CN116240164A - One-step culture solution - Google Patents

One-step culture solution Download PDF

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CN116240164A
CN116240164A CN202310019081.9A CN202310019081A CN116240164A CN 116240164 A CN116240164 A CN 116240164A CN 202310019081 A CN202310019081 A CN 202310019081A CN 116240164 A CN116240164 A CN 116240164A
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culture solution
step culture
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董亮亮
郭常亮
杨琴
胡文平
邓康为
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Jinbao Medical Technology Shenzhen Co ltd
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Abstract

A one-step culture solution is prepared from physiological salt, energy substance, energy matrix, D-calcium pantothenate, recombinant human insulin and ethylenediamine tetraacetic acid. The energy substance contains D-glucose, L-calcium lactate and sodium pyruvate. The physiological salt contains sodium chloride, potassium chloride, monopotassium phosphate, magnesium sulfate heptahydrate and calcium chloride. The one-step culture solution can remove harmful cytokines and metabolic wastes secreted by cells in the culture process, and can retain the cytokines beneficial to the culture in the one-step culture solution, and meanwhile, the one-step culture solution can effectively reduce manual operation and reduce mechanical damage and stress reaction, so that the one-step culture solution has higher blastula rate and transplanting success rate. The one-step culture solution can reduce the variety of the culture solution and reduce the cost of in vitro fertilization-embryo transfer.

Description

One-step culture solution
Technical Field
The invention relates to the technical field of auxiliary reproduction, in particular to a one-step culture solution.
Background
Assisted reproductive technologies are short for human assisted reproductive technologies (Assisted Reproductive Technology, ART) and include two broad categories, artificial insemination and in vitro fertilization-embryo transfer and derivatives thereof. In the auxiliary reproduction process, the nutrient requirements corresponding to each reproduction culture stage are different, and at present, more than ten liquid such as egg taking liquid culture solution, fertilization liquid culture solution, embryo culture solution and the like exist, and the substances in the culture solution are very various and have complex proportion. In addition, only a little culture liquid is needed for culturing each embryo transfer operation, however, most culture liquids provided by suppliers for saving cost are large-sized culture liquids, and the validity period of the culture liquid is very short (3 months-6 months are different). Then multiple large format culture solutions would need to be purchased for each full process embryo culture, which adds significant cost to the embryo transfer process.
In the culture process of each stage of auxiliary reproduction, cells can be continuously exchanged with a culture solution, absorb energy and discharge cytokines and metabolic wastes, ethical restrictions and current technical level restrictions exist for human embryo research, and the components of the cytokines secreted by the embryo in the culture process and the mechanism of the cytokines secreted by the embryo cannot be completely understood. When the culture solution is frequently replaced, not only the embryo is damaged, but also some beneficial cytokines are replaced together, so that the blastocyst rate and the transplanting success rate of the embryo are reduced finally.
So that the embryo is cultured at different stages at present, and the corresponding culture solution still needs to be replaced. For example, CN103173403a, a chinese patent of a branched embryo culture solution and a method for preparing the same, in which different culture solution components are required to be provided at early and later stages of embryo, and the problem of low blastocyst formation rate caused by mechanical damage possibly received during embryo transfer and stress reaction caused by different culture environments is increased during operation.
If all the nutrients required during the whole culture phase are mixed directly, this in turn affects the pH value, osmotic pressure and the culture effect of the culture broth. At present, some culture solutions suitable for several stages are disclosed, such as CN108251354, one-step embryo culture solution and Chinese patent invention patent publication No. CN108251354, and the one-step embryo culture solution is still essentially required to be egg taking solution, semen receiving solution, one-step embryo culture solution and transplanting solution in the embryo culture process. Since conventional embryo culture solutions are not suitable for fertilization of sperm and ovum, semen-receiving liquid is usually required to be additionally prepared, IVF success rate and conception rate are not high, and the reason is many, wherein the reason is that the culture solution used by IVF damages embryos more or less. As can be seen, there is no one-step embryo culture that truly enables the culture of fertilized to embryo stages.
