Cell transplantation methods
The described cell transplantation method addresses issues of cell loss and concentration variation by using a centrifuged capillary with a tapered end to concentrate and transplant cells efficiently.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- NAT UNIV CORP EHIME UNIV
- Filing Date
- 2022-03-10
- Publication Date
- 2026-07-01
AI Technical Summary
Existing cell transplantation methods using microinjection result in decreased cell numbers, variations in cell concentration, and increased loss due to contamination and centrifugation, leading to inconsistent cell transplantation.
A method involving a capillary with one closed, tapered end and one open end, centrifuging the capillary to separate culture medium from cells, forming a hole in the tapered tip, and transplanting concentrated cells through the pores.
This method simplifies the process, reduces contamination, and increases cell concentration for transplantation, minimizing cell loss.
Smart Images
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Abstract
Description
Technical Field
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[0001] The present invention relates to a new method for cell transplantation.
Background Art
[0002] In recent years, mainly in the field of life sciences, injection methods such as the microinjection method have been applied to introduce and transplant new cells and the like into objects to be introduced such as individuals and biological tissues (hereinafter also referred to as transplantation objects). The microinjection method is a method of piercing a fine hollow glass needle (capillary) filled with cells to be transplanted into a transplantation object and injecting the cells to be transplanted into the transplantation object through the capillary. Therefore, generally, the tip of the capillary that pierces the transplantation object is sharp, and the other end has a released shape in order to fill the cells (Patent Document 1).
Prior Art Documents
Patent Documents
[0003]
Patent Document 1
Summary of the Invention
Problems to be Solved by the Invention
[0004] However, in order to fill the capillary with the cells to be transplanted, since the cells dispersed in the culture solution are filled from the released end, the culture solution is also filled at the same time in that case. As a result, there are problems such as a decrease in the number of cells to be transplanted, variations in cell concentration within the capillary, and different numbers of transplanted cells for each individual of the transplantation object.
[0005] In addition, when the capillary is filled after removing the culture solution by centrifugation, there is also a problem that the loss in the operation before filling the capillary increases.
[0006] The present invention aims to provide a cell transplantation method that is simple, minimizes contamination of the culture medium, and increases the concentration of cells for transplantation. [Means for solving the problem]
[0007] The objective of the present invention was achieved as follows. 1. 1) A step of preparing a capillary in which one end is a closed, tapered shape for injection into the target object, and the other end is open. 2) Filling the capillary with a culture medium containing cells for transplantation from the other open end. 3) Centrifuging the capillary filled with the cells with the tapered end facing outwards to separate the culture medium. 4) A step of removing the separated culture medium and obtaining a capillary filled with a cell concentrate containing concentrated cells. 5) A step of forming a hole in the tapered tip of a capillary filled with the cell concentrate for injecting and transplanting cells, and 6) A step of transplanting the concentrated cells into other cells through the formed pores, A method for transplanting cells having the following characteristics. [Effects of the Invention]
[0008] According to the present invention, it is possible to provide a cell transplantation method that is simple, can reduce contamination of the culture medium, and can increase the concentration of cells for transplantation. [Brief explanation of the drawing]
[0009] [Figure 1] This is a schematic diagram illustrating a conventional transplantation method. [Figure 2] This is a schematic diagram of the capillary used in the present invention. [Figure 3] This is a schematic diagram of a capillary filled with cells dispersed in a culture medium, used in each step of the present invention. [Figure 4] This is a schematic diagram of a capillary tube with a hole formed at its tip according to the present invention. [Figure 5] This is a schematic diagram of the process for transplanting concentrated cells according to the present invention. [Figure 6] This is a schematic diagram of the centrifuge used in the centrifugal separation process of the present invention. [Modes for carrying out the invention]
[0010] Embodiments of the present invention will be described in detail below.
[0011] Figure 1 shows a conventional transplantation method. Cells to be transplanted, dispersed in culture medium, are filled into a capillary with an open end, using the larger end of the capillary, while leaving a small amount of air at the tip. Transplantation to other cells is performed by removing the air from the tapered tip of the capillary, inserting it into the other cell, and injecting the filled cells.
[0012] In this method, the transplanted cells are dispersed at the same concentration as they were in the culture medium, resulting in variations in cell concentration.
[0013] The present invention will be explained with reference to schematic diagrams 2 to 6 illustrating the steps of the present invention. It is preferable that the following operations be performed under conditions that allow the cells to be transplanted to survive (4°C to 30°C in the case of fish cells).
[0014] First, prepare the capillary shown in Figure 2 in step 1) preparing the capillary. One end S has a closed, tapered shape for injection into cells, and the other end O is open for filling with cells dispersed in culture medium. This capillary can be made at home using conventional methods, or it can be purchased commercially. A capillary tube has a tapered section of length L1, a cylindrical section of length L2, and an inner diameter of Φ. Preferably, L1 is 3-6 mm, L2 is 30-100 mm, and Φ is 0.8-1.2 mm.
[0015] In step 2), cells dispersed in the culture solution are introduced into the capillary from the open end O of the capillary prepared in step 1). Fig. 3-1 shows a capillary filled with cells dispersed in the culture solution.
