CN105052892A - Cryoprotectant free of DMSO (dimethyl sulfoxide) and ovary cryopreservation method - Google Patents

Abstract

The invention relates to the field of hypothermal medicine of biotechnology, in particular to a cryoprotectant free of DMSO (dimethyl sulfoxide). The cryoprotectant comprises 200 mmol/L of trehalose, 0.3 g/mL of PVP (polyvinylpyrrolidone) 40, 200 mu g/L of cholesterol and 200 mu g/L of lecithin. The cryoprotectant is injected into a newly obtained ovary; all macroscopic follicles on the surface of the ovary are punctured and sucked, so that content in the follicles is removed; then the ovary is put in a freezing bag to be cooled. The cryoprotectant doesn't contain DMSO, does not have cytotoxicity and is easy to remove, simple to prepare and good in cryoprotective effect, raw materials are easy to obtained, the rate of normal primordial follicles is high, and the number of dead cells is small; an ovary cryopreservation method is simple and easy to operate and good in cryoprotective effect; the histological structure can be better conserved after the whole ovary of sheep is frozen.

Description

A kind of method of cryoprotector without DMSO and freezen protective ovary

Technical field

The present invention relates to biotechnology cryogenic medicine field, particularly a kind of cryoprotector without DMSO, also relate to the method for the freezen protective ovary using this cryoprotector.

Background technology

Along with the progress of Treatment and diagnosis means, breeding time, the survival rate of female cancer patients significantly promoted.But anti-cancer therapies (as chemicotherapy) may cause ovarian function to decline even exhaustion.Ovary cortex is organized technique of cryopreservation to deposit cash to be applied to clinical as the method for preserving fertility before chemicotherapy, but be limited to tissue ischemia damage and a large amount of ovarian follicle loss after transplanting, and intact ovaries transplanting can recover blood confession by anastomosis of blood vessel, this difficult problem can be overcome in theory, now transplant and the report of lamb of giving a birth after existing sheep intact ovaries freeze thawing.

Intact ovaries is freezing similar to cortex cryo; the cryoprotectors such as normal employing dimethyl sulfoxide (DMSO) (DMSO), glycerine, animal blood serum; but the infiltration of cryoprotector and remove comparatively that cortex block is more difficult, particularly DMSO, has cytotoxicity more than 4 DEG C.Therefore, study a kind of nontoxic and cryoprotector that cell survival rate is high, just become an ovarian freezing field problem urgently to be resolved hurrily.Trehalose has avirulence and preserves cytoactive, has become cryoprotector area research focus in recent years.

Summary of the invention

In order to solve, cryoprotector in above prior art is toxic, infiltration and remove the problem of difficulty, the invention discloses a kind of containing DMSO nontoxic, easy removal, better can preserve the cryoprotector without DMSO of Histological Study.

Present invention also offers the method using the above-mentioned cryoprotector freezen protective ovary without DMSO.

The present invention is obtained by following steps:

Without a cryoprotector of DMSO, containing following component:

Trehalose 200mmol/l, PVP400.3g/mL, cholesterol 200ug/l, lecithin 200ug/l.

Described cryoprotector, also containing hyclone 0.1g/mL and RMPI-1640.

Described cryoprotector, is mixed and made into sterile suspensions by trehalose, PVP40, cholesterol, lecithin, hyclone (FBS), RMPI-1640.

A method for freezen protective ovary, comprises the following steps:

(1) pour into: to newly plucking ovary, fixed by the arteria ovarica intubate silk thread that walks abreast, after ligation arteria ovarica branch, be connected with device for casting, starting drive is with the speed of 0.5-5mL/min, to the cryoprotector according to any one of ovary perfusion claim 1-3, perfusion duration amounts to 10-60min;

(2) frozen: after perfusion; by any for Ovarian surface macroscopic immature follicle puncture; suction exenterates; then ovary is put into and the freezer bag that respective right requires the frozen solution according to any one of 1-3 is housed; lower the temperature; be set as 1. 4 DEG C of balances; 2. be down to 0 DEG C with the speed of 1 DEG C/min, and keep 10min, be 3. down to-18 DEG C with the speed of 2 DEG C/min; keep 15min; 4. be down to-45 DEG C with 2 DEG C/min speed, keep 15min, be 5. down to-90 DEG C with the speed of 5 DEG C/min again; keep 5min, drop in liquid nitrogen subsequently and deposit.

