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 particularly to a method for transporting cells.

Background Art

[0002] Cell therapy is widely used in clinical research on immune diseases, cancer, genetic diseases, etc., which involve steps such as cell collection, culture amplification, gene modification, and cell infusion / transplantation therapy, as a safe and effective treatment method. Since the implementation locations and times of each step do not match, in many cases, it is necessary to transport the cells used for treatment between different locations in a timely manner. One of the important factors affecting the success of cell therapy is to efficiently solve the problem of cell transportation.

[0003] Currently, there are mainly two types of cell transportation methods: cryopreservation transportation and liquid-filled room temperature transportation. Among these, cryopreservation transportation is carried out by freezing the cells and then placing them in a special container filled with liquid nitrogen or dry ice. The preservation effect is high, but the procedure is complex and not suitable for long-term transportation. Generally, air transportation is used, resulting in high costs. Liquid-filled room temperature transportation is carried out by filling a culture flask with cells adhered to it with cell culture medium. It is simple and practical, but during transportation, the cells are easily detached due to the upwelling of the culture medium, which has a significant impact on the cell activity.

[0004] Therefore, there is an urgent need for a cell transportation method that can be implemented simply and easily at low cost and has little impact on the activity of cells during transportation.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The technical problem to be solved by the present invention is to provide a cell transportation method that can be implemented simply and easily at low cost and has little impact on the activity of cells during transportation. s

Means for Solving the Problems

[0006] The present invention (1) Remove the biomaterial aqueous solution from a refrigerator at 4°C, aspirate an appropriate amount of the biomaterial aqueous solution, and mix it uniformly with the cells.

[0007] (2) A step in which the cell / material mixture from step (1) is placed at 37°C to form a hydrogel structure containing the cells.

[0008] (3) A step of transporting the hydrogel structure from step (2) with cell culture medium.

[0009] (4) A method for transporting cells, characterized by sequentially including the steps of placing the hydrogel structure transported in step (3) at 4°C, removing the material, and separating the cells by centrifugation.

[0010] (1) Remove the biomaterial aqueous solution from a refrigerator at 4°C, aspirate an appropriate amount of the biomaterial aqueous solution, and mix it uniformly with the cells.

[0011] (2) A step in which the cell / material mixture from step (1) is placed at 37°C to form a hydrogel structure containing the cells.

[0012] (3) The hydrogel structure from step (2) is placed in a cell culture medium, and three-dimensional cell culture is performed in a cell incubator.

[0013] (4) A step of transporting the hydrogel structure cultured in step (3) with cell culture medium.

[0014] (5) This is a cell transport method that sequentially includes the steps of placing the hydrogel structure transported in step (4) at 4°C, removing the material, and separating the cells by centrifugation.

[0015] The present invention relates to a method for transporting cells in which the biomaterial in each method comprises at least one of the following: poloxamer, collagen, peptide-based materials, chitosan and its derivatives, starch-based materials, serum albumin, clucopaine, 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, alginates and their 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.% in each method.

[0017] In this 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 cell transport is controlled within the range of 4 to 37°C.

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

[0021] The present invention provides a method for transporting cells in which a cell-containing hydrogel structure can be directly used without removing the material after cell transport.

[0022] The cell transportation method of the present invention, compared with the prior art, forms a three-dimensional hydrogel structure by encapsulating cells with a biomaterial under mild conditions, immerses the hydrogel in a cell culture solution for transportation, and after transportation, gently removes the biomaterial to achieve cell separation. It can be implemented simply and easily at low cost and has little impact on the activity of cells during transportation.

Brief Description of the Drawings

[0023] [Figure 1] It is a diagram showing the effect of live / dead cell staining of cells before and after transportation in the hydrogel structure of Example 1 (Figure 1A is the sample before transportation, and Figure 1B is the sample after transportation). [Figure 2] It is a diagram showing the effect of live / dead cell staining of cells before and after transportation in the hydrogel structure of Example 2 (Figure 2A is the sample before transportation, and Figure 2B is the sample after transportation).

Modes for Carrying Out the Invention

[0024] Example 1 This example is (1) Taking out an aqueous solution of a biomaterial containing 1% poloxamer and 1% collagen from a refrigerator at 4°C, aspirating 1 mL and uniformly mixing it with mouse aortic vascular smooth muscle cells to make the cell density 1×10 6 cells / mL.

[0025] (2) Placing the cell / material mixture in step (1) at 37°C to form a hydrogel structure encapsulating the cells.

[0026] (3) Placing the hydrogel structure in step (2) into a high-glucose DMEM cell culture medium containing 10% fetal bovine serum and transporting it at room temperature.

[0027] (4) A cell transportation method that sequentially includes the steps of placing the hydrogel structure after 5 days of transportation in step (3) at 4°C, removing the material, and separating the cells by centrifugation until the cell viability reaches 90%.

[0028] Cells within the hydrogel structure were labeled as live or dead before and after transport using AO / PI double staining reagents. Cell nuclei were then labeled using DAPI reagent, and cell viability was recorded by fluorescence microscopy. Acridine orange (AO) passed through the complete cell membrane and was taken up by the nuclei of all cells (live and dead), emitting green fluorescence. Propidium iodide (PI) passed through the incomplete cell membrane, i.e., only the cell membrane of dead cells, and was taken up by the nuclei of all dead cells, emitting red fluorescence. DAPI passed through the cell membrane, bound to double-stranded DNA, and emitted blue fluorescence. The results of the experiment are shown in Figure 1. The experiment revealed that cells exhibited a large amount of green fluorescence signal within the hydrogel structure before and after transport, indicating that the cells maintained a high viability.

