CN113755429A - Composition for preparing cleavage culture solution, cleavage culture solution and preparation method - Google Patents
Composition for preparing cleavage culture solution, cleavage culture solution and preparation method Download PDFInfo
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- CN113755429A CN113755429A CN202111149515.4A CN202111149515A CN113755429A CN 113755429 A CN113755429 A CN 113755429A CN 202111149515 A CN202111149515 A CN 202111149515A CN 113755429 A CN113755429 A CN 113755429A
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- culture solution
- cleavage
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- cleavage culture
- sodium
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a composition for preparing a cleavage culture solution, the cleavage culture solution and a preparation method thereof, wherein the composition comprises acetyl L-carnitine and hyaluronic acid; the cleavage culture solution comprises the composition, inorganic salt buffer solution, nonessential amino acid, ethylene diamine tetraacetic acid, sodium pyruvate, alanyl glutamine, taurine, glucose, sodium lactate, human serum albumin and a bactericide. The invention adds acetyl L-carnitine and hyaluronic acid into the cleavage culture solution as a composition, has the effects of scavenging oxygen free radicals in vivo to play an antioxidation role, protecting mitochondrial function, regulating energy metabolism, providing nutrition and energy for cell maturation and embryo development through catabolism and the like, has higher ratio of blastocysts and higher form and score of the blastocysts than those obtained by singly using any one of the compositions.
Description
Technical Field
The invention relates to the field of embryo in-vitro culture, and particularly relates to a composition for preparing a cleavage culture solution, the cleavage culture solution and a preparation method.
Background
In the process of embryo in vitro culture, the early embryo is very sensitive to oxidative damage, and the concentration of active oxygen in vitro culture is higher than that in vivo development environment. The antioxidant is added into the cleavage culture solution, so that the concentration of Reactive Oxygen Species (ROS) in the in-vitro culture process of the early embryo can be reduced, and the in-vitro culture is closer to the oxygen environment for in-vivo development of the embryo. In addition, the existing auxiliary reproduction technology in China mainly depends on imported finished products, the cost is high, the effectiveness of the solution cannot be well expressed, and if the culture solution is still used after going bad, irreversible results are caused to the experiment or clinical operation.
The world health organization makes statistics of the onset of infertility in developed countries, and the results show that up to 15% of women of childbearing age suffer from infertility. Although reliable data capable of reflecting the disease condition of infertility of couples of childbearing age in China are lacked at present, the disease has high disease rate, more influence people and wide range, which is undoubted. Human Assisted Reproductive Technology (ART) is the best method for treating infertility, and can effectively solve the fertility problem of most infertility patients. The currently used ART assisted pregnancy methods include In Vitro Fertilization-Embryo Transfer (IVF-ET), intracytoplasmic sperm injection (ICSI), Embryo freezing-Freezing Embryo Transfer (FET) matched with the IVF-ET, and Preimplantation Genetic Diagnosis (PGD), which have good and definite curative effects on many infertility patients, and IVF-ET, ICSI and FET are widely used clinically.
In Vitro Fertilization-Embryo Transfer (IVF-ET), commonly known as "tube baby", refers to taking out ovum and sperm, completing Fertilization and early development of pregnant ovum under artificial environment condition, moving the Embryo developed to a certain period back to mother uterus, and allowing it to grow to term for birth. Since the first "test tube" infant in the world, born in the uk on 1978, 7-25, IVF brought gospel to the world's barrenned couples. However, years of clinical practice have not significantly improved the success rate of IVF, and one of the important reasons is the quality of IVF embryo culture fluid, which is one of the most important determinants of IVF embryo quality.
In early embryo in vitro culture, Reactive Oxygen Species (ROS) are considered to be an important factor affecting embryo development. The early embryo is very sensitive to oxidative damage, the concentration of active oxygen in vitro culture is higher than that in vivo development environment, and the reduction of the concentration of active oxygen in the in vitro culture process of the early embryo can enable the in vitro culture to be closer to the oxygen environment for in vivo development of the embryo. Under normal physiological conditions, the development of in vivo embryos is regulated by various antioxidant substances, so that active oxygen reaches a balanced state, such as taurine, VC, superoxide dismutase, glutathione peroxidase, glutamylcysteine synthetase and the like. However, the negative effects caused by active oxygen are still difficult to overcome by a plurality of existing cleavage culture solutions, and in addition, the existing cleavage culture solutions in China mainly depend on import, and have the defects of high cost, long product transportation time, short service cycle and the like.
