Cryopreservation method and application of human urine cells

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a cryopreservation method and application of human urine cells. The improved cell freezing medium is obtained by compounding and combining the REGM culture medium, the FBS and the DMSO according to a specific proportion, and the freezing medium can remarkably improve the activity and the multiplication capacity of urine cells separated from human urine and effectively prolong the storage time of the cells; moreover, the cryopreservation method disclosed by the invention is simple and convenient to operate, low in cost and noninvasive, and the high-activity urine cells obtained by separation can be further induced and converted into induced pluripotent stem cells (iPSCs) through a reprogramming technology, so that a reliable cell source is provided for a disease model.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a cryopreservation method and application of human urine cells. Background Art

[0002] Urine cell separation technology has important applications in biomedical research, especially in extracting cells from urine for disease detection, gene analysis, early diagnosis, etc. Currently, the commonly used urine cell separation method is rapid separation by centrifugation. Usually, fresh human urine is used, and the cells in the urine are precipitated by centrifugal force to obtain urine cells. However, this method has certain limitations, resulting in poor cell viability and insufficient proliferation ability of the separated cells. Specifically, using the traditional centrifugal separation technology, although cells can be extracted from urine in a short time, due to the high centrifugal force generated during centrifugation and its physical damage to cells, the separated cells often suffer a certain degree of damage, leading to a significant decrease in their viability. In addition, the cells in urine are often prone to aging and have poor proliferation ability after extraction, which affects subsequent cell culture, analysis, and experimental effects; the aging and insufficient proliferation ability of cells also reduce the application value of these cells in disease diagnosis, gene detection, etc.

[0003] Therefore, the prior art cannot well maintain the activity and proliferation ability of cells during urine cell separation, and there is an urgent need to develop new separation methods and optimize the existing technology to improve the quality and usability of the separated urine cells, so as to provide more reliable cell samples for clinical research and diagnosis. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a cryopreservation method and application of human urine cells to effectively extend the survival time of urine cells and enhance cell activity and proliferation ability.

[0005] To achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a cell cryopreservation solution that can improve the activity and proliferation ability of human urine cells. The cell cryopreservation solution includes the following components: REGM medium, FBS, and DMSO, and the volume ratio of the REGM medium, FBS, and DMSO is (2 - 6):(3 - 7):1.

[0007] The present invention has obtained an improved cell cryopreservation solution through a compound combination of REGM medium, FBS, and DMSO in a specific ratio. The cryopreservation solution can effectively improve the activity and proliferation ability of human urine cells. In the cryopreservation solution combination, the REGM medium provides the basic nutrients required for cell growth, while FBS supplements cell growth factors and other necessary components. DMSO serves as a cryoprotectant, helping cells reduce freezing damage during cryopreservation, and can improve the permeability of the cell membrane, promoting the balance of substances inside and outside the cell, thereby improving cell viability to a certain extent.

[0008] Through experimental exploration, it has been found that too much or too little DMSO may cause toxic effects on cells, while improper ratios of REGM or FBS may lead to insufficient nutrient supply or imbalance of cell growth factors, thereby affecting cell survival and proliferation. Therefore, precisely controlling the dosage ratios of these three components has an important impact on optimizing the activity and proliferation ability of the isolated urine cells, thereby improving the reliability of urine cells in subsequent research and clinical applications. If the ratios of the three components exceed the scope defined in the present invention or a certain component is missing, the activity and proliferation ability of urine cells will be significantly decreased. Therefore, the innovation of the present invention lies precisely in precisely regulating the ratio of the components to ensure that cells maintain a high activity during the cryopreservation and separation process, thereby providing higher-quality cell samples for further research and clinical applications.

[0009] Preferably, the volume ratio of the REGM medium, FBS, and DMSO is (4 - 7):(2 - 5):1.

[0010] More preferably, the volume ratio of the REGM medium, FBS, and DMSO is 6:3:1.

[0011] Through experimental exploration, it has been found that the cell cryopreservation solution prepared by compounding the three components with the above optimal ratio can make the cell viability and proliferation ability of the isolated urine cells the best.

[0012] In a second aspect, the present invention provides the application of the cell cryopreservation solution in the separation and culture of urine cells.

