CN109971821A - A kind of safety detecting method of supplementary reproduction liquid reagent - Google Patents
A kind of safety detecting method of supplementary reproduction liquid reagent Download PDFInfo
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- CN109971821A CN109971821A CN201910326431.XA CN201910326431A CN109971821A CN 109971821 A CN109971821 A CN 109971821A CN 201910326431 A CN201910326431 A CN 201910326431A CN 109971821 A CN109971821 A CN 109971821A
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Abstract
A kind of safety detecting method of supplementary reproduction liquid reagent, method therefor is according to international 1- cell stage method, correlation technique is improved, in detection method used, use PVA and the method combined without haemocyanin culture solution, carry out the statistics of blastaea quantity and blastocyst rate or the blastaea rate of spread respectively using the mice embryonic of removal oolemma, double standards reflect the quality of testing liquid reagent, to improve the Mass sen- sitivity and reliability of this method verifying embryo's liquid reagent.
Description
Technical field
The present invention relates to a kind of safety detecting methods of supplementary reproduction liquid reagent, belong to biological medicine material Quality Control
Field.
Background technique
The safety of assisted reproductive technology (Assisted Reproductive Technology, ART) is to be related to it
The normal development of gamete and embryo, the important guarantee of clinical effectiveness and offspring's long-term health.MEA(Mouse Embryo
It Assay is) using the external routine culture system of mice embryonic, in the incubation from fertilized eggs to blastaea, according to product to be checked
Function and characteristic, pass through observation using the leaching liquor of liquid type product to be checked or utensil class product in accordingly culture link
Developmental state of the body early embryo from fertilized eggs to blastaea evaluates product to be checked to the genotoxic potential of embryonic development.1- cell mouse
Embryo's measuring method (MEA) is the quality detecting method of most widely used culture solution and other processing liquid in the world.ART training
Nutrient solution and gamete treatment fluid need to carry out quality control test for preclinical, provide effective risk assessment.Normal practice is
One cell of Mouse Eggs after fertilization is taken out from internal, is cultivated 96 hours in culture solution to be tested, if high-quality blastaea number
Reach and be greater than 80%, then proves the requisite quality of the culture solution.
But since its biological nature causes mice embryonic to be more resistant to, to the insensitivity of micro nuisance, wherein cultivating
Fail effectively to be checked with potential harmful substances many in liquid.All kinds of liquid reagents tested by existing MEA
There is potential toxicity, this unknown toxicity also brings hidden danger to the success rate of assisted reproductive technology and embryo quality, because
This needs a kind of safer, more sensitive to toxicity detection method, improves detection sensitivity.
The present invention can be used for the supplementary reproduction detection of various culture solutions, such as spilting of an egg culture solution, blastocyst culture liquid etc.；Simultaneously
It can be used for the supplementary reproduction detection of various gamete treatment fluids, such as take ovum liquid, Sperm treatment liquid etc..
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of safety detection sides of supplementary reproduction liquid reagent
Method, comprising the following steps:
1) promote female mice ovulation, fertilization after being fertilized successfully, takes cell mass from fallopian tubal pot portion；
2) granular cell for removing step 1) cell mass, collects 1- cell mouse embryo；
3) oolemma for the 1- cell mouse embryo that further removal step 2) obtains；
4) the 1- cell mouse embryo that step 3) obtains is added in the liquid reagent and is handled, handle Time Dependent
In the purposes of the liquid reagent；
5) the 1- cell mouse embryo for obtaining step 4) carries out the microlayer model culture solution culture of single embryo, microlayer model culture
Polyvinyl alcohol is added in liquid, covers culture mineral oil, cultivates the statistics of laggard luggage embryo quantity and blastocyst rate.
Preferably, if the liquid reagent is embryo medium, directly the 1- cell mouse embryo in step 3) is added
Enter to carry out the culture of individual drops culture solution in the liquid reagent (not needing subsequent without the drop culture of haemocyanin culture solution
Step), polyvinyl alcohol is added in the drop reagent, covers mineral oil, cultivates the statistics of laggard luggage embryo quantity and blastocyst rate.
