Although different cell culture colloids have been developed, such as Taiwan Patent No. TWI746069B, which provides a set of 3D cell culture colloids that can be used for in vitro 3D cell culture, and Taiwan Patent No. TWI774661B, which provides a culture medium composition that can be used for suspended cultured cells; however, the application of these culture media is currently limited to in vitro cultured cells. Summary of the invention
[0004] The present invention relates to an allogeneic transplantation colloid kit and a method of using the same. The allogeneic transplantation colloid kit comprises a colloid material and a colloid solution.
[0005] The colloidal material of the present invention comprises 0.1-5wt% sodium alginate, 1-30wt% gelatin, 0.5-2wt% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) buffer, 1-800U / mL heparin sodium and the remaining percentage of pure water. The colloidal solution comprises 0.2-10wt% divalent cation salts, 0.01-1wt% HEPES buffer and the remaining percentage of pure water.
[0006] In one embodiment, the colloid material includes 5-15 wt % gelatin.
[0007] In one embodiment, the colloidal material includes 0.5-1 wt % HEPES buffer.
[0008] In one embodiment, the colloid material comprises 400-800 U / mL heparin sodium.
[0009] In one embodiment, the divalent cation salt of the colloidal solution includes at least one of strontium chloride, calcium phosphate, calcium chloride, and calcium sulfate.
[0010] In one embodiment, the colloidal solution further comprises at least one growth factor.
[0011] In one embodiment, the growth factor comprises fibroblast growth factor, transforming growth factor-β, insulin-like growth factor, tumor-derived colony stimulating factor-1, epidermal growth factor, stromal cell-derived factor-1, platelet-derived growth factor, or glial cell line derived neurotrophic factor.
[0012] The present invention also relates to a method for using the above-mentioned allogeneic transplantation colloid kit, comprising: preparing a colloid material, a colloid solution and a cell culture solution; mixing the cell culture solution and the colloid solution evenly to obtain a first mixed solution; and mixing the first mixed solution and the colloid material evenly to obtain a second mixed solution.
[0013] In one embodiment, the cell culture solution contains at least one cell to be cultured.
[0014] In one embodiment, the cell density in the second mixed solution is between 0.1×10 6 ~5×10 7 cells / mL.
[0015] Thus, the components of the xenotransplantation colloid material of the present invention are synthetic raw materials, which are simple and non-biotoxic. Moreover, the xenotransplantation colloid kit of the present invention can be added with different cell culture materials according to the needs of individual experiments, such as adding different growth factors. The hardness of the culture colloid formed by the xenotransplantation colloid kit of the present invention will vary depending on the proportion of the materials, and the applicable cell lines are wide. In addition, the cell culture colloid kit of the present invention can be operated at room temperature, and the operation steps are simple. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 :Analysis of tumor cell mass growth in experimental mice (I).
[0017] Figure 2 :Photograph of stained tissue sections of experimental mouse tumor cell clusters (a).
[0018] Figure 3 :Analysis of tumor cell mass growth in experimental mice (II).
[0019] Figure 4 :Photograph of stained tissue sections of experimental mouse tumor cell clusters (II). DETAILED DESCRIPTION
[0020] In order to have a more complete and clear understanding of the technical means of the present invention and the effects that can be achieved, a detailed description is now given as follows, and please refer to the drawings of the present invention.
[0021] 1. Colloidal materials
[0022] The colloidal material of the present invention comprises 0.1-5wt% sodium alginate, 1-30wt% gelatin, 0.5-2wt% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, 1-800U / mL heparin sodium, and the remaining percentage of pure water. In one embodiment, the colloidal material comprises 5-15wt% gelatin; in another embodiment, the colloidal material comprises 0.5-1wt% HEPES buffer; in another embodiment, the colloidal material comprises 400-800U / mL heparin sodium.
[0023] Please refer to Table 1, which shows 10 different colloidal material formulations, which contain different proportions of sodium alginate, gelatin, HEPES buffer and sodium heparin; when used, the colloidal material is mixed with a colloidal solution, the obtained solution is placed at room temperature, and the state of colloid formation is observed after 5 minutes; the above-mentioned colloidal solution contains 0.2-10wt% of divalent cation salts, 0.01-1wt% of HEPES buffer and the remaining percentage of pure water; wherein the divalent cation salt includes at least one of strontium chloride, calcium phosphate, calcium chloride, and calcium sulfate.
[0024] As shown in Table 1, formulas 1 to 9 can form colloids, but formula 10 cannot form colloids. The colloid described here refers to a state in which the solution does not flow freely and presents a gel state.
[0025] Table 1
[0026]
[0027] Next, use a 1mL syringe to extract the colloid formed by the above formula materials, and then use a 24G or 26G needle to perform an injection test to observe whether the colloid can be injected with a 24G or 26G needle. Please refer to Table 2, all colloids formed by the formula can be injected with a 24G needle; and except for the colloid of formula 1, which cannot be injected with a 26G needle, the colloids of other formulas can be injected with a 26G needle.
[0028] Table 2
[0029]
[0030] 2. Application of allogeneic transplantation colloid kit
[0031] The allogeneic transplantation colloid kit of the present invention mainly comprises the above-mentioned colloid material and colloid solution.
[0032] When using the cell culture colloid kit of the present invention, a cell culture solution containing cells is first mixed with a colloid solution to obtain a cell-colloid solution; then the cell-colloid solution and the colloid material are mixed evenly at a volume ratio of 1:1 to obtain a first solution; a required volume of the first solution is drawn with a syringe and left to stand at room temperature for at least 10 minutes; then, the first solution is injected into an experimental animal, and the cell growth in the experimental animal is observed.
[0033] The following examples are the use of the allogeneic transplantation colloid kit of the present invention to perform cell allogeneic transplantation in an animal model; the following examples only provide one use of the present invention and are not intended to limit the scope of protection of the present invention.
[0034] First, a cell culture colloid containing cells is prepared by the following steps 1 to 4.
[0035] Step 1: The refrigerated colloidal material that has formed a gel block is warmed up to 37° C., for example, by placing it in a 37° C. water bath or in a 37° C. cell culture incubator to melt the colloidal material into a gel-like colloid.
[0036] Step 2: Preheat a cell-free cell culture medium at 37°C, and then mix the cell culture medium with the colloidal solution at a volume ratio of 1:1 to 3:1 to prepare a 0.25X to 0.5X colloidal solution; the mixing ratio of the colloidal solution to the cell culture medium is related to the hardness of the cell culture colloid prepared subsequently. If the content ratio of the colloidal solution increases, the hardness of the prepared cell culture colloid will be higher.
[0037] Step 3: Take a cell seed solution, centrifuge it, remove the supernatant, and dissolve the precipitated cell aggregates with the 0.25X to 0.5X colloidal solution of step 2, and evenly disperse the cells therein to obtain a cell-colloid solution; in this step, the concentration of the cells dissolved in the colloidal solution can be between 1×10 6 ~4×10 7 cells / mL, for example 4×10 6 cells / mL.
[0038] Step 4: The colloid material warmed up in step 1 and the cell-colloid solution in step 3 are mixed evenly at a volume ratio of 1:1 to obtain a cell culture colloid. The cell density in the cell culture colloid is between 0.5×10 6 ~2×10 7 cells / mL, for example 2×10 6 cells / mL. The cell culture colloid obtained in this step can be applied to animal models, such as cell allogeneic transplantation.
[0039] The following is an example of applying the above-mentioned cell culture colloid to allogeneic transplantation.
[0040] In this embodiment, the colloidal materials used are respectively Formula 4, Formula 5 and Formula 7 in Table 1, and the cell line used is HCT116 cells, which is a human colon cancer cell line. The cells used in this embodiment were purchased from the Biological Resources Conservation and Research Center, and the cell number is BCRC 60349. The difference between the three formula colloidal materials is the different content of heparin sodium.
[0041] HCT116 cells were prepared with the above steps to a cell concentration of 2×10 6cells / mL of cell culture colloid, and draw 0.2mL of cell culture colloid with a syringe equipped with a 24G needle or a syringe without a needle; let the syringe filled with cell culture colloid stand at room temperature for at least 10 minutes before cell injection.
[0042] This example uses C.B17 / Icr-Prkdcscid / CrlNarl strain mice, which are 5-6 weeks old female mice; the cell culture gel is injected into the mouse by subcutaneous injection, and the growth of HCT116 cells in the mouse is regularly observed after injection. The cell culture gel containing HCT116 will grow into a mass similar to tumor tissue at the injection site of the mouse.
[0043] See also Figure 1 (A) shows the size of cell clusters on the experimental mice 1 to 4 weeks after the injection of cell culture colloids. The cell culture colloids prepared with the three formulas can grow into tumor-like cell clusters at the injection site of the experimental mice. Among them, the cell culture colloid prepared with the colloid material of formula 4 has the largest volume of cell clusters, and its average cell cluster volume is 966.8mm 3 The cell agglomerates grown from the cell culture colloid prepared by formula 5 were the second largest, with an average cell agglomerate volume of 649.6 mm 3 The cell culture colloid prepared by formula 7 had the smallest volume of cell aggregates, with an average cell aggregate volume of 513.3 mm 3 ;again Figure 1 (B) is a photograph showing the appearance of cell aggregates on experimental mice four weeks after injection of cell culture colloid.
[0044] Four weeks after the injection, the experimental mice were sacrificed and the cell aggregates grown in their bodies were collected, stained with hematoxylin and eosin, and observed under a microscope; see Figure 2 The staining results showed that the cells injected into the experimental mice did grow into clumps similar to tumor tissue, and almost no residual cell culture colloid was observed.
[0045] III. Xenotransplantation experiments with different cell culture colloids
[0046] In this example, different cell culture colloids and the cell culture colloid prepared by formula 4 of the present invention were used to inject the same number of HCT116 cells into experimental mice by subcutaneous injection, and the growth of tumor-like cell clusters was observed regularly after injection. In this experiment, the cell concentration in the cell culture colloid was 2×10 6 cells / mL.
[0047] This trial consists of four groups:
[0048] (a) Negative control group: cells were dissolved in 1X PBS buffer and then transplanted.
[0049] (b) Group M: Cell allogeneic transplantation was performed using a culture medium containing Matrigel.
[0050] (c) Group V: Cell allogeneic transplantation was performed using the culture medium of animal-free hydrogel Vitrogel.
[0051] (d) The present invention: Cell culture colloid prepared by formula 4 in Table 1 is used for cell allogeneic transplantation.
[0052] See also Figure 3 (A) is a graph showing the growth analysis of cell clusters in the four groups of experimental mice. Four weeks after injection, the average volume of cell clusters in the experimental mice in group M was the largest, at 946.2 mm. 3 However, the size of the cell clumps in each experimental mouse was quite different, so the standard error after statistics was large; the average volume of the cell clumps in the cell culture colloid group of formula 4 of the present invention was slightly smaller than that of the M group, which was 796.7mm 3 However, the size of the cell clusters in each experimental mouse was relatively consistent, so the statistical standard error was small; the average volume of the cell clusters in the negative control group of experimental mice was 153.3mm 3 The average volume of cell aggregates in group V experimental mice was 28.8 mm 3 . Figure 3 (B) is a photograph of the appearance of cell clusters in each group of experimental mice four weeks after cell injection.
[0053] Figure 4 The following are photos of hematoxylin-eosin staining of cell mass tissue sections of experimental mice in each group; the experimental mice in the negative control group, M group and the cell culture colloid group of formula 4 of the present invention were sacrificed four weeks after injection, while the experimental mice in group V were sacrificed six weeks after injection. Although a large amount of HCT116 cells were observed growing in the mass of experimental mice in group M, residual gel matrix was also observed; in addition to a large amount of HCT116 cells growing in the experimental mice in the cell culture colloid group of the present invention, there was no residual colloid in the cell mass; and in the cell mass of experimental mice in group V, the cell growth was poor and a large amount of residual cell culture colloid was also observed.
[0054] In addition, the xenotransplantation colloid kit of the present invention can be further added with different growth factors, such as fibroblast growth factor, transforming growth factor, insulin-like growth factor, tumor-derived colony stimulating factor, epidermal growth factor, stromal cell-derived factor, platelet-derived growth factor or neurotrophic factor derived from glial cell line, etc., so as to be applied to the culture of different cells.
[0055] In summary, the allogeneic transplantation colloid kit of the present invention has the following advantages:
[0056] 1. The present invention uses synthetic biological raw materials for preparation, the ingredients are simple and there is no risk of pathogens and endotoxins that may be present when using animal raw materials in the past.
[0057] 2. The hardness of the prepared colloid of the allogeneic transplantation colloid kit of the present invention can be adjusted according to the needs so as to be applied to the culture of various types of cells; in addition, the colloid material of the present invention takes a short time to form a gel, and the whole process can be operated at room temperature, which is very convenient to use.
[0058] 3. When the allogeneic transplant colloid of the present invention is used for cell allogeneic transplantation, the allogeneic cells can grow well in the experimental animal without residual culture colloid; in addition, the cell clusters of allogeneic cells grown in the experimental animal are similar in size, which can reduce experimental errors and improve experimental reproducibility.
Claims
1. A colloidal material comprising 0.1-5wt% sodium alginate, 1-30wt% gelatin, 0.5-2wt% HEPES buffer, 1-800U / mL heparin sodium and the remaining percentage of pure water.
2. An allogeneic transplantation colloid kit, comprising a cell colloid material and a colloid solution, wherein: The cell colloid material comprises 0.1-5wt% sodium alginate, 1-30wt% gelatin, 0.5-2wt% HEPES buffer, 1-800U / mL heparin sodium and the remaining percentage of pure water, and the colloid solution comprises 0.2-10wt% divalent cation salts, 0.01-1wt% HEPES buffer and the remaining percentage of pure water.
3. The allogeneic transplant colloid kit according to claim 2, wherein: The divalent cation salt of the colloidal solution includes at least one of strontium chloride, calcium phosphate, calcium chloride, and calcium sulfate.
4. The allogeneic transplant colloid kit according to claim 2, wherein: The colloidal solution further comprises at least one growth factor.
5. The allogeneic transplant colloid kit according to claim 4, wherein: The growth factor comprises fibroblast growth factor, transforming growth factor, insulin-like growth factor, tumor-derived colony stimulating factor, epidermal growth factor, stromal cell-derived factor, platelet-derived growth factor or glial cell line-derived neurotrophic factor.
6. A method for culturing cells using the allogeneic transplantation colloid kit as claimed in claim 2, comprising: preparing a colloid material, a colloid solution and a cell culture solution; mixing the cell culture solution and the colloid solution evenly to obtain a first mixed solution; and mixing the first mixed solution and the colloid material evenly to obtain a second mixed solution.
7. The method of claim 6, wherein: The cell culture solution contains at least one cell to be cultured.
8. The method of claim 7, wherein: The cell density in the second mixed solution is between 0.1×10 6 ~5×10 7 cells / mL.
9. The method of claim 6, wherein: The mixing ratio of the cell culture solution to the colloid solution is 1:1 to 3:1.