CN108251353A - A kind of Human embryo in vitro culture liquid and culture systems - Google Patents
A kind of Human embryo in vitro culture liquid and culture systems Download PDFInfo
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- C12N2500/00—Specific components of cell culture medium
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Abstract
The present invention provides a kind of Human embryo in vitro culture liquid and preparation method thereof, belong to human ancillary reproductive field.The culture solution mainly includes：0.01~0.05g/L of gentamicin sulphate,Phenol red 0.005~0.01g/L,0~0.01mM/L of disodium ethylene diamine tetraacetate,90~116mM/L of sodium chloride,2.5~10.0mM/L of potassium chloride,0~1.8mM/L of calcium chloride dihydrate,0~7.16mM/L of potassium dihydrogen phosphate,0.2~1.51M/L of sulfate dihydrate magnesium,20~25mM/L of sodium bicarbonate,0.1~7.27mM/L of Sodium Pyruvate,0~20mM/L of sodium lactate,0~3.25mM/L of glucose,0~1.0mM/L of sodium citrate,0~1.0mM/L of alanyl-glutamine,0~2.7mM/L of inositol,0.05~5mM/L of taurine,Human albumin 5g/L,And essential amino acid and nonessential amino acid.
Description
Technical field
The invention belongs to human ancillary reproductive fields, and in particular to a kind of embryonic cell Vitro Culture Techniques, including one kind
Mankind's early embryonic development can be supported to the culture solution of blastaea and its culture systems of culture apparatus.
Background technology
Mankind's first case (IVF) baby in vitro fertilization is born in 1978, flourishes from this supplementary reproduction, is born in succession
Ovum intracytoplasmic sperm injection (ICSI), preimplantation genetic diagnosis (PGD), the technologies such as ovum or embryo freezing are greatly promoted
The development of supplementary reproduction industry.
Embryo medium provides nutrition needed for development for preimplantation embryo, for specific period embryo transfer needed for clinic
(ET) it or preimplantation genetic diagnosis (PGD) etc. operations is carried out provides vitro, be that supplementary reproduction field is clinical or experiment
Indispensable reagent in research.The domestic following defect of culture systems generally existing at present：(1) use sequential culture liquid more,
Incubation need to change liquid halfway；(2) most domestic buying import reagent, price high procurement cycle are long；(3) overall process embryo is cultivated
Tire ware bottom is kept flat, the microenvironment of no support embryo cutting；For this purpose, there must be a set of new culture systems to solve above-mentioned ask
Topic.
Invention content
It is described it is an object of the present invention to provide a kind of consistent formula culture solution for human embryonic cell's in vitro culture
Culture solution includes following component：0.01~0.05g/L of antibacterial composition gentamicin sulphate；PH indicator phenol red 0.005~
0.01g/L；Ion chelating agent disodium ethylene diamine tetraacetate (EDTA) 0~0.01mM/L；90~116mM/ of nutrition sodium chloride
L, 2.5~10.0mM/L of potassium chloride, 0~1.8mM/L of calcium chloride dihydrate, 0~7.16mM/L of potassium dihydrogen phosphate, sulfate dihydrate magnesium
0.2~1.51M/L, 20~25mM/L of sodium bicarbonate, 0.1~7.27mM/L of Sodium Pyruvate, 0~20mM/L of sodium lactate, glucose 0
~3.25mM/L, 0~1.0mM/L of sodium citrate, 0~1.0mM/L of alanyl-glutamine, 0~2.7mM/L of inositol, taurine
0.05~5mM/L, human albumin 5g/L, it is necessary to amino acid 1 .43~3.11% (m/m), nonessential amino acid 0.37~
0.72% (m/m).
Wherein, essential amino acid of the present invention is the l-cysteine selected from L-arginine, L-Histidine, l-Isoleucine,
L-Leu, L-lysine, l-methionine, L-phenylalanine, L-threonine, L-Trp, l-tyrosine, Valine
It is one or more of.
