A granulocyte dissociation solution and a preparation method

Abstract

The application belongs to the field of human assisted reproductive technology, and provides a granular cell stripping solution and a preparation method. The granular cell stripping solution takes water as a solvent and comprises the following components: sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium bicarbonate, hydroxyethylpiperazine ethanesulfonic acid, hyaluronidase, sodium pyruvate, fibroblast growth factor, ursodeoxycholic acid, human blood albumin, phenol red indicator, and gentamicin sulfate. The granular cell stripping solution can provide a stable buffer system and sufficient energy for oocytes and surrounding granular cells during stripping operation, and improve the fertilization rate of subsequent fertilization treatment.

Description

Technical Field

[0001] This invention relates to the field of human assisted reproductive technology, and in particular to a granular cell stripping solution and its preparation method. Background Technology

[0002] Before intracytoplasmic sperm injection (ICSI), it is necessary to remove the cumulus oophorus and granulosa cells surrounding the oocyte to expose the zona pellucida surface, allowing for easy gripping and injection with micro-instruments. Previously, the most common method for removing cumulus oophorus and granulosa cells was mechanical. However, mechanical methods require highly skilled operators, and improper handling can cause significant cell damage and poor removal efficiency. Therefore, granulosa cell removal solutions have been developed. Currently, most granulosa cell removal solutions are imported, have complex compositions, and are expensive, but their effectiveness is not significant, affecting subsequent fertilization and embryonic development. Therefore, to overcome the shortcomings of the above techniques, there is a need for a granulosa cell removal solution that is simple in composition, inexpensive, and highly effective. Summary of the Invention

[0003] The purpose of this invention is to provide a granulosa cell stripping solution and its preparation method. The granulosa cell stripping solution of this invention has simple and readily available raw material components and a simple preparation method. It can provide a stable buffer system and sufficient energy for oocytes and surrounding granulosa cells during the stripping operation, thereby improving the fertilization rate of subsequent fertilization treatment.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a granulocyte stripping solution, using water as a solvent, comprising the following components at final concentrations: sodium chloride 100–105 mmol / L, potassium chloride 4–5 mmol / L, magnesium sulfate 0.1–0.3 mmol / L, calcium chloride 2–2.4 mmol / L, sodium bicarbonate 3–5 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 20–25 mmol / L, hyaluronidase 20–25 mmol / L, sodium pyruvate 0.3–0.5 mmol / L, fibroblast growth factor 2.5–4 mmol / L, ursodeoxycholic acid 5–10 mmol / L, human serum albumin 2–8 mg / mL, phenol red indicator 0.01–0.1 mg / L, and gentamicin sulfate 20–25 mg / L.

[0006] Preferably, the granule cell stripping solution uses water as a solvent and comprises the following components at final concentrations: sodium chloride 101–104 mmol / L, potassium chloride 4.2–4.8 mmol / L, magnesium sulfate 0.15–0.25 mmol / L, calcium chloride 2.1–2.3 mmol / L, sodium bicarbonate 3.5–4.5 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 21–24 mmol / L, hyaluronidase 21–24 mmol / L, sodium pyruvate 0.35–0.45 mmol / L, fibroblast growth factor 2.8–3.8 mmol / L, ursodeoxycholic acid 6–9 mmol / L, human serum albumin 3–7 mg / mL, phenol red indicator 0.02–0.08 mg / L, and gentamicin sulfate 21–24 mg / L.

[0007] Preferably, the granule cell stripping solution uses water as a solvent and comprises the following components at final concentrations: sodium chloride 102–103 mmol / L, potassium chloride 4.4–4.6 mmol / L, magnesium sulfate 0.18–0.22 mmol / L, calcium chloride 2.15–2.25 mmol / L, sodium bicarbonate 3.8–4.2 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 22–23 mmol / L, hyaluronidase 22–23 mmol / L, sodium pyruvate 0.38–0.42 mmol / L, fibroblast growth factor 3–3.5 mmol / L, ursodeoxycholic acid 7–8 mmol / L, human serum albumin 4–6 mg / mL, phenol red indicator 0.04–0.06 mg / L, and gentamicin sulfate 22–23 mg / L.

[0008] The present invention also provides a method for preparing the granule cell stripping solution described in any one of the above claims, comprising the following steps:

[0009] Sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium bicarbonate, and sodium pyruvate are mixed with water. Then, gentamicin sulfate, phenol red indicator, and hydroxyethylpiperazine ethanesulfonic acid are added sequentially. After mixing, fibroblast growth factor, ursodeoxycholic acid, human serum albumin, and hyaluronidase are added to obtain a solution. The solution is filtered, and the filtrate is collected as granulocyte exfoliation solution.