[0013] In a third aspect, the present invention provides a method for cryopreserving human urine cells, and the cryopreservation method includes the following steps:

[0014] S1. Collect midstream urine, add 90 - 110x double antibody P / S, centrifuge and discard the supernatant, then resuspend with PBS solution containing 40 - 60x double antibody P / S, centrifuge again and discard the supernatant, resuspend with PBS solution containing 40 - 60x double antibody P / S, centrifuge, and discard the supernatant until ≤1 mL to obtain cell precipitate;

[0015] S2. Resuspend the cell pellet obtained in step S1 with the aforementioned cell cryopreservation solution. After mixing evenly, transfer it into a cell cryopreservation tube, and then place it in a cell cryopreservation box for cryopreservation at -80 to -100 °C;

[0016] S3. After cryopreservation, quickly thaw the cell cryopreservation solution, then resuspend it with urine cell culture medium, centrifuge, aspirate and discard the supernatant, and then resuspend it with urine cell culture medium containing 10 - 30x double antibody P / S to obtain a resuspended solution;

[0017] S4. Add the resuspended solution obtained in step S3 into a coated 6-well plate, and culture it in an environment of 37 °C and 5% CO2, then human urine cells are separated and obtained.

[0018] Preferably, in step S2, the cryopreservation time using the cell cryopreservation solution is 1 - 72 h.

[0019] Preferably, in step S2, the cryopreservation time using the cell cryopreservation solution is 1 - 24 h.

[0020] Traditional urine cell separation techniques usually use centrifugation for rapid separation, which will result in poor cell viability of the separated cells. The cells are prone to rapid aging during storage, thus leading to a short storage time of the separated cells and being unfavorable for subsequent cell culture and research. However, using the cell cryopreservation solution and separation method provided by the present invention can effectively extend the storage time of urine cells, realize long-distance transportation at -80 °C within 24 h, and at the same time can significantly improve the cell viability and proliferation ability, thereby providing a more reliable cell sample for subsequent research.

[0021] Preferably, in step S2, the volume ratio of the cell pellet to the cell cryopreservation solution is 1:(1 - 2).

[0022] In step S1, the centrifugation conditions are all: centrifuge at 300 - 500 g for 8 - 12 min;

[0023] Preferably, in step S3, the centrifugation conditions are: centrifuge at 200 - 400 g for 2 - 4 min.

[0024] Preferably, in step S3, the urine cell culture medium includes the following components: REGM medium and FBS, and the volume ratio of the REGM medium to the FBS is 1:(1 - 2).

[0025] Preferably, in step S1, the collected midstream urine should be separated within 0 - 3 h after collection. If the urine cells are not separated in time, they should be placed in an environment of 4 °C to ensure the activity of the separated urine cells and be beneficial for subsequent culture.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention obtains an improved cell cryopreservation solution through a compound combination of REGM medium, FBS, and DMSO in a specific ratio. The cryopreservation solution can significantly improve the viability and proliferation ability of urine cells isolated from human urine, and effectively extend the storage time of cells. Moreover, the cryopreservation method of the present invention is simple to operate, low in cost, and non-invasive. It not only avoids the trauma risk in traditional cell separation methods but also provides a safer cell acquisition method for patients by non-invasively collecting urine samples. In addition, the highly active urine cells isolated and cryopreserved by the present invention can be further induced and transformed into induced pluripotent stem cells (iPSCs) through reprogramming technology, thereby providing a reliable cell source for disease models. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic flow chart of the urine cell separation process in the group without adding cryopreservation solution in Example 1;

[0029] Figure 2 It is a schematic flow chart of the urine cell separation process in the group with different ratios of cryopreservation solution added in Example 1;

[0030] Figure 3 It is the cell morphology (bright field objective 5×) on Day 3 in the urine cell separation process in the group without adding cryopreservation solution and the group with different ratios of cryopreservation solution added in Example 1;

[0031] Figure 4 It is the cell morphology (bright field objective 5×) on Day 6 in the urine cell separation process in the group without adding cryopreservation solution and the group with different ratios of cryopreservation solution added in Example 1;

[0032] Figure 5 It is the cell morphology (bright field objective 5×) on Day 15 in the urine cell separation process in the group without adding cryopreservation solution and the group with different ratios of cryopreservation solution added in Example 1;

[0033] Figure 6 It is a result diagram of analyzing the proportion of EdU-positive cells by immunofluorescence technology (objective 10×) on Day 16 of the urine cells separated with or without adding cryopreservation solution in Example 1;

[0034] Figure 7 It is a result diagram of analyzing the proportion of EdU-positive cells by flow cytometry on Day 16 of the urine cells separated with or without adding cryopreservation solution in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0035] The following specific implementation methods in the form of examples are used to further elaborate on the above content of the present invention in detail. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples.