Therefore, a reliable solution is needed to solve the problems of the current cleavage culture solution.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a composition for preparing a cleavage culture solution, a cleavage culture solution and a preparation method thereof, aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: a composition for preparing a cleavage culture fluid comprising: acetyl L-carnitine and hyaluronic acid.
Acetyl L-carnitine is white powder, is easy to dissolve in water and is insoluble in acetone. Acetyl L-carnitine is a natural substance present in the body, and is particularly abundant in muscle, brain and testis; in muscle, 1/5, the L-carnitine reserve is present in the form of acetyl L-carnitine. Acetyl L-carnitine is an acetyl compound of L-carnitine and participates in a series of important metabolic processes in a human body; is a high-energy form of L-carnitine and plays a role in energy transfer in human bodies.
The germ cell membrane is particularly vulnerable to Reactive Oxygen Species (ROS) due to the rich polyunsaturated fatty acids, which causes embryo development defects and growth retardation, and damages cell membrane and DNA. ROS can interfere with the dynamic balance of calcium ions by directly influencing the storage of calcium ions in cells, so that meiosis of oocytes is stopped and degraded, apoptosis is generated, and adverse effects are caused on oocyte maturation and subsequent embryo development.
The balance between ROS and antioxidants has a major impact on oocyte maturation, fertilization and early embryo development. Acetyl L-carnitine is a powerful antioxidant, can effectively prevent mitochondrial damage and mitochondrial-dependent apoptosis caused by oxidative stress, and can scavenge free radicals, superoxide anions, hydrogen peroxide and the like. In the in vitro culture process, ROS can be generated in embryos and other external factors, and the generation of ROS is reduced by adding a free radical scavenger, namely acetyl-L-carnitine, so that the fertility potential of an assisted reproduction technology is improved. The acetyl L-carnitine can effectively promote the in vitro maturation and embryonic development of the ovum, the oocyte and the embryo provide essential auxiliary factors by utilizing fatty acid, and the output of the embryo can be improved when the acetyl L-carnitine is added into the in vitro maturation and early embryonic development of the oocyte.
Hyaluronic Acid (HA) is a physiological substance widely present in the human body, is a mucopolysaccharide secreted by mammalian granulosa cells, cumulus cells and epithelial cells, is distributed throughout connective tissues, and also HAs a high content in many organs and tissues such as skin, joint synovial fluid, vitreous eye, umbilical cord, and the like.
HA is widely present in the reproductive system of animals and plays an important role in reproductive reproduction of animals, and researches show that HA plays an important role in mouse blastocyst implantation, ectoderm proliferation and embryo structure differentiation and can promote proliferation and migration of cells. It is one of the most abundant GAGs in the uterus, oviduct and follicular fluid of animals and humans. Hyaluronic acid from cumulus cells, oviduct cells and oviduct fluid in vivo can support embryonic development to the blastocyst stage. GAGs have been shown to delay oocyte death and prevent oocyte breakage, with the most active factor being a substance closely related to HA. Hyaluronic acid is thought to play a role in cell migration early in embryonic development. In addition, HA HAs been shown to support the development of 1-cell and 2-cell embryos up to the blastocyst stage. Hyaluronic acid not only improves the physical properties of serum-free media, but also provides nutrients and energy for cell maturation and embryo development through catabolism.
Preferably, the concentration of the acetyl L-carnitine is 0.1-10 mu mol/L, and the concentration of the hyaluronic acid is 0.02-0.5 g/L.
Preferably, the concentration of the acetyl L-carnitine is 1-10 mu mol/L, and the concentration of the hyaluronic acid is 0.05-0.5 g/L.
