CN104004708A - Method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells - Google Patents
Method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells Download PDFInfo
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Abstract
The invention relates to a method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells. The method includes the steps that various solutions are prepared, 2-3 g of tissue blocks of the seminiferous tubule are taken out from the testis of a sheep of 3-4 mouths of age, single-cell suspension is prepared through enzymolysis and digestion of two steps, purified spermatogonia stem cells are separated according to the difference adherence method, the long-term passage cultivation of the sheep spermatogonia stem cells is achieved through an SSCM culture solution, and the sheep spermatogonia stem cells are cryopreserved and recovered after the long-term passage cultivation. By the technical scheme, the sheep spermatogonia stem cells with high purity can be obtained without using specific sheep spermatogonia stem cell antibodies, a long-term passage cultivation system of the sheep spermatogonia stem cells is built, and the method for preserving the sheep spermatogonia stem cells for a long term through a liquid nitrogen freezing method is successfully provided. The sheep spermatogonia stem cells achieving separation, purification and passage according to the method are cultivated at least for more than 40 generations, and the longest cultivation period reaches four years if the cryopreservation time is included.
Description
Technical field
The present invention relates to the biological technical fields such as domestic animal germplasm resource conservation and improved seeds are bred, tissue and organ regeneration, be specifically related to the method for the separation and purification of a kind of sheep stem spermatogonium, the long-term cultivation that goes down to posterity, frozen and recovery.
Background technology
Stem spermatogonium (SSCs) is one of most important adult stem cell.Stem spermatogonium not only has the Genetic Function of producing a male heir to continue the family line, and recently studies show that, stem spermatogonium is easy to produce multipotential stem cell by moving back differentiation, and this makes stem spermatogonium operative technique obtain showing appreciation for somebody of life science, agricultural sciences and medical science each road expert.Since spermatogonial stem cell transplantation technology is set up, the research of stem spermatogonium starts to accelerate, and after 2000, has set up the external long-period culture method of Mouse and rat stem spermatogonium, and stem spermatogonium fundamental research and applied research have all obtained development rapidly.On the one hand, the stem spermatogonium of cultivation is transplanted to acceptor testis again through genetic modification, produces genetically modified sperm, and then breed transgenic animal by during spermatogenesis.On the other hand, the stem spermatogonium of cultivation produces reproduction multipotential stem cell by moving back differentiation, for regenerative medicine research, can be applied to the Regeneration and Repair of the histoorgan of damage.Therefore stem spermatogonium biology of reproduction research, transgenic animal technical study, move back induction produce multipotential stem cell research, animal germ plasm resource preserve study and histoorgan clinical rehabilitation medical research on significant.
In genetic breeding field, paternal inherited character is by stem spermatogonium, to break up the sperm producing to pass to offspring's by fertilization.The in vitro study of sheep breeding in the past and sheep variety improvement (or claiming test tube research) concentrates on ovocyte.Along with closely during the last ten years, to stem spermatogonium progress of research, sheep stem spermatogonium propagation and the function and significance of differentiation research in sheep breeding and sheep variety Upgrading are more and more obvious.By spermatogonial stem cell transplantation, will provide new technological line for the prepotent utilization of sheep hybrid.Because often there are some practical difficulties in the practical application of crossbreeding technology, such as must first forming and retain a large amount of populations (kind or strain) independently separately, this needs a large amount of work and cost, if but adopted the stem spermatogonium of the outstanding ram of good character as variety source, so just problem reduction.As long as prepare stem spermatogonium with this ram testis, breed also freezing preservation, Here it is a good sheep variety.While needing, just its recovery is cultivated, then acceptor ram is carried out to spermatogonial stem cell transplantation, will produce the sperm of the good sheep of our needs.As long as retain the stem spermatogonium of good ram, just retained a good sheep variety, saved the work of a large amount of populations of reservation of time and effort consuming.In addition, another difficult problem of cross-breeding work is, natural condition or raising condition between native breed and external improved seeds differ greatly, be difficult to be cross-breeding, if but prepare acceptor in local sheep variety, with external improved seeds, prepare stem spermatogonium, then the stem spermatogonium of external good sheep variety is injected to local sheep acceptor testis, by local acceptor ram, produced the sperm of external improved seeds, for hybridizing with native breed ewe, expection can the good sheep hybrid generation of acquired character.
But the separation and purification of sheep stem spermatogonium and long-term cultivation remain world-famous puzzle so far.Stem spermatogonium is studied once published thesis for 2008 foundation of large animal stem spermatogonium long-period culture method of study group that foremost expert Brinster leads also needs the research of 5 to 10 years.Although the stem spermatogonium long-period culture method of muroid is set up, due to race differential, gene pool difference, commercially available antibody can not be general etc. the existence of many factors, the long-period culture method of sheep stem spermatogonium can not be indiscriminately imitated the method for muroid.In view of sheep stem spermatogonium long-term cultivation technical meaning great, not only there is theory value, in husbandry with medically also have that Economic Application is worth and social value, beautiful, Europe, day, Australia, Han Deng state all has scientific research organization to be devoted to the research of sheep stem spermatogonium long-term cultivation, once the purifying that had sheep stem spermatogonium, the report of evaluation aspect, but also exist separating and purifying technology complicated, the stem spermatogonium being separated to is few, the problem that purity is not high, particularly a whole set of sheep stem spermatogonium separation and purification, long-term cultivation goes down to posterity, technological operation system frozen and recovery is not also successfully reported.