Wherein, nonessential amino acid of the present invention is selected from l-Alanine, L-Aspartic acid, L- tianmenine, L-
Glutamic acid, L- glycine, L-PROLINE, the one or more of Serine.
Culture solution of the present invention prepares solvent used as ultra-pure water, and pH value is 7.3 ± 0.1, and osmotic pressure is
257~273mOsm.
It is described it is a further object to provide a kind of method for preparing human embryonic cell's in vitro culture liquid
Method includes the following steps：
(1) using ultra-pure water as solvent, be proportionally added into respectively sodium chloride of the present invention, potassium chloride, potassium dihydrogen phosphate,
Calcium chloride dihydrate, sulfate dihydrate magnesium, disodium ethylene diamine tetraacetate, it is necessary to amino acid and nonessential amino acid, glucose, inositol,
Sodium citrate, taurine, Sodium Pyruvate, sodium lactate are made into basal liquid A；
(2) gentamicin sulphate of the present invention, phenol red is proportionally added into basal liquid A, is eventually adding bicarbonate
Sodium is made into basal liquid B；
(3) above-mentioned basal liquid B is put into 5% carbon dioxide, the incubator inner equilibrium of 5% oxygen and 90% nitrogen overnight after
It is pumped in super-clean bench with vacuum filter and stoste is obtained by filtration through 0.2 μm of filter membrane；
(4) above-mentioned stoste pH value 7.3 ± 0.1 is adjusted, 257~273mOsm of osmotic pressure adds in human serum albumins and mixes
It is even, it dispenses, seal in super-clean bench, finished product, and 4 DEG C of preservations are obtained after packaging.
Further, the preparation method of human embryos in vitro culture liquid of the present invention, which is additionally included in, prepares the culture solution
Before, the step of used utensil high-temperature sterilization will be prepared and be dried for standby.And it prepares overall process and is meeting industry requirement
It is carried out in dust proof workshop.
A further object of the present invention is to provide a kind of vitro culture system for cultivating human embryonic cell, the external training
The system of supporting includes human embryonic cell's in vitro culture liquid of the present invention and places the culture apparatus of the culture solution.Wherein
The culture apparatus preferred cell culture dish.Culture apparatus of the present invention most preferably China Patent No. is
Culture dish described in ZL201621262089.X.Wherein, the utility model of Patent No. ZL201621262089.X is this Shen
The first achievement in research asked someone.The bottom of wherein described culture dish be equipped with cultivation region, the cultivation region include the first cultivation region and
Second cultivation region, first cultivation region be equipped with the first culture hole, second cultivation region be equipped with the second culture hole, described first
Culture hole is tip hole, and the bottom of second culture hole is flat, and the oral area internal diameter of second culture hole is more than in bottom
Diameter.Wherein, whether second culture hole is developed normally for the 0-1 days culture embryos, and the first culture hole is really to cultivate
The place of embryo for the culture of the 1-5 days, using this technical solution, embryo is put into culture hole and is cultivated, cultivated
Culture solution of the present invention is added in ware and carries out drop culture, each embryo discharged in incubation some biotic factors into
Enter in culture dish, act on each other, play the role of promoting growth.
The beneficial effects of the invention are as follows:1st, it solves Embryo Culture reagent in the current supplementary reproduction in China and relies on import for a long time
Problem, and overcome reagent from during external import for a long time transport caused by influence；2nd, embryo support is from fertilized eggs to blastaea
Each period In vitro culture in stage overcomes the problem of Embryo Culture needs midway to change culture solution in the process, reduces operation
Step thereby reduces the influence to embryonic development, substantially increases the culture effect of embryonic cell.
Description of the drawings
Fig. 1 is the schematic diagram of preferably culture dish of the invention.
Wherein, 1 is culture dish；2 be the first cultivation region；3 be the second cultivation region；4 be that cell rinses boss；21 be the first training
Support hole；31 be the second culture hole.