Further preferably, the concentration of the acetyl L-carnitine is 5-10 mu mol/L, and the concentration of the hyaluronic acid is 0.05-0.2 g/L.
Still more preferably, the concentration of the acetyl L-carnitine is 5 or 10 mu mol/L, and the concentration of the hyaluronic acid is 0.1 g/L. The phenomenon of slow cell development, low embryo formation rate, poor morphology and the like in the embryo development process is caused by the over-low content of acetyl L-carnitine; if too high, the embryo will fail to divide normally and die. Too low a hyaluronic acid content leads to late cell lysis and too high a hyaluronic acid content leads to low embryo viability. Therefore, the configuration of the acetyl L-carnitine and the hyaluronic acid at proper concentration has important beneficial effect on the development of embryos.
The invention also provides an egg lysis culture solution, which comprises a base solution and the composition, wherein the base solution comprises: inorganic salt buffer solution, nonessential amino acid, ethylene diamine tetraacetic acid, sodium pyruvate, alanyl glutamine, taurine, glucose, sodium lactate, human serum albumin and bactericide.
Preferably, the inorganic salt buffer comprises one or more of sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, and sodium bicarbonate.
Preferably, the nonessential amino acid is one or more of L-alanine, L-glutamic acid, glycine, L-proline, L-serine, L-aspartic acid and L-asparagine.
Preferably, the bactericide is gentamicin.
Preferably, ultrapure water is used as a solvent for the cleavage culture solution.
Preferably, the pH value of the cleavage culture solution is 7.20-7.40, and the osmotic pressure is 260-290 mOsm/Kg.
Preferably, the cleavage culture solution comprises 80-100mmol/L of sodium chloride, 3.5-7.5mmol/L of potassium chloride, 0.2-2.0mmol/L of magnesium sulfate, 0.8-2.8mmol/L of calcium chloride, 0.05-1.5mmol/L of sodium dihydrogen phosphate, 15-30mmol/L of sodium bicarbonate, 0.1-1.0mmol/L of non-essential amino acid, 0.005-0.20mmol/L of ethylene diamine tetraacetic acid, 0.1-1.0mmol/L of sodium pyruvate, 0.1-1.0mmol/L of alanylglutamine, 0.01-10.0mmol/L of taurine, 0.05-5.0mmol/L of glucose, 5.0-20.0mmol/L of sodium lactate, 1-10.0g/L of human serum albumin and 9.5-10.5 mu g/L of gentamicin.
Preferably, the preparation method of the cleavage culture solution comprises the following steps:
1) preparing a base liquid:
1-1) weighing each raw material of the base liquid according to the proportion, and separately placing;
1-2) dissolving sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, non-essential amino acids, ethylenediamine tetraacetic acid, sodium pyruvate, alanyl glutamine, taurine, glucose and sodium lactate in ultrapure water;
1-3) adding gentamicin, sodium bicarbonate, acetyl L-carnitine and hyaluronic acid into the solution obtained in the step 1-2) to obtain a base solution;
1-4) placing the base liquid obtained in the step 1-3) at 37 ℃ with 5% CO2Balancing in an incubator for 6-12 hours;
2) preparing a cleavage culture solution stock solution:
2-1) filtering and sterilizing the basic solution obtained in the step 1-4) by using a filter membrane of 0.22 mu m under an ultra-clean workbench to obtain a filtrate A;
2-2) adding human serum albumin into the filtrate A, and shaking or stirring and mixing uniformly to obtain a cleavage culture solution stock solution; or omitting the step, taking the filtrate A as the stock solution of the cleavage culture solution, and adding human serum albumin according to the requirement when in use;
3) subpackaging and sealing the raw liquid of the cleavage culture solution prepared in the step 2) in an aseptic environment to obtain the cleavage culture solution, and storing the cleavage culture solution in a refrigerator or a cold storage;
4) detecting a cleavage culture solution:
and (3) detecting the packed cleavage culture solution in the step 3), and judging whether the cleavage culture solution is qualified, wherein the detection items comprise pH value, osmotic pressure, cytotoxicity, intradermal stimulation, sensitization, sterility, pyrogen, bacterial endotoxin and in-vitro rat embryo test.