[0016] Step 3) is a step of centrifuging the capillary filled with cells in step 2) with the tapered end facing outward to separate the culture solution. Fig. 3-2 shows the state after centrifuging the capillary filled with cells dispersed in the culture solution. Centrifugation is performed using a device as shown in Fig. 6, with the tip S facing outward. Then the cells are concentrated on the tip S side, and the excess culture solution is separated to the other end O side. Either a fixed-type or a swing-arm type centrifuge can be used.
[0017] Step 4) is a step of removing the centrifuged culture solution and obtaining a capillary filled with a cell concentrate containing the concentrated cells. Fig. 3-3 shows the state where the excess culture solution has been removed from the open end O. The cells to be transplanted are filled at a high concentration.
[0018] Step 5) is a step of forming a hole for injecting and transplanting cells at the tapered tip of the capillary filled with the cell concentrate. Fig. 4 shows the step of obliquely cutting and forming a hole for injecting into the cells to be transplanted at the tapered tip S. The position and shape of the hole can be appropriately selected according to the type of cells to be transplanted. For example, it may be formed at a horizontal position slightly away from the tip S. As the shape, it can be cut perpendicular to the longitudinal direction of the capillary or obliquely. The cut surface is preferably shaped to facilitate insertion into the cells to be transplanted.
[0019] Step 6) is the process of transplanting concentrated cells into other cells through a formed pore. Figure 5 shows the injection of cells to be transplanted through a capillary with a pore formed at its tip S. The tip S with the formed pore is inserted into the cells to be transplanted, and the highly concentrated cells, which have been centrifuged, are injected directly without contact with any other manipulative means. In this invention, since the cells are transplanted immediately after centrifugation, the loss of transplanted cells is extremely small.
[0020] Figure 6 shows a device for centrifuging a capillary. The device shown in Figure 6 has two capillary mounting points symmetrically positioned with respect to the centrifugal axis. There may be one or more mounting points, and they may be located above or below each other. It may be either a fixed or swing-arm type. The present invention will be described in detail below with reference to examples. [Examples]
[0021] The present invention will be described in detail below with reference to examples. Unless otherwise noted, the following experiments were conducted under conditions of 23°C and 50% RH.
[0022] The results of using this technique for spermatogonial cell transplantation into the peritoneal cavity of hatched zebrafish (Danio rerio) larvae are shown in the examples. The testes of genetically modified adult zebrafish, in which germ cells were labeled with red fluorescent protein, were excised, finely shredded with a surgical scalpel, and then germ cells were isolated by shaking culture in 0.1% trypsin dissolved in PBS(-).
[0023] Spermatogonia were concentrated by density gradient centrifugation using Percoll solutions adjusted to 30% and 35%, and then resuspended in PBS(-) containing 0.1% bovine serum albumin. The suspended cells were counted using a cell counter, and the concentration was calculated. In this example, cells were prepared to a concentration of approximately 50,000 / μl.
[0024] Next, a microcapillary (L1: 5mm, L2: 50mm, Φ: 1mm) was fabricated (step 1). Step (2) involved aspirating 10 μl of the isolated and concentrated spermatogonial cell saturation using a pipette and loading it into a prepared microcapillary.
[0025] The microcapillaries filled with this cell saturation were fixed to a holder (Figure 6) that was fabricated using a 3D printer so that the tapered ends faced outwards, and centrifuged at approximately 800xg for 1 minute using a manual centrifuge (step 3).
[0026] After centrifugation, excess culture medium was removed from the microcapillary as much as possible using a micropipette. The cell-enriched microcapillary was fixed to the injector holder, and after matching the internal pressure of the microcapillary to the ambient pressure, the needle tip was immersed in a petri dish filled with PBS placed on a stereomicroscope stage (step 4).
[0027] Using tweezers, the tip of the microcapillary was broken off under a stereomicroscope so that the opening was approximately 10 μm (step 5). The tip of a microcapillary was inserted into the peritoneal cavity of zebrafish hatched larvae (10 days old) that had been pre-sterilized by microinjection of morpholino nucleotides into the dnd gene, and approximately 100,000 cells were transplanted per individual (step 6).
[0028] Two weeks after transplantation, observation using a fluorescence microscope revealed that in 9 out of 10 transplanted individuals, the implantation of germ cells into the gonadal primordia, visualized by red fluorescence derived from donor cells, was confirmed. As described above, the present invention is a simple and low-loss transplantation method. [Explanation of Symbols]
[0029] 1 capillary 2. Cells to be transplanted 3 Culture solution 4. Cell loading device for transplantation 5 holes S Capillary with a closed, tapered end O The open end of the capillary Φ Inner diameter of the capillary
Claims
[Claim 1] 1) A step of preparing a capillary in which one end is a closed, tapered shape for injection into the target object, and the other end is open. 2) Filling the capillary with a culture medium containing cells for transplantation from the other open end, 3) Centrifuging the capillary filled with the cells with the tapered end facing outwards to separate the culture medium. 4) A step of removing the separated culture medium and obtaining a capillary filled with a cell concentrate containing concentrated cells. 5) A step of forming a hole in the tapered tip of the capillary filled with the cell concentrate for injecting and transplanting cells, and 6) A step of transplanting the concentrated cells into the target object through the formed pores using a microinjection method. A method for transplanting cells having the following characteristics (excluding medical procedures on humans).