Described method, in step (1), irrigation rate is 1.3mL/min, perfusion duration 30min.

Described method, described ovary is sheep ovary.

Beneficial effect of the present invention:

1) cryoprotector of the present invention is not containing DMSO, and no cytotoxicity, easily removes, and raw material is easy to get, and preparation is simple, and cryoprotective effect is good, and normal morphology primordial follicle ratio is high, and Apoptosis quantity is few;

2) method of freezen protective ovary of the present invention, simple to operation, and controlled-rate freezing is good, better can preserve Histological Study after frozen to sheep ovary entirety.

Accompanying drawing explanation

The primordial follicle of typical normal morphology and abnormal morphology in Fig. 1 freeze thawing Hou Yang ovary cortex district,

(A) (→ place) shows normal morphology primordial follicle, spherical egg mother cell, and cell nucleus is complete and kernel is clear, and the granular cell be around evenly distributed holds.Bar=100 μm (B) (→ place) shows abnormal morphology ovarian follicle, and endochylema has included cavity (↓ place), and neighboring particles cell arrangement is disorderly, Bar=100 μm.;

Each group sheep ovary medulla regional representativeness picture (HE dyeing) Bar=100 μm under Fig. 2 (A) light microscopic, (B) respectively group stroma cell picture count quantitative analysis ^*represent and compare with fresh control group p<0.05;

Fig. 3 (A) respectively organizes the representative picture of cell of TUNEL reacting positive, and blueness is dyeed by DAPI for cell nucleus, and green is that apoptotic cell is marked colour developing by TUNEL, Bar=100 μm; (B) the cell image quantitative analysis of each group TUNEL reacting positive ^*represent and compare with fresh control group p<0.05, △ represent and to compare with trehalose group p<0.05;

Fig. 4 BAX gene and the expression of CIRP gene mRNA in each group.

Embodiment

By the following examples formulation principle of the present invention and preparation method are described further.Following embodiment is for sheep ovary.

the screening of embodiment 1 cryoprotection agent prescription

Cellular morphology complete, the integrity degree of cell membranous structure is in other words the basis ensureing cytoactive and function.People's bleeding of the umbilicus MNC is freezing, dry with rehydration experimentation, and cell membranous structure can be subject to solute in various degree because material phase transformation reason is inevitable, ice crystal damages.The cell freezing protectants such as DMSO, trehalose, PVP can reduce solute, ice crystal damage amplitude, thus Cell protection film-type structure.I sums up experimental group previous experiments, and the popularity of conjecture Cell model structure can have certain influence to the cytoactive in freezing, dry and reconstitution process.Given this, the formula of cryoprotector is screened.

experiment reagent:

Ficoll, trehalose, PVP, cholesterol, lecithin, trypan blue, Apoptosis detects AnnexinVPI double-staining kit

Experimental procedure:

One, bleeding of the umbilicus extracts

1, the 200mL duplex Cord blood collecting bag including 28mL sodium citrate anticoagulant is adopted to collect Cord Blood of Neonates.

2, Ficoll centrifugation:

A, get bleeding of the umbilicus and divide and be filled to 50mL centrifuge tube, the centrifugal 15min of 2200rpm,

B, get middle tunica albuginea layer, i.e. leukocytic cream, mix with physiological saline according to 1:2 volume ratio,

C, according to volume ratio 1:1 ratio, be filled in the 50mL centrifuge tube of ready lymphocyte separation medium, keep interface,

The centrifugal 20min of d, 2200rpm, stops without brake, ensures various aspect boundary,

E, leave and take tunica albuginea layer, physiological saline cleans 2 times, obtains bleeding of the umbilicus MNC.

