Menstrual blood preservation solution, method of making and use thereof

Abstract

The application provides a blood transfusion preservation solution and a preparation method and application thereof, relates to the technical field of biology, and provides the blood transfusion preservation solution, which selects specific lactate ciprofloxacin, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate and amphotericin B as effective components of an antibiotic base solution, can guarantee that the blood transfusion is not polluted by the outside world in the process of collection, transportation and preservation, and the pollution rate of mesenchymal stem cell seeds separated from the blood transfusion preservation solution is also reduced accordingly; on the other hand, the blood transfusion preservation solution can effectively maintain the activity of stem cells in the blood transfusion and improve the efficiency of obtaining stem cells from the blood transfusion.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a menstrual blood preservation solution, its preparation method, and its application. Background Technology

[0002] Menstrual blood mesenchymal stem cells (MSCs) are derived from the menstrual blood of healthy female donors. Endometrial stromal cells (MSCs) are responsible for the high regenerative capacity of the endometrium, and they are shed during menstruation and mixed into the menstrual blood. Men-MSCs are derived from menstrual blood collected from healthy female donors using a non-invasive method (menstrual cup collection). Adult stem cells shed during menstruation from the endometrium are extracted from the menstrual blood via density gradient centrifugation, and then aseptically cultured in vitro to expand to appropriate cell passages. The expansion and culture process is used to assess the presence of MSCs exhibiting adherent proliferation, multi-lineage differentiation potential, and the expression of phenotypic proteins characteristic of MSCs, indicating their biological activity. Currently, both literature and patents describe the preparation and storage of menstrual blood-derived MSCs by isolating them from menstrual blood, purifying and culturing them, and then cryopreserving them.

[0003] Because the vagina naturally contains bacteria and fungi, menstrual blood inevitably becomes contaminated as it flows through the vagina. Currently, the only way to solve the problem of cell contamination during the later separation and culture of stem cells from menstrual blood samples is to add antibiotics to the menstrual blood preservation solution and culture medium to inhibit the growth of bacteria and fungi. Penicillin and streptomycin are commonly used antibiotics for cell culture; however, laws and regulations prohibit their direct use in clinical-grade cell preparations, so penicillin- and streptomycin-free menstrual blood preservation solutions must be used.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a menstrual blood preservation solution to at least solve one of the technical problems existing in the prior art.

[0006] The second objective of this invention is to provide a method for preparing the above-mentioned menstrual blood preservation solution.

[0007] The third objective of this invention is to provide the application of the above-mentioned menstrual blood preservation solution.

[0008] The fourth objective of this invention is to provide a menstrual blood collection kit containing the aforementioned menstrual blood preservation solution.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0010] This invention provides a menstrual blood preservation solution, comprising: an antibiotic base solution, an anticoagulant, and a basal culture medium;

[0011] The antibiotic base solution includes ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B.

[0012] Further, the concentrations of ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B are 1–5 μg / ml, 50–70 μg / ml, 80–120 μg / ml, 1–5 μg / ml, and 100–150 μg / ml, respectively, preferably 2 μg / ml, 60 μg / ml, 100 μg / ml, 2.7 μg / ml, and 120 μg / ml.

[0013] Furthermore, the anticoagulant includes one or more of heparin, sodium citrate, or ethylenediaminetetraacetic acid;

[0014] Preferably, the effective concentration of the anticoagulant is 50-70 U / ml, and more preferably 60 U / ml.

[0015] Furthermore, the basal culture medium includes at least one of DMEM, DMEM / F12, RPMI 1640, IMDM, MCDB 201, and MCDB 131.

[0016] Furthermore, the solvents for the antibiotic base solution and the anticoagulant each independently include a buffer solution, preferably a PBS buffer solution.

[0017] Furthermore, it includes 1% to 10% by volume of antibiotic base solution, 1% to 5% of anticoagulant, and the remainder of basal culture medium;

[0018] Preferably, it comprises 5% by volume of antibiotic base solution, 2% of anticoagulant and 93% of basal culture medium.

[0019] The present invention also provides a method for preparing the above-mentioned menstrual blood preservation solution, comprising mixing the components of the formula in equal amounts to obtain the menstrual blood preservation solution.

[0020] The present invention also provides the application of the above-mentioned menstrual blood preservation solution in menstrual blood collection, menstrual blood preservation and menstrual blood transportation.

[0021] Furthermore, the temperature for menstrual blood preservation and transportation is 2–25°C.

