CN109628386A - A kind of the In-vitro maturation liquid and preparation method thereof and cultural method of human oocyte - Google Patents
A kind of the In-vitro maturation liquid and preparation method thereof and cultural method of human oocyte Download PDFInfo
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- CN109628386A CN109628386A CN201910049865.XA CN201910049865A CN109628386A CN 109628386 A CN109628386 A CN 109628386A CN 201910049865 A CN201910049865 A CN 201910049865A CN 109628386 A CN109628386 A CN 109628386A
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Abstract
The present invention discloses the In-vitro maturation liquid and preparation method thereof and cultural method of a kind of human oocyte, include following operating procedure: serum production: blood being put into carrying out centrifugally operated in centrifuge, and it is spare to take out sera components, the production of antibiotic liquid: 100ug/ml penicillin, 100ug/ml streptomysin, 50ug/ml kanamycins and 25ug/ml nystatin are sufficiently mixed with the ratio of 1:2:1:3 respectively, it is spare that antibiotic liquid is made with this.The In-vitro maturation liquid and preparation method thereof and cultural method of the human oocyte, use a variety of salting liquids and antibiotic as assisting growth agent, based on more universal calf serum, the pH value of culture solution is adjusted by the way of adding NaHCO3 and HEPES solution simultaneously, the growth of miscellaneous bacteria is inhibited while providing life condition to egg mother cell, it is studied caused by avoiding due to miscellaneous bacteria infection difficult, the maturation in vitro rate of egg mother cell is improved simultaneously, manufacturing cost is reduced, the use for the person of being convenient to use while reducing research difficulty.
Description
Technical field
The present invention relates to technical field of cell culture, the In-vitro maturation liquid preparation side of specially a kind of human oocyte
Method and cultural method.
Background technique
Egg mother cell carries out the oogonium of meiosis in Process of oogenesis, is divided into first oocyte, secondary
Egg mother cell and mature egg mother cell, they are generation, first time subtrahend after oogonium differentiation and DNA replication dna division respectively
The product of division and second meiotic division needs to carry out in vitro culture behaviour to egg mother cell in egg mother cell research process
Make.
In the prior art, used culture solution be suitable for multiple types miscellaneous bacteria existence, therefore be easy to appear due to
Study difficult caused by miscellaneous bacteria infection, while reducing the maturation in vitro rate of egg mother cell, used culture solution it is required
It is mostly that ready-made biological fluid extraction is process, therefore manufacturing cost is higher, is made while difficulty is studied in increase in material
The use of user is made troubles.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the present invention provides a kind of In-vitro maturation liquid and preparation method thereof of human oocyte and trainings
The method of supporting, solves in the prior art, and used culture solution was suitable for the miscellaneous bacteria existence of multiple types, therefore was easy to appear
It is difficult due to being studied caused by miscellaneous bacteria infection, while the maturation in vitro rate of egg mother cell is reduced, used culture solution
The extraction of mostly ready-made biological fluid is process in material requested, therefore the higher problem of manufacturing cost.
(2) technical solution
In order to achieve the above object, the present invention is achieved by the following technical programs:
A kind of In-vitro maturation liquid and preparation method thereof of human oocyte includes following operating procedure:
S1, serum production: blood is put into carrying out centrifugally operated in centrifuge, and it is spare to take out sera components；
S2, antibiotic liquid production: respectively by 100ug/ml penicillin, 100ug/ml streptomysin, 50ug/ml kanamycins and
25ug/ml nystatin is sufficiently mixed with the ratio of 1:2:1:3, and it is spare that antibiotic liquid is made with this；
S3, ionic liquid production: respectively by 75IU/L rFSH, 150IU/L rHCG, 20% SPS, 2mg/L EGF and 25mmol/L
Sodium Pyruvate is sufficiently mixed with the ratio of 1:3:2:2, and it is spare that ionic liquid is made with this；
S4, solution mixing: the ionic liquid that the obtained antibiotic liquid of serum that step S1 is obtained, step S2 and step S3 are obtained with
The ratio of 5:1:2 mixes, and is sufficiently stirred, and obtains nutrient solution with this；
S5, pH value debugging: the pH value of nutrient solution obtained in detecting step S4, the NaHCO3 and HEPES by addition 3.7% are molten
Liquid carries out the debugging of pH value, and makes ph value of mixture (even pH value is then added less than 7.2 in range 7.2-7.4
NaHCO3 adds HEPES solution, until when pH value is in 7.2-7.4 if pH value is greater than 7.4), culture solution is completed with this
Production.
