Cell transport methods

Abstract

The present invention relates to biotechnology, and in particular to a method for transporting cells. The present invention can directly transport cells at room temperature after mixing with a material, or can transport cells at room temperature after mixing with a material and performing three-dimensional culture, during which the cells maintain a high viability of 80% or more, and after transportation, the cell / material mixture can be used directly, or the cells can be separated and used.

Description

[Technical field]

[0001] The present invention relates to biotechnology, and in particular to a method for transporting cells. [Background technology]

[0002] As a safe and effective treatment, cell therapy is widely used in clinical research on immune diseases, cancer, genetic diseases, etc., which involves steps such as cell collection, culture amplification, gene modification, and cell infusion / transplantation therapy. Since the locations and times of each step are not consistent, cells used in therapy often need to be transported between different locations in a timely manner. One of the key factors that determine the success or failure of cell therapy is to efficiently solve the cell transportation.

[0003] Currently, there are two main methods for transporting cells: cryopreservation transport and liquid-filled room temperature transport. In cryopreservation transport, cells are frozen and then placed in special containers filled with liquid nitrogen or dry ice, which has a high preservation effect, but the procedure is complicated and not suitable for long-term transport, so air transport is generally used, which is expensive. In liquid-filled room temperature transport, the culture flasks to which the cells have adhered are filled with cell culture medium, which is simple and practical, but the culture medium may bubble up during transport, making the cells prone to detachment, which has a significant impact on the activity of the cells.

[0004] Therefore, there is an urgent need for a method for transporting cells that is low-cost, simple and easy to implement, and that has little effect on the activity of the cells during transport. Summary of the Invention [Problem to be solved by the invention]

[0005] The technical problem to be solved by the present invention is to provide a method for transporting cells which is simple and easy to carry out at low cost and which has little effect on the activity of the cells during transport. [Means for solving the problem]

[0006] The present invention relates to (1) removing the aqueous biomaterial solution from a 4°C refrigerator, aspirating an appropriate amount of the aqueous biomaterial solution, and uniformly mixing it with the cells;

[0007] (2) placing the cell / material mixture of step (1) at 37° C. to form a cell-encapsulating hydrogel structure;

[0008] (3) transporting the hydrogel structure of step (2) in a cell culture medium;

[0009] (4) A method for transporting cells, comprising the steps of: placing the hydrogel structure transported in step (3) at 4°C; removing the material; and isolating the cells by centrifugation.

[0010] (1) removing the aqueous biomaterial solution from a 4°C refrigerator, aspirating an appropriate amount of the aqueous biomaterial solution, and uniformly mixing it with the cells;

[0011] (2) placing the cell / material mixture of step (1) at 37° C. to form a cell-encapsulating hydrogel structure;

[0012] (3) placing the hydrogel structure of step (2) in a cell culture medium and performing three-dimensional cell culture in a cell incubator;

[0013] (4) transporting the hydrogel structure cultured in step (3) in a cell culture medium;

[0014] (5) placing the hydrogel structure transported in step (4) at 4° C., removing the material, and isolating the cells by centrifugation.

[0015] The present invention is a method for transporting cells, wherein the biomaterial in each method includes at least one of poloxamer, collagen, peptide-based materials, chitosan and its derivatives, starch-based materials, serum albumin, clupein, polylysine, carbomer, polymethacrylic acid, poly N-isopropylacrylamide, polyacrylamide, acrylic acid, gelatin and its derivatives, hyaluronic acid, gellan gum, polyvinyl alcohol, fibrinogen, keratin, fibronectin, reduced keratin, cellulose-based materials, alginate and its derivatives, and xylan.

[0016] The present invention relates to a method for transporting cells, in which the concentration of the biomaterial is in the range of 0.001 to 20 wt. %.

[0017] In the present invention, in step (1) of each method, the density of cells in the aqueous biomaterial solution is 1×10 5 ~1×10 9 This is a method for transporting cells in the range of cells / mL.

[0018] The present invention relates to a method for transporting cells, in which the three-dimensional cell culture time is in the range of 3 to 30 days.

[0019] The present invention relates to a method for transporting cells, in which the temperature during transport of the cells is controlled in the range of 4 to 37°C.

[0020] The present invention relates to a method for transporting cells, in which the cell viability is 70% or more and 100% or less, and preferably 80% or more and 95% or less, in each method.

[0021] The present invention is a method for transporting cells that allows the cell-containing hydrogel structure to be used directly without removing the material after the delivery of the cells in each method.