[0036] Unless otherwise specified, the reagents used in the examples are all conventional reagents existing in the art and can be obtained through commercial channels. The experimental operations not specifically described in the examples are all conventional operations in the art or can be understood or known by those skilled in the art based on the existing technology or common general knowledge they have mastered.

[0037] Example 1

[0038] This example provides a method for cryopreserving human urine cells. A control group (without cryopreservation during the entire separation process), Group A (the cryopreservation solution ratio is REGM:FBS:DMSO = 0:9:1, that is, without adding REGM medium), Group B (the cryopreservation solution ratio is REGM:FBS:DMSO = 2:7:1), Group C (the cryopreservation solution ratio is REGM:FBS:DMSO = 4:5:1), Group D (the cryopreservation solution ratio is REGM:FBS:DMSO = 6:3:1), Group E (the cryopreservation solution ratio is REGM:FBS:DMSO = 7:2:1), and Group F (the cryopreservation solution ratio is REGM:FBS:DMSO = 9:0:1, that is, without adding the FBS component) are respectively set to determine the optimal cryopreservation solution ratio for still being able to isolate urine cells after cryopreserving urine precipitates.

[0039] The method for isolating urine cells from fresh urine includes the following steps:

[0040] (1) Preparation before urine cell separation (Day 0): Prepare 250 mL sterile collection bottles, and strictly collect about 200 mL of midstream urine for each group, and add 100x double antibody P / S (Gibco brand, product number 15140 - 122) in advance. Use Gelatin (STEMCELL Technologies brand, product number 07903) to coat the 6-well plates for at least 30 minutes in advance.

[0041] (2) Prepare cryopreservation solutions with different ratios: Add 900 μL of FBS (Viva Cell brand, catalog number C04001-500) and 100 μL of DMSO (Merck Sigma-Aldrich brand, catalog number D1435) respectively to form the cryopreservation solution for Group A; add 200 μL of REGM (Lonza brand, catalog number cc-3190), 700 μL of FBS, and 100 μL of DMSO respectively to form the cryopreservation solution for Group B; add 400 μL of REGM, 500 μL of FBS, and 100 μL of DMSO respectively to form the cryopreservation solution for Group C; add 600 μL of REGM, 300 μL of FBS, and 100 μL of DMSO respectively to form the cryopreservation solution for Group D; add 700 μL of REGM, 200 μL of FBS, and 100 μL of DMSO respectively to form the cryopreservation solution for Group E; add 900 μL of REGM and 100 μL of DMSO respectively to form the cryopreservation solution for Group F.

[0042] (3) Urine cell separation (Day 0): Aliquot 200 mL of urine from each of Groups A, B, C, D, E, and F into 4 tubes of 50 mL sterile centrifuge tubes, and centrifuge at 400 g for 10 minutes at room temperature. Aspirate and discard the supernatant until 1 - 2 mL remains, then combine the 4 tubes of solution into 2 tubes of solution, and then resuspend with 40 mL of PBS solution containing 50x double antibody P / S, mix well and wash, centrifuge at 400 g for 10 minutes at room temperature, and aspirate and discard the supernatant until 1 - 2 mL remains. Finally, combine the 2 tubes of solution into 1 tube of solution, add 40 mL of PBS solution containing 50x double antibody P / S again to resuspend, mix well and wash. Centrifuge at 400 g for 10 minutes at room temperature, and aspirate and discard the supernatant for the last time to ≤1 mL.

[0043] The control group directly resuspends the precipitate with 1 mL of urine cell culture medium containing 1x P / S double antibody (REGM:10% FBS = 1:1), adds it to a six-well plate pre-coated with gelatin in advance, supplements 1 mL of the above urine cell culture medium, and places it in a 37°C, 5% constant temperature incubator for culture. For experimental Groups A, B, C, D, E, and F, resuspend the precipitate with the corresponding cryopreservation solution, add it to a 2 mL cell cryopreservation tube, place it in a cell cryopreservation box, and store it in a -80°C environment. This cryopreserved cell can be transported over a long distance under the condition of -80°C within 24 hours.