Specific embodiment
Below by way of specific embodiment, further the present invention will be described, it is impossible to be not understood as the limit to the present invention
It makes, material, the reagent used in embodiment, instrument and equipment is commercially available unless otherwise specified.
【Embodiment 1】Embryo Culture formula of liquid and preparation method：
A, formula of liquid is cultivated：
B, preparation method：
A, used utensil high-temperature sterilization will be prepared and be dried for standby, prepare overall process in satisfactory dust proof workshop
Middle progress；
B, add load weighted ultra-pure water, then be separately added into sodium chloride, the chlorine of each group of formula in the formula table through precise
Change potassium, potassium dihydrogen phosphate, calcium chloride dihydrate, sulfate dihydrate magnesium, disodium ethylene diamine tetraacetate, it is necessary to amino acid and non-essential amino
Acid, glucose, inositol, sodium citrate, taurine, Sodium Pyruvate, sodium lactate start blender until solid matter completion is molten
Solution, is made into basal liquid A；
C, gentamicin sulphate, the phenol of each group of formula in the formula table through precise are separately added into above-mentioned basal liquid A
It is red, sodium bicarbonate is eventually adding, starts blender until solid matter completion dissolving, is made into basal liquid B；
E, above-mentioned basal liquid B is put into 5% carbon dioxide, the incubator inner equilibrium of 5% oxygen and 90% nitrogen overnight after
Aforesaid liquid is filtered into membrane filtration through 0.2 μm with vacuum filter pump in super-clean bench, obtains stoste；
F, stoste pH value 7.3 ± 0.1,257~273mOsm/kg of osmotic pressure are adjusted, adds in human serum albumins and mixing,
Coating-dividing sealing, packaging in super-clean bench obtain finished product, 4 DEG C of preservations after packaging.
【Embodiment 2】The detection of embryo medium
PH value detects
Take appropriate sample to be tested pH meter measurement three times, the average value for taking three data of gained is as a result, 7.3 ± 0.1
It is considered as qualification.
Osmotic pressure detects
After osmometer correction, take in 50 μ l samples to be tested to testing tube and start to test, data are read after numerical stability,
3 data are measured by the above process, are averaged as a result, being considered as qualification in 257~273mOsm/kg.
Detection of bacterial endotoxin
According to 2015 editions requirements of Pharmacopoeia of People's Republic of China, it is detected with reagents gel method, as a result≤0.25EU/
Ml is considered as qualification.
Cytotoxicity
It is detected by the regulation of GB/T 16886.5, cytotoxicity score is not to be exceeded 1 point.
Sensitization detects
It is detected by the regulation of GB/T 16886.10, it should be without sensitivity response.
Sterility test
According to Pharmacopoeia of People's Republic of China, 2015 editions will require, and with membrane-filter procedure, sample to be tested is through filter
Direct inoculation medium culture 14 days after membrane filtration, observation is as a result, should be sterile daily.
Skin and flesh stimulates
It is detected by the regulation of GB/T 16886.10, it should be without intradermal stimulate the reaction.
Pyrogen
According to 2015 editions requirements of Pharmacopoeia of People's Republic of China, apyrogeneity is answered to react.
Mice embryonic in vitro culture is tested
(1) mouse superfecundation
4~6 week old B6D2 10IU/ PMSG Injections of Strains of Mouse are chosen, 10IU/ only injects hCG after 48h, has injected hCG
It mates afterwards with the male mouse that same strain has mating ability.
(2) prepare In vitro culture ware
Sterile 35mm wares are taken, do few drops of 50 μ l drops by the use of sample to be tested drips as test group culture, with endogenous toxic material containing 70IU/ml
The M16 culture solutions of element do few drops of 50 μ l drops as positive controls, and few drops of 50 μ l drops are done as the moon by the use of common M16 culture solutions
Property control group, covering culture mineral oil in surface puts 5%CO2, and 5%O2,90%N2 are put down overnight in the incubator of saturated humidity
Weighing apparatus.