[0022] In addition, the present invention also provides a menstrual blood collection kit, including sterile rubber surgical gloves, a mask, a surgical cap, alcohol swabs, skin disinfection wipes, a menstrual cup, sealing film, a label, a temperature recorder, a cold storage agent, and the aforementioned menstrual blood preservation solution.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The menstrual blood preservation solution provided by this invention selects a specific combination of ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B as the effective components of the antibiotic base solution. On the one hand, this ensures that menstrual blood is protected from external contamination during collection, transportation, and preservation, and the contamination rate of mesenchymal stem cell seeds isolated from the menstrual blood preservation solution is correspondingly reduced. On the other hand, the menstrual blood preservation solution provided by this invention can also effectively maintain the activity of stem cells in menstrual blood, thereby improving the efficiency of obtaining stem cells from menstrual blood. Detailed Implementation

[0025] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0026] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] According to one aspect of the present invention, a menstrual blood preservation solution is provided, comprising: an antibiotic base solution, an anticoagulant, and a basal culture medium; wherein the antibiotic base solution comprises ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B.

[0029] The inventors of this invention conducted extensive experimental verification, including antibiotic susceptibility testing on Streptococcus equi, Staphylococcus epidermidis, Staphylococcus epidermidis [MRS strain], Streptococcus agalactiae, and Staphylococcus aureus [β-Lac strain] that broke out during menstrual blood sample culture. This allowed them to screen out a specific combination of ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B as the effective components of the antibiotic base solution for the menstrual blood preservation solution of this invention. This allows the menstrual blood preservation solution to effectively maintain the activity of the target stem cells in the menstrual blood while reducing the probability of in vitro contamination.

[0030] To further balance the antibacterial ability and the maintenance of stem cell activity, in some preferred embodiments, the concentrations of ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B are 1–5 μg / ml, 50–70 μg / ml, 80–120 μg / ml, 1–5 μg / ml, and 100–150 μg / ml, respectively.

[0031] The optimal balance between antibacterial activity and antimicrobial activity was achieved when the concentrations of ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B were 2 μg / ml, 60 μg / ml, 100 μg / ml, 2.7 μg / ml, and 120 μg / ml, respectively.

[0032] As an optional implementation, the anticoagulant includes one or more of heparin, sodium citrate, or ethylenediaminetetraacetic acid (EDTA). All of these anticoagulants can achieve an anticoagulant effect without damaging the activity of the target stem cells. The effective concentration of the anticoagulant can be 50–70 U / ml, preferably 60 U / ml.

[0033] In some embodiments, the solvents for the antibiotic base solution and the anticoagulant each independently comprise a buffer solution, such as, but not limited to, HEPES, PBS, DPBS, or TBST, preferably a PBS buffer solution.

[0034] The basic culture medium used in the menstrual blood preservation solution provided by the present invention is not limited. Any basic culture medium that can be used to culture stem cells in routine cell experiments is acceptable, such as, but not limited to, at least one of DMEM, DMEM / F12, RPMI1640, IMDM, MCDB 201 and MCDB 131.

[0035] In some preferred embodiments, the menstrual blood preservation solution comprises 1% to 10% (v / v) of antibiotic base solution, 1% to 5% of anticoagulant, and the remainder being basal culture medium. The preservation solution exhibits superior performance when it comprises 5% (v / v) of antibiotic base solution, 2% of anticoagulant, and 93% of basal culture medium.

[0036] According to a second aspect of the present invention, a method for preparing the above-mentioned menstrual blood preservation solution is provided, comprising mixing an antibiotic base solution, an anticoagulant and a basal culture medium in a uniform manner to obtain the menstrual blood preservation solution.

[0037] This method is simple in process, easy to operate, and suitable for widespread application.

[0038] Specifically, it can be prepared by the following method:

[0039] Preparation of antibiotic base solution: This includes the following antibiotics: ciprofloxacin lactate injection; vancomycin hydrochloride injection; kanamycin sulfate injection; gentamicin sulfate injection; and amphotericin B injection. The concentrations used are 2 μg / ml, 60 μg / ml, 100 μg / ml, 2.7 μg / ml, and 120 μg / ml, respectively. For a specific 50 ml solution, use 1 ml of ciprofloxacin lactate injection, 600 μl of vancomycin hydrochloride injection, 400 μl of kanamycin sulfate injection, 3 ml of gentamicin sulfate injection, and 270 μg of amphotericin B injection. Mix the above antibiotics thoroughly according to the dosages used, and add PBS to make up to 50 ml to prepare the antibiotic base solution.