In addition, current stem spermatogonium separation purification method mainly contains: the methods such as cryptorchidism operation concentration method, enzymolysis process, Percoll density gradient centrifugation, flow cytometry separating method, immunological magnetic bead sorting method, difference adherent method etc.Flow cytometry separating method and immunological magnetic bead sorting method effect in muroid stem spermatogonium is better, but two kinds of methods all need to have the antibody with the fine combination of stem spermatogonium surface antigen specificity of studied species, and the antibody that is applicable at present sheep stem spermatogonium is uncommon, therefore in the research of sheep stem spermatogonium, be not suitable for using flow cytometry separating method and immunological magnetic bead sorting method.And cryptorchidism operation concentration method and Percoll density gradient centrifugation all can not obtain highly purified stem spermatogonium, and find that in practice Percoll density gradient centrifugation has impact to the vigor of stem spermatogonium, successfully in stem spermatogonium culture technique, also do not adopting the report of these two kinds of methods up to now.
At present existing art technology researchist uses the report of the separated stem spermatogonium of enzymolysis process+difference adherent method success, but the key of using enzymolysis process+difference adherent method is the stem spermatogonium containing in the single-cell suspension liquid of preparation and wants purity high, quantity is many, active good, and the weave construction of different animals Testicular Seminiferous Epithelium cell each is variant, use enzymolysis process digestion perienchyma, while discharging sustenticular cell and stem spermatogonium, the kind of digestive ferment and concentration, action intensity (rotating speed), digestion time, it is all the factor that affects digestible degree that damping fluid becomes to grade, any one factor is improper, all affect the digestion of seminiferous epithelium and discharge stem spermatogonium, cause the stem spermatogonium quantity in single-cell suspension liquid few of poor quality, finally by difference adherent method purifying, can not obtain the good stem spermatogonium of vigor of sufficient amount, even if prepared high-quality single-cell suspension liquid, in the operating process of purifying stem spermatogonium, the concrete operation methods such as separation sequence, disengaging time, washing time, flushing dynamics are all again the factors of the last stem spermatogonium quality and quantity of collecting of impact, grasp when bad and can not obtain the stem spermatogonium of high vigor in the same old way.Therefore, although enzymolysis process+difference adherent method is fairly simple from principle, the apparatus and the reagent that use are also all more conventional, but because species are different, the requirement of each operation link is different, experimenter's operating habit is different, can directly affect the separation and purification effect of stem spermatogonium.The operational details of visible each step is all very important, a thousand li of the least bit mistake of difference, Success Determined by Details.Exactly because do not catch details, cause by the method for enzymolysis process+difference adherent method success separation and purification sheep stem spermatogonium and also successfully do not report.In the scientific and technical literature of certain species stem spermatogonium of use enzymolysis process+difference adherent method separation and purification of having reported both at home and abroad at present, only emphasize the mechanism of action of enzymolysis process and difference adherent method, and ignore each operational details, but stem spermatogonium is to have bioactive cell, any externalities all can discharge to the separation of cell, keep active or dead etc. and have a direct impact, therefore with reference to these scientific and technical literatures, often carry out test of many times, be also difficult to successfully separation and purification and go out stem spermatogonium.
Summary of the invention
For addressing the above problem, the present invention proposes a kind of method of sheep stem spermatogonium separation and purification of refinement, the long-term cultivation that goes down to posterity, frozen and recovery, aim to provide a kind of specific sheep stem spermatogonium antibody that do not need, use the method system of enzymolysis process+difference adherent method separation and purification sheep stem spermatogonium, therefrom the stem spermatogonium purity of acquisition is high, quantity is many, vigor good, be easy to cultivation, on this basis to sheep stem spermatogonium go down to posterity long-term cultivation, frozen and recovery, thereby set up a whole set of operative technique system.