Two, experiment grouping:

Following component is contained respectively in every 100mL cryoprotector in each group

1,200mmol/L trehalose+0.3g/mLPVP40,

2,200mmol/L trehalose+0.3g/mLPVP40+ cholesterol 200ug/L,

3,200mmol/L trehalose+0.3g/mLPVP40+ lecithin 200ug/L,

4,200mmol/L trehalose+0.3g/mLPVP40+ cholesterol 200ug/L+ lecithin 200ug/L,

4, the MNC will left and taken, add the cell freezing protection liquid of same volume, piping and druming mixing, divides and is filled to frozen bottle,

5, cell freezing box is freezing:

4 DEG C of balance 10min;-20 DEG C of 30min;-80 DEG C are spent the night;

Three bleeding of the umbilicus MNC related activity detect

Flow cytomery: purchase cell apoptosis detection kit (two dye), differentiates the cell division cycle and cell survival rate that detect and analyze frozen front and back.

Four, experimental result: recovering experiment fluidic cell apoptosis testing result

Every bottle to add medium 3mL centrifugal, heavily revolves adding medium 3mL, the results are shown in Table 1.

Table 1

	Reclaim cell	Living cells %	Apoptosis ing%	Downright bad %	Apoptosis %
						1	0.6×10 6	28.67	51.43	0.19	19.71
2	0.8×10 6	35.16	40.68	0.19	23.98
						3	0.8×10 6	37.40	5.76	45.09	11.45
4	0.8×10 6	44.97	4.42	44.41	8.99

Preliminary conclusion and analysis

Result after the freezing bleeding of the umbilicus MNC of experimental group recovers, display: group 1,2 living cells and be just respectively 80.10%, 75.84% in apoptotic cell ratio summation, is wherein just respectively 51.43%, 40.68% at the cell proportion of apoptosis; Organize 3,4 living cells and be just respectively 43.16%, 49.39% in apoptotic cell ratio summation, being wherein just respectively 5.76%, 4.42% at the cell proportion of apoptosis.Group 3,4 Apoptosis ratio significantly reduces, its mechanicalness till death or natural non-viable non-apoptotic cell ratio comparatively improve, organize in addition 4 live cell fraction comparatively 1,2,3 group significantly improve, bring up to 40% section by 30% section.Can infer thus, lecithin has the effect of inhibited apoptosis (mechanism is temporarily indefinite, need further experiment analysis), and cholesterol and lecithin conbined usage can play the effect of Cell protection membrane structure.

embodiment 2

(1) laboratory animalusing 28 sheep ovaries with arteria ovarica and aortal vascular pedicle as object, be placed in containing liquaemin and antibiotic that (final concentration is respectively liquaemin 0.01IU/L, penicillin 1IU/L, streptomycin 1IU/L, amphotericin B 0.25mg/L) physiological saline in, in 12 DEG C of insulating box 1h, transport laboratory back.

(2) main agentstrehalose is purchased from Chemical Reagent Co., Ltd., Sinopharm Group, and dimethyl sulfoxide (DMSO) (DMSO), PVP (Polyvinylpyrrolidone40, PVP40), lecithin are purchased from sigma company of the U.S..Cholesterol is purchased from Amresco company of the U.S..Hyclone (FetalBovineSerum, FBS) is purchased from Hyclone company of the U.S..Glycerine is purchased from Tianjin Fu Yu Fine Chemical Co., Ltd.In situ cell apoptosis detection kit (InSituCellDeathDetectionKit, POD) is purchased from ROCHE company of the U.S., and Trizol is purchased from LifeTechnology company of the U.S., and reverse transcription and amplification kit are purchased from Japanese TOYOBO company.

(3) experimental technique

3.1 grouping

A group is fresh control group, does not pour into cryoprotector; B group be trehalose group (containing trehalose 200mmol/L, PVP400.3g/mL, cholesterol 200ug/L, lecithin 200ug/L, hyclone 0.1g/mL and RMPI-1640.); C group is glycerine protectant group, and perfusion liquid is that 15% glycerine is linearly increased in 60% glycerine, and glycerine is dissolved in distilled water, and perfusion liquid also contains the NAHCO that final concentration is the glucose of 32.5g/L, 0.85g/L ₃, the KH of the KCI of 2.125g/L, 0.975g/L ₂pO ₄, the MgCl of 0.2g/L ₂6H ₂o; D group is DMSO group, and perfusion liquid is the RMPI-1640 liquid containing 10%DMSO and 10%FBS.28 sheep ovaries that experiment obtains are assigned in four groups at random, often organize each 7 ovaries.