[0040] Preparation of anticoagulant (60U / ml) (taking 20ml as an example): Take 10ml of stock solution and add PBS to make up to 20ml.

[0041] The final formula for menstrual blood preservation solution is as follows (taking 30ml as an example): 1.5ml of antibiotic base solution, 0.6ml of anticoagulant, and 27.9ml of DMEM / F12 culture medium are mixed evenly to prepare menstrual blood preservation solution.

[0042] Based on the beneficial effects of the menstrual blood preservation solution provided by the present invention, a third aspect of the present invention also provides the application of the above-mentioned menstrual blood preservation solution in menstrual blood collection, menstrual blood preservation and menstrual blood transportation.

[0043] The ideal temperature for storage and transportation is 2–25°C.

[0044] In addition, the present invention provides a menstrual blood collection kit, including sterile rubber surgical gloves, a mask, a surgical cap, alcohol swabs, skin disinfection wipes, a menstrual cup, sealing film, a label, a temperature recorder, a cold storage agent, and the menstrual blood preservation solution of the present invention.

[0045] When using this menstrual blood collection kit to collect menstrual blood from women, the vulva is cleaned and disinfected. The sterile menstrual cup in the prepared collection kit is used to collect the menstrual blood, which is then stored in a menstrual blood preservation solution to maintain the activity of stem cells in the menstrual blood.

[0046] Specifically, the menstrual blood sample collection process is as follows:

[0047] After receiving the complete and intact kit, the blood donor removes the blood sample collection tube, blue ice, and temperature recorder from the kit.

[0048] Preparation before collection: Prepare the items needed for inserting the menstrual cup, including sterile rubber surgical gloves, mask, surgical cap, alcohol swabs, skin disinfection wipes, and sterile packaged menstrual cups; before inserting the menstrual cup, wear a mask and surgical cap, then wash your hands, dry your hands, put on sterile rubber surgical gloves, disinfect your hands with alcohol swabs, tear open the skin disinfection wipes packaging, and clean the vulva.

[0049] Open the disposable sterile packaging of the menstrual cup, remove the cup, fold it, and gently push it into the vagina until it reaches the cervix, holding the bottom. Rotate the cup to ensure it is fully open and fits tightly against the vaginal wall. The cup should be inserted to the appropriate length, with the bottom of the stem flush with the vaginal opening and without any foreign body sensation.

[0050] Collect once when you feel or notice the menstrual cup is full, or collect once every 3-8 hours after inserting the menstrual cup into the vagina.

[0051] After blood collection, pour the collected menstrual blood into a menstrual blood sample collection tube and seal the tube opening with sealing film. Simultaneously, affix a label to the collection tube indicating the batch number, expiration date, and menstrual blood sample number, and record the start and end times of the menstrual blood sample collection for that tube. After completion, store the menstrual blood sample collection tube and temperature recorder at 2-25°C for temporary storage and transportation.

[0052] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0053] Example 1

[0054] This embodiment provides a menstrual blood preservation solution, comprising an antibiotic base solution, an anticoagulant, and a basal culture medium; wherein,

[0055] Preparation of antibiotic base solutions: Ciprofloxacin lactate injection, vancomycin hydrochloride injection, kanamycin sulfate injection, gentamicin sulfate injection, and amphotericin B injection, with concentrations of 2 μg / ml, 60 μg / ml, 100 μg / ml, 2.7 μg / ml, and 120 μg / ml, respectively. For a 50 ml solution, use 1 ml of ciprofloxacin lactate injection, 600 μl of vancomycin hydrochloride injection, 400 μl of kanamycin sulfate injection, 3 ml of gentamicin sulfate injection, and 270 μg of amphotericin B injection. Mix the above antibiotics thoroughly according to the required dosage, and add PBS to make up to 50 ml to prepare the antibiotic base solution.

[0056] Preparation of anticoagulant (60U / ml): The volume ratio of heparin stock solution to PBS is 1:1.

[0057] The final formula of the menstrual blood preservation solution provided in this embodiment is as follows (taking 30ml as an example): 1.5ml of antibiotic base solution, 0.6ml of anticoagulant, and 27.9ml of DMEM / F12 culture medium are mixed evenly to prepare the menstrual blood preservation solution.

[0058] Example 2

[0059] This embodiment provides a menstrual blood preservation solution, which differs from Example 1 in that the concentrations of ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B are 1 μg / ml, 70 μg / ml, 80 μg / ml, 5 μg / ml, and 100 μg / ml, respectively.