[0022] Compared to conventional techniques, the cell transport method of the present invention forms a three-dimensional hydrogel structure by encapsulating cells in a biomaterial under mild conditions, transports the hydrogel by immersing it in a cell culture medium, and after transport, separates the cells by gently removing the biomaterial, which can be performed simply and easily at low cost and with little effect on the activity of the cells during transport. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1A is a diagram of the effect of live / dead cell staining before and after transportation in the hydrogel structure of Example 1 (FIG. 1A is the sample before transportation, and FIG. 1B is the sample after transportation). [Diagram 2] FIG. 2 is a diagram of the effect of live / dead cell staining before and after transportation in the hydrogel structure of Example 2 (FIG. 2A is the sample before transportation, and FIG. 2B is the sample after transportation). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Example 1 In this embodiment, (1) An aqueous solution of biomaterial containing 1% poloxamer and 1% collagen was taken out of a 4°C refrigerator, 1 mL of the solution was aspirated and mixed uniformly with mouse aortic vascular smooth muscle cells to a cell density of 1 × 10 6 a step of making the concentration per mL;

[0025] (2) placing the cell / material mixture of step (1) at 37° C. to form a cell-encapsulating hydrogel structure;

[0026] (3) placing the hydrogel structure of step (2) in high-sugar DMEM cell culture medium containing 10% fetal bovine serum and transporting at room temperature;

[0027] (4) A method for transporting cells, comprising the steps of placing the hydrogel structure transported in step (3) at 4°C for 5 days, removing the material, and isolating the cells by centrifugation, so that the cell viability is 90%.

[0028] The cells in the hydrogel structure were labeled as live / dead cells using AO / PI dual staining reagent before and after the cell transport, respectively, and the cell nuclei were labeled with DAPI reagent, and the cell viability was recorded by observation under a fluorescent microscope. Among them, Acridine Orange (AO) passed through the complete cell membrane and was incorporated into the cell nuclei of all cells (live and dead cells) and emitted green fluorescence, Propidium iodide (PI) passed through only the incomplete cell membrane, i.e., the cell membrane of dead cells, and was incorporated into the cell nuclei of all dead cells and emitted red fluorescence, and DAPI passed through the cell membrane, bound to double-stranded DNA, and emitted blue fluorescence. The test results are shown in Figure 1. The test revealed that the cells showed a large amount of green fluorescent signal in the hydrogel structure before and after the transport, and the cells maintained a high viability.

[0029] Example 2 (1) Take out the biomaterial aqueous solution containing 5% polyacrylamide and 0.1% polylysine from the refrigerator at 4 °C, aspirate 2 mL, and mix it evenly with human umbilical vein endothelial cells to adjust the cell density to 1 × 10 5 a step of making the concentration per mL;

[0030] (2) placing the cell / material mixture of step (1) at 37° C. to form a cell-encapsulating hydrogel structure;

[0031] (3) placing the hydrogel structure of step (2) in RPMI-1640 cell culture medium containing 10% fetal bovine serum, performing three-dimensional cell culture in a cell incubator, and culturing for 10 days;

[0032] (4) placing the hydrogel structure of step (3) in RPMI-1640 cell culture medium containing 10% fetal bovine serum and transporting at 37° C.;

[0033] (5) A method for transporting cells, comprising the steps of placing the hydrogel structure transported in step (4) at 4°C for 3 days, removing the material, and isolating the cells by centrifugation, so that the cell viability is 80%.

[0034] In the cell transportation method of the present invention, the time period for the three-dimensional cell culture in step (3) can be any period from 3 to 30 days, in addition to 10 days.

[0035] Example 2 differs from Example 1 in that the hydrogel structure is first placed in a cell culture medium and three-dimensional cell culture is performed in a cell incubator before being placed in the cell culture medium and transported in step (2).Whether or not to perform three-dimensional cell culture before transporting the cells is selected taking into consideration the following two main points.

[0036] 1. Whether the density of cells encapsulated in the hydrogel structure can meet the cell demand required in the use process after transportation. If the cell density before transportation can meet the actual cell demand after transportation, the cells can be used directly after transportation without three-dimensional cell culture before transportation; if the cell density before transportation cannot meet the actual cell demand after transportation, the cells can be used directly after transportation by three-dimensional cell culture before transportation and increasing the cell density in the hydrogel structure by the proliferation behavior in the hydrogel structure to meet the actual cell demand.