3.2 perfusions are to each group of ovary; fixed by the arteria ovarica intubate silk thread that walks abreast; after ligation arteria ovarica branch; with start the gradient concentration mixing Programmed freezing organ perfusion instrument reaching 4 DEG C that freezes in advance and be connected; starting drive and quantitatively liquid feeding pump, according to above-mentioned grouping, with the speed of 1.3mL/min; pour into corresponding cryoprotector to each group of ovary, perfusion duration amounts to 30min.Irrigation rate can be 2mL/min,

3.3 frozen before, by any for Ovarian surface macroscopic immature follicle puncture, suction exenterates.Then the ovary be disposed is put into 50mL freezer bag (U.S. sky Ni that corresponding frozen solution is housed, Germany) in, and then insert sequencing frigorimeter (ThermoScientificForma, the U.S.) in, cooling process (ControledRateFreezingVersion1.0, the U.S.) be set as 1. 4 DEG C of balances, 2. 0 DEG C is down to the speed of 1 DEG C/min, and keep 10min, 3.-18 DEG C are down to the speed of 2 DEG C/min, keep 15min, 4.-45 DEG C are down to 2 DEG C/min speed, keep 15min, 5.-90 DEG C are down to the speed of 5 DEG C/min again, keep 5min.Subsequently the frozen bag that ovary is housed is dropped into and deposit at least one Zhou Houhang in liquid nitrogen and recover.

3.4 recovery samples are placed on 37 DEG C of water-baths from liquid nitrogen taking-up and are melted up to mixture of ice and water, moment shake prevents local temperature too high, again arteria ovarica intubate is connected with device for casting after melting completely, control perfusion liquid temperature and be 4 DEG C and Continuous Perfusion contains the RMPI-1640 liquid of 10%FBS, the velocity perfusion 30min of 1.3mL/min.

(4) index determining and analytical method

B, C, D group ovary after A group and recovery is placed in formalin and fixes by 4.1 morphological analysis, 4 μm of thick serial section after transparent, the paraffin embedding of dewatering, row HE dyeing and TUNEL staining analysis.According to Gougeon criteria for classification, the simple squamous granular cell that primordial follicle is held by a first oocyte and surrounding forms.Under microscope, HE section observed and counted primordial follicle, normal morphology primordial follicle and abnormal morphology primordial follicle can be divided into according to its form.It is spherical in shape that normal morphology primordial follicle shows as egg mother cell, includes the cell nucleus of complete circle, and the monolayer of particles cell be around evenly distributed holds; Paramorph ovarian follicle shows as karyopyknosis, and basilar memebrane is imperfect, and neighboring particles cell arrangement is disorderly.Often open random counter 10 visuals field under high power lens of cutting into slices, often organize counting 7 section and amount to primordial follicle sum in 70 visuals field and normal morphology primordial follicle number, normal morphology primordial follicle ratio is: normal morphology primordial follicle number/primordial follicle sum × 100%.Ovary medulla part equally under high power lens to often opening section random shooting 5 high power fields, its stroma cell density is by ImageJ1.42q (download of http://rsbweb.nih.gov/ij/index.html official website) count fine karyon.

The typical picture of frozen rear normal morphology primordial follicle and abnormal morphology primordial follicle is shown in Fig. 1, and the primordial follicle number of each group normal morphology and normal morphology ovarian follicle ratio are in table 1.Fresh control group normal morphology primordial follicle ratio (93.46 ± 2.30%) is significantly higher than glycerine group (88.03 ± 4.80%) and DMSO group (80.85 ± 2.28%), difference have statistical significance ( p<0.05).Trehalose group (90.29 ± 2.30%) normal morphology primordial follicle ratio and fresh control group there was no significant difference ( p>0.05).