Therefore, aiming at the defects of the prior art, the one-step culture solution is provided to overcome the defects of the prior art.
Disclosure of Invention
The invention aims to avoid the defects of the prior art and provide a one-step culture solution which can culture embryos in the whole auxiliary reproduction process.
The above object of the present invention is achieved by the following technical measures:
a one-step culture medium is provided, which comprises physiological salt, energy substance, energy matrix, D-calcium pantothenate, recombinant human insulin and ethylenediamine tetraacetic acid.
Preferably, the energy matrix comprises glycine, L-asparagine monohydrate, L-aspartic acid, L-glutamic acid, L-proline, L-serine, alanylglutamine, L-tyrosine, L-arginine hydrochloride, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan, L-valine, L-cystine dihydrochloride, L-threonine, L-lysine hydrochloride.
Preferably, the energy substance contains D-glucose, L-calcium lactate and sodium pyruvate.
The one-step culture solution also comprises a bactericide, a pH indicator and a pH buffer.
Preferably, the physiological salt contains sodium chloride, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and calcium chloride.
The concentration ratio of the raw materials of the one-step culture solution is as follows:
physiological salt: 88.00 to 105.50mmol/L of sodium chloride, 2.00 to 3.30mmol/L of potassium chloride, 0.20 to 0.40mmol/L of monopotassium phosphate and 0.15 to 0.32mmol/L of magnesium sulfate;
energy substance: 0.45-0.75 mmol/L of D-glucose, 0.15-0.30 g/L of L, L-calcium lactate and 0.30-0.45 mmol/L of sodium pyruvate;
energy matrix: 0.65 g/L-0.85 g/L-1.20 g/L-1.65 g/L-1.80 g/L-6.80 g/L-L, L-histidine hydrochloride monohydrate 1.50 g/L-2.50 g/L-1.30 g/L-2.85 g/L-L, L-leucine 2.20 g/L-2.95 g/L, L-methionine 0.60 g/L-0.85 g/L-1.20 g/L, alanyl glutamine 0.80 mmol/L-1.20 mmol/L-L, L-tyrosine 1.50 g/L-1.98 g/L, L-arginine hydrochloride 5.80 g/L-6.80 g/L-L, L-histidine hydrochloride monohydrate 1.50 g/L-2.50 g/L-L, L-isoleucine 2.30 g/L-2.85 g/L-L, L-leucine 2.20 g/L-2.95 g/L, L-methionine 0.60 g/L-0.85 g-37.38-phenylalanine hydrochloride 1.80 g-37.80 g-1.80 g/L-1.98 g/L-37.80 g-6.80 g/L-37.92-histidine hydrochloride;
calcium D-pantothenate: 5.00 mu g/L-15.00 mu g/L;
recombinant human insulin: 5.00 mu g/L to 50.00 mu g/L;
ethylenediamine tetraacetic acid: 0.005mmol/L to 0.020mmol/L;
a bactericide: 6.0mg/L to 15.0mg/L;
pH indicator: 0.0005g/L to 0.0200g/L;
pH buffer: 22.00 mmol/L-30.00 mmol/L.