If or the liquid reagent be embryo's treatment fluid or gamete treatment fluid, by the 1- cell mouse embryo in step 3)
It is added in the liquid reagent and is handled, handle Time Dependent in measured fluid nutrient medium purposes (for example, gamete treatment fluid
The conventional treatment time be 5-15 minute), after the completion of processing, 1- cell mouse embryo is subjected to the training of independent microlayer model culture solution
It supports, polyvinyl alcohol is added in drop culture solution, cover mineral oil, cultivate the statistics of laggard luggage embryo quantity and blastocyst rate.(at this time
Drop culture solution be commercially available or verified qualification embryo medium, 80%) MEA blastocyst rate reaches
In the above-mentioned methods, microlayer model (10 μ l-20 μ l) culture solution culture for having used single embryo of applicant's creativeness,
This belongs to the non routine operation of MEA, and conventional method is 50 μ L-200 μ L drop cultures of polyembryony tire, applicant's previous experiments card
It is bright, be based on this method, using the drop culture of polyembryony tire, can significantly reduce this method sensitivity Detection degree (or even with tradition
MEA method susceptibility cannot be distinguished from).Applicant is speculated as more Embryo Cultures and is easy to produce interaction, secretes and protects into environment
Shield property substance forms gregarious effect, the fitness to adverse circumstances is improved, to reduce the susceptibility of entire method.
In above-mentioned safety detecting method, the female mice selects 5-7 week old mouse.
In above-mentioned safety detecting method, fertilization procedures, which include that intraperitoneal injection pregnant mare serum induction is super, ovulates, and 48
Human chorionic gonadotrophin induces ovulation, female mice and male mouse mating after hour, and mate good authentication.
In above-mentioned safety detecting method, the granular cell preferably clear matter acid enzyme removed on step 1) cell mass is gone
It removes.
In above-mentioned safety detecting method, the preferred 0.5-1.5mg/ml of hyaluronidase concentration.
In above-mentioned safety detecting method, the polyvinyl alcohol concentration is 0.01mg/mL-0.025mg/mL.
The safety detection mentioned in this experiment, safety refer to potentially harmful substance pair in supplementary reproduction liquid reagent
In the influence that embryo or gamete develop.Safety detection, which refers to, carries out supplementary reproduction liquid reagent using mice embryonic method
Detection, observes the developmental state of mice embryonic, according to Mouse Blastocysts number and blastocyst rate or the minor change of the blastaea rate of spread, reflection
The genotoxic potential size and its quality of liquid reagent.
It is (including commercial or prepare to be divided into embryo medium and embryo or gamete treatment fluid for signified liquid reagent in this method
Product), source may be from the preparation of the manufacturers such as Sigma, or the related liquid prepared according to Embryo Culture reference book, such as " small
Mouse embryo operation laboratory manual " Cold Spring Harbor Laboratory Press, the 3rd edition.
The dosage concentration of polyvinyl alcohol is 0.01-0.025mg/ml, lower than the 0.05mg/ml-0.1mg/ml of conventional amount used
(journal of Shandong university, 42 (3), 2004).The MEA of polyvinyl alcohol is used at present, there is no final conclusion, nobody is for caused by it
Embryo's resistance is studied, in particular, the influence whether culture medium can generate Mouse Embryo Development is added in polyvinyl alcohol.
Applicant is by early-stage study discovery, in the test of single factor test polyvinyl alcohol dosage, for example with conventional amount used polyvinyl alcohol
When the susceptibility comparative study of 0.04mg/ml and conventional H SA culture medium, carried out using the M16 culture solution without haemocyanin
It cultivates, is separately added into above-mentioned comparison, do not remove in removal oolemma group and oolemma group, susceptibility gap is unobvious.It is lower than
0.01mg/ml, then influence the development of embryo, and is easy to happen embryo's adhesion.When polyvinyl alcohol concentration be lower than 0.03mg/ml,
Desired effect can occur in this detection, therefore applicant's preferred concentration is 0.01mg/ml-0.025mg/ml, and in 0.01mg/ml
When, reach preferable sensitivity technique effect.