The invention has the beneficial effects that:
according to the cleavage culture solution provided by the invention, the active oxygen concentration in the in-vitro culture process of the early embryo can be reduced by adding acetyl L-carnitine, so that the in-vitro culture is closer to the oxygen environment for in-vivo development of the embryo, and the development and viability of the embryo are improved; by adding hyaluronic acid into the culture solution, nutrition and energy are provided for cell maturation and embryo development through degradation and metabolism;
the acetyl L-carnitine and the hyaluronic acid are added into the cleavage culture solution as the composition, so that the acetyl L-carnitine and the hyaluronic acid can play a complementary synergistic enhancement effect, have the effects of clearing oxygen radicals in vivo to play an antioxidation role, protecting mitochondrial functions, regulating energy metabolism, providing nutrition and energy for cell maturation and embryo development through degradation and metabolism and the like, have a higher ratio of blastocysts than that of the blastocysts obtained by singly using any one of the compositions, and have a higher form and score of the blastocysts;
the cleavage culture solution provided by the invention is expected to solve the problem that embryo culture reagents in assisted reproduction in China are imported for a long time at present, and can overcome the defect that the quality of the cleavage culture solution is reduced due to long-time transportation of the reagents imported from abroad.
Drawings
FIG. 1 is a schematic diagram of blastocysts cultured with the culture solution obtained in example 1;
FIG. 2 is a schematic diagram of blastocysts cultured with the culture solution obtained in example 2;
FIG. 3 is a schematic diagram of blastocysts cultured with the culture solution prepared in comparative example 1;
FIG. 4 is a schematic diagram of blastocysts cultured with the culture solution prepared in comparative example 2;
FIG. 5 is a schematic diagram of blastocysts cultured with the culture solution prepared in comparative example 3;
FIG. 6 is a schematic diagram of blastocysts cultured with the culture solution prepared in comparative example 4.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description. The experimental methods without specific conditions noted in the examples are generally performed according to conventional conditions, and the examples are given for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1
An egg lysis culture solution comprises the following raw material components:
the preparation method of the cleavage culture solution comprises the following steps:
1) preparing a base liquid:
1-1) weighing each raw material of the base liquid according to the proportion, and separately placing;
1-2) dissolving sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, non-essential amino acids, ethylenediamine tetraacetic acid, sodium pyruvate, alanyl glutamine, taurine, glucose and sodium lactate in ultrapure water;
1-3) adding gentamicin, sodium bicarbonate, acetyl L-carnitine and hyaluronic acid into the solution obtained in the step 1-2) to obtain a base solution;
1-4) placing the base liquid obtained in the step 1-3) at 37 ℃ with 5% CO2Balancing in an incubator for 6-12 hours;
2) preparing a cleavage culture solution stock solution:
2-1) filtering and sterilizing the basic solution obtained in the step 1-4) by using a filter membrane of 0.22 mu m under an ultra-clean workbench to obtain a filtrate A;
2-2) adding human serum albumin into the filtrate A, and shaking or stirring and mixing uniformly to obtain a cleavage culture solution stock solution;
3) subpackaging and sealing the stock solution of the cleavage culture solution prepared in the step 2) in an aseptic environment to obtain the cleavage culture solution, and storing the cleavage culture solution in a refrigerator or a cold storage.
Example 2
An egg cleavage culture solution which differs from example 1 only in that: in this example, the concentration of acetyl L-carnitine was 10. mu. mol/L.
The preparation method is the same as that of example 1.
Comparative example 1
An egg cleavage culture solution which differs from example 1 only in that: the concentration of acetyl L-carnitine in this example was 0.1. mu. mol/L.
The preparation method is the same as that of example 1.
Comparative example 2
An egg cleavage culture solution which differs from example 1 only in that: acetyl L-carnitine and hyaluronic acid were not added in this example.
The preparation method is the same as that of example 1.
Comparative example 3
An egg cleavage culture solution which differs from example 1 only in that: in this example, acetyl-L-carnitine was not added.