The method of a kind of sheep stem spermatogonium of the present invention separation and purification, comprises the following steps:
The first step: obtain solution:
1) antibacterial-anti-mycotic agent that is 0.1% by volume percent adds in DMEM/F12 nutrient solution makes nutrient solution A, in two weeks, uses;
2) IV Collagenase Type is added and in DMEM low sugar nutrient solution, makes IV Collagenase Type storing solution ,-20 ℃ of preservations with the concentration of 20mg/ml;
3) Unidasa is added in DMEM low sugar nutrient solution and makes Unidasa storing solution ,-20 ℃ of preservations with the concentration of 10mg/ml;
4) DnaseI is added to DMEM low sugar nutrient solution with the concentration of 5mg/ml, then to add volume percent be 20% glycerine, make DnaseI storing solution ,-20 ℃ of preservations;
5) Trypsin that is 0.5% by volume percent and concentration are that 2mM EDTA is dissolved in and in PBS damping fluid, makes Trypsin/EDTA storing solution ,-20 ℃ of preservations;
6) foetal calf serum that is 10% by volume percent and 0.1% antibacterial-anti-mycotic agent adds and in DMEM/F12 nutrient solution, makes nutrient solution C, in two weeks, use;
7) horse serum that is 5.5% by volume percent, 2.4% foetal calf serum and 0.1% antibacterial-anti-mycotic agent adds in DMEM/F12 nutrient solution and makes nutrient solution B, in two weeks, use;
8) mercaptoethanol of 30 μ Μ is added in nutrient solution C, filtration sterilization is made nutrient solution D, now with the current;
9) bovine serum albumin that is 0.52% by volume percent is dissolved in PBS damping fluid, and filtration sterilization is made PBS-BSA solution, now with the current;
Second step: utilize sheep testicle tissue preparation single-cell suspension liquid, under aseptic condition, operation steps is as follows successively:
1) from the sheep testicle at 3-4 monthly age, get 2-3g convoluted seminiferous tubule tissue block, put into the 100ml culture dish that is loaded with 2.5ml nutrient solution A, with tweezers, remove albuginea testis in convoluted seminiferous tubule tissue block and larger blood vessel;
2) last tissue block is put into 20ml bottle, with ophthalmology scissors straight, shred, continue to cut 3 minutes;
3) tissue block shredding is transferred in 100ml jar with cover, added nutrient solution A to 7.5ml;
4) carry out the first step enzymic digestion, concrete operations are as follows: in above-mentioned 100ml jar with cover, add the IV Collagenase Type storing solution of 0.63ml, be placed in 37 ℃ of shaking baths, with the rotating speed of 90rpm, sway 40min; Add subsequently the Unidasa storing solution of 0.825ml and the DnaseI storing solution of 0.125ml, continue to be placed in 37 ℃ of shaking baths, with the rotating speed of 90rpm, sway 20min, with 10ml transfer pipet, leniently inhale and blow suspended substance 5 times up and down; Collagenase digesting 40min is first used in this operation, then adds Unidasa, and two kinds of enzyme acting in conjunction 20min, can obtain good digestion effect, can make the membranin of convoluted seminiferous tubule obtain the digestion of larger limit, can not cause larger damage to pipe inner cell again;
5) from shaking bath, take out in 100ml jar with cover, with 10ml transfer pipet, leniently inhale and blow suspended substance 5 times up and down;
6) suspension in above-mentioned 100ml jar with cover is proceeded to 50ml centrifuge tube, the centrifugal 6min of rotating speed with 2000rpm, removes supernatant liquor;
7) in step 6) add the PBS damping fluid of 12ml in described centrifuge tube, shake up and make throw out Eddy diffusion, the centrifugal 6min of rotating speed with 2000rpm, removes supernatant liquor;
8) operation of repeating step 7 once;
9) in step 8) add the nutrient solution A Eddy diffusion throw out of 0.75ml in described centrifuge tube, add again the Trypsin/EDTA storing solution of 1ml and the DNaseI storing solution of 0.25ml, be placed in 37 ℃ of shaking baths, rotating speed with 90rpm sways 10min, with 10ml transfer pipet, leniently inhales and blows suspended substance 5 times up and down; Can the peptic cell outer attachment proteins of Trypsin/EDTA, DNaseI can remove long-chain DNA molecular, reduces the viscosity of cell suspending liquid, and these two kinds of solution actings in conjunction are conducive to form single-cell suspension liquid;
10) immediately in step 9) add the nutrient solution C of 10ml in described centrifuge tube, with 10ml transfer pipet leniently inhale up and down blow suspended substance until significantly block disappear; Foetal calf serum in nutrient solution C can stop enzymic digestion reaction, stops damaged membrane, maintains sexual cell vigor;
11) by step 10) described centrifuge tube is with the centrifugal 6min of rotating speed of 2000rpm, removes supernatant liquor, then uses 2ml nutrient solution C re-suspended cell; Guarantee that digestion reaction thoroughly stops, guarantee sexual cell vigor;
12) first with 200 orders, then with 300 order nylon filters, distinguish filtration steps 11) described cell suspending liquid; Remove and do not digest fragment of tissue and cell mass completely;
13) by step 12) cell suspending liquid after described filtration leniently inhales and blows 5-10 time with 10ml transfer pipet, carries out cell counting, then adds nutrient solution C adjustment cell density to 1.25x10
6cell/ml, makes single-cell suspension liquid; In single-cell suspension liquid now, be mainly somatocyte and sexual cell;
The 3rd step: purifying stem spermatogonium from the single-cell suspension liquid of separation and Culture described in second step, operation steps is as follows successively:
1) by 10ml second step step 13) described single-cell suspension liquid is laid on the coated culture dish of gelatin that diameter is 100mm, is placed on 32.5 ℃, 5.5%CO
2, in the cell culture incubator of saturated humidity, cultivate 2 days half to 3 day half;
2) with 5ml transfer pipet from step 1) described in leniently remove all nutrient solutions culture dish;