In above-mentioned safety detecting method, the polyvinyl alcohol concentration 0.01mg/ml.
In above-mentioned safety detecting method, the removal 1- cell mouse embryo oolemma includes by 1- cell mouse embryo
Tire is put into the culture solution containing 0.5% pronase to be digested 5 minutes in 37 DEG C, is removed the oolemma of embryo, is used buffer
The remaining pronase of cleaning in (such as PBS solution).
Existing research is studied for removing the MEA method of oolemma, it is believed that removal oolemma is for the ovum in MEA
The rate of splitting does not have significant impact.But applicant of the present invention is using the MEA method of removal oolemma, it is therefore an objective to further to improve
Embryo cooperates the culture solution that PVA is added and independent embryo's droplet culture to the sensibility of adverse circumstances, tired using multi-layer effect
Meter mode, successively amplifies the susceptibility of this method, the pleasantly surprised discovery of applicant, the mice embryonic group of lensless Fourier transform holography in embodiment 1,
In non-toxic culture solution, 96 hours blastocyst rates are extremely close with control group, it was demonstrated that this method can't reduce embryo close to 80%
Tire for the adaptability of nontoxic environment, support by the significant data for becoming this method establishment.
In above-mentioned safety detecting method, the drop culture solution is without haemocyanin culture solution.
It is described to be selected from what Sigma-Aldrich was provided without haemocyanin culture solution in above-mentioned safety detecting method
M16, KSOM or HTF cultivate formula of liquid, remove wherein serum protein composition.
KSOM embryo medium and HTF (Human Tubal Fluid) culture solution are that culture solution is commonly used in mice embryonic culture,
With commercial product or " Mouse Embryo laboratory manual " Cold Spring Harbor Laboratory Press is referred to,
3rd edition is prepared.
Mineral oil used is commercial product, preferably Sigma-Aldrich, M8410, guarantee MEA blastocyst rate reach 80% with
On.
The step 4) cultivates the statistics of 96 or 144 hours laggard luggage embryo quantity and blastocyst rate or the blastaea rate of spread.
Conventional incubation time is 96 hours.In the method, in order to further increase the susceptibility of method, culture when
Between preferably 96-144 hours.When using the serum protein free of super low concentration PVA, incubation time also utmost importance has
96 hours cultivations of Shi Chuantong may be not enough to show small toxicity gap therein, therefore this detection method will expand the time
Exhibition is 144 hours, further the presence of prominent genotoxic potential.
The utility model has the advantages that
Applicant's first passage removes the oolemma (zona pellucida) of mice embryonic, and with microlayer model, independent embryo is trained
Feeding method improves its sensitivity, in addition, human or animal's haemocyanin in culture solution has the function of absorption harmful substance, influences to training
The detection of feeding liquid quality, but if removal haemocyanin, mice embryonic will be unable to be developed.For the first time using unconventional low
Density polyethylene alcohol (poly-vinyl alcohol) is used as substitute, safe and non-toxic, has good biocompatibility, is that can replace
Do not have for function of the haemocyanin in culture solution but the organic matter for adsorbing other harmful substance functions, and people can be improved
Class supplementary reproduction culture liquid reagent quality testing sensitivity, it is more reliable to guarantee that it is used safer.
Specific embodiment
Preferred embodiment below is only used to illustrate the technical scheme of the present invention and not to limit it, although passing through following preferred implementations
Example is described in detail the present invention, however, those skilled in the art should understand that, can be right in the form and details
It makes reasonable change, without departing from claims of the present invention limited range.
Embodiment 1
The present embodiment is Experimental comparison's example, and artificial preparation is formulated to be measured without containing haemocyanin based on Sigma M16
Embryo medium carries out sensitivity verifying, it was demonstrated that safety detection method improves the detection sensitivity of traditional MEA.