[0010] Preferably, the step of adjusting the osmotic pressure and pH value before the filtration treatment is further included, wherein the osmotic pressure is adjusted to 260-290 mOsm / kg and the pH value is adjusted to 7.2-7.4.

[0011] Preferably, the filtration is performed using a 0.22μm filter membrane.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] The granulosa cell stripping solution of this invention can effectively remove oocytes and granulosa cells, providing a stable buffer system and sufficient energy during this operation. The granulosa cell stripping solution of this invention contains Na. + K + Cl - Ca 2+ Mg 2+ Plasma is an important component for maintaining osmotic pressure and regulating pH; sodium pyruvate provides energy in the early stages of cell development; hydroxyethylpiperazine ethanesulfonic acid can be used to maintain the pH stability of the solution; fibroblast growth factor can promote the survival of granulosa cells and regulate their proliferation; the addition of antibiotics can reduce contamination during cell culture; human serum albumin can stabilize and protect cell membranes by binding to fatty acids and inhibiting lipid peroxidation, and can also bind to various antibodies, heavy metals, lipoproteins and unidentified toxic substances to maintain the stability of colloidal osmotic pressure.

[0014] The raw materials of the granule cell stripping fluid of this invention are simple, and its use can improve the fertilization rate of oocytes, providing a new direction for the further development of assisted reproductive technology. Detailed Implementation

[0015] This invention provides a granulocyte stripping solution, using water as a solvent, comprising the following components at final concentrations: sodium chloride 100–105 mmol / L, potassium chloride 4–5 mmol / L, magnesium sulfate 0.1–0.3 mmol / L, calcium chloride 2–2.4 mmol / L, sodium bicarbonate 3–5 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 20–25 mmol / L, hyaluronidase 20–25 mmol / L, sodium pyruvate 0.3–0.5 mmol / L, fibroblast growth factor 2.5–4 mmol / L, ursodeoxycholic acid 5–10 mmol / L, human serum albumin 2–8 mg / mL, phenol red indicator 0.01–0.1 mg / L, and gentamicin sulfate 20–25 mg / L.

[0016] In this invention, the granule cell stripping solution uses water as a solvent and includes sodium chloride at a concentration of 100–105 mmol / L, preferably 101–104 mmol / L, and more preferably 102–103 mmol / L.

[0017] In this invention, the granule cell stripping solution uses water as a solvent and includes 4-5 mmol / L potassium chloride, preferably 4.2-4.8 mmol / L, and more preferably 4.4-4.6 mmol / L.

[0018] In this invention, the granule cell stripping solution uses water as a solvent and includes magnesium sulfate at 0.1–0.3 mmol / L, preferably 0.15–0.25 mmol / L, and more preferably 0.18–0.22 mmol / L.

[0019] In this invention, the granule cell stripping solution uses water as a solvent and includes calcium chloride at 2-2.4 mmol / L, preferably 2.1-2.3 mmol / L, and more preferably 2.15-2.25 mmol / L.

[0020] In this invention, the granule cell stripping solution uses water as a solvent and includes sodium bicarbonate at 3-5 mmol / L, preferably 3.5-4.5 mmol / L, and more preferably 3.8-4.2 mmol / L.

[0021] In this invention, the granule cell stripping solution uses water as a solvent and includes 20-25 mmol / L of hydroxyethylpiperazine ethanesulfonic acid, preferably 21-24 mmol / L, and more preferably 22-23 mmol / L.

[0022] In this invention, the granule cell stripping solution uses water as a solvent and contains 20-25 mmol / L of hyaluronidase, preferably 21-24 mmol / L, and more preferably 22-23 mmol / L.

[0023] In this invention, the granule cell stripping solution uses water as a solvent and includes sodium pyruvate at 0.3-0.5 mmol / L, preferably 0.35-0.45 mmol / L, and more preferably 0.38-0.42 mmol / L.

[0024] In this invention, the granule cell stripping solution uses water as a solvent and contains 2.5–4 mmol / L of fibroblast growth factor, preferably 2.8–3.8 mmol / L, and more preferably 3–3.5 mmol / L.

[0025] In this invention, the granule cell stripping solution uses water as a solvent and includes 5-10 mmol / L ursodeoxycholic acid, preferably 6-9 mmol / L, and more preferably 7-8 mmol / L.

[0026] In this invention, the granule cell stripping solution uses water as a solvent and contains 2-8 mg / mL of human serum albumin, preferably 3-7 mg / mL, and more preferably 4-6 mg / mL.