The concentration ratio of the raw materials of the one-step culture solution is as follows:
physiological salt: 91.20 to 100.70mmol/L of sodium chloride, 2.37 to 3.02mmol/L of potassium chloride, 0.30 to 0.36mmol/L of monopotassium phosphate and 0.18 to 0.24mmol/L of magnesium sulfate;
energy substance: 0.51-0.68 mmol/L of D-glucose, 0.19-0.24 g/L of L, L-calcium lactate and 0.35-0.41 mmol/L of sodium pyruvate;
energy matrix: glycine 0.71 g/L-0.79 g/L, L-asparagine monohydrate 1.43 g/L-1.58 g/L, L-aspartic acid 1.26 g/L-1.40 g/L, L-glutamic acid 1.40 g/L-1.54 g/L, L-proline 1.09 g/L-1.21 g/L, L-serine 1.00 g/L-1.10 g/L0.9 to 1.1mmol/L alanyl glutamine 1.71 to 1.89g/L L, L-tyrosine 1.48 g/L L, L-arginine hydrochloride 6.00g/L to 6.64g/L, L-histidine hydrochloride monohydrate 2.00g/L to 2.21g/L, L-isoleucine 2.49g/L to 2.76g/L L, L-leucine 2.49g/L to 2.75g/L, L-methionine 0.72g/L to 0.79g/L L, L-phenylalanine 1.57g/L to 1.73g/L, L-tryptophan 0.48g/L to 0.54g/L L, L-valine 2.22g/L to 2.46g/L, L-cystine dihydrochloride 1.49g/L to 1.64g/L, L-threonine 2.26g/L to 2.50g/L, L-lysine hydrochloride 2.22g/L to 2.46g/L;
calcium D-pantothenate: 8.00 mu g/L to 12.00 mu g/L;
recombinant human insulin: 10.00 mu g/L to 25.00 mu g/L;
ethylenediamine tetraacetic acid: 0.012mmol/L to 0.014mmol/L;
a bactericide: 9.50mg/L to 10.50mg/L;
pH indicator: 0.0010g/L to 0.0100g/L;
pH buffer: 26.30 mmol/L-28.25 mmol/L.
The one-step culture solution also contains human serum albumin.
The concentration ratio is 0.01mL/L to 15.00mL/L of human serum albumin.
Further according to concentration ratio, the human serum albumin is 0.5mL/L
10.20mL/L。
Preferably, the bactericide is gentamicin sulfate.
Preferably, the pH indicator is phenol red sodium salt.
The one-step culture solution of the invention comprises physiological salt, energy substances, energy matrix, D-calcium pantothenate, recombinant human insulin and ethylenediamine tetraacetic acid. The energy matrix comprises glycine, L-asparagine monohydrate, L-aspartic acid, L-glutamic acid, L-proline, L-serine, alanylglutamine, L-tyrosine, L-arginine hydrochloride, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan, L-valine, L-cystine dihydrochloride, L-threonine, L-lysine hydrochloride; the energy substance contains D-glucose, L-calcium lactate and sodium pyruvate. The physiological salt contains sodium chloride, potassium chloride, monopotassium phosphate, magnesium sulfate heptahydrate and calcium chloride. The one-step culture solution can remove harmful cytokines and metabolic wastes secreted by cells in the culture process, and can retain the cytokines beneficial to the culture in the one-step culture solution, and meanwhile, the one-step culture solution can effectively reduce manual operation and reduce mechanical damage and stress reaction, so that the one-step culture solution has higher blastula rate and transplanting success rate. The one-step culture solution can culture embryos in the whole auxiliary reproduction process, which is equivalent to the egg taking solution, the semen receiving solution, the early-stage embryo culture solution and the later-stage embryo culture solution in the prior art, or the egg taking solution, the semen receiving solution and the traditional one-step embryo culture solution, so that the variety of the culture solution can be reduced, and the cost of in vitro fertilization-embryo transplantation can be reduced.
Drawings
The invention is further illustrated by the accompanying drawings, which are not to be construed as limiting the invention in any way.
FIG. 1 is a photograph of blastocysts cultured using the one-step culture broth of sample 8.
FIG. 2 is a photograph of blastocysts cultured using the one-step culture solution of comparative example 1.
Detailed Description
The technical scheme of the present invention is further described with reference to the following examples, but the present invention is not limited in any way by the examples. Unless otherwise indicated, all starting reagents used in the examples of the present invention were conventionally purchased.