3) first with nutrient solution A, wash cell: from the edge of culture dish, slowly add the nutrient solution A of 4ml preheating, leniently rock back and forth culture dish 1 time, then with transfer pipet, remove nutrient solution; This operation is for washing away the dead cell of culture dish;
4) with PBS damping fluid, wash cell twice again: from the edge of culture dish, slowly add the PBS damping fluid of 4ml preheating, leniently rock back and forth culture dish 1 time, with transfer pipet, remove supernatant liquor immediately, then repetitive operation once; This operation continues to wash away the dead cell in culture dish; Now somatocyte is adsorbed by gelatin, and sexual cell is hung on somatocyte;
5) add 4ml nutrient solution A to culture dish, with transfer pipet suction, blow nutrient solution A and rinse gently the whole surface of culture dish about 2 times; Flushing ability by nutrient solution A to sexual cell, can be flushed to the sexual cell being hung on somatocyte in washing fluid, so now somatocyte is still adsorbed by gelatin, and in washing fluid, major part is sexual cell;
6) by step 5) washing fluid in described culture dish transfers in aseptic 50ml centrifuge tube;
7) by step 6) described centrifuge tube is in the centrifugal 6min of 2000rpm, removes supernatant liquor, retains cell precipitation;
8) in step 7) add the nutrient solution B re-suspended cell of 4ml in described centrifuge tube;
9) in the coated culture dish of collagen I, add 4ml nutrient solution B, repave step 8) described cell suspending liquid, then culture dish is put into 32.5 ℃, 5.5 ﹪ CO
2, saturated humidity cell culture incubator in hatch 2 hours;
10) from incubator, take out culture dish, with 5ml transfer pipet, leniently on culture dish surface, repeatedly inhale and blow 5-10 time, make cytomixis, then culture dish is put back in incubator and continued to cultivate 2 hours;
11) from incubator, take out after culture dish, with 5ml transfer pipet, in culture dish, leniently inhale and blow cell suspending liquid, until rinsed whole culture dish surface, then from culture dish, collect the non-adsorbable cell suspending liquid of collagen I with 5ml transfer pipet, all transfer in 50ml centrifuge tube; The cell major part of at this moment being adsorbed by collagen I is the sexual cell of differentiation, and the major part not being adsorbed in cell suspending liquid is stem spermatogonium;
12) by step 11) described centrifuge tube centrifugal 6min under 2000rpm, sedimentation cell, removes supernatant liquor, with 4ml nutrient solution D suspension cell, then draws cell suspending liquid by 200 order nylon filters, with 50ml test tube, collects filtrate; In nutrient solution D, added beta-mercaptoethanol, there is antioxygenation, stem spermatogonium has been had to provide protection;
13) promptly by step 12) filtrate that obtains blows 4-5 time with the suction of 5ml transfer pipet gentleness, then the filtrate mixing is spread to the 12 coated well culture plates into Laminin by every hole 1ml, wherein said culture plate is prepared in advance, facing the used time first removes the supernatant liquor in each hole, add again 1ml nutrient solution A to clean, just spread into above-mentioned filtrate after removing all nutrient solution A; The stem spermatogonium that this step operation must could protect separation to obtain rapidly better;
14) by step 13) described culture plate is placed in 32.5 ℃, 5.5 ﹪ CO
2in the cell culture incubator of saturated humidity, hatch 20-40 minute, then take out, with the suction as mild as a dove of 1ml micropipet, blow surperficial one time of the culture hole of described culture plate, cell suspending liquid levels in hole is mixed, then put back to rapidly and in incubator, continue to hatch 10-30 minute; Incubation time is very crucial, the long or short quality and quantity that all can directly affect the stem spermatogonium of final purification acquisition;
15) by step 14) described culture plate taking-up, with 1ml micropipet, inhale and blow once, cell suspending liquid in hole is mixed, now the cell in suspension is not adherent cell of Laminin, these cell suspending liquids are removed from culture hole, then, in culture hole, added immediately the freshly prepared nutrient solution D of 1ml to wash culture hole surface one time, then remove the nutrient solution D in culture hole, again in hole, add the freshly prepared nutrient solution D of 1ml immediately.Nutrient solution D wants gentle and drips equably the whole surface in hole, and the cell adhering in hole is Laminin adherent cell; Now Laminin adherent cell is exactly stem spermatogonium, and the Laminin removing not adherent cell be the sexual cell having broken up;
16) with 1ml micropipet from step 15) remove nutrient solution D described culture hole, then in culture hole, add 1ml PBS-BSA solution immediately, then culture plate is put into 32.5 ℃, 5.5 ﹪ CO
2, in the cell culture incubator of saturated humidity, hatch 3-6 minute, in the sterile test tube of a 50ml, add 4ml nutrient solution D standby simultaneously;
17) by step 16) described culture plate taking-up, use immediately 1ml transfer pipet gentle pressure-vaccum PBS-BSA solution 5-10 time in culture hole, make Laminin adherent cell from the de-wall of culture hole, the PBS-BSA solution that collection contains Laminin adherent cell, transfers to the PBS-BSA solution that contains Laminin adherent cell in the 50ml centrifuge tube that contains nutrient solution D; PBS-BSA solution can be from Laminin flat board the stem spermatogonium that adheres to of wash-out;
18) by step 17) described centrifuge tube centrifugal 6min under 2000rpm rotating speed, remove supernatant liquor, reversing test tube, leniently removes remaining liquid;
19) in step 18) add 1ml nutrient solution D Eddy diffusion cell in described centrifuge tube, with cell counting count board, measure cell concn;
20) by dyeing immunofluorescence cell method authentication step 19) cell in described cell suspending liquid is exactly sheep stem spermatogonium.