[0027] In this invention, the granule cell stripping solution uses water as a solvent and includes 0.01-0.1 mg / L of phenol red indicator, preferably 0.02-0.08 mg / L, and more preferably 0.04-0.06 mg / L.

[0028] In this invention, the granule cell stripping solution uses water as a solvent and includes 20-25 mg / L of gentamicin sulfate, preferably 21-24 mg / L, and more preferably 22-23 mg / L.

[0029] The present invention also provides a method for preparing the granule cell stripping solution described in any one of the above claims, comprising the following steps:

[0030] Sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium bicarbonate, and sodium pyruvate are mixed with water. Then, gentamicin sulfate, phenol red indicator, and hydroxyethylpiperazine ethanesulfonic acid are added sequentially. After mixing, fibroblast growth factor, ursodeoxycholic acid, human serum albumin, and hyaluronidase are added to obtain a solution. The solution is filtered, and the filtrate is collected as granulocyte exfoliation solution.

[0031] In this invention, the mixing method can be any conventional mixing method in the art. In one specific embodiment of this invention, the mixing method is preferably carried out using a magnetic stirring device.

[0032] In this invention, the step of adjusting the osmotic pressure and pH value before the filtration treatment is further included. The osmotic pressure is adjusted to 260-290 mOsm / kg, preferably 265-285 mOsm / kg, and more preferably 270-280 mOsm / kg; the pH value is adjusted to 7.2-7.4, preferably 7.25-7.35, and more preferably 7.28-7.32.

[0033] In this invention, the filtration is performed using a 0.22μm filter membrane.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] This embodiment provides a granulocyte stripping solution, using water as a solvent, comprising the following components at final concentrations: sodium chloride 100 mmol / L, potassium chloride 5 mmol / L, magnesium sulfate 0.1 mmol / L, calcium chloride 2.4 mmol / L, sodium bicarbonate 3 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 25 mmol / L, hyaluronidase 20 mmol / L, sodium pyruvate 0.5 mmol / L, fibroblast growth factor 2.5 mmol / L, ursodeoxycholic acid 10 mmol / L, human serum albumin 2 mg / mL, phenol red indicator 0.1 mg / L, and gentamicin sulfate 20 mg / L.

[0037] The preparation method of the granule cell stripping solution is as follows: Weigh the above raw materials, mix sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium bicarbonate, sodium pyruvate and water, stir evenly with a magnetic stirrer, then add gentamicin sulfate, phenol red indicator and hydroxyethylpiperazine ethanesulfonic acid in sequence, stir evenly with a magnetic stirrer, then add fibroblast growth factor, ursodeoxycholic acid, human serum albumin and hyaluronidase, adjust the osmotic pressure of the solution to 270 mOsm / kg and the pH value to 7.3, and obtain the granule cell stripping solution after filtration and sterilization through a 0.22 μm filter membrane.

[0038] Example 2

[0039] This embodiment provides a granulocyte stripping solution, using water as a solvent, comprising the following components at final concentrations: sodium chloride 105 mmol / L, potassium chloride 4 mmol / L, magnesium sulfate 0.3 mmol / L, calcium chloride 2 mmol / L, sodium bicarbonate 5 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 20 mmol / L, hyaluronidase 25 mmol / L, sodium pyruvate 0.3 mmol / L, fibroblast growth factor 4 mmol / L, ursodeoxycholic acid 5 mmol / L, human serum albumin 8 mg / mL, phenol red indicator 0.01 mg / L, and gentamicin sulfate 25 mg / L.

[0040] The preparation method of the granule cell stripping solution is as follows: Weigh the above raw materials, mix sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium bicarbonate, sodium pyruvate and water, stir evenly with a magnetic stirrer, then add gentamicin sulfate, phenol red indicator and hydroxyethylpiperazine ethanesulfonic acid in sequence, stir evenly with a magnetic stirrer, then add fibroblast growth factor, ursodeoxycholic acid, human serum albumin and hyaluronidase, adjust the osmotic pressure of the solution to 280 mOsm / kg and the pH value to 7.3, and obtain the granule cell stripping solution after filtration and sterilization through a 0.22 μm filter membrane.

[0041] Example 3

[0042] This embodiment provides a granulocyte stripping solution, using water as a solvent, comprising the following components at final concentrations: sodium chloride 102 mmol / L, potassium chloride 4.5 mmol / L, magnesium sulfate 0.2 mmol / L, calcium chloride 2.2 mmol / L, sodium bicarbonate 4 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 22 mmol / L, hyaluronidase 23 mmol / L, sodium pyruvate 0.4 mmol / L, fibroblast growth factor 3 mmol / L, ursodeoxycholic acid 8 mmol / L, human serum albumin 5 mg / mL, phenol red indicator 0.05 mg / L, and gentamicin sulfate 22 mg / L.