The normal morphology rate of table 1 four groups of primordial follicles compares

Group	Primordial follicle sum	Normal morphology primordial follicle number	Normal morphology primordial follicle ratio (%)
				Fresh group	243	227	93.46±2.30%
Trehalose group	220	198	90.29±2.30%
				Glycerine group	246	217	88.03±4.80%*
DMSO group	255	206	80.85±2.28%**

^*represent and compare with fresh control group p<0.05, ^*represent and compare with fresh control group p<0.01

Stroma cell density is as Fig. 2, by ImageJ software, it is quantized, fresh control group (1114.68 ± 184.24/400 × visual field) stroma cell density higher than B, C, D tri-groups frozen group, itself and DMSO group (906.14 ± 304.78/400 × visual field) difference have statistical significance ( p<0.05).Trehalose group (1020.83 ± 278.39/400 × visual field) and glycerine group (1089.38 ± 573.13/400 × visual field) stroma cell density and fresh group of no significant difference ( p> 0.05).

4.2tdT-mediated-dUTP nick end labeling technology (TUNEL) detects apoptotic cell dyeing, is detected by In situ cell apoptosis detection kit (InSituCellDeathDetectionKit, POD).According to the requirement of kit specification, ovary tissue section dewaxing aquation is placed in the 10mMTris-HCl solution containing 100g/L, hatches 30min for 37 DEG C, adds TUNEL reaction mixture after PBS washes, hatch 60min for 37 DEG C, negative control replaces TdT enzyme solutions with PBS.And redye cell nucleus by DAPI.In fluorescence microscopy Microscopic observation, detect within the scope of green fluorescence, apoptotic cell is green fluorescence, normal cell redgreen fluorescence excitation.Detect within the scope of blue-fluorescence, cell nucleus all shows blueness.Often open section and get 5 high power field pictures at random, the apoptosis degree of each group ovary tissue is evaluated by ImageJ software counting apoptotic cell cell nucleus.

From Fig. 3 (A), after ovary tissue freeze thawing, apoptosis mainly concentrates on ovary cortex part.Can be found the cell counting of TUNEL reacting positive by fluorescence microscope and ImageJ software, see Fig. 3 (B), fresh control group (13.86 ± 13.22/400 × visual field) apoptosis number is obviously less than three groups freezing group, difference has statistical significance (P<0.05), in frozen group, trehalose group (73.14 ± 32.01/400 × visual field) apoptosis cell is less than glycerine group (166.43 ± 60.88/400 × visual field) and DMSO(250.29 ± 85.90/400 × visual field) group, difference has statistical significance (P<0.05).

4.3real-time fluorescence quantitative PCR detects expression design of primers and the synthesis of BAX and CIRP gene: reference gene selects GAPDH, and each gene searches gene order by GenBank, and design and the synthesis of primer are completed by Shanghai Sheng Gong biotechnology Co., Ltd.Primer sequence is as follows: GAPDH:Forward:5 '-ATCGCTCTCTTTGTCGGAAG-3 ', Reverse:5 '-GCAGAGCATTTATTCCGTCCC-3 ', BAX:Forward:5 '-GAGTGGCGGCTGAAATGTT-3 ', Reverse:5 '-AAGTCCAAGGCAGTTGATGG-3 ', CIRP:Forward:5 '-ATCGCTCTCTTTGTCGGAAG-3 ', Reverse:5 '-TCTACTCTGCCTGCCTCAAG-3 '.Total RNAs extraction and cDNA synthesis: under the environment without RNA enzyme, extract total tissue RNA by Trizol method, add the mixing of each reagent by the specification of cDNA Reverse Transcriptase kit in each pipe.Reaction condition: 37 DEG C of 15min, 98 DEG C of 5min, 4 DEG C of preservations.Real-time quantitative PCR: 10 μ LSYBRGreenRealtimePCRMasterMix, a upstream and downstream primer 0.8 μ L, 2.0 μ LcDNA templates, 6.4 μ L tri-distilled water sterilizings.Adopt ABI company quantitative real time PCR Instrument (ABI7500, the U.S.) amplification, 7500SystemSDSSoftware(Version1.4, the U.S.) analysis result.Reaction condition: 95 DEG C of 60s; 95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C 55s40 circulation; 95 DEG C of 15S, 60 DEG C of 1min, 90 DEG C of 15S.