The preparation method is the same as that of example 1.
Comparative example 4
An egg cleavage culture solution which differs from example 1 only in that: in this example, hyaluronic acid was not added.
The preparation method is the same as that of example 1.
Example 3
The cleavage culture solutions prepared in examples 1-2 and comparative examples 1-4 were tested, and the test items included pH, osmotic pressure, cytotoxicity, intradermal stimulation, sensitization, sterility, pyrogen, bacterial endotoxin, and in vitro rat embryo test, and the test results were used to determine whether the cleavage culture solution was acceptable.
(I) pH value detection
And (3) taking a certain amount of finished products, detecting and recording the value of the finished products for three times by using a pH meter, and taking the average value as the pH value of the final liquid, wherein the final liquid is qualified in the range.
Osmotic pressure (II)
Taking 600 mu L of liquid, dripping the liquid into a sample tube of an osmometer (freezing point osmometer), pushing a probe into the sample tube to wait for the stability of a detection value, reading out a numerical value, detecting for three times by the same method, taking an average value, namely the osmotic pressure of the liquid, and determining the liquid to be qualified in a range.
(III) cytotoxicity
The cytotoxicity score should not exceed 1 point, as specified in GB/T16886.5.
(IV) intradermal stimulation
The method is carried out according to the regulation of GB/T16886.10, and has no subcutaneous stimulation response.
(V) sensitization
The method is carried out according to the regulation of GB/T16886.10, and sensitization reaction is avoided.
(VI) sterility testing
The membrane filtration method of sterility test in Chinese pharmacopoeia is adopted, and the filtered filter membrane is cultured in a culture medium of bacteria and fungi, and the sterile growth is qualified.
(VII) pyrogen
The in vitro receptor should be pyrogen-free according to the method specified in appendix XI in the second part of the pharmacopoeia of the people's republic of China, 2010 edition.
(VIII) detection of bacterial endotoxins
Adopting a limulus reagent gel method in the detection method of the endotoxin in Chinese pharmacopoeia, judging that the content is less than 1.0EU/mL and the product is qualified;
(nine) in vitro mouse embryo assay
1. Superovulation
Selecting 6-8 weeks old female mice, and injecting 10 IU/female mouse by abdominal cavity; after 48h, hCG10 IU/mouse was injected intraperitoneally, and female mice and male mice of the same strain were housed overnight on the same day after hCG injection.
2. Preparing culture dish
Before culturing embryos, preparing a certain amount of liquid microdroplets with the size of 30-50 mu L in a cell culture dish, covering the surface with culture oil, and adding CO2Pre-balancing the inside of the incubator for 4-18 hours.
3. Collection of mouse embryos
And checking the mating condition in the morning of the next day of cage combination, and selecting the mice with emboli for later use.
1-cell embryo collection: the female embolus was sacrificed 18 to 22 hours after hCG injection and 1-cell embryos were collected at the ampulla of the oviduct. And placing the collected flocculent fertilized egg masses into hyaluronidase preheated at 37 ℃, immediately transferring and cleaning cumulus and granular cells around the embryos after digestion and separation, selecting fertilized embryos in normal forms, transferring the fertilized embryos into test article cleavage culture solution droplets, and using the fertilized embryos for a 1-cell mouse embryo detection test.
4. In vitro culture
Culturing by microdroplet method, randomly dividing collected mouse embryo into a positive control group, a negative control group and a test sample group, placing in balanced culture solution, and culturing at 37 deg.C with 5% CO2And culturing in an incubator with saturated humidity. The number of mouse embryos in each group is not less than 100.
5. Test results
1-cell embryos the number of blastocysts was recorded after 96 hours of in vitro culture.
Observation indexes are as follows: and (5) observing the morphology of the blastula.
Acceptance criteria:
1) the blastocyst formation rate of the positive control group is remarkably lower than that of the negative control group through statistical analysis;
2) the blastocyst formation rate of the negative control group is more than or equal to 80 percent.