[0037] 2. After transportation, the requirement for cell viability during use. If the requirement for cell viability after transportation is not high, for example, the cell viability in the present invention is 70%, the cells do not need to be three-dimensional cell cultured before transportation, and the hydrogel structure mainly provides three-dimensional physical support for the transportation of cells, and does not provide a suitable microenvironment for cell proliferation, so that the cell viability during transportation is not affected; if the requirement for cell viability after transportation is high, for example, the cell viability in the present invention is 95%, the cells are three-dimensional cell cultured before transportation, and continue to proliferate during the culture, and also continue to secrete extracellular matrix, which builds a suitable microenvironment for cell proliferation, so that it is favorable to maintain high cell viability during transportation; however, the length of the three-dimensional cell culture time is related to factors such as different cell culture environments, cell proliferation conditions, the desired cell viability, and the use form of cells after transportation, and the appropriate culture time is selected according to different usage requirements.

[0038] The cells in the hydrogel structure were labeled as live / dead cells using AO / PI dual staining reagent before and after the cell transport, respectively, and the cell nuclei were labeled with DAPI reagent, and the cell viability was recorded by observation under a fluorescent microscope. Among them, Acridine Orange (AO) passed through the complete cell membrane and was incorporated into the cell nuclei of all cells (live and dead cells) and emitted green fluorescence, Propidium iodide (PI) passed through the incomplete cell membrane, i.e., only the cell membrane of dead cells, and was incorporated into the cell nuclei of all dead cells and emitted red fluorescence, and DAPI passed through the cell membrane, bound to double-stranded DNA, and emitted blue fluorescence. The test results are shown in Figure 2. The test revealed that the cells showed a large amount of green fluorescent signal in the hydrogel structure before and after the transport, and the cells maintained a high viability.

[0039] The present invention provides a method for transporting cells, which involves forming a three-dimensional hydrogel structure by encapsulating cells in a biomaterial under mild conditions, transporting the hydrogel by immersing it in a cell culture medium at 4 to 37°C, and then gently removing the biomaterial after transport to achieve cell separation (or directly using the cell-containing hydrogel structure without removing the material). This method can be performed simply and easily at low cost, with little effect on the activity of cells during transport, and tests have confirmed that, provided that the temperature control range during cell transport is maintained at 4 to 37°C, the cell viability is 80% or more and 95% or less after transport using this method.

[0040] The above examples are merely illustrative of the preferred embodiments of the present invention, and various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the design of the present invention shall all fall within the protection scope determined by the claims of the present invention. [Industrial Applicability]

[0041] The present invention provides a cell transport method that is simple and easy to implement at low cost, has little effect on the activity of the cells during transport, and can be applied to cell therapy, and is key to successfully implementing cell therapy procedures in the fields of immune diseases, cancer, genetic diseases, etc.

Claims

1. (1) removing the aqueous biomaterial solution from a 4° C. refrigerator, aspirating an appropriate amount of the aqueous biomaterial solution, and uniformly mixing it with the cells; (2) placing the cell / material mixture of step (1) at 37° C. to form a cell-encapsulating hydrogel structure; (3) transporting the hydrogel structure of step (2) in a cell culture medium; (4) placing the hydrogel structure delivered in step (3) at 4° C., removing the material, and isolating the cells by centrifugation. Methods for transporting cells.

2. (1) removing the aqueous biomaterial solution from a 4° C. refrigerator, aspirating an appropriate amount of the aqueous biomaterial solution, and uniformly mixing it with the cells; (2) placing the cell / material mixture of step (1) at 37° C. to form a cell-encapsulating hydrogel structure; (3) placing the hydrogel structure of step (2) in a cell culture medium and performing three-dimensional cell culture in a cell incubator; (4) transporting the hydrogel structure cultured in step (3) in a cell culture medium; (5) placing the hydrogel structure delivered in step (4) at 4° C., removing the material, and isolating the cells by centrifugation. Methods for transporting cells.

3. The biomaterial is characterized in that it contains at least one of poloxamer, collagen, peptide-based materials, chitosan and its derivatives, starch-based materials, serum albumin, clupein, polylysine, carbomer, polymethacrylic acid, poly N-isopropylacrylamide, polyacrylamide, acrylic acid, gelatin and its derivatives, hyaluronic acid, gellan gum, polyvinyl alcohol, fibrinogen, keratin, fibronectin, reduced keratin, cellulose-based materials, alginate and its derivatives, and xylan. A method for transporting cells according to claim 1 or 2.

4. The concentration of the biomaterial is in the range of 0.001 to 20 wt. %. A method for transporting cells according to claim 3.

5. In step (1), the density of cells in the aqueous biomaterial solution is 1×10 ５ ~1×10 ９ The range of the number of particles / mL A method for transporting cells according to claim 1 or 2.

6. In step (3), the time for the three-dimensional cell culture is in the range of 3 to 30 days. A method for transporting cells according to claim 2.

7. The temperature during cell transport is controlled in the range of 4 to 37°C. A method for transporting cells according to claim 1 or 2.

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