[0060] Example 3

[0061] This embodiment provides a menstrual blood preservation solution, which differs from Example 1 in that the concentrations of ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B are 5 μg / ml, 50 μg / ml, 120 μg / ml, 1 μg / ml, and 150 μg / ml, respectively.

[0062] Example 4

[0063] This embodiment provides a menstrual blood preservation solution, which differs from Example 1 in that 0.5 ml of antibiotic base solution and 1 ml of anticoagulant are mixed evenly with 28.5 ml of DMEM / F12 culture medium.

[0064] Comparative Example 1

[0065] This comparative example provides a menstrual blood preservation solution, which differs from Example 1 in that the antibiotics are selected from ciprofloxacin lactate injection, vancomycin hydrochloride for injection, and kanamycin sulfate injection. Specifically, a 50ml solution is prepared by using 4ml of ciprofloxacin lactate injection, 800μl of vancomycin hydrochloride for injection, and 470μl of kanamycin sulfate injection. The antibiotics are mixed thoroughly according to the dosage used, and PBS is added to make up to 50ml to prepare the antibiotic base solution.

[0066] Comparative Example 2

[0067] This comparative example provides a menstrual blood preservation solution, which differs from Example 1 in that the antibiotics are selected from 2× penicillin, 5× streptomycin and 40 μg / ml kanamycin.

[0068] Experiment Example 1: Aseptic analysis of menstrual blood samples mixed with menstrual blood preservation solution at different time points.

[0069] 1. Experimental Procedure

[0070] Sample preservation and sampling: Samples without mixed menstrual blood preservation solution and samples diluted 10 times were retained for testing. The menstrual blood preservation solutions provided in Examples 1-4 and Comparative Examples 1 and 2 were mixed with menstrual blood samples at a ratio of 1:1 and stored in a refrigerator at 4 degrees Celsius. They were used for sterility testing at 2h, 4h, 8h, 24h, 48h, and 72h.

[0071]

[0072] The √ indicates that sterility testing was performed at this time point, and - indicates that sterility testing was not performed.

[0073] 2. Sampling aseptic testing

[0074] Aseptically, 100 μl of the test sample was quantitatively aspirated and inoculated into a plate. Two plates were inoculated at each sampling point to check the uniformity of the sample. The plates were incubated upright in a 35°C incubator for 1 hour, then inverted for 24-96 hours. The results were then observed.

[0075] Sample colony count table and analysis

[0076]

[0077]

[0078]

[0079] The results showed that mixing the same menstrual blood sample with different menstrual blood preservation solutions resulted in varying bactericidal effects. Generally, the longer the menstrual blood sample remained in contact with the solution, the better the bactericidal effect. Specifically, the menstrual blood preservation solutions provided in Examples 1-3 demonstrated good bactericidal effects on the samples. However, the ratio of antibiotic base solution and anticoagulant in Example 4 was outside the preferred range of this invention, and the antibiotics in Comparative Example 1 did not include gentamicin sulfate and amphotericin B, which are present in the antibiotic base solution provided by this invention, resulting in poor bactericidal effects. As for Comparative Example 2, although it also showed some bactericidal effect after 72 hours, its use did not comply with legal regulations.

[0080] To save on experimental costs, samples of menstrual blood mixed with the menstrual blood from Application Example 1, which showed better sterilization effects, were used for subsequent experiments.

[0081] Experiment Example 2: Study on the antibacterial ability of antibiotics in menstrual blood preservation solution

[0082] 1. Experimental Procedure

[0083] Four menstrual blood samples collected from different donors and at different time points were placed at various temperatures (2℃, 15℃, 25℃, and 37℃) to simulate transportation and temporary storage temperatures. After 48 hours, the menstrual blood samples mixed with the menstrual blood preservation solution provided in Example 1 of this invention were filtered separately in a biosafety cabinet using the filter of a disposable blood transfusion set. The bacterial count of the filtrate was detected using the plate inoculation method (rapid test) to evaluate the antibacterial ability of the menstrual blood preservation solution at different temperatures.

[0084] Four samples were collected: samples 1, 2, 3, and 4. The ratio of menstrual blood to menstrual blood preservation solution was 5ml to 30ml:20ml. Specifically, the ratio was 5ml:20ml for sample 1, 10ml:20ml for sample 2, 15ml:20ml for sample 3, and 30ml:20ml for sample 4. The blood was filtered through a transfusion set filter, dispensed, and stored at various temperatures for 48 hours before being collected for aerobic bacteria, mold, and yeast culture on petri dishes. Specific sampling points are shown in the table below.