The detection results of the items are as follows:
example 4
100 fertilized eggs were cultured using the culture solutions of examples 1 to 2 and comparative examples 1 to 4, and blastocyst formation results were recorded as follows:
1. fertilized eggs were cultured in the culture solution prepared in example 1, and the results were observed, and the following three sets of experimental data were repeated:
| number of fertilized eggs | Number of blastocysts formed |
| 100 | 90 |
| 100 | 91 |
| 100 | 93 |
2. Fertilized eggs were cultured in the culture solution prepared in example 2, and the results were observed, and the following three sets of experimental data were repeated:
| number of fertilized eggs | Number of blastocysts formed |
| 100 | 97 |
| 100 | 97 |
| 100 | 96 |
3. Fertilized eggs were cultured in the culture solution prepared in comparative example 1, and the results were observed, and the following three sets of experimental data were repeated:
| number of fertilized eggs | Number of blastocysts formed |
| 100 | 82 |
| 100 | 84 |
| 100 | 85 |
4. Comparative example 2 fertilized eggs were cultured in a culture solution prepared without adding the key material A, B, and the results were observed as three sets of data of repeated experiments as follows:
| number of fertilized eggs | Number of blastocysts formed |
| 100 | 83 |
| 100 | 85 |
| 100 | 82 |
5. Comparative example 3 fertilized eggs were cultured without the culture solution prepared from the key material a, and the results were observed as three sets of repeated experimental data:
6. comparative example 4 fertilized eggs were cultured without the culture solution prepared from the key material B, and the results were observed as three sets of repeated experimental data:
| number of fertilized eggs | Number of blastocysts formed |
| 100 | 88 |
| 100 | 86 |
| 100 | 86 |
According to the experimental results, the influence of the key materials A and B on the development of embryos is large in the culture process of the culture solution, particularly the content of the key material A is directly related to the blastocyst formation rate, and each material is linearly related in a certain range; the culture solution without the key materials A and B has low blastocyst rate, and the key materials A and B have obvious physiological effect on the formation of the blastocyst. The blastocyst forming rate of the cleavage culture solution is high and can reach 97%.
Referring to FIG. 1, blastocysts cultured in the culture medium (A: 5. mu. mol/L, B: 0.1g/L) obtained in example 1 were: most of the blastocysts have good development, morphological analysis shows that the grading level is higher, the blastocyst cavities are completely filled with embryos, the total volume of the embryos is increased, the zona pellucida is thinned, the number of inner cell masses is large, and the arrangement is compact; trophoblasts are high in number and form a tightly packed epithelial cell layer.
Referring to FIG. 2, blastocysts cultured in the culture medium (A: 10. mu. mol/L, B: 0.1g/L) obtained in example 2 were: the embryos basically develop to blastula, and individual embryos are in the blastula expansion stage, so that the total volume of the embryos is increased, the zona pellucida is thinned, the number of inner cell masses is large, and the arrangement is compact; trophoblasts are high in number and form a tightly packed epithelial cell layer.
Referring to FIG. 3, there is a blastocyst cultured with the culture solution (A: 0.1. mu. mol/L, B: 0.1g/L) prepared in comparative example 1, wherein: the blastula rate is 86.67%, the development of a few embryos is slow, the blastula cavity volume is smaller than 1/2 of the total volume of the embryos, and the embryo score is low.
Referring to FIG. 4, there is shown a blastocyst cultured in the culture solution (without addition of A and B) prepared in comparative example 2, wherein: the blastula rate is 80%, a few embryos are stunted, and the embryo score is low.
Referring to FIG. 5, blastocysts cultured in the culture solution prepared in comparative example 3 (without addition of A) were observed: most embryos developed better with higher scores. A few embryos develop slowly and fail to form dilated blastocysts.
Referring to FIG. 6, there is a blastocyst cultured with the culture solution (without B) prepared in comparative example 4, wherein: 86.67% of the embryos developed well, with a few embryos developing slowly.