Example 1
A one-step culture solution comprises physiological salt, energy substance, energy matrix, hyaluronic acid, D-calcium pantothenate, recombinant human insulin, ethylenediamine tetraacetic acid, bactericide, pH indicator and pH buffer.
Wherein the physiological salt contains sodium chloride, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and calcium chloride. The physiological salt of the present invention is used to ensure the osmotic pressure of the culture solution, and the energy substance is used to supply energy to the various stages of culture.
Wherein the energy substance comprises D-glucose, L-calcium lactate and sodium pyruvate. It should be noted that, the nutrients required for different stages of embryo are different, for example, the main energy sources in the early stage of embryo are pyruvic acid and lactic acid, and the main energy source in the later stage is glucose.
Wherein the energy matrix comprises glycine, L-asparagine monohydrate, L-aspartic acid, L-glutamic acid, L-proline, L-serine, alanylglutamine, L-tyrosine, L-arginine hydrochloride, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan, L-valine, L-cystine dihydrochloride, L-threonine, L-lysine hydrochloride.
It should be noted that only a small amount of essential amino acids are required in the cleavage stage, whereas more essential amino acids and non-essential amino acids are required in the blastocyst stage. The absence of amino acids in the energy matrix described above in embryo parts such as cleavage stages is culturable, but the presence of the energy matrix does not have an effect on cell development in these stages; but are indispensable in the later stages (such as blastula stage) and affect the development of the embryo and even cause the embryo to stop developing.
Wherein the bactericide is gentamicin sulfate, and the pH indicator is phenol red sodium salt.
The pH value of the culture solution is monitored in real time under the action of the pH indicator, so that the abnormality of the pH value of the culture solution can be found early.
The effect of the D-calcium pantothenate, the recombinant human insulin and the ethylenediamine tetraacetic acid is to remove harmful substances secreted by embryo cells, so that the culture solution can be kept to meet the requirements of embryo production environment for a long time.
The embryo cells can continuously exchange substances with the culture solution in the culture process, absorb energy, discharge cytokines and metabolic waste, wherein the metabolic waste contains heavy metals or ammonium decomposed by amino acids, the components of the cytokines are not clear based on the prior study, some cytokines and metabolic waste can generate toxicity to the embryo cells and are harmful to the cells, and some substances are beneficial to the further growth of the cells. The D-calcium pantothenate, the recombinant human insulin and the ethylenediamine tetraacetic acid have the effects of removing harmful cytokines and metabolic wastes and retaining favorable substances.
The one-step culture solution in the invention can also be added with human serum albumin, and the function of the human serum albumin is to keep the solution stable and protect cells.
The raw material dosage formulation table of various samples of the one-step culture solution of the invention is shown in table 1:
TABLE 1 dosage formulation of one-step culture fluid samples of the invention
Figure SMS_1
Figure SMS_2
The invention relates to a preparation method of a one-step culture solution, which comprises the following steps:
firstly, adding physiological salt into water for injection, and then adding a pH buffer, an energy substance, an energy matrix, human serum albumin, a bactericide, hyaluronic acid, a pH indicator and a pH buffer; finally, adding D-calcium pantothenate, recombinant human insulin and ethylenediamine tetraacetic acid to obtain a primary culture solution;
and (2) filtering and sterilizing the primary culture solution obtained in the step (1) through a filter membrane to obtain a one-step culture solution. Wherein the filter membrane is a 0.2 μm filter membrane.
The one-step culture solution can remove harmful cytokines and metabolic wastes secreted by cells in the culture process, and can keep the beneficial cytokines in the one-step culture solution for culture, and meanwhile, the one-step culture solution can effectively reduce manual operation and mechanical damage and stress reaction, so that the one-step culture solution has higher blastula rate and transplanting success rate. The one-step culture solution can culture embryos in the whole auxiliary reproduction process, which is equivalent to the egg taking solution, the semen receiving solution, the early-stage embryo culture solution and the later-stage embryo culture solution in the prior art, or the egg taking solution, the semen receiving solution and the traditional one-step embryo culture solution, so that the variety of the culture solution can be reduced, and the cost of in vitro fertilization-embryo transplantation can be reduced.