This experiment adopt Real-timePCR method detect respectively fresh group, trehalose group, glycerine group and DMSO group ovary tissue BAX and CIRP mrna expression situation (see figure 4), result shows: the expression of fresh control group and the relative GAPDH of trehalose group BAXmRNA is respectively 0.016 ± 0.019 and 0.026 ± 0.024, be starkly lower than the relative expression quantity 0.081 ± 0.075,0.099 ± 0.125 of glycerine group and DMSO group, difference have statistical significance ( p<0.05), the expression of the relative GAPDH of mRNA of the CIRP gene of three groups frozen group all higher than fresh control group (0.164 ± 0.121), have significant difference ( p<0.05), and the mRNA relative expression quantity (0.512 ± 0.226) of trehalose group CIRP gene is apparently higher than glycerine group (0.252 ± 0.129) and DMSO group (0.224 ± 0.093), difference have statistical significance ( p<0.05). ^*represent and compare with fresh control group p<0.05, ^*represent and compare with fresh control group p<0.01, △ represent and to compare with trehalose group p<0.05.

(5) statistical analysis technique

Experiment the data obtained all adopts mean ± standard deviation to represent, ImageJ software counting cells, statistics software SPSS19.0 carries out data analysis, between the multisample that variance is neat, mean compares employing one-way analysis of variance, heterogeneity of variance person adopts rank test, is all that difference has statistical significance with P<0.05.

Conbined usage of the present invention trehalose, PVP40; add the use of cholesterol and lecithin, do not have cytotoxicity containing DMSO(), application trehalose associating cholesterol and lecithin; the lipid on better promotion Cell protection film surface and carbohydrate, make cell membrane reach a kind of stable poised state.And PVP and trehalose use in conjunction, by the hydrogen bond action between carbohydrate, change the vitrification point of carbohydrate.

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not by the restriction of embodiment; other is any do not deviate from Spirit Essence of the present invention and principle under make change, modification, combination, substitute, simplify and all should be equivalent substitute mode, be included within protection scope of the present invention.

Claims (6)

1., without a cryoprotector of DMSO, it is characterized in that containing following component:

Trehalose 200mmol/L, PVP400.3g/mL, cholesterol 200ug/L, lecithin 200ug/L.

2. cryoprotector according to claim 1, is characterized in that also containing hyclone 0.1g/mL and RMPI-1640.

3. cryoprotector according to claim 1, is characterized in that trehalose, PVP40, cholesterol, lecithin, hyclone and RMPI-1640 to be mixed and made into sterile suspensions.

4. a method for freezen protective ovary, is characterized in that comprising the following steps:

(1) pour into: to newly plucking ovary, fixed by the arteria ovarica intubate silk thread that walks abreast, after ligation arteria ovarica branch, be connected with device for casting, starting drive is with the speed of 0.5-5mL/min, to the cryoprotector according to any one of ovary perfusion claim 1-3, perfusion duration amounts to 10-60min;

(2) frozen: after perfusion; by any for Ovarian surface macroscopic immature follicle puncture; suction exenterates; then ovary is put into and the freezer bag that respective right requires the frozen solution according to any one of 1-3 is housed; lower the temperature; be set as 1. 4 DEG C of balances; 2. be down to 0 DEG C with the speed of 1 DEG C/min, and keep 10min, be 3. down to-18 DEG C with the speed of 2 DEG C/min; keep 15min; 4. be down to-45 DEG C with 2 DEG C/min speed, keep 15min, be 5. down to-90 DEG C with the speed of 5 DEG C/min again; keep 5min, drop in liquid nitrogen subsequently and deposit.

5. method according to claim 4, is characterized in that in step (1), irrigation rate is 1.3mL/min, perfusion duration 30min.

6. method according to claim 4, is characterized in that described ovary is sheep ovary.