Preferably, the blood in the step S1 is calf arterial blood.
Preferably, the used time being sufficiently stirred in the step S4 is 30-40 minutes.
Preferably, the gross mass of the NaHCO3 and HEPES solution added in the step S5 must not exceed former nutrient solution matter
The 3% of amount.
A kind of cultural method of human oocyte includes following operating procedure:
The preparation of S1, culture dish: the culture dish being suitble to according to the scale selection capacity of culture, and selected culture dish is carried out clear
It washes and sterilizing operation；
S2, culture solution addition: in an aseptic environment, culture solution is made an addition in culture dish；
S3, cell addition: cell solution is instilled to culture solution, and guarantees that every drop cell solution does not contact with each other after drippage
Then cover the lid of culture dish；
S4, cell culture: cultivating under culture dish obtained in step S3 is put into 23-28 DEG C of temperature of gnotobasis, and every
Week observation is primary, and in observation, the lid of culture dish is opened 5-10 minutes, carries out culture operation with this.
Preferably, the range that the amount of culture solution is added in the step S2 is the 50%-70% of culture dish capacity used.
Preferably, the requirement in the step S3 to environment are as follows: it is sterile, it is 23-28 DEG C of temperature, relative humidity 40%-50%, empty
Oxygen content is greater than 20% in gas.
(3) beneficial effect
The present invention provides the In-vitro maturation liquid and preparation method thereofs and cultural method of a kind of human oocyte.Have following beneficial
Effect:
The In-vitro maturation liquid and preparation method thereof and cultural method of the human oocyte, use a variety of salting liquids and antibiotic as
Assisting growth agent, based on the more universal calf serum of use, while by the way of adding NaHCO3 and HEPES solution
The pH value for adjusting culture solution inhibits the growth of miscellaneous bacteria, avoids due to miscellaneous bacteria while providing life condition to egg mother cell
Difficulty is studied caused by infection, while improving the maturation in vitro rate of egg mother cell, reduces manufacturing cost, and it is difficult to reduce research
The use for the person of being convenient to use while spending.
Specific embodiment
Described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
A kind of In-vitro maturation liquid and preparation method thereof of human oocyte includes following operating procedure:
S1, serum production: blood is put into carrying out centrifugally operated in centrifuge, and takes out sera components spare (blood is ox
Calf arterial blood)；
S2, antibiotic liquid production: respectively by 100ug/ml penicillin, 100ug/ml streptomysin, 50ug/ml kanamycins and
25ug/ml nystatin is sufficiently mixed with the ratio of 1:2:1:3, and it is spare that antibiotic liquid is made with this；
S3, ionic liquid production: respectively by 75IU/L rFSH, 150IU/L rHCG, 20% SPS, 2mg/L EGF and 25mmol/L
Sodium Pyruvate is sufficiently mixed with the ratio of 1:3:2:2, and it is spare that ionic liquid is made with this；
S4, solution mixing: the ionic liquid that the obtained antibiotic liquid of serum that step S1 is obtained, step S2 and step S3 are obtained with
The ratio of 5:1:2 mixes, and is sufficiently stirred, and obtains nutrient solution with this (used time being sufficiently stirred is 30-40 minute)；
S5, pH value debugging: the pH value of nutrient solution obtained in detecting step S4, the NaHCO3 and HEPES by addition 3.7% are molten
Liquid carries out the debugging of pH value, and makes ph value of mixture (even pH value is then added less than 7.2 in range 7.2-7.4
NaHCO3 adds HEPES solution, until when pH value is in 7.2-7.4 if pH value is greater than 7.4), culture solution is completed with this
Production (gross mass of the NaHCO3 and HEPES solution of addition must not exceed former nutrient solution quality 3%).
A kind of cultural method of human oocyte includes following operating procedure:
The preparation of S1, culture dish: the culture dish being suitble to according to the scale selection capacity of culture, and selected culture dish is carried out clear
It washes and sterilizing operation；
S2, culture solution addition: in an aseptic environment, culture solution makes an addition in culture dish to (range of the amount of addition culture solution is
The 50%-70% of culture dish capacity used)；
S3, cell addition: cell solution is instilled to culture solution, and guarantees that every drop cell solution does not contact with each other after drippage
Then cover the lid (requirement to environment are as follows: sterile, 23-28 DEG C of temperature, relative humidity 40%-50%, air includes of culture dish
20%) oxygen amount is greater than；
S4, cell culture: cultivating under culture dish obtained in step S3 is put into 23-28 DEG C of temperature of gnotobasis, and every
Week observation is primary, and in observation, the lid of culture dish is opened 5-10 minutes, carries out culture operation with this.
