Glass slide for assisting single circulating tumor cell separation
By setting up polylysine and bovine serum albumin layers on the slide, the problem of excessive adhesion of circulating tumor cells to the slide caused by failure of picking is solved, and efficient separation and picking of cells are achieved.
Patent Information
- Application Number
- CN202421610433.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-09
- Publication Date
- 2025-05-13
- Estimated Expiration
- 2034-07-09
AI Technical Summary
Prior Art During the isolation of circulating tumor cells, cells adhere too tightly to the slide, resulting in failure of picking and cell loss.
A slide is designed with a surface of circulating tumor cell assisted separation zone, covered with a cell adhesion layer and an adhesion regulation layer. The cell adhesion layer is a polylysine layer, and the adhesion regulation layer is a bovine serum albumin layer, which has a negative charge, neutralizing the positive charge of the cell adhesion layer, and reducing the cell adsorption on the slide.
By reducing the adsorption force between cells and slides, circulating tumor cells are easily separated, avoiding picking failure and cell loss, and improving the success rate of cell picking.
Smart Images
Figure CN222866690U_ABST
Abstract
Description
Technical Field
[0001] The utility model relates to the field of experimental equipment, in particular to a glass slide for assisting the separation of single circulating tumor cells. Background Art
[0002] CTC (circulating tumor cells) are obtained through blood-based liquid biopsies, and because of their minimal invasiveness, cancer can be monitored in real time. In recent years, numerous technical platforms for CTC detection have emerged, mainly based on the immune affinity and physical properties of CTC (size and density of tumor cells) for CTC detection. The platform for CTC detection based on the physical properties of CTC has become a widely used CTC separation method due to its reliability and cheapness, but its ability to eliminate leukocyte contamination is limited, resulting in low purity, and therefore, it is usually used as an initial enrichment step. In order to further purify a single CTC, it is necessary to first throw the enriched CTC onto a glass slide, and then manually pick the single CTC from the glass slide by capillary mouth suction or pump suction. When aspirating cells with a capillary, the cells often fail to pick up due to too tight adhesion to the glass slide, resulting in the loss of CTC. Utility Model Content
[0003] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a glass slide for assisting in the separation of single circulating tumor cells, so as to solve the problems in the prior art.
[0004] To achieve the above-mentioned purpose and other related purposes, the utility model provides a glass slide for assisting the separation of a single circulating tumor cell, wherein the glass slide has a glass slide body, a circulating tumor cell auxiliary separation area is provided on the surface of the glass slide body, and the circulating tumor cell auxiliary separation area is covered with a cell adhesion layer and an adhesion regulation layer.
[0005] Preferably, the cell adhesion layer is a poly-lysine layer.
[0006] Preferably, the adhesion regulating layer is a bovine serum albumin layer.
[0007] As described above, the glass slide for assisting the separation of a single circulating tumor cell of the utility model has the following beneficial effects: the glass slide for assisting the separation of a single circulating tumor cell provided by the utility model has an adhesion regulating layer, and the bovine serum albumin in the adhesion regulating layer carries a negative charge, which is used to neutralize the positive charge carried by the cell adsorption layer, thereby reducing the adsorption effect of the glass slide on the cells, and avoiding the circulating tumor cells in the prior art that adhere too tightly to the glass slide, resulting in failure of picking and causing loss of circulating tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 Shown is a top view schematic diagram of the slide provided by the utility model.
[0009] Figure 2 Shown is a front view schematic diagram of the slide provided by the utility model.
[0010] Figure 3 Shown is a schematic diagram of the structure of the auxiliary separation area of circulating tumor cells in the glass slide provided by the utility model.
[0011] Figure 4 Shown is a front view schematic diagram of a stack of glass slides provided by the present invention.