Comparative example 1
The culture solution, other raw materials and the mixture ratio are the same as those of the sample 8, except that D-calcium pantothenate, recombinant human insulin and ethylenediamine tetraacetic acid are not added.
The method comprises the following steps:
firstly, adding physiological salt into water for injection, and then adding a pH buffer, an energy substance, an energy matrix, a bactericide, hyaluronic acid, a pH indicator and a pH buffer to obtain a primary culture solution;
and (2) filtering and sterilizing the primary culture solution obtained in the step (1) through a 0.2 mu m filter membrane to obtain the culture solution of the comparative example 1.
Comparative example 2
The culture solution, other raw materials and the mixture ratio are the same as those of sample 8, except that D-calcium pantothenate and ethylenediamine tetraacetic acid are not added.
The method comprises the following steps:
firstly, adding physiological salt into water for injection, then adding a pH buffer, an energy substance, an energy matrix, a bactericide, hyaluronic acid, a pH indicator and a pH buffer, and finally adding recombinant human insulin to obtain a primary culture solution;
and (2) filtering and sterilizing the primary culture solution obtained in the step (1) through a 0.2 mu m filter membrane to obtain the culture solution of the comparative example 2.
Comparative example 3
The culture solution, other raw materials and the mixture ratio are the same as those of sample 8, except that alanylglutamine, L-phenylalanine and L-cystine dihydrochloride are not added.
The method comprises the following steps:
firstly, adding physiological salt into water for injection, and then adding a pH buffer, an energy substance, an energy matrix, human serum albumin, a bactericide, hyaluronic acid, a pH indicator and a pH buffer; finally, adding D-calcium pantothenate, recombinant human insulin and ethylenediamine tetraacetic acid to obtain a primary culture solution;
and (2) filtering and sterilizing the primary culture solution obtained in the step (1) through a filter membrane to obtain the culture solution of the comparative example 3.
Detection, experiment and results
1. Culture fluid detection
The one-step culture solutions prepared by proportioning the raw materials of samples 1 to 8 in table 1 of example 1 and the culture solutions of comparative examples 1 to 3 were subjected to pH, osmotic pressure and endotoxin detection according to the measurement method of the chinese pharmacopoeia 2020.
2. Animal experiment
Animal experiments were carried out using the one-step culture solutions obtained by proportioning the raw materials of samples 1 to 8 in table 1 of example 1 and the culture solutions of comparative examples 1 to 3 according to the in vitro mouse embryo test of the medical device of the industry standard YY/T1434-2016 human in vitro assisted reproduction technology. Specifically, 3-4 weeks C57/6J female mice are superarranged, ovum and sperm are taken, the culture solution obtained by mixing the raw materials of samples 1-8 in table 1 is fertilized in the culture solution of comparative examples 1-3, then the culture is carried out, when the culture is carried out to two cells, 50 embryos are taken for transplantation, and the remaining 50 embryos are continuously cultured in vitro for 5 days, and then the blastula ratio is calculated.
3. Detection and experimental results
Table 2 shows the pH, osmotic pressure, endotoxin detection results and animal test results of the one-step culture solutions obtained according to the raw material ratios of samples 1 to 8 and the culture solutions of comparative examples 1 to 3, as follows:
TABLE 2 pH, osmotic pressure and endotoxin test results and animal test results
Figure SMS_3
Figure SMS_4
Remarks: in the calculation of blastocyst rate and graft success rate, the total number of fertilized eggs used for each sample was 50, respectively.