1.5~7 week old female mices (C57Bl, Nanjing treasury medicine health) are in intraperitoneal injection 5IU pregnant mare serum (PMSG)
(Sigma-Adrich, G4877) super ovulation.After 48 hours with 5IU human chorionic gonadotrophin (hCG) (Sigma-Alrich,
C1063 ovulation, and the mating single cage raising of female mice hero mouse) are induced.After 18-20 hours, by the appearance for checking female rat vagina plug
Confirmation mates successfully.1- cell mouse embryo is collected from fallopian tubal pot portion, removes granular cell with the hyaluronidase of 1mg/ml.
Note: mating the next morning inspection mating situation, and selection is shown in that bolt mouse is spare；1- cell stage is collected: injection hCG
It is put to death after 18 to 22 hours afterwards and sees bolt female mice, collect 1- cell stage in ampulla of uterine tube；The cotton-shaped cell mass that will be collected into
37 DEG C are placed into advance in preheated hyaluronidases, transfer immediately after the granular cell around embryo is digested separation,
After cleaning, the fertilized embryo of normal morphology is picked out, is transferred in Medium drop, tested for the detection of 1- cell mouse embryo,
2. 1- cell mouse embryo is put into the culture containing 0.5% pronase (Sigma-Aldrich P8811)
It is digested 5 minutes in liquid (M2 culture solution, Sigma-Aldrich, M7167) in 37 DEG C, the oolemma of embryo is removed, in buffer
Clean remaining pronase.
3. the 1- cell mouse embryo for removing oolemma will be individually in 96 well culture plates, 10 μ LM16 culture solution droplets
(Sigma-Aldrich, M7292 provide formula and prepare, and are free of haemocyanin culture solution), surface covers dedicated mineral in vitro fertilization
Oily (Sigma-Aldrich, M8410): prepare 96 well culture plates on the day before Embryo Culture, and in 37 DEG C and 5-6%CO2Environment
Incubator in balance overnight, to keep standard pH 7.4-7.6.Mice embryonic will be in control group (M16+PVA) (Sigma-
Aldrich, P8136) or the middle culture of detection reagent (formaldehyde of M16+PVA+ various concentration) 96 hours, with different dense in this test
Degree formaldehyde represents harmful substance that may be present, counts Blastocyst formation number and keeps the blastaea of extended mode after 144 hours
Number.
4. untreated one cell of mouse of oolemma with it is above-mentioned 3) in similarity condition under cultivate 96 hours, count capsule
Embryogenesis number and the blastaea number that extended mode is kept after 144 hours.
Test result and basic index observation:
Blastocyst formation rate: 1- cell stage records blastaea quantity-quality after cultivating 96 hours respectively in vitro, to formation blastaea
Fertilized eggs ratio calculated.
Blastocyst formation rate (%)=blastaea number/1- cell stage embryo number × 100%
Mouse embryo pass the test standard: 1- cell stage records blastaea quantity after cultivating 96 hours respectively in vitro；Observe blastaea
Form.The Blastocyst formation rate of detection reagent group is substantially less than negative control group through statistical analysis, and (oolemma is complete for negative control group
Whole mouse embryo, M16 culture solution group) Blastocyst formation rate >=80%.
The mice embryonic of the complete mice embryonic of 1- cell oolemma and lensless Fourier transform holography is compared in table 1 in various concentration
0 hour to 144 hours developmental state in formaldehyde environment.
Table 1: influence of the formaldehyde to the Mouse Embryo Development of the complete mice embryonic of oolemma and lensless Fourier transform holography in culture solution
Interpretation of result:
1. usually in normal nontoxic culture solution growing environment (either detection group and there is complete oolemma group), 1-
Cell mouse embryo was having 80% or so cell that can form blastaea by culture in 96 hours, in extension culture in 144 hours
The blastaea extended afterwards keeps extended mode.
2. the complete mice embryonic of oolemma is than the condition of culture that the mice embryonic of lensless Fourier transform holography optimizes more resistant against Asia.Blood
Albumin has the function of absorption heavy metal and other harmful substances, improves the tolerance of cell, shields to harmful substance
Detection.The sensibility improved with polyvinyl alcohol alternative serum albumen, is effectively detected in culture environment that there are low concentrations
Noxious material.