[0012] Component number description
[0013] 1. Labeling area; 2. Slide body; 3. Air interface; 4. Circulating tumor cell auxiliary separation area; a. Adhesion regulation layer; b. Cell adhesion layer DETAILED DESCRIPTION
[0014] See also Figures 1 to 4 It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the utility model, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the utility model without affecting the effects and purposes that can be achieved by the utility model. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the utility model. The change or adjustment of their relative relationship should also be regarded as the scope of the implementation of the utility model without substantial changes in the technical content.
[0015] like Figure 1 As shown, an embodiment of the utility model provides a glass slide for assisting in the separation of a single circulating tumor cell, wherein the glass slide has a glass slide body 2, a surface of the glass slide body 2 is provided with a circulating tumor cell assisting separation region 4, and the circulating tumor cell assisting separation region 4 is covered with a cell adhesion layer b and an adhesion regulating layer a.
[0016] In some specific embodiments, the cell adhesion layer b is disposed between the surface of the glass slide body 2 and the adhesion regulating layer a.
[0017] In some specific embodiments, a marking area 1 is provided at the other end of the glass slide, and the marking area (1) is adjacent to the circulating tumor cell auxiliary separation area (4). The marking area 1 is used to make a mark on the glass slide to distinguish different glass slides with different samples.
[0018] In some specific embodiments, the area of the circulating tumor cell auxiliary separation zone 4 is larger than the marking area 1.
[0019] In some specific embodiments, the cell adhesion layer b is a polylysine layer, and the amino acid residues of polylysine in the polylysine layer carry positive charges to interact with the negative charges on the cell surface to help the cells adhere.
[0020] In some specific embodiments, the adhesion regulating layer a is a bovine serum albumin layer, in which the bovine serum albumin carries a negative charge, which is used to neutralize the positive charge carried by the amino acid residues of poly-lysine and reduce the adsorption effect of the poly-lysine layer on cells.
[0021] In some specific embodiments, particles for maintaining the activity of bovine serum albumin are distributed in the adhesion regulating layer a, and the particles are phosphate and / or hydrogen phosphate particles.
[0022] In some specific embodiments, the thickness of the cell adhesion layer b is 0.01-1 μm. Specifically, the thickness is 0.01-0.05 μm, 0.05-0.1 μm, 0.1-0.2 μm, 0.2-0.4 μm, 0.4-0.6 μm, 0.6-0.8 μm or 0.8-1 μm.
[0023] In some specific embodiments, the thickness of the adhesion regulating layer a is 0.01-1 μm, specifically, the thickness is 0.01-0.05 μm, 0.05-0.1 μm, 0.1-0.2 μm, 0.2-0.4 μm, 0.4-0.6 μm, 0.6-0.8 μm or 0.8-1 μm.
[0024] In some specific embodiments, the glass slide is prepared by the following method:
[0025] 1) Mix bovine serum albumin (BSA) with phosphate buffered saline (such as Gibco TM No. 10010023) and filtered to obtain phosphate-bovine serum albumin (PBSA) solution;
[0026] 2) Covering the surface of a poly-lysine glass slide (Shitai, REF.188105) with the PBSA solution and incubating the surface, and removing the PBSA solution remaining on the surface of the glass slide to obtain the glass slide.
[0027] In some specific embodiments, in step 1), based on the mass of the PBSA solution, the mass / volume percentage of BSA is 0.005-1%. Specifically, the mass / volume percentage of BSA is 0.005-0.008%, 0.008-0.01%, 0.01-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.12%, 0.12-0.3%, 0.3-0.5%, 0.5-0.7% or 0.7-1%. Preferably, the mass / volume percentage of BSA is 0.08-0.12%.
[0028] As shown in the figure, the utility model provides a glass slide for assisting the separation of a single circulating tumor cell, the glass slide comprising a glass slide body 2 and a circulating tumor cell auxiliary separation region 4 arranged at one end of the glass slide, the circulating tumor cell auxiliary separation region 4 being provided with a cell adhesion layer b and an adhesion regulation layer a in sequence from bottom to top on the surface of the glass slide body 2.