In conclusion the In-vitro maturation liquid and preparation method thereof and cultural method of the human oocyte, molten using a variety of salt
Liquid and antibiotic are as assisting growth agent, based on the more universal calf serum of use, while using addition NaHCO3 and
The mode of HEPES solution adjusts the pH value of culture solution, and the growth of miscellaneous bacteria is inhibited while providing life condition to egg mother cell,
Difficulty is studied caused by avoiding due to miscellaneous bacteria infection, while improving the maturation in vitro rate of egg mother cell, reduces manufacture
Cost, the use for the person of being convenient to use while reducing research difficulty.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (7)
1. a kind of In-vitro maturation liquid and preparation method thereof of human oocyte, it is characterised in that: include following operating procedure:
S1, serum production: blood is put into carrying out centrifugally operated in centrifuge, and it is spare to take out sera components；
S2, antibiotic liquid production: respectively by 100ug/ml penicillin, 100ug/ml streptomysin, 50ug/ml kanamycins and
25ug/ml nystatin is sufficiently mixed with the ratio of 1:2:1:3, and it is spare that antibiotic liquid is made with this；
S3, ionic liquid production: respectively by 75IU/L rFSH, 150IU/L rHCG, 20% SPS, 2mg/L EGF and 25mmol/L
Sodium Pyruvate is sufficiently mixed with the ratio of 1:3:2:2, and it is spare that ionic liquid is made with this；
S4, solution mixing: the ionic liquid that the obtained antibiotic liquid of serum that step S1 is obtained, step S2 and step S3 are obtained with
The ratio of 5:1:2 mixes, and is sufficiently stirred, and obtains nutrient solution with this；
S5, pH value debugging: the pH value of nutrient solution obtained in detecting step S4, the NaHCO3 and HEPES by addition 3.7% are molten
Liquid carries out the debugging of pH value, and makes ph value of mixture (even pH value is then added less than 7.2 in range 7.2-7.4
NaHCO3 adds HEPES solution, until when pH value is in 7.2-7.4 if pH value is greater than 7.4), culture solution is completed with this
Production.
2. a kind of In-vitro maturation liquid and preparation method thereof of human oocyte according to claim 1, it is characterised in that: institute
Stating the blood in step S1 is calf arterial blood.
3. a kind of In-vitro maturation liquid and preparation method thereof of human oocyte according to claim 1, it is characterised in that: institute
Stating the used time being sufficiently stirred in step S4 is 30-40 minutes.
4. a kind of In-vitro maturation liquid and preparation method thereof of human oocyte according to claim 1, it is characterised in that: institute
The gross mass for stating NaHCO3 the and HEPES solution added in step S5 must not exceed the 3% of former nutrient solution quality.
5. a kind of cultural method of human oocyte, it is characterised in that: include following operating procedure:
The preparation of S1, culture dish: the culture dish being suitble to according to the scale selection capacity of culture, and selected culture dish is carried out clear
It washes and sterilizing operation；
S2, culture solution addition: in an aseptic environment, culture solution is made an addition in culture dish；
S3, cell addition: cell solution is instilled to culture solution, and guarantees that every drop cell solution does not contact with each other after drippage
Then cover the lid of culture dish；
S4, cell culture: cultivating under culture dish obtained in step S3 is put into 23-28 DEG C of temperature of gnotobasis, and every
Week observation is primary, and in observation, the lid of culture dish is opened 5-10 minutes, carries out culture operation with this.
6. a kind of cultural method of human oocyte according to claim 5, it is characterised in that: added in the step S2
The range of the amount of culture solution is the 50%-70% of culture dish capacity used.
7. a kind of cultural method of human oocyte according to claim 5, it is characterised in that: to ring in the step S3
The requirement in border are as follows: sterile, 23-28 DEG C of temperature, relative humidity 40%-50%, oxygen content is greater than 20% in air.
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Cited By (3)
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| CN110643569A (en) * | 2019-11-29 | 2020-01-03 | 广州达瑞生殖技术有限公司 | Granular cell stripping liquid and preparation method thereof |
| CN110684723A (en) * | 2019-11-21 | 2020-01-14 | 东北农业大学 | Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof |
| CN110760473A (en) * | 2019-11-29 | 2020-02-07 | 东北农业大学 | Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof |
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