As long as operate in strict accordance with above-mentioned sheep stem spermatogonium separation purification method, the researchist of any Cell Lab can successful separation and purification go out stem spermatogonium, and through different experiments chamber different operating personnel, the operating practice that comprises professor and common postgraduate shows, adopt above method all can successful separation and purification go out sheep stem spermatogonium, the sheep stem spermatogonium purity of preparation is more than 90%.
The long-period culture method that goes down to posterity to above-mentioned sheep stem spermatogonium, comprises the following steps:
The first step: obtain solution:
1) foetal calf serum that is 7% by volume percent adds makes STO cell culture fluid in DMEM/F12 nutrient solution;
2) foetal calf serum that is 20% by volume percent, 10% dimethyl sulfoxide (DMSO) add makes STO cells frozen storing liquid in DMEM/F12 nutrient solution;
3) in lmlSSCB solution, the anti-mycotic agent storage liquid 1 μ l of Streptomycin sulphate storage liquid 1 μ l, 2.5mg/ml of penicillin storage liquid 1 μ l, 100mg/ml of glial cellline-derived neurotrophic factor receptor alpha 1 sample protein antibodies storage liquid 1 μ l, 100kU/ml of Prostatropin storing solution 1 μ l, 100ng/ μ l that adds respectively glial cell line-derived neurotrophic factor storing solution 1 μ l-2 μ l, the 20ng/ μ l of 20ng/ μ l, mixes and makes SSCM nutrient solution; SSCM is the nutrient solution of preparing for domestic animal stem spermatogonium specially, and long-term practice proof can effectively be cultivated stem spermatogonium;
The component that wherein said 1 liter of SSCB solution comprises is:
Second step: preparation trophocyte, successively operation as follows:
1) trophocyte's is prefabricated: with STO cell culture fluid, cultivate STO cell, when cell degree of converging reaches 70%-80%, by final concentration, 15 μ g/ml add mitomycin, put into cell culture incubator, at 37 ℃, 5%CO
2, saturated humidity condition under cultivate 2 hours, then remove the nutrient solution that contains mitomycin, and wash three times with 10mlPBS, with Trypsin/EDTA Digestive system described in 1ml claim 1, make cell take off wall again, and the digestion that stops Trypsin with nutrient solution C described in 5ml claim 1, collect postdigestive cell, the centrifugal 5min of 2000rpm, getting the cell of precipitation, is 6 * 10 by concentration
5the concentration of cell/mL adds STO cells frozen storing liquid, and liquid nitrogen is preserved; After processing like this, STO cell has just lost differentiation capability, can only survive for some time, for stem spermatogonium provides nutrient environment (trophoderm), then just dies, and can not survive together with stem spermatogonium.