[0044] After 24 hours, take out the cells of Groups A, B, C, D, E, and F from the -80°C environment, quickly place them in a 37°C water bath for thawing. At this time, pay attention to rapid thawing to reduce the impact of the cryopreservation solution on cell viability. Resuspend with 5 mL of urine cell culture medium, centrifuge at 300 g for 3 minutes at room temperature. Aspirate and discard the supernatant until 1 mL remains. Add 1 mL of urine cell culture medium to each group, add the resuspended solution to a pre-coated 6-well plate, and then supplement 1 mL of urine cell culture medium, and place it in a 37°C, 5% constant temperature incubator for culture.

[0045] (4) Observe the urine cell separation situation (Day3 - Day15): Incubate statically for the first 3 days without moving the 6 - well plate to allow more cells to adhere to the wall. Observe the cells on Day3. It is found that a small number of cells adhere to the wall in the control group and groups A, B, C, D, and E, and no cells adhere to the wall in group F. The results are referred to Figure 3 . At the same time, supplement 1 mL of urine cell culture medium. On Day5, continue to supplement 1 mL of urine cell culture medium. On Day6, change the urine cell culture medium. Obvious cell clumps can be seen in the control group and groups A, B, C, D, and E, and no cells adhere to the wall in group F. The results are referred to Figure 4 . After that, change the urine cell culture medium every other day. On Day15, the urine cells grow to a density of 80% - 90%. The results are referred to Figure 5 .

[0046] (5) Urine cell digestion and passage (Day15): Use 0.05% trypsin - EDTA (Gibco brand, catalog number 25300054) to digest the cells. After digesting at 37°C for 2 minutes, add 500 μL of 10% FBS (DMEM:FBS = 9:1) to terminate the digestion. After centrifuging at 300 g at room temperature for 3 minutes, aspirate the supernatant. Seed the control group cells and cells in groups A, B, C, D, and E into a 12 - well plate coated with gelatin 30 minutes in advance.

[0047] (6) Detection of urine cell proliferation ability (Day16): Use an EdU proliferation detection kit (Beyotime brand, catalog number C0071S) to detect the cell proliferation ability. Dilute EdU (10 mM) at a ratio of 500:1 with urine cell culture medium to obtain a 2xEdU working solution. Add the pre - warmed 2xEdU working solution and urine cell culture medium in equal volumes at a ratio of 1:1 to the 12 - well plate and incubate at 37°C for 2 hours.

[0048] After the EdU-labeled cells were completed, the culture medium was removed, 1 mL of 4% paraformaldehyde was added, and the cells were fixed at room temperature for 15 minutes. The fixing solution was removed, and the cells in each well were washed 3 times with 1 mL of washing solution for 3 minutes each time. The washing solution was removed, and each well was permeabilized with 1 mL of PBS containing 0.3% TritonX-100 and incubated at room temperature for 15 minutes. The permeabilizing solution was removed, and the cells in each well were washed 2 times with 1 mL of PBS for 3 minutes each time. Subsequently, the Click reaction solution was prepared, and 250 μL of the Click reaction solution was added to each well and incubated in the dark for 30 minutes. The Click reaction solution was washed off and washed 3 times with PBS for 3 minutes each time. Hoechst 33342 was diluted at a ratio of 1:1000, and 500 μL of the diluted Hoechst 33342 solution was added to each well and incubated for 10 minutes. The 1X Hoechst 33342 solution was aspirated and washed 3 times with PBS for 3 minutes each time. Immunofluorescence and flow cytometry were performed on each group, and the results were referred to Figures 6 - 7 The results showed that when the cell cryopreservation solution prepared at a ratio of REGM:FBS:DMSO = 6:3:1 in group D was used to cryopreserve urine cells, the proliferation ability and cell viability of urine cells after 24 hours of cryopreservation were significantly improved compared with those of the control group that was directly cultured without cryopreservation. When the cryopreservation solutions prepared at ratios of REGM:FBS:DMSO = 2:7:1 and REGM:FBS:DMSO = 4:5:1 in groups B and C were used to cryopreserve cells, the viability and proliferation ability of the urine cells also showed a certain degree of improvement after 24 hours of storage compared with the control group, indicating that the cell cryopreservation solution provided by the present invention can effectively extend the storage time of urine cells, realize long-distance transportation under the condition of -80 °C within 24 hours, and at the same time can effectively improve the cell viability and proliferation ability, especially when the ratio of the cryopreservation solution is REGM:FBS:DMSO = 6:3:1, the effect is the best; when the REGM medium was absent in group A, the viability of the isolated urine cells decreased significantly after 24 hours of cryopreservation. When the cell cryopreservation solution prepared at a ratio of REGM:FBS:DMSO = 7:2:1 in group E was used to cryopreserve urine cells, the proliferation ability and cell viability of urine cells after 24 hours of cryopreservation decreased slightly compared with those in group D, indicating that the ratio of the cryopreservation solution in group D of REGM:FBS:DMSO = 6:3:1 had the best effect. When the FBS component was absent in group F, there was no cell adhesion growth directly, and the separation and culture effect was the worst.