[0043] The preparation method of the granule cell stripping solution is as follows: Weigh the above raw materials, mix sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium bicarbonate, sodium pyruvate and water, stir evenly with a magnetic stirrer, then add gentamicin sulfate, phenol red indicator and hydroxyethylpiperazine ethanesulfonic acid in sequence, stir evenly with a magnetic stirrer, then add fibroblast growth factor, ursodeoxycholic acid, human serum albumin and hyaluronidase, adjust the osmotic pressure of the solution to 270 mOsm / kg and the pH value to 7.3, and obtain the granule cell stripping solution after filtration and sterilization through a 0.22 μm filter membrane.

[0044] Comparative Example 1

[0045] The difference between this comparative example and Example 1 is that it does not include ursodeoxycholic acid, while the concentrations of the remaining raw materials and the preparation methods are the same as in Example 1.

[0046] Comparative Example 2

[0047] The difference between this comparative example and Example 1 is that it does not include fibroblast growth factor, while the concentrations of the remaining raw materials and the preparation methods are the same as in Example 1.

[0048] Comparative Example 3

[0049] The difference between this comparative example and Example 1 is that it does not include ursodeoxycholic acid and fibroblast growth factor, while the concentrations of the remaining raw materials and the preparation methods are the same as in Example 1.

[0050] Comparative Example 4

[0051] The difference between this comparative example and Example 1 is that the amount of fibroblast growth factor used is replaced with 8 mmol / L, while the concentrations of other raw materials and the preparation methods are the same as in Example 1.

[0052] Experimental Example 1

[0053] This experiment tested the performance indicators of the granular cell stripping solutions prepared in Examples 1-3, and the testing methods are as follows:

[0054] 1. pH measurement: Take 1 mL of the granule cell stripping solution from Examples 1 to 3, measure it three times with a pH meter, and take the average of the three data as the pH result.

[0055] 2. Osmotic pressure measurement: After calibrating the freezing point osmometer, take 0.5 mL of the granulocyte exfoliation solution from Examples 1-3 into the test tube to begin the test. Measure three data points and take the average value as the result. An osmotic pressure between 260 and 290 mOsm / kg is considered acceptable.

[0056] 3. Measurement of bacterial endotoxin content: According to the requirements of the 2015 edition of the Pharmacopoeia of the People's Republic of China, the Limulus amebocyte lysate (LAL) reagent gel method was used for detection, and the result <0.25 EU / mL was considered qualified.

[0057] 4. Sterility test: In accordance with the requirements of the 2015 edition of the Pharmacopoeia of the People's Republic of China, the membrane filtration method is used. After filtration through the filter membrane, the culture medium is directly inoculated and cultured for 14 days. The results are observed daily. If sterility is observed, it is considered qualified.

[0058] 5. Mouse embryo in vitro culture test: Treat mouse 1-cell embryos with the granule cell stripping solution of Examples 1-3, and then culture them in vitro for 96 hours. If the blastocyst expansion rate is ≥80%, it is considered qualified.

[0059] The test results are shown in Table 1:

[0060] Table 1. Performance indicators of granule cell stripping solution

[0061]

[0062]

[0063] As shown in Table 1, the pH and osmotic pressure of the granular cell stripping solutions prepared in Examples 1-3 are stable, and the bacterial endotoxin and bacterial content of the prepared products are very low, indicating that the granular cell stripping solution product system prepared by the present invention is stable and meets the product requirements.

[0064] Experiment Example 2

[0065] This experiment investigated the effect of the granulosa cell stripping solutions prepared in Examples 1-3 and Comparative Examples 1-4 on the removal of surrounding granulosa cells after treating oocytes. The steps are as follows:

[0066] Mouse oocyte-cumulus complexes (OCCs) were obtained using conventional oocyte retrieval methods. 500 OOCs were divided into 5 groups: Examples 1-3 and Comparative Examples 1-4. Granulocyte-cell stripping solutions prepared for each group were pre-equilibrated to 37°C and distributed to culture dishes for each group. The OOCs were then placed in the granulosa cell stripping solution for no more than 1 minute. Oocytes were transferred to washing culture medium using a minimal volume of solution, and the remaining OOCs were aspirated using a pipette to remove them. The stripped OOCs were washed until complete. Following standard mouse IVF procedures, capacitated sperm were added to the OOCs in each group for fertilization. The fertilization rate was observed and tallied under a microscope 17 hours after fertilization. The results are shown in Table 2.