(3) fertilization ovum collecting
Rear the next morning inspection bolt is mated, sees that bolt female rat is put to death after choosing, its fallopian tubal is taken to be put in and is preheated to 37 DEG C of M2 behaviour
Make in liquid, ampulla of uterine tube is expanded place punctures, and collects during cotton-shaped fertilized eggs group drips to 37 DEG C of hyaluronidases and digests particle
Fertilized eggs after granular cell is completely fallen off are transferred in 37 DEG C of M2 and clean for several times by cell, and picking fertilized embryo is for use.
(4) Embryo Culture
Above-mentioned embryo's stochastic averagina points three groups, every group is no less than 50 pieces, and one group is put in test group and cultivates, and one group is put in sun
Property control grade culture, one group is put in negative control group culture, and culture dish is put in 5%CO2,5%O2,90%N2, saturated humidity
96h is cultivated in incubator.
(5) test result
Each group blastaea number is recorded after cultivating 96h
Acceptable standard：A, positive controls blastaea number is statistically significantly less than negative control group blastaea number
B, negative control group blastaea number >=80%
(6) testing result
The Embryo Culture efficiency comparative invented using body：
Claims (10)
1. a kind of human embryonic cells in vitro culture liquid, the culture solution includes 0.01~0.05g/ of antibacterial composition gentamicin sulphate
L；Phenol red 0.005~the 0.01g/L of pH indicator；Ion chelating agent disodium ethylene diamine tetraacetate (EDTA) 0~0.01mM/L；Nutrition
Composition 90~116mM/L of sodium chloride, 2.5~10.0mM/L of potassium chloride, 0~1.8mM/L of calcium chloride dihydrate, potassium dihydrogen phosphate 0~
7.16mM/L, sulfate dihydrate 0.2~1.51M/L of magnesium, 20~25mM/L of sodium bicarbonate, 0.1~7.27mM/L of Sodium Pyruvate, lactic acid
0~20mM/L of sodium, 0~3.25mM/L of glucose, 0~1.0mM/L of sodium citrate, 0~1.0mM/L of alanyl-glutamine, flesh
0~2.7mM/L of alcohol, 0.05~5mM/L of taurine, human albumin 5g/L, it is necessary to amino acid 1 .43~3.11% (m/m), it is non-
Essential amino acid 0.37~0.72% (m/m).
2. the culture solution as described in right 1, wherein the essential amino acid is selected from L-arginine, l-cysteine, L-Histidine,
L-Isoleucine, L-Leu, L-lysine, l-methionine, L-phenylalanine, L-threonine, L-Trp, L- junket ammonia
Acid, the one or more of Valine.
3. the culture solution as described in right 1, wherein the nonessential amino acid is L- days selected from l-Alanine, L-Aspartic acid
Door propylhomoserin, Pidolidone, L- glycine, L-PROLINE, the one or more of Serine.
4. the culture solution as described in any one of right 1 to 3, wherein it is ultra-pure water to prepare the solvent used in the culture solution.
5. a kind of method for preparing human embryonic cell's in vitro culture liquid as described in claim 1, includes the following steps：
(1) using ultra-pure water as solvent, it is proportionally added into the sodium chloride, potassium chloride, potassium dihydrogen phosphate, two water chlorinations respectively
Calcium, sulfate dihydrate magnesium, disodium ethylene diamine tetraacetate, it is necessary to amino acid and nonessential amino acid, glucose, inositol, sodium citrate,
Taurine, Sodium Pyruvate, sodium lactate are made into basal liquid A；
(2) gentamicin sulphate, phenol red is proportionally added into basal liquid A, sodium bicarbonate is eventually adding, is made into basis
Liquid B；
(3) above-mentioned basal liquid B is put into 5% carbon dioxide, the incubator inner equilibrium of 5% oxygen and 90% nitrogen overnight after ultra-clean
It is pumped in platform with vacuum filter and stoste is obtained by filtration through 0.2 μm of filter membrane；
(4) above-mentioned stoste pH value 7.3 ± 0.1 is adjusted, 257~273mOsm of osmotic pressure adds in human serum albumins and mixing,
It is dispensed in super-clean bench, sealing, finished product, and 4 DEG C of preservations is obtained after packaging.