From Table 2, it is understood that the one-step culture solution using physiological salt, energy substance, energy matrix, hyaluronic acid, D-calcium pantothenate, recombinant human insulin and ethylenediamine tetraacetic acid as the main raw materials of the present invention has a blastocyst rate of more than 80% for mice, and a graft success rate of more than 38%. The results of the one-step culture solution of sample 8 were the highest in blastula rate and graft success rate, 84% and 42%. The one-step culture broth of sample 5, to which no human serum albumin was added, had a blastocyst rate of 80% and a grafting success rate of 38%, was relatively low compared to the other one-step culture broth of the present invention, but still much higher than the comparative example. The blastocyst rates and the implantation success rates of samples 1 to 8 were far higher than those of comparative examples 1 and 2 without the addition of D-calcium pantothenate, recombinant human insulin or ethylenediamine tetraacetic acid. For comparative example 3, in which alanylglutamine, L-phenylalanine and L-cystine dihydrochloride were not added, the blastula rate and the success rate of transplantation were the lowest.
4. Blastula state
Under otherwise identical conditions, microphotographs were performed on blasts cultured using the one-step culture broth of sample 8 and blasts cultured using the culture broth of comparative example 1. As can be seen from fig. 1 and 2, the blastula state of sample 8 is significantly better than that of comparative example 1.
In summary, the one-step culture solution of the invention contains energy and nutrient substances required by the whole culture process from fertilized eggs to embryos through the selection of various raw material compositions and corresponding dosage, meanwhile, the pH value is kept between 7.20 and 7.40, the osmotic pressure is within the range of 260-290 mOsm/Kg, and the endotoxin detection result is less than 15EU/mL. The one-step culture solution has higher blastula rate and transplanting success rate and better embryo culture effect, so that the substances of the raw materials in the one-step culture solution prove that the one-step culture solution can remove harmful cytokines and metabolic wastes secreted by cells in the culture process, and the cytokines beneficial to the culture can be reserved in the one-step culture solution. The one-step culture solution of the invention truly realizes one-step culture from fertilization to embryo stage.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (9)

1. A one-step culture solution, which is characterized in that: the raw materials comprise physiological salt, energy substance, energy matrix, D-calcium pantothenate, recombinant human insulin and ethylenediamine tetraacetic acid;
the energy matrix comprises glycine, L-asparagine monohydrate, L-aspartic acid, L-glutamic acid, L-proline, L-serine, alanylglutamine, L-tyrosine, L-arginine hydrochloride, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan, L-valine, L-cystine dihydrochloride, L-threonine, L-lysine hydrochloride;
the energy substance contains D-glucose, L-calcium lactate and sodium pyruvate;
the physiological salt contains sodium chloride, potassium chloride, monopotassium phosphate, magnesium sulfate heptahydrate and calcium chloride.
2. The one-step culture broth of claim 1, wherein: further comprises a germicide, a pH indicator and a pH buffer.
3. The one-step culture broth of claim 2, wherein: the concentration ratio of the raw materials is as follows:
physiological salt: 88.00 to 105.50mmol/L of sodium chloride, 2.00 to 3.30mmol/L of potassium chloride, 0.20 to 0.40mmol/L of monopotassium phosphate and 0.15 to 0.32mmol/L of magnesium sulfate;
energy substance: 0.45-0.75 mmol/L of D-glucose, 0.15-0.30 g/L of L, L-calcium lactate and 0.30-0.45 mmol/L of sodium pyruvate;
energy matrix: 0.65 g/L-0.85 g/L-1.20 g/L-1.65 g/L-1.80 g/L-6.80 g/L-L, L-histidine hydrochloride monohydrate 1.50 g/L-2.50 g/L-1.30 g/L-2.85 g/L-L, L-leucine 2.20 g/L-2.95 g/L, L-methionine 0.60 g/L-0.85 g/L-1.20 g/L, alanyl glutamine 0.80 mmol/L-1.20 mmol/L-L, L-tyrosine 1.50 g/L-1.98 g/L, L-arginine hydrochloride 5.80 g/L-6.80 g/L-L, L-histidine hydrochloride monohydrate 1.50 g/L-2.50 g/L-L, L-isoleucine 2.30 g/L-2.85 g/L-L, L-leucine 2.20 g/L-2.95 g/L, L-methionine 0.60 g/L-0.85 g-37.38-phenylalanine hydrochloride 1.80 g-37.80 g-1.80 g/L-1.98 g/L-37.80 g-6.80 g/L-37.92-histidine hydrochloride;
calcium D-pantothenate: 5.00 mu g/L-15.00 mu g/L;
recombinant human insulin: 5.00 mu g/L to 50.00 mu g/L;
ethylenediamine tetraacetic acid: 0.005mmol/L to 0.020mmol/L;
a bactericide: 6.0mg/L to 15.0mg/L;
pH indicator: 0.0005g/L to 0.0200g/L;
pH buffer: 22.00 mmol/L-30.00 mmol/L.