[0049] In summary, the present invention obtains an improved cell cryopreservation solution through a compound combination of a REGM medium, FBS, and DMSO in a specific ratio. The cryopreservation solution can significantly enhance the viability and proliferation ability of urine cells isolated from human urine, effectively extend the storage time of cells, and ensure a high survival rate of cells during cryopreservation and resuscitation. When a certain component is missing, the separation and culture effect of urine cells significantly decreases, and the cell proliferation ability is poor, which is not conducive to subsequent research and applications.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A cell freezing solution capable of improving the activity and proliferation of human urine cells, characterized in that: The cell freezing solution comprises the following components: REGM culture medium, FBS and DMSO, and the volume ratio of the REGM culture medium, FBS and DMSO is (2-7): (2-7):

1.

2. The cell freezing solution according to claim 1, characterized in that The volume ratio of the REGM culture medium, FBS and DMSO is (4-7): (2-5):

1.

3. The cell freezing solution according to claim 2, characterized in that The volume ratio of the REGM medium, FBS and DMSO is 6:3:

1.

4. Use of the cell freezing solution as described in any one of claims 1 to 3 in urine cell separation and culture.

5. A method for freezing human urine cells, characterized in that: The freezing method comprises the following steps: S1. Collect midstream urine, add 90-110x double-antibody P / S, centrifuge and discard the supernatant, then add PBS solution containing 40-60x double-antibody P / S to resuspend, centrifuge again and discard the supernatant, add PBS solution containing 40-60x double-antibody P / S to resuspend, centrifuge, discard the supernatant to ≤1mL, and obtain cell pellet; S2, resuspending the cell precipitate obtained in step S1 with the cell freezing solution according to claim 1 or 2, transferring the mixture to a cell freezing tube after mixing, and then freezing the mixture in a cell freezing box at -80 to -100°C; S3. After freezing, the cell freezing solution is quickly thawed, and then urine cell culture medium is added to resuspend, centrifuge, the supernatant is discarded, and then urine cell culture medium containing 10-30x double antibody P / S is added to resuspend to obtain a resuspension; S4. Add the resuspension obtained in step S3 to the coated 6-well plate and culture it in an environment of 37° C. and 5% CO 2 to isolate and obtain human urine cells.

6. The cryopreservation method according to claim 5, characterized in that: In the step S2, the cell freezing solution is used for freezing for 1 to 72 hours.

7. The cryopreservation method according to claim 6, characterized in that: In step S2, the cell freezing solution is used for freezing for 1 to 24 hours.

8. The cryopreservation method according to claim 5, characterized in that: In the step S2, the volume ratio of the cell precipitate to the cell freezing solution is 1:(1-2).

9. The cryopreservation method according to claim 5, characterized in that: In step S1, the centrifugation conditions are: centrifugation at 300-500 g for 8-12 min; In step S3, the centrifugation condition is: centrifugation at 200-400g for 2-4 minutes.

10. The cryopreservation method according to claim 5, characterized in that: In step S3, the urine culture medium includes the following components: REGM culture medium and FBS, and the volume ratio of the REGM culture medium to FBS is 1:(1-2).

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