[0085]

[0086] Four whole blood samples were mixed thoroughly and transferred to corresponding new centrifuge tubes, then centrifuged at 2800 rpm. The blood cell pellet was resuspended in a certain volume of PBS (Phosphate Buffered Saline), and the total number of WBCs (White Blood Cells) in the whole blood was counted. The cell suspension was then slowly added dropwise to the surface of Ficoll (Ficoll:blood cell suspension volume ratio = 1:2), and the cells were centrifuged at 700g to separate mononuclear cells. After centrifugation, the white membrane layer was collected and washed twice with PBS. After washing, the supernatant was discarded, and the cells were resuspended in complete culture medium. The total number of WBCs was counted again, and the cell viability of each group was detected by trypan blue staining. The experimental data were recorded as follows: the total number of WBCs in the whole blood during primary isolation and culture, and the total number of WBCs after Ficoll isolation. The cell yield was calculated using the formula: Cell yield (%) = Total number of WBCs after Ficoll isolation / Total number of WBCs in whole blood × 100%.

[0087] 2. Experimental Results

[0088] Specific aerobic bacteria test results are detailed in the table. Aerobic bacteria test results:

[0089]

[0090] Results of mold and yeast tests:

[0091]

[0092] The mean values ​​of the data measured for each sample under each temperature condition are as follows:

[0093]

[0094] 3. Experimental Conclusions

[0095] The total number of aerobic bacteria was suppressed at all simulated temperatures, and the overall trend of the total number of aerobic bacterial colonies decreased, indicating that the menstrual blood preservation solution showed a good antibacterial effect against aerobic bacteria. Molds and yeasts were not detected after filtration of the menstrual blood; therefore, this experiment did not demonstrate the antibacterial ability of the menstrual blood preservation solution against molds and yeasts.

[0096] The isolation yield of menstrual blood samples is affected by storage temperature under certain conditions. When menstrual blood samples are stored at 2℃–25℃ for 48 hours, the isolation yield of white blood cells (WBCs) is relatively high, but there is no significant difference in yield among the 2℃, 15℃, and 25℃ groups. The WBC isolation yield is low when stored at 37℃ for 48 hours, significantly lower than the 2℃–25℃ experimental group. Within the temperature range of 2℃–37℃, the survival rate of WBCs isolated from all groups is above 95%, indicating that within this temperature range, the WBC cell activity of menstrual blood samples stored for 48 hours is good and unaffected by temperature changes. Based on the results and analysis of this experiment, a temperature control range of 2℃–25℃ is recommended for the storage and transportation of menstrual blood samples.

[0097] Experiment 3: Analysis of sterility of menstrual blood samples stored at 2℃ for 24 hours and study on changes in surface markers of menstrual blood stem cells.

[0098] 1. Experimental procedure:

[0099] The centrifuge tubes containing menstrual blood samples (mixed with the menstrual blood preservation solution provided in Example 1 of this invention) were sealed in self-sealing bags and stored in a 2°C refrigerator. After 24 hours, the real-time temperature recorder was removed, and the temperature change curve over 24 hours was exported via computer. The menstrual blood samples were filtered using a disposable transfusion set filter in a biosafety cabinet, blood cells were counted, and the filtrate was centrifuged (600g, 10min). The blood cell pellet was resuspended in PBS (phosphate-buffered saline solution) and centrifuged using Ficoll (Ficoll: blood cell suspension volume ratio = 1:2) (700g, 14min) to obtain mononuclear cells. The cells were washed twice with PBS (first wash centrifugation conditions: 800g, 6min; second wash centrifugation conditions: 300g, 6min). After washing, the supernatant was discarded, and the cell pellet was kept for later use. The cells were resuspended in an appropriate amount of complete culture medium and counted. Depending on the number of cells, the pellet was placed in a CO2 incubator and incubated conventionally until passage P5. Samples were taken and tested at the corresponding stages of separation and culture according to subsequent steps. This experiment performed sterility and phenotypic testing on four batches of samples.