3. when there are the formaldehyde of low concentration (a kind of volatile organic matter) ratio 0.025mM, 0.05mM in culture environment, and
The mice embryonic of the complete mice embryonic of 0.075mM, 1- cell oolemma and lensless Fourier transform holography is 96 in culture solution containing polyvinyl alcohol
Hour and grow within 144 hours it is close with blastula stage percentage is reached, with the increase of concentration of formaldehyde, the mice embryonic of lensless Fourier transform holography
Growth show bigger sensibility: when the concentration of formaldehyde reaches 0.125mM, no mice embryonic reaches blastula stage, is shown in Table 1.
Claims (10)
1. a kind of safety detecting method of supplementary reproduction liquid reagent, which comprises the following steps:
1) promote female mice ovulation, fertilization after being fertilized successfully, takes cell mass from fallopian tubal pot portion；
2) granular cell for removing step 1) cell mass, collects 1- cell mouse embryo；
3) oolemma for the 1- cell mouse embryo that further removal step 2) obtains；
4) the 1- cell mouse embryo that step 3) obtains is added in the liquid reagent and is handled, handle Time Dependent in institute
State the purposes of liquid reagent；
5) the 1- cell mouse embryo obtained step 4) carries out the microlayer model culture solution culture of single embryo, in microlayer model culture solution
Polyvinyl alcohol is added, covers culture mineral oil, cultivates the statistics of laggard luggage embryo quantity and blastocyst rate.
2. safety detecting method according to claim 1, which is characterized in that the female mice selects 5-7 week old small
Mouse.
3. safety detecting method according to claim 1, which is characterized in that fertilization procedures include that intraperitoneal injection gestation is female
The super ovulation of horse serum induction, human chorionic gonadotrophin induces ovulation, female mice and male mouse mating after 48 hours, mates and successfully tests
Card.
4. safety detecting method according to claim 1, which is characterized in that of step 2) removal step 1) cell mass
The removal of granulocyte preferably clear matter acid enzyme.
5. safety detecting method according to claim 1, which is characterized in that the step 5) is cultivated 96 or 144 hours
The statistics of laggard luggage embryo quantity and blastocyst rate or the blastaea rate of spread.
6. safety detecting method according to claim 1, which is characterized in that the polyvinyl alcohol concentration is 0.01mg/
mL-0.025mg/mL。
7. safety detecting method according to claim 6, which is characterized in that the polyvinyl alcohol concentration 0.01mg/ml.
8. safety detecting method according to claim 1, which is characterized in that the removal 1- cell mouse embryo is transparent
Band includes being put into 1- cell mouse embryo in the embryo medium containing 0.5% pronase to digest 5 minutes in 37 DEG C, is gone
Except the oolemma of embryo, with cleaning remaining pronase in buffer.
9. safety detecting method according to claim 1, which is characterized in that the microlayer model culture solution is without serum
Albumen culture solution.
10. safety detecting method according to claim 9, which is characterized in that described to be selected without haemocyanin culture solution
M16, KSOM or the HTF provided from Sigma-Aldrich cultivates formula of liquid, removes wherein serum protein composition.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111272517A (en) * | 2020-02-26 | 2020-06-12 | 东蕴医疗科技(上海)有限公司 | Mouse embryo blastocyst staining method for quality control of human assisted reproduction consumable product |
-
2019
- 2019-04-23 CN CN201910326431.XA patent/CN109971821A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| ALESSANDRA J. AINSWORTH等: "Improved detection of mineral oil toxicity using an extended mouse embryo assay", 《J ASSIST REPROD GENET》 * |
| JUDITH A. FLEETHAM等: "The mouse embryo culture system: improving the sensitivity for use as a quality control assay for human in vitro fertilization", 《FERTILITY AND STERILITY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111272517A (en) * | 2020-02-26 | 2020-06-12 | 东蕴医疗科技(上海)有限公司 | Mouse embryo blastocyst staining method for quality control of human assisted reproduction consumable product |
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