Uterine blood collection liquid

Abstract

The invention provides a uterine blood collection liquid, and belongs to the field of biology. The uterine blood collection liquid is composed of levofloxacin, amphotericin B, heparin sodium and DMEM-F12, the concentration of the levofloxacin is 30-50 [mu] g / mL, the concentration of the amphotericin B is 1.5-3.0 [mu] g / mL, the concentration of the heparin sodium is 50-70 U / mL, and the DMEM-F12 is a basic liquid. The invention provides a uterine blood collection liquid, the uterine blood collection liquid is composed of levofloxacin, amphotericin B, heparin sodium and DMEM-F12, the collection liquid uses few types of antibiotics, the preparation is simple, and the cost of the collection liquid is low.

Description

Technical Field

[0001] The present disclosure relates to the biological field, and in particular to a uterine blood collection fluid. Background Art

[0002] Uterine blood stem cells are stem cells with the ability to self-replicate and differentiate. They originate from the endometrium and are excreted from the body through uterine blood. The storage of uterine blood is crucial; improper storage can affect the activity and proliferation potential of these stem cells.

[0003] Existing uterine blood collection fluids contain many types of antibiotics and are complicated to prepare, which makes the cost of the uterine blood collection fluids high. In order to reduce the cost of the uterine blood collection fluids, a low-cost uterine blood collection fluid is urgently needed.

[0004] Public content In order to solve the problems of the prior art, the present disclosure provides a uterine blood collection solution. The technical solution is as follows: The present disclosure provides a uterine blood collection solution, which is composed of levofloxacin, amphotericin B, heparin sodium and DMEM-F12, wherein the concentration of the levofloxacin is 30-50 μg / mL, the concentration of the amphotericin B is 1.5-3.0 μg / mL, the concentration of the heparin sodium is 50-70 U / mL, and the DMEM-F12 is used as a base solution.

[0005] Specifically, the concentration of levofloxacin is 45 μg / mL, the concentration of amphotericin B is 2.7 μg / mL, and the concentration of heparin sodium is 60 U / mL.

[0006] The technical solutions provided by the embodiments of the present disclosure provide the following beneficial effects: The present disclosure provides a uterine blood collection solution composed of levofloxacin, amphotericin B, heparin sodium, and DMEM-F12. The collection solution uses a limited number of antibiotics and is simple to prepare, resulting in a low cost. Furthermore, the collection solution can reduce bacterial proliferation during the storage of uterine blood, reduce the probability of contamination of mesenchymal stem cell seeds isolated from the uterine blood, and effectively maintain the activity of stem cells in the uterine blood, thereby improving the efficiency of stem cell extraction from the uterine blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0008] Figure 1This is a morphological diagram of cells of generation P0 of the comparative example provided in Example 1 of the present disclosure; Figure 2 This is a morphological diagram of P0 generation cells provided in Example 1 of the present disclosure; Figure 3 This is a morphological diagram of cells of generation P1 of the comparative example provided in Example 1 of the present disclosure; Figure 4 This is a morphological diagram of P1 generation cells provided in Example 1 of the present disclosure; Figure 5 This is a morphological diagram of cells of generation P0 of the comparative example provided in Example 2 of the present disclosure; Figure 6 This is a morphological diagram of P0 cells provided in Example 2 of the present disclosure; Figure 7 This is a morphological diagram of cells of generation P1 of the comparative example provided in Example 2 of the present disclosure; Figure 8 This is a morphological diagram of P1 generation cells provided in Example 2 of the present disclosure. DETAILED DESCRIPTION

[0009] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0010] Comparative Example The collection fluid provided in this comparative example includes: DMEM-F12 as a basal fluid, ciprofloxacin 2 μg / mL, vancomycin 60 μg / mL, kanamycin 100 μg / mL, amphotericin B: 2.7 μg / mL, gentamicin 120 μg / mL, and heparin sodium 60 U / mL.

[0011] Example 1 An embodiment of the present disclosure provides a uterine blood collection fluid, which is composed of levofloxacin, amphotericin B, heparin sodium and DMEM-F12. DMEM-F12 is a base fluid purchased from Thermo Fisher Scientific. The concentration of levofloxacin is 30 μg / mL, the concentration of amphotericin B is 2.7 μg / mL, and the concentration of heparin sodium is 60 U / mL.

[0012] The following is a brief introduction to the preparation method of the uterine blood collection fluid, which specifically includes: using DMEM-F12 as the base fluid, successively adding 30 μg / mL levofloxacin, 2.7 μg / mL amphotericin B, and 60 U / mL heparin sodium, and mixing them for later use.

[0013] After the collected uterine blood samples were divided equally (the uterine blood samples used in this embodiment were obtained from qualified donors of appropriate age who met the physical examination standards and whose menstrual blood was collected with a menstrual cup and stored in a collection solution at 4°C), they were respectively placed in the collection solution provided in the comparative example and Example 1, and allowed to stand for more than 24 hours. The uterine blood samples were treated by the red lysis method to obtain cells to be cultured. The cells to be cultured were resuspended in a mesenchymal stem cell-specific culture medium and seeded at the same density in a culture flask with T25 culture medium. The comparative example collection solution was cultured for 9 days. After 5 minutes of digestion, the morphology of the P0 generation cells (uterine blood mesenchymal stem cells, MSCs) was observed, as shown in FIG. Figure 1 The uterine blood stem cells extracted from the collection solution provided in Example 1 were cultured for 5 days, and after 4 minutes of digestion, the morphology of the P0 generation cells was observed. Figure 2 As shown. Figure 1 and Figure 2 It can be seen that the P0 generation cells treated with the collection solution provided in Example 1 are more numerous. The P0 generation cells obtained by the two collection solutions were measured for the collected cell amounts, and the collected cell amount obtained in the comparative example was 4.5×10 5 The amount of cells collected by the collection solution provided in Example 1 is 5.5×10 5 , thus it can be seen that the collection solution provided in Example 1 collects more cells.