[0100] 2. Experimental Results

[0101] 2.1 Total bacterial count of the sample

[0102]

[0103] 2.2 Total number of fungal colonies in the sample

[0104]

[0105] 2.3 Surface marker detection results

[0106] 1-P1 ON ON ON 0.1 ON ON 99.0 ON 1-P2 ON ON ON 0.1 ON ON 99.9 ON 1-P3 0.1 0.0 0.0 0.2 99.8 99.9 100.0 0.0 1-P4 1.3 0.0 0.0 0.1 99.9 100.0 100.0 0.0 1-P5 0.4 0.0 0.2 0.0 99.7 99.9 98.7 0.1 2-P2 0.0 0.0 0.0 0.0 99.9 99.8 99.7 0.0 2-P3 0.0 0.0 0.0 0.0 100.0 99.8 100.0 0.0 2-P4 0.0 0.0 0.0 0.1 99.9 99.5 99.9 0.0 2-P5 0.4 0.0 0.0 0.0 99.9 99.8 99.9 0.0 4-P2 1.8 0.4 0.8 0.4 99.5 99.7 95.9 0.8 4-P3 1.9 0.5 0.4 0.7 100.0 99.9 99.4 0.4 4-P4 2.0 0.3 0.3 0.4 100.0 99.9 99.2 0.2 4-P5 0.3 0.3 0.1 0.3 100.0 99.9 99.4 0.2

[0107] 3. Results Analysis

[0108] 3.1 Aseptic analysis:

[0109] Three out of the four batches of samples had a colony count of <10 cfu / plate in the supernatant after the first culture at P0. No contamination was found during subsequent culture. Only one sample was contaminated. No fungi were detected in any of the samples. This indicates that storing menstrual blood samples at 2°C for 24 hours (when the bactericidal ability of the menstrual blood preservation solution is at its weakest) can also be used as a condition for transport and preservation.

[0110] 3.2 Analysis of Surface Marker Detection Results:

[0111] The results of cell surface marker detection in all three batches of samples met the ISCT requirements for stem cell surface markers: CD73, CD90, CD105 ≥ 95%, CD11b, CD19, CD34, CD45, HLA-DR ≤ 2%. This experiment preliminarily shows that P2 generation menstrual blood stem cells have met the purification requirements.

[0112] In conclusion, the results of this experiment demonstrate that 2℃ can be used as the minimum temperature for transporting and storing menstrual blood samples, and the temperature conditions for transporting and storing menstrual blood samples can be between 2℃ and 25℃.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A menstrual blood preservation solution, characterized in that, include: The volume percentage of the solution is 1% to 10% antibiotic base solution, 1% to 5% anticoagulant, and the remainder is basal culture medium; The antibiotic base solution includes ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B. The concentrations of ciprofloxacin lactate, vancomycin hydrochloride, kanamycin sulfate, gentamicin sulfate, and amphotericin B were 2 μg / ml, 60 μg / ml, 100 μg / ml, 2.7 μg / ml, and 120 μg / ml, respectively. The basal culture medium includes at least one of DMEM, DMEM / F12, RPMI 1640, IMDM, MCDB 201 and MCDB 131.

2. The menstrual blood preservation solution according to claim 1, characterized in that, The anticoagulant includes one or more of heparin, sodium citrate, or ethylenediaminetetraacetic acid; The effective concentration of the anticoagulant is 50~70 U / ml.

3. The menstrual blood preservation solution according to claim 2, characterized in that, The effective concentration of the anticoagulant is 60 U / ml.

4. The menstrual blood preservation solution according to claim 1, characterized in that, The solvents for the antibiotic base solution and the anticoagulant each independently include a buffer solution.

5. The menstrual blood preservation solution according to claim 4, characterized in that, The solvents for the antibiotic base solution and the anticoagulant are each independently PBS buffer.

6. The menstrual blood preservation solution according to any one of claims 1-5, characterized in that, It includes 5% antibiotic base solution, 2% anticoagulant and 93% basal culture medium by volume.

7. The method for preparing the menstrual blood preservation solution according to any one of claims 1-6, characterized in that, The process involves mixing the prescribed amounts of antibiotic base solution, anticoagulant, and basal culture medium evenly to obtain the menstrual blood preservation solution.

8. The use of the menstrual blood preservation solution according to any one of claims 1-6 in menstrual blood collection, preservation and transportation.

9. The application according to claim 8, characterized in that, The temperature for menstrual blood preservation and transportation is 2~25℃; The volume ratio of menstrual blood to menstrual blood preservation solution is 5~30:

20.

10. A menstrual blood collection kit, characterized in that, Includes sterile rubber surgical gloves, masks, surgical caps, alcohol swabs, skin disinfection wipes, menstrual cups, sealing films, labels, temperature recorders, cold storage agents, and menstrual blood preservation solutions as described in any one of claims 1-6.

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