2) prepare trophocyte: by step 1) the STO cell processed of described mitomycin, with 2 * 10
5the final concentration of cell/mL is seeded in cell in the 12 porocyte culture plates of being processed by gelatin in advance, at 37 ℃, and 5%CO
2, under the condition of saturated humidity, cultivate 12-24 hour;
The 3rd step: the long-term cultivation that goes down to posterity sheep stem spermatogonium, operation steps is as follows:
1) the former culture of stem spermatogonium: remove second step step 2) in described 12 porocyte culture plates in STO trophoderm containing serum free culture system liquid, and wash twice with PBS damping fluid, then use SSCM nutrient solution resuspended the good stem spermatogonium of above-mentioned separation and purification, with 5 * 10
4the concentration of cell/mL is inoculated into be completed in the trophoblastic 12 porocyte culture plates of STO, then culture plate is placed on to 37 ℃, 5%CO
2, saturated humidity cell culture incubator in, second day changes SSCM nutrient solution one time, changes one time SSCM nutrient solution every 2 days later; In culture plate, be now the stem spermatogonium of former culture;
2) long-term cultivation that goes down to posterity of stem spermatogonium: by step 1) stem spermatogonium of described former culture is cultivated 6-9 days, take out culture plate, remove SSCM nutrient solution, remove floating cell, the SSCM nutrient solution that adds the fresh configuration of 1ml in culture hole, and with 1mL pipette pressure-vaccum gently, stem spermatogonium is departed from from trophocyte, stem spermatogonium suspension is transferred to 50ml centrifuge tube, repetitive operation 1-2 time, to the centrifugal 5min of 2000rpm for centrifuge tube of stem spermatogonium suspension be housed, abandon supernatant, with SSCM nutrient solution re-suspended cell, be inoculated on new STO trophocyte, first three after former culture time goes down to posterity and carries out with the ratio of 1:1 or 1:1.5, go down to posterity afterwards and carry out with the ratio of 1:2-1:3, every 2 days, change one time SSCM nutrient solution, cultured stem spermatogonium carries out the frozen or cultivation that continues to go down to posterity as required.
Sheep stem spermatogonium to the above-mentioned long-term cultivation that goes down to posterity carries out frozen method, comprise the following steps: by the stem spermatogonium of the above-mentioned long-term cultivation that goes down to posterity, with the leniently piping and druming gently of 1mL pipette, stem spermatogonium is departed from from STO trophocyte, collect stem spermatogonium to 50ml centrifuge tube, carry out cell counting, then the centrifugal 5min of 2000rpm, abandon supernatant, with Sigma serum-free cell frozen storing liquid re-suspended cell and adjust concentration to 5 * 10
4-2 * 10
5cell/ml, divides and installs in cryopreservation tube, places 30min for 4 ℃, places 24h, proceeds to afterwards the medium-term and long-term preservation of liquid nitrogen container for-80 ℃.
The method that above-mentioned frozen sheep stem spermatogonium is recovered, comprise the following steps: above-mentioned cryopreservation tube is taken out from liquid nitrogen container, in 37 ℃ of water-baths, melt 3 minutes fast, cell is proceeded in the 50ml centrifuge tube that contains 10mlSSCM nutrient solution to centrifugal 6 minutes of 2000rpm in Bechtop, abandon supernatant liquor, throw out is resuspended in 1-3mlSSCM nutrient solution, transfers to 12 well culture plates, every hole 1ml, be placed in 37 ℃, 5%CO
2, in the cell culture incubator of saturated humidity, every 2 days, change one time SSCM nutrient solution, cultivate 2-3 days.
Beneficial effect of the present invention is:
1. do not need to use the antibody of specific sheep stem spermatogonium surface antigen just can obtain the sheep stem spermatogonium that purity is higher: to adopt separation purification method provided by the invention, avoided carrying out purifying stem spermatogonium by flow cytometry sorting and immunomagnetic beads method, therefore do not need the antibody of sheep stem spermatogonium surface antigen, solved a difficult problem that can not find antibody while adopting prior art purifying sheep stem spermatogonium, from purification effect, adopt separation purification method of the present invention, can obtain the stem spermatogonium of purity 90% left and right.
2. simple to operate easy to learn: the present invention adopts two step enzymolysis processs to prepare after testis single cell suspension, with difference adherent method, add that the method for ln specific combination stem spermatogonium carrys out separation and purification sheep stem spermatogonium, these methods are all the conventional methods in cytobiology field, simple to operate easy to learn, required reagent and apparatus are all the conventional reagent of field of biology and apparatus.
3. operational details is easy to control: RESEARCH ON CELL-BIOLOGY is focused on operational details very much, the inside and outside scientific and technical literature of host country is all only reported emphatically the mechanism of action of stem cell separating and purifying, if but actual going operates, result but varies, many times just set up in theory, actually operating is difficult to successfully, technical scheme provided by the invention is all used the time by every single stepping, sequentially, concentration, dynamics, the parameters such as number of times define, make whole operation be easy to control, only need operate in strict accordance with above-mentioned sheep stem spermatogonium separation purification method, be the personnel of cell research, no matter be professor or common postgraduate can successful separation and purification go out sheep stem spermatogonium, the sheep stem spermatogonium purity of preparation is more than 90%.
4. set up the sheep stem spermatogonium long-term cultivation system that goes down to posterity: the present invention successfully processes trophocyte by domestic mitomycin, and according to test of many times result of study, invented applicable sheep stem spermatogonium nutrient solution, determined the cytokine that in nutrient solution, needs add, make the not differentiation of sheep stem spermatogonium of external long-term cultivation.
5. successfully set up the method that liquid nitrogen freezing is preserved sheep stem spermatogonium for a long time: sheep stem spermatogonium cryopreservation methods provided by the invention, can make stem spermatogonium after Cryopreservation goes down to posterity repeatedly, the sheep stem spermatogonium that recovery is cultivated still keep marker gene expression characterization and differentiation capability constant, more than the sheep stem spermatogonium that uses method separation and purification provided by the invention and go down to posterity has at least been cultivated and had been reached for 40 generations, if add the frozen time, the longest incubation period has reached 4 years.