[0014] P0 cells were seeded at a rate of 4.5×10 5 The cells were inoculated into T75 medium at a concentration of 1:1 and cultured for 8 days. Figure 3 The P1 generation cells obtained from the collection solution provided in Example 1 were cultured for 7 days to obtain P1 generation cells. The P1 generation cells were digested for 5 minutes and the morphology of the P1 generation cells was observed. Figure 4 As shown. Figure 3 and Figure 4 It can be seen that the P1 generation cells treated with the collection solution provided in Example 1 have more cells. The P1 generation cells obtained by the two collection solutions were measured for the collected cell amount, and the cell collection amount obtained in the comparative example was 1.96×10 6 , the yield was 4.3 times, and the amount of cells collected by the collection solution provided in Example 1 was 3.29×10 6 , the yield was 6.5 times, which shows that the collection solution provided in Example 1 collected more cells.

[0015] Comparison was performed through flow cytometry data. The comparison ratio was CD73: 100%, CD90: 99.9%, CD105: 99.9%; the present embodiment was CD73: 100%, CD90: 99.9%, CD105: 99.9%.

[0016] Example 2 The present disclosure provides a uterine blood collection fluid, which consists of levofloxacin, amphotericin B, heparin sodium and DMEM-F12, wherein DMEM-F12 is a base fluid, the concentration of levofloxacin is 45 μg / mL, the concentration of amphotericin B is 1.5 μg / mL, and the concentration of heparin sodium is 50 U / mL.

[0017] After the collected uterine blood samples were divided equally (the uterine blood samples used in this embodiment were derived from qualified donors of appropriate age who met the physical examination criteria, and menstrual blood was collected using a menstrual cup and stored in a collection fluid at 4°C), they were respectively placed in the uterine blood collection fluid provided in the comparative example and Example 2, and allowed to stand for more than 24 hours. The uterine blood samples were treated by the red lysis method to obtain cells to be cultured. The cells to be cultured were resuspended in a mesenchymal stem cell-specific culture medium and seeded at the same density in a culture flask with T25 culture medium. The comparative example collection fluid was cultured for 8 days. After 5 minutes of digestion, the morphology of the P0 generation cells (MSCs) was observed, as shown in FIG. Figure 5 The collected solution provided in Example 2 was cultured for 6 days, and after 5 minutes of digestion, the morphology of the P0 generation cells was observed. Figure 6 As shown. Figure 5 and Figure 6 It can be seen that the P0 generation cells treated with the collection solution provided in Example 2 are more numerous. The P0 generation cells obtained by the two collection solutions were measured for the collected cell amounts, and the collected cell amount obtained in the comparative example was 3.6×10 5 The amount of cells collected by the collection solution provided in Example 2 is 4×10 5 , thus it can be seen that the uterine blood collection fluid provided in Example 2 collects more cells.

[0018] The P0 generation cells were inoculated in T75 at the same density for culture. The P1 generation cells obtained by the comparative sample collection solution were cultured for 9 days to obtain the P1 generation cells. Figure 7 The P1 generation cells obtained from the uterine blood collection fluid provided in Example 2 were cultured for 7 days to obtain the P1 generation cells. Figure 8 As shown. Figure 7 and Figure 8 It can be seen that the P1 generation cells treated with the uterine blood collection solution provided in Example 2 are more numerous. The P1 generation cells obtained by the two collection solutions were measured for the collected cell amounts, and the collected cell amount obtained in the comparative example was 2.2×10 6 , the yield was 6.1 times, and the amount of cells collected by the uterine blood collection solution provided in Example 2 was 2.8×10 6 , the yield is 7 times, which shows that the uterine blood collection fluid provided in Example 2 collects more cells.

[0019] Comparing the flow cytometry data, the control showed CD73: 99.9%, CD90: 99.9%, and CD105: 99.9%, while the present embodiment showed CD73: 100%, CD90: 100%, and CD105: 99.9%. This comparison demonstrates that the positive parameters in the flow cytometry data provided by this embodiment do not decrease cell stemness due to changes in antibiotics, demonstrating that the culture data from the collected fluid provided by this embodiment meets the requirements for stem cell characteristics.

[0020] Example 3 The present disclosure provides a uterine blood collection fluid, which consists of levofloxacin, amphotericin B, heparin sodium and DMEM-F12, wherein DMEM-F12 is a base fluid, the concentration of levofloxacin is 50 μg / mL, the concentration of amphotericin B is 3.0 μg / mL, and the concentration of heparin sodium is 70 U / mL.

[0021] The above description is merely an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure.

Claims

1. A uterine blood collection solution, characterized in that: The uterine blood collection fluid consists of levofloxacin, amphotericin B, heparin sodium and DMEM-F12, wherein the concentration of the levofloxacin is 30-50 μg / mL, the concentration of the amphotericin B is 1.5-3.0 μg / mL, the concentration of the heparin sodium is 50-70 U / mL, and the DMEM-F12 is the base fluid.

2. The uterine blood collection solution according to claim 1, characterized in that The concentration of the levofloxacin is 45 μg / mL, the concentration of the amphotericin B is 2.7 μg / mL, and the concentration of the heparin sodium is 60 U / mL.

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