4. The one-step culture solution according to claim 3, wherein: the concentration ratio of the raw materials is as follows:
physiological salt: 91.20 to 100.70mmol/L of sodium chloride, 2.37 to 3.02mmol/L of potassium chloride, 0.30 to 0.36mmol/L of monopotassium phosphate and 0.18 to 0.24mmol/L of magnesium sulfate;
energy substance: 0.51-0.68 mmol/L of D-glucose, 0.19-0.24 g/L of L, L-calcium lactate and 0.35-0.41 mmol/L of sodium pyruvate;
energy matrix: glycine 0.71 g/L-0.79 g/L, L-asparagine monohydrate 1.43 g/L-1.58 g/L, L-aspartic acid 1.26 g/L-1.40 g/L, L-glutamic acid 1.40 g/L-1.54 g/L, L-proline 1.09 g/L-1.21 g/L, L-serine 1.00 g/L-1.10 g/L0.9 to 1.1mmol/L alanyl glutamine 1.71 to 1.89g/L L, L-tyrosine 1.48 g/L L, L-arginine hydrochloride 6.00g/L to 6.64g/L, L-histidine hydrochloride monohydrate 2.00g/L to 2.21g/L, L-isoleucine 2.49g/L to 2.76g/L L, L-leucine 2.49g/L to 2.75g/L, L-methionine 0.72g/L to 0.79g/L L, L-phenylalanine 1.57g/L to 1.73g/L, L-tryptophan 0.48g/L to 0.54g/L L, L-valine 2.22g/L to 2.46g/L, L-cystine dihydrochloride 1.49g/L to 1.64g/L, L-threonine 2.26g/L to 2.50g/L, L-lysine hydrochloride 2.22g/L to 2.46g/L;
calcium D-pantothenate: 8.00 mu g/L to 12.00 mu g/L;
recombinant human insulin: 10.00 mu g/L to 25.00 mu g/L;
ethylenediamine tetraacetic acid: 0.012mmol/L to 0.014mmol/L;
a bactericide: 9.50mg/L to 10.50mg/L;
pH indicator: 0.0010g/L to 0.0100g/L;
pH buffer: 26.30 mmol/L-28.25 mmol/L.
5. The one-step culture solution according to any one of claims 1 to 4, wherein: also contains human serum albumin.
6. The one-step culture solution according to claim 5, wherein: the concentration ratio is 0.01mL/L to 15.00mL/L of human serum albumin.
7. The one-step culture solution according to claim 5, wherein: the concentration ratio is 0.5-10.20 mL/L of human serum albumin.
8. The one-step culture solution according to any one of claims 2 to 4, wherein: the bactericide is gentamicin sulfate.
9. The one-step culture solution according to any one of claims 2 to 4, wherein: the pH indicator is phenol red sodium salt.
CN202310019081.9A 2023-01-06 2023-01-06 One-step culture solution Pending CN116240164A (en)

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