[0029] Example 2 (1) Remove an aqueous solution of biomaterial containing 5% polyacrylamide and 0.1% polylysine from a refrigerator at 4°C, aspirate 2 mL, and homogeneously mix it with human umbilical vein endothelial cells, and set the cell density to 1 × 10⁻⁶. 5 The process of reducing the number of cells / mL

[0030] (2) A step in which the cell / material mixture from step (1) is placed at 37°C to form a hydrogel structure containing the cells.

[0031] (3) Place the hydrogel structure from step (2) into RPMI-1640 cell medium containing 10% fetal bovine serum, perform three-dimensional cell culture in a cell incubator, and culture for 10 days.

[0032] (4) The hydrogel structure from step (3) is placed in RPMI-1640 cell medium containing 10% fetal bovine serum and transported at 37°C.

[0033] (5) This cell transport method includes the steps of placing the hydrogel structure, which has been transported in step (4) for 3 days, at 4°C, removing the material, separating the cells by centrifugation, and achieving a cell viability of 80%.

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

[0035] Example 2 differs from Example 1 in that, before transporting the hydrogel structure in the cell culture medium in step (2), three-dimensional cell culture is first performed in a cell incubator. The decision of whether or not to perform three-dimensional cell culture before transporting the cells is mainly based on the following two points.

[0036] 1. Can the density of cells encapsulated within the hydrogel structure meet the cell demand required for use after transport? If the cell density before transport can meet the actual cell demand after transport, the cells can be used directly after transport without performing three-dimensional cell culture before transport. If the cell density before transport cannot meet the actual cell demand after transport, the cells can be cultured in three dimensions before transport, and the proliferation behavior within the hydrogel structure can increase the cell density, thereby meeting the actual cell demand after transport.

[0037] 2. Requirements for cell viability after transport. If the requirements for cell viability after transport are not high, for example, if the cell viability in this invention is 70%, the cells do not need to undergo three-dimensional cell culture before transport. The hydrogel structure mainly provides three-dimensional physical support for cell transport and does not provide a microenvironment suitable for cell proliferation, thus not affecting the cell viability during transport. If the requirements for cell viability after transport are high, for example, if the cell viability in this invention is 95%, the cells undergo three-dimensional cell culture before transport, continue to proliferate during culture, and continue to secrete extracellular matrix, thus building a microenvironment suitable for cell proliferation, which is advantageous for maintaining a high cell viability during transport. However, the length of the three-dimensional cell culture time is related to factors such as the culture environment of different cells, the cell proliferation state, the expected cell viability, and the form of cell use after transport, and an appropriate culture time should be selected according to different usage requirements.

[0038] Cells within the hydrogel structure were labeled as live or dead before and after transport using AO / PI double staining reagents. Cell nuclei were then labeled using DAPI reagent, and cell viability was recorded by fluorescence microscopy. Acridine orange (AO) passed through the complete cell membrane and was taken up by the nuclei of all cells (live and dead), emitting green fluorescence. Propidium iodide (PI) passed through the incomplete cell membrane, i.e., only the cell membrane of dead cells, and was taken up by the nuclei of all dead cells, emitting red fluorescence. DAPI passed through the cell membrane, bound to double-stranded DNA, and emitted blue fluorescence. The results of the experiment are shown in Figure 2. The experiment revealed that cells exhibited a large amount of green fluorescence signal within the hydrogel structure before and after transport, indicating that the cells maintained a high viability.

[0039] The present invention provides a method for transporting cells that forms a three-dimensional hydrogel structure by encapsulating cells in a biomaterial under mild conditions, transports the hydrogel by immersion in a cell culture medium at 4-37°C, and then gently removes the biomaterial after transport to achieve cell separation (or directly uses the cell-containing hydrogel structure without removing the material). This method is low-cost, simple, and easy to implement, and has little effect on the activity of cells during transport. Tests have confirmed that, assuming the temperature control range during cell transport is maintained at 4-37°C, the cell viability after transport by this method is between 80% and 95%.

[0040] The above embodiments are merely descriptions of preferred embodiments of the present invention, and any modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the design of the present invention shall all be included within the scope of protection as defined by the claims of the present invention. [Industrial applicability]

[0041] The present invention provides a method for transporting cells that is low-cost, simple, and easy to implement, has little effect on the activity of cells during transport, and can be applied to cell therapy. This method is key to the successful and smooth implementation of cell therapy in fields such as immunological diseases, cancer, and genetic diseases.

Claims

[Claim 1] (1) Take an aqueous solution of biomaterial containing 1% poloxamer and 1% collagen out of a refrigerator at 4°C, aspirate 1 mL of the aqueous biomaterial solution, and mix it uniformly with mouse aortic vascular smooth muscle cells to achieve a cell density of 1 × 10⁶ cells / mL. (2) A step in which the mouse aortic vascular smooth muscle cell / biomaterial mixture from step (1) is placed at 37°C to form a hydrogel structure that encloses the cells. (3) The hydrogel structure from step (2) is placed in a high-sugar DMEM cell culture medium containing 10% fetal bovine serum and transported at room temperature. (4) The hydrogel structure that has been transported in step (3) for 5 days is placed at 4°C, the biomaterial is removed, and mouse aortic vascular smooth muscle cells are separated by centrifugation, a step that is characterized by including these steps sequentially. A method for transporting mouse aortic vascular smooth muscle cells.

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