The results show that the key materials A (acetyl L-carnitine) and B (hyaluronic acid) have important effects on embryonic development, and the key materials A (acetyl L-carnitine) and B (hyaluronic acid) can play complementary synergistic enhancement effects.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (10)
1. A composition for preparing a cleavage culture fluid, comprising: acetyl L-carnitine and hyaluronic acid.
2. The composition for preparing a cleavage culture solution of claim 1, wherein the concentration of the acetyl L-carnitine is 0.1-10 μmol/L, and the concentration of the hyaluronic acid is 0.02-0.5 g/L.
3. The composition for preparing a cleavage culture solution of claim 2, wherein the concentration of the acetyl L-carnitine is 1-10 μmol/L and the concentration of hyaluronic acid is 0.05-0.5 g/L.
4. An egg lysis solution comprising a base solution and a composition according to any one of claims 1 to 3, said base solution comprising: inorganic salt buffer solution, nonessential amino acid, ethylene diamine tetraacetic acid, sodium pyruvate, alanyl glutamine, taurine, glucose, sodium lactate, human serum albumin and bactericide.
5. The cleavage culture fluid of claim 4, wherein the inorganic salt buffer comprises one or more of sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, and sodium bicarbonate.
6. The cleavage culture fluid of claim 5, wherein the non-essential amino acid is one or more of L-alanine, L-glutamic acid, glycine, L-proline, L-serine, L-aspartic acid, and L-asparagine.
7. The cleavage culture fluid of claim 6, wherein the bactericide is gentamicin.
8. The cleavage culture solution as claimed in claim 7, wherein the pH of the cleavage culture solution is 7.20-7.40, and the osmotic pressure is 260-290 mOsm/Kg.
9. The cleavage culture solution of claim 8, the cleavage culture solution comprises 80-100mmol/L of sodium chloride, 3.5-7.5mmol/L of potassium chloride, 0.2-2.0mmol/L of magnesium sulfate, 0.8-2.8mmol/L of calcium chloride, 0.05-1.5mmol/L of sodium dihydrogen phosphate, 15-30mmol/L of sodium bicarbonate, 0.1-1.0mmol/L of non-essential amino acid, 0.005-0.20mmol/L of ethylene diamine tetraacetic acid, 0.1-1.0mmol/L of sodium pyruvate, 0.1-1.0mmol/L of alanylglutamine, 0.01-10.0mmol/L of taurine, 0.05-5.0mmol/L of glucose, 5.0-20.0mmol/L of sodium lactate, 1-10.0g/L of human serum albumin and 9.5-10.5 mu g/L of gentamycin.
10. The cleavage culture solution of any one of claims 7 to 9, wherein the preparation method of the cleavage culture solution comprises the following steps:
1) preparing a base liquid:
1-1) weighing each raw material of the base liquid according to the proportion, and separately placing;
1-2) dissolving sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, non-essential amino acids, ethylenediamine tetraacetic acid, sodium pyruvate, alanyl glutamine, taurine, glucose and sodium lactate in ultrapure water;
1-3) adding gentamicin, sodium bicarbonate, acetyl L-carnitine and hyaluronic acid into the solution obtained in the step 1-2) to obtain a base solution;
1-4) placing the base liquid obtained in the step 1-3) at 37 ℃ with 5% CO2Balancing in an incubator for 6-12 hours;
2) preparing a cleavage culture solution stock solution:
2-1) filtering and sterilizing the basic solution obtained in the step 1-4) by using a filter membrane of 0.22 mu m under an ultra-clean workbench to obtain a filtrate A;
2-2) adding human serum albumin into the filtrate A, and shaking or stirring and mixing uniformly to obtain a cleavage culture solution stock solution;
3) subpackaging and sealing the raw liquid of the cleavage culture solution prepared in the step 2) in an aseptic environment to obtain the cleavage culture solution, and storing the cleavage culture solution in a refrigerator or a cold storage;
4) detecting a cleavage culture solution:
and (3) detecting the packed cleavage culture solution in the step 3), and judging whether the cleavage culture solution is qualified, wherein the detection items comprise pH value, osmotic pressure, cytotoxicity, intradermal stimulation, sensitization, sterility, pyrogen, bacterial endotoxin and in-vitro rat embryo test.
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