[0029] When in use, a solution containing circulating tumor cells and other cells is placed in the circulating tumor cell auxiliary separation area 4, the positively charged polylysine in the cell adhesion layer b can adsorb the cells with negative charges on the cell surface in the solution, so that the cells are close to the surface of the slide body 2, and the bovine serum albumin in the adhesion regulation layer a combines with the positively charged polylysine in the cell adhesion layer b to neutralize the electrical properties of the cell adhesion layer b to reduce the adsorption strength of the cell adhesion layer b on the cells, so that the cells can be easily separated by the operator manually using capillary mouth suction or pump suction under a microscope.
[0030] Effect of BSA coating on the success rate of cell picking
[0031] 1% BSA in PBS: weigh 1g BSA dry powder, dilute to 100ml with PBS, and filter sterilize with a 0.22μm mesh;
[0032] Preparation of PBSA solutions of different concentrations: dilute 1% BSA in PBS by 2 times, 10 times, and 100 times, and filter with a 0.22 μm mesh for sterilization.
[0033] Drop 200μl PBSA solution on a glass slide (Shitai, REF.188105), place it on the table at room temperature for 5 minutes, remove the PBSA solution, add 150μl of cell suspension containing CTC (first take 4.5ml blood, separate PBMC by gradient centrifugation, and further sort PBMC to obtain cell suspension containing CTC), centrifuge at 260g for 5 minutes, place it on a microscope and pick it with a capillary. Use 0%, 1%, 0.5%, 0.1%, and 0.01% BSA to coat the slide, pick 20 cells, and count the success rate. Successful picking means successful picking of a single cell, and failure to aspirate and mistakenly aspirate surrounding cells are counted as failed picking. The cell picking success rate is as follows:
[0034] BSA concentration Successfully picking single cells Failed to pick Success rate 0％ 2 20 10％ 1％ 10 10 50％ 0.5％ 13 7 65％ 0.1％ 18 2 90％ 0.01％ 5 15 25％
[0035] It can be seen from the above table that with the increase of coating concentration, the picking success rate shows a trend of first rising and then falling. The uncoated glass slides cannot be aspirated due to their strong adhesion. When the coating BSA concentration reaches 0.5%, the adhesion of the glass slide decreases significantly, resulting in the mistaken aspiration of surrounding cells during the aspiration process, leading to picking failure. When the coating BSA concentration is 0.1%, the picking success rate is the highest.
[0036] In summary, the utility model provides an adhesion regulating layer on the glass slide to facilitate the separation of circulating tumor cells under a microscope, so the utility model effectively overcomes various shortcomings of the prior art and has a high industrial utilization value.
[0037] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed in the present invention shall still be covered by the claims of the present invention.
Claims
1. A slide for assisting in the separation of a single circulating tumor cell, characterized in that: The glass slide comprises a glass slide body (2), a circulating tumor cell auxiliary separation area (4) is provided on the surface of the glass slide body (2), and the circulating tumor cell auxiliary separation area (4) is covered with a cell adhesion layer (b) and an adhesion regulating layer (a).
2. The slide glass according to claim 1, characterized in that The cell adhesion layer (b) is arranged between the surface of the glass slide body (2) and the adhesion regulating layer (a).
3. The slide glass according to claim 1, characterized in that The cell adhesion layer (b) is a poly-lysine layer.
4. The slide glass according to claim 1, characterized in that The adhesion regulating layer (a) is a bovine serum albumin layer.
5. The slide glass according to claim 1, characterized in that: The surface of the glass slide body (2) is also provided with a marking area (1), and the marking area (1) is adjacent to the circulating tumor cell auxiliary separation area (4).
6. The slide glass according to claim 5, characterized in that: The area of the circulating tumor cell auxiliary separation zone (4) is larger than the marking area (1).
7. The slide glass according to claim 1, characterized in that: The thickness of the cell adhesion layer (b) is 0.01-1 μm.
8. The slide glass according to claim 1, characterized in that: The thickness of the adhesion regulating layer (a) is 0.01-1 μm.