[0067] Table 2. Fertilization status of oocytes in each group

[0068] Example 1 100 79 79 Example 2 100 85 85 Example 3 100 88 88 Comparative Example 1 100 62 62 Comparative Example 2 100 58 58 Comparative Example 3 100 40 40 Comparative Example 4 100 45 45

[0069] As shown in Table 2, the granulosa cell stripping solution prepared according to this invention effectively separates the oocyte from the surrounding granulosa cells after treatment of the oocyte-cumulus complex, thus improving the fertilization rate. Furthermore, the difference analysis between the fertilization results of Examples 1-3 and Comparative Examples 1-4 revealed a significant difference (P<0.05). Therefore, the granulosa cell stripping solution prepared according to this invention can provide a more stable buffer system for oocytes, thereby improving their fertilization rate.

[0070] As can be seen from the above embodiments, the present invention provides a granulosa cell stripping solution and a preparation method. The raw materials of the granulosa cell stripping solution of the present invention are simple and readily available, and the preparation method is simple. It can provide a stable buffer system and sufficient energy for oocytes and surrounding granulosa cells during the stripping operation, thereby improving the fertilization rate of subsequent fertilization treatment.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A granular cell stripping solution, characterized in that, Using water as a solvent, the following components are present in final concentrations: sodium chloride 100–105 mmol / L, potassium chloride 4–5 mmol / L, magnesium sulfate 0.1–0.3 mmol / L, calcium chloride 2–2.4 mmol / L, sodium bicarbonate 3–5 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 20–25 mmol / L, hyaluronidase 20–25 mmol / L, sodium pyruvate 0.3–0.5 mmol / L, fibroblast growth factor 2.5–4 mmol / L, ursodeoxycholic acid 5–10 mmol / L, human serum albumin 2–8 mg / mL, phenol red indicator 0.01–0.1 mg / L, and gentamicin sulfate 20–25 mg / L.

2. The granular cell stripping solution according to claim 1, characterized in that, Using water as a solvent, the following components are present in final concentrations: sodium chloride 101–104 mmol / L, potassium chloride 4.2–4.8 mmol / L, magnesium sulfate 0.15–0.25 mmol / L, calcium chloride 2.1–2.3 mmol / L, sodium bicarbonate 3.5–4.5 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 21–24 mmol / L, hyaluronidase 21–24 mmol / L, sodium pyruvate 0.35–0.45 mmol / L, fibroblast growth factor 2.8–3.8 mmol / L, ursodeoxycholic acid 6–9 mmol / L, human serum albumin 3–7 mg / mL, phenol red indicator 0.02–0.08 mg / L, and gentamicin sulfate 21–24 mg / L.

3. The granular cell stripping solution according to claim 2, characterized in that, Using water as a solvent, the following components are present in final concentrations: sodium chloride 102–103 mmol / L, potassium chloride 4.4–4.6 mmol / L, magnesium sulfate 0.18–0.22 mmol / L, calcium chloride 2.15–2.25 mmol / L, sodium bicarbonate 3.8–4.2 mmol / L, hydroxyethylpiperazine ethanesulfonic acid 22–23 mmol / L, hyaluronidase 22–23 mmol / L, sodium pyruvate 0.38–0.42 mmol / L, fibroblast growth factor 3–3.5 mmol / L, ursodeoxycholic acid 7–8 mmol / L, human serum albumin 4–6 mg / mL, phenol red indicator 0.04–0.06 mg / L, and gentamicin sulfate 22–23 mg / L.

4. The method for preparing the granular cell stripping solution according to any one of claims 1 to 3, characterized in that, Includes the following steps: Sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium bicarbonate, and sodium pyruvate are mixed with water. Then, gentamicin sulfate, phenol red indicator, and hydroxyethylpiperazine ethanesulfonic acid are added sequentially. After mixing, fibroblast growth factor, ursodeoxycholic acid, human serum albumin, and hyaluronidase are added to obtain a solution. The solution is filtered, and the filtrate is collected as granulocyte exfoliation solution.

5. The preparation method according to claim 4, characterized in that, The filtration process also includes steps to adjust the osmotic pressure and pH value, with the osmotic pressure adjusted to 260–290 mOsm / kg and the pH value adjusted to 7.2–7.

4.

6. The preparation method according to claim 5, characterized in that, The filtration process uses a 0.22μm filter membrane.

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