6. method as claimed in claim 5 is additionally included in before preparing the culture solution, the utensil high temperature for preparing used is gone out
Bacterium and the step of be dried for standby.
7. a kind of human embryonic vitro culture system of culture, the vitro culture system include the mankind described in claim 1
Embryo in-vitro culture solution and the culture apparatus for placing the culture solution.
8. vitro culture system as claimed in claim 7, wherein the culture apparatus is culture dish.
9. vitro culture system as claimed in claim 8, wherein the bottom of the culture dish is equipped with cultivation region, the cultivation region
Including the first cultivation region and the second cultivation region, first cultivation region is equipped with the first culture hole, and second cultivation region is equipped with the
Two culture holes, first culture hole are tip hole, and the bottom of second culture hole is flat, the mouth of second culture hole
Portion's internal diameter is more than bottom inner diameter.
10. vitro culture system as claimed in claim 9, wherein second culture hole is embryo's training for the 0-1 days
It supports, the first culture hole is used for the Embryo Culture of the 1-5 days.
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Cited By (8)
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| CN109628380A (en) * | 2018-12-25 | 2019-04-16 | 深圳韦拓生物科技有限公司 | A kind of people's liquid in vitro fertilization and preparation method thereof |
| CN109628384A (en) * | 2018-12-29 | 2019-04-16 | 深圳韦拓生物科技有限公司 | A kind of ovum flushing liquor and preparation method thereof |
| CN109628379A (en) * | 2018-12-18 | 2019-04-16 | 西安交通大学 | A kind of animal cloning embryo medium and cultural method |
| CN109679895A (en) * | 2018-12-18 | 2019-04-26 | 西安交通大学 | The embryo medium and Embryo Culture method of the excretion body of source containing fallopian tubal |
| CN110669724A (en) * | 2019-11-08 | 2020-01-10 | 广州达瑞生殖技术有限公司 | Saccharum embryo culture solution and preparation method thereof |
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| CN112048467A (en) * | 2020-09-11 | 2020-12-08 | 江苏集萃药康生物科技有限公司 | Application of KSOM-AA culture solution in-vitro culture of NOD (non-specific oligonucleotide) background mouse embryos |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109628379A (en) * | 2018-12-18 | 2019-04-16 | 西安交通大学 | A kind of animal cloning embryo medium and cultural method |
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| CN109628380A (en) * | 2018-12-25 | 2019-04-16 | 深圳韦拓生物科技有限公司 | A kind of people's liquid in vitro fertilization and preparation method thereof |
| CN109628384A (en) * | 2018-12-29 | 2019-04-16 | 深圳韦拓生物科技有限公司 | A kind of ovum flushing liquor and preparation method thereof |
| CN110669724A (en) * | 2019-11-08 | 2020-01-10 | 广州达瑞生殖技术有限公司 | Saccharum embryo culture solution and preparation method thereof |
| CN110669725A (en) * | 2019-11-08 | 2020-01-10 | 广州达瑞生殖技术有限公司 | Cleavage embryo culture solution and preparation method thereof |
| CN112048467A (en) * | 2020-09-11 | 2020-12-08 | 江苏集萃药康生物科技有限公司 | Application of KSOM-AA culture solution in-vitro culture of NOD (non-specific oligonucleotide) background mouse embryos |
| CN115812695A (en) * | 2022-12-30 | 2023-03-21 | 广州赛莱拉干细胞科技股份有限公司 | Bone marrow preservation solution for efficiently obtaining hematopoietic stem cells and using method thereof |
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