Accompanying drawing explanation
Fig. 1 is that the sheep stem spermatogonium of separation and purification is schemed by the evaluation of dyeing immunofluorescence cell method;
Fig. 2 goes down to posterity that the sheep stem spermatogonium of long-term cultivation is identified by OCT4/CDH1 immunofluorescence double labelled staining method and double-tagging result Merge fusion is schemed;
Fig. 3 goes down to posterity that the sheep stem spermatogonium of long-term cultivation is identified by PLZF/Gfra1 immunofluorescence double labelled staining method and double-tagging result Merge fusion is schemed;
The sheep stem spermatogonium of Fig. 4 after for recovery entered by Scp3 immunofluorescence staining, to identify after vitro differentiation inducing culture and Merge fusion is schemed.
Embodiment
By specific embodiment, further illustrate technical scheme of the present invention below, but technical scheme of the present invention is not limited with embodiment.
The method of a kind of sheep stem spermatogonium of the present invention separation and purification, comprises the following steps:
The first step: obtain solution:
1) antibacterial-anti-mycotic agent that is 0.1% by volume percent adds in DMEM/F12 nutrient solution makes nutrient solution A, in two weeks, uses;
2) IV Collagenase Type is added and in DMEM low sugar nutrient solution, makes IV Collagenase Type storing solution ,-20 ℃ of preservations with the concentration of 20mg/ml;
3) Unidasa is added in DMEM low sugar nutrient solution and makes Unidasa storing solution ,-20 ℃ of preservations with the concentration of 10mg/ml;
4) DnaseI is added to DMEM low sugar nutrient solution with the concentration of 5mg/ml, then to add volume percent be 20% glycerine, make DnaseI storing solution ,-20 ℃ of preservations;
5) Trypsin that is 0.5% by volume percent and concentration are that 2mM EDTA is dissolved in and in PBS damping fluid, makes Trypsin/EDTA storing solution ,-20 ℃ of preservations;
6) foetal calf serum that is 10% by volume percent and 0.1% antibacterial-anti-mycotic agent adds and in DMEM/F12 nutrient solution, makes nutrient solution C, in two weeks, use;
7) horse serum that is 5.5% by volume percent, 2.4% foetal calf serum and 0.1% antibacterial-anti-mycotic agent adds in DMEM/F12 nutrient solution and makes nutrient solution B, in two weeks, use;
8) mercaptoethanol of 30 μ Μ is added in nutrient solution C, filtration sterilization is made nutrient solution D, now with the current;
9) bovine serum albumin that is 0.52% by volume percent is dissolved in PBS damping fluid, and filtration sterilization is made PBS-BSA solution, now with the current;
Second step: utilize sheep testicle tissue preparation single-cell suspension liquid and carry out separation and Culture, under aseptic condition, operation steps is as follows successively:
1) from the sheep testicle at 3 monthly ages, get 3g convoluted seminiferous tubule tissue block, put into the 100ml culture dish that is loaded with 2.5ml nutrient solution A, with tweezers, remove albuginea testis in convoluted seminiferous tubule tissue block and larger blood vessel;
2) last tissue block is put into 20ml bottle, with ophthalmology scissors straight, shred, continue to cut 3 minutes;
3) tissue block shredding is transferred in 100ml jar with cover, added nutrient solution A to 7.5ml;
4) carry out the first step enzymic digestion, concrete operations are as follows: in the above 100ml jar with cover, add the IV Collagenase Type storing solution of 0.63ml, be placed in 37 ℃ of shaking baths, with the rotating speed of 90rpm, sway 40min; Add subsequently the Unidasa storing solution of 0.825ml and the DnaseI storing solution of 0.125ml, continue to be placed in 37 ℃ of shaking baths, with the rotating speed of 90rpm, sway 20min, with 10ml transfer pipet, leniently inhale and blow suspended substance 5 times up and down;
5) from shaking bath, take out 100ml jar with cover, with 10ml transfer pipet, leniently inhale and blow suspended substance 5 times up and down;
6) by step 5) suspension in described 100ml jar with cover proceeds to 50ml centrifuge tube, and the centrifugal 6min of rotating speed with 2000rpm, removes supernatant liquor;
7) in step 6) add the PBS damping fluid of 12ml in described centrifuge tube, shake up and make throw out Eddy diffusion, the centrifugal 6min of rotating speed with 2000rpm, removes supernatant liquor;
8) operation of repeating step 7 once;
9) in step 8) add the nutrient solution A Eddy diffusion throw out of 0.75ml in described centrifuge tube, add again the Trypsin/EDTA storing solution of 1ml and the DNaseI storing solution of 0.25ml, be placed in 37 ℃ of shaking baths, rotating speed with 90rpm sways 10min, with 10ml transfer pipet, leniently inhales and blows suspended substance 5 times up and down;
10) immediately in step 9) add the nutrient solution C of 10ml in described centrifuge tube, with 10ml transfer pipet leniently inhale up and down blow suspended substance until significantly block disappear;
11) by step 10) described centrifuge tube is with the centrifugal 6min of rotating speed of 2000rpm, removes supernatant liquor, then uses 2ml nutrient solution C re-suspended cell;
12) first with 200 orders, then with 300 order nylon filters, distinguish filtration steps 11) described cell suspending liquid;
13) by step 12) cell suspending liquid after described filtration leniently inhales and blows 5-10 time with 10ml transfer pipet, carries out cell counting, then adds nutrient solution C adjustment cell density to 1.25x10
6cell/ml, makes single-cell suspension liquid;
The 3rd step: purifying stem spermatogonium from the single-cell suspension liquid of separation and Culture described in second step, operation steps is as follows successively:
1) by 10ml second step step 13) described single-cell suspension liquid is laid on the coated culture dish of gelatin that diameter is 100mm, is placed on 32.5 ℃, 5.5%CO
2, in the cell culture incubator of saturated humidity, cultivate 3 days;
2) with 5ml transfer pipet from step 1) described in leniently remove all nutrient solutions culture dish;
3) first with nutrient solution A, wash cell: from the edge of culture dish, slowly add the nutrient solution A of 4ml preheating, leniently rock back and forth culture dish 1 time, then with transfer pipet, remove nutrient solution;
4) with PBS damping fluid, wash cell twice again: from the edge of culture dish, slowly add the PBS damping fluid of 4ml preheating, leniently rock back and forth culture dish 1 time, with transfer pipet, remove supernatant liquor immediately, then repetitive operation once;
5) add 4ml nutrient solution A to culture dish, with transfer pipet suction, blow nutrient solution A and rinse gently the whole surface of culture dish about 2 times;
6) by step 5) washing fluid in described culture dish transfers in aseptic 50ml centrifuge tube;
7) by step 6) described centrifuge tube is in the centrifugal 6min of 2000rpm, removes supernatant liquor, retains cell precipitation;
8) in step 7) add the nutrient solution B re-suspended cell of 4ml in described centrifuge tube;
9) in the coated culture dish of collagen I, add 4ml nutrient solution B, repave step 8) described cell suspending liquid, then culture dish is put into 32.5 ℃, 5.5 ﹪ CO
2, saturated humidity cell culture incubator in hatch 2 hours;
10) from incubator, take out culture dish, with 5ml transfer pipet, leniently on culture dish surface, repeatedly inhale and blow 10 times, make cytomixis, then culture dish is put back in incubator and continued to cultivate 2 hours;
11) from incubator, take out after culture dish, with 5ml transfer pipet, in culture dish, leniently inhale and blow cell suspending liquid, until rinsed whole culture dish surface, then from culture dish, collect the non-adsorbable cell suspending liquid of collagen I with 5ml transfer pipet, all transfer in 50ml centrifuge tube;
12) by step 11) described centrifuge tube centrifugal 6min under 2000rpm, sedimentation cell, removes supernatant liquor, with 4ml nutrient solution D suspension cell, then draws cell suspending liquid by 200 order nylon filters, with 50ml test tube, collects filtrate;
13) promptly by step 12) filtrate that obtains blows 5 times with the suction of 5ml transfer pipet gentleness, then the filtrate mixing is spread to the 12 coated well culture plates into Laminin by every hole 1ml, wherein said culture plate is prepared in advance, facing the used time first removes the supernatant liquor in each hole, add again 1ml nutrient solution A to clean, just spread into above-mentioned filtrate after removing all nutrient solution A;
14) by step 13) described culture plate is placed in 32.5 ℃, 5.5 ﹪ CO
2in the cell culture incubator of saturated humidity, hatch 30 minutes, then take out, with the suction as mild as a dove of 1ml micropipet, blow surperficial one time of the culture hole of described culture plate, cell suspending liquid levels in hole is mixed, then put back to rapidly in incubator and continue to hatch 20 minutes;
15) by step 14) described culture plate taking-up, with 1ml micropipet, inhale and blow once, cell suspending liquid in hole is mixed, now the cell in suspension is not adherent cell of Laminin, these cell suspending liquids is removed from culture hole, then, in culture hole, add immediately the freshly prepared nutrient solution D of 1ml to wash culture hole surface one time, with the nutrient solution D in mix aperture, then remove the nutrient solution D in culture hole, again in hole, add the freshly prepared nutrient solution D of 1ml immediately.Nutrient solution D wants gentle and drips equably the whole surface in hole, and the cell adhering in hole is Laminin adherent cell;
16) with 1ml micropipet from step 15) remove nutrient solution D described culture hole, then in culture hole, add the freshly prepared PBS-BSA solution of 1ml immediately, then culture plate is put into 32.5 ℃, 