Xenotransplantation colloid set and use method thereof

Abstract

A xenotransplantation colloid set and a use method thereof. The xenotransplantation colloid set comprises a colloid material and a colloid solution; the colloid material comprises sodium alginate, a gelatin, and HEPES; the colloid solution comprises a divalent cation salt. After the colloid material and the colloid solution are mixed with cells, a xenotransplantation colloid is obtained, and the xenotransplantation colloid can be used to establish an animal experimental model. Components of the xenotransplantation colloid material are synthesized raw materials, and the components are simple and do not have biotoxicity. The hardness of culture colloids formed by the xenotransplantation colloid set varies according to different material ratios, and thus, the present invention is applicable to a wide range of cell lines; the present invention can be operated at room temperature, and has simple operation steps. When the xenotransplantation colloid is used for cell xenotransplantation, xenogeneic cells can well grow in experimental animal bodies, and no culture colloid remains.

Description

Allograft colloid kit and method of use thereof

[0001] Cross-reference information

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on June 14, 2023, with application number 202310702331.9 and invention name “Allogeneic transplant colloid kit and method of use thereof”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention relates to an allograft colloid kit and a method of using the same. Background Art

[0004] In biomedical research, isolated cell lines are often used as research models to study various phenomena and biological mechanisms within organisms. However, in real organisms, various cells interact with each other in complex ways, so the observed phenomena and biological mechanisms often differ from the results obtained from cell models. Therefore, animal experiments are a very important part of biomedical research.

[0005] However, when conducting animal experiments, such as animal experiments related to tumors, human tumor cells are not easy to grow in the bodies of experimental animals. Therefore, a culture method has been developed to culture cells in a gel culture medium and then transplant them into the bodies of experimental animals. However, the raw materials used in the gel culture medium currently used include animal raw materials, and 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 suspension culture of cells, the application of these culture media is currently limited to in vitro cell culture.

[0006] Summary of the Invention

[0007] The present invention relates to an allograft colloid kit and a method of using the same. The allograft colloid kit comprises a colloid material and a colloid solution.

[0008] The colloidal material of the present invention comprises 0.1-5 wt% sodium alginate, 1-30 wt% gelatin, 0.5-2 wt% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, 1-800 U / mL heparin sodium, and the remainder pure water. The colloidal solution further comprises 0.2-10 wt% divalent cation salt, 0.01-1 wt% HEPES buffer, and the remainder pure water.

[0009] In one embodiment, the colloidal material comprises 5-15 wt % of gelatin.

[0010] In one embodiment, the colloidal material includes 0.5-1 wt % HEPES buffer.

[0011] In one embodiment, the colloid material comprises 400-800 U / mL heparin sodium.

[0012] 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.

[0013] In one embodiment, the colloidal solution further comprises at least one growth factor.

[0014] 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.

[0015] The present invention also relates to a method for using the aforementioned allogeneic transplant colloid kit, comprising: preparing a colloid material, a colloid solution, and a cell culture solution; uniformly mixing the cell culture solution and the colloid solution to obtain a first mixed solution; and uniformly mixing the first mixed solution and the colloid material to obtain a second mixed solution.

[0016] In one embodiment, the cell culture solution contains at least one cell to be cultured.

[0017] In one embodiment, the cell density in the second mixed solution is between 0.1×10 6 ~5×10 7 cells / ml.

[0018] Thus, the xenograft colloid material of the present invention is composed of synthetic raw materials, which are simple and non-biotoxic. Furthermore, the xenograft colloid kit of the present invention can be supplemented with different cell culture materials, such as different growth factors, according to the needs of individual experiments. The hardness of the culture colloid formed by the xenograft colloid kit of the present invention varies depending on the ratio of the materials, and is applicable to a wide range of cell lines. 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

[0019] Figure 1: Analysis of tumor cell cluster growth in experimental mice (I).

[0020] Figure 2: Photograph of stained tissue sections of tumor cell clusters in experimental mice (I).

[0021] Figure 3: Analysis of tumor cell cluster growth in experimental mice (II).

[0022] Figure 4: Photograph of stained tissue sections of tumor cell clusters in experimental mice (II). DETAILED DESCRIPTION

[0023] 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 given below, and please refer to the drawings of the present invention.

[0024] 1. Colloidal materials

[0025] The colloidal material of the present invention comprises 0.1-5 wt% sodium alginate, 1-30 wt% gelatin, 0.5-2 wt% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, 1-800 U / mL heparin sodium, and the remainder pure water. In one embodiment, the colloidal material comprises 5-15 wt% gelatin; in another embodiment, the colloidal material comprises 0.5-1 wt% HEPES buffer; and in yet another embodiment, the colloidal material comprises 400-800 U / mL heparin sodium.

[0026] Please refer to Table 1, which shows 10 different colloidal material formulations containing different proportions of sodium alginate, gelatin, HEPES buffer, and sodium heparin. During use, the colloidal material is mixed with a colloidal solution, the resulting solution is left at room temperature, and the state of colloid formation is observed after 5 minutes. The colloidal solution contains 0.2-10 wt% of a divalent cation salt, 0.01-1 wt% of HEPES buffer, and the remaining percentage is pure water. The divalent cation salt includes at least one of strontium chloride, calcium phosphate, calcium chloride, and calcium sulfate.

[0027] As shown in Table 1, formulas 1 to 9 all formed colloids, but formula 10 did not. The colloid described here refers to a solution that does not flow freely and appears to be in a gel state.

[0028] Table 1

[0029] Next, a 1mL syringe was used to draw up the colloid formed from the above formulations and then injected using a 24G or 26G needle to test whether the colloid could be injected using a 24G or 26G needle. As shown in Table 2, all colloids formed from the formulations could be injected using a 24G needle. With the exception of Formulation 1, which could not be injected using a 26G needle, all other formulations could be injected using a 26G needle.

[0030] Table 2

[0031] 2. Application of Allograft Colloid Kit

[0032] The allograft colloid kit of the present invention mainly comprises the above-mentioned colloid material and colloid solution.

[0033] 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. The cell-colloid solution is then mixed with a colloid material at a volume ratio of 1:1 to obtain a first solution. The required volume of the first solution is drawn up with a syringe and allowed to stand at room temperature for at least 10 minutes. The first solution is then injected into an experimental animal, and the cell growth in the experimental animal is observed.

[0034] The following examples illustrate the use of the allogeneic transplantation colloid kit of the present invention in performing cell allogeneic transplantation in an animal model. The following examples merely provide one use of the present invention and are not intended to limit the scope of protection of the present invention.

[0035] First, a cell culture colloid containing cells is prepared by the following steps 1 to 4.

[0036] Step 1: The refrigerated colloidal material that has formed a gel block is warmed to 37°C, for example, by placing it in a 37°C water bath or a 37°C cell culture incubator to melt the colloidal material into a gel-like colloid.

[0037] Step 2: Preheat a cell-free cell culture medium to 37°C, 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. As the content ratio of the colloidal solution increases, the hardness of the cell culture colloid prepared will be higher.

[0038] 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 in it to obtain a cell-colloid solution. In this step, the concentration of cells dissolved in the colloidal solution can be between 1×10 6 ~4×10 7 cells / mL, for example 4×10 6 cells / mL.

[0039] Step 4: The colloid material returned to temperature in step 1 was mixed evenly with the cell-colloid solution in step 3 at a volume ratio of 1:1 to obtain a cell culture colloid. The cell density in the cell culture colloid was 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.

[0040] The following is an example of applying the above-mentioned cell culture colloid to xenotransplantation.

[0041] In this example, the colloidal materials used were Formulations 4, 5, and 7 in Table 1. The cell line used was HCT116, a human colon cancer cell line. The cells used in this example were purchased from the Bioresource Conservation and Research Center, with the cell number BCRC 60349. The three colloidal material formulations differed in their sodium heparin content.

[0042] HCT116 cells were prepared with the above steps to a cell density of 2×10 6 cells / 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.

[0043] This example used 5-6-week-old female mice of the C.B17 / Icr-Prkdcscid / CrlNarl strain. Cell culture gel was injected subcutaneously into the mice, and the growth of HCT116 cells in the mice was regularly observed after injection. The HCT116-containing cell culture gel grew into tumor-like masses at the injection site.

[0044] Please refer to Figure 1 (A), which shows the size of cell clumps in experimental mice 1 to 4 weeks after injection of the cell culture colloid. The cell culture colloids prepared with the three formulations all grew into tumor-like cell clumps at the injection site of the experimental mice. Among them, the cell culture colloid prepared with the colloid material of Formula 4 produced the largest cell clumps, with an average cell clump volume of 966.8 mm. 3 The cell culture colloid prepared by formula 5 produced the second largest cell agglomerates, 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 ; Figure 1 (B) is a photograph of the appearance of cell clumps on experimental mice four weeks after injection of cell culture colloid.

[0045] Four weeks after injection, the mice were sacrificed, and the cell clumps grown in their bodies were collected and stained with hematoxylin and eosin for microscopic observation. See Figure 2 . The staining results show that the cells injected into the mice had indeed grown into tumor-like clumps, with virtually no residual cell culture colloid observed.

[0046] 3. Xenotransplantation Experiments with Different Cell Culture Colloids

[0047] In this example, different cell culture colloid compositions and the cell culture colloid prepared according to Formula 4 of the present invention were used to subcutaneously inject the same number of HCT116 cells into experimental mice. The growth of tumor-like cell clusters was observed periodically after injection. In this experiment, the cell concentration in the cell culture colloid was 2×10 6 cells / mL.

[0048] This trial consists of four groups:

[0049] (a) Negative control group: cells were dissolved in 1X PBS buffer and then transplanted.

[0050] (b) Group M: Cell allografts were performed using a culture medium containing Matrigel.

[0051] (c) Group V: Cell allografts were performed using the culture medium of animal-free hydrogel Vitrogel.

[0052] (d) The present invention: Cell allotransplantation was performed using the cell culture colloid prepared using Formula 4 in Table 1.

[0053] Please refer to Figure 3 (A), which shows the cell mass growth analysis of the four groups of experimental mice. Four weeks after injection, the average volume of cell mass 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 796.7 mm 3 However, the size of the cell clusters in each experimental mouse was relatively consistent, so the standard error after statistics was small; the average volume of the cell clusters in the negative control group of experimental mice was 153.3mm 3 , and the average volume of cell aggregates in group V mice was 28.8 mm 3 Figure 3 (B) shows the appearance of cell aggregates in the experimental mice of each group four weeks after cell injection.

[0054] Figure 4 shows hematoxylin-eosin-stained photographs of cell aggregate tissue sections from each group of experimental mice. Mice in the negative control group, Group M, and the group receiving the cell culture colloid of Formula 4 of the present invention were sacrificed four weeks after injection, while mice in Group V were sacrificed six weeks after injection. While abundant HCT116 cells were observed growing in the aggregates of mice in Group M, residual gel matrix was also observed. In addition to the abundant HCT116 cells growing in the cell culture colloid of the present invention, no residual gel was observed within the aggregates. Furthermore, in the cell aggregates of mice in Group V, cell growth was poor, and a large amount of residual cell culture colloid was observed.

[0055] In addition, the xenotransplantation colloid kit of the present invention can be further supplemented 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, so as to be applied to the culture of different cells.

[0056] In summary, the allogeneic transplant colloid kit of the present invention has the following advantages:

[0057] 1. The present invention uses synthetic biological raw materials, which are simple in composition and do not have the risks of pathogens and endotoxins that may be present when using animal raw materials.

[0058] 2. The hardness of the colloid prepared by the allograft colloid kit of the present invention can be adjusted as needed, so it can be used for the culture of various cell types. In addition, the colloid material of the present invention takes a short time to form a gel, and the entire process can be operated at room temperature, making it very convenient to use.

[0059] 3. When allogeneic cell transplantation is performed using the allogeneic transplant colloid of the present invention, allogeneic cells can grow well in the body of the experimental animal without any residual culture colloid. In addition, the cell clumps of allogeneic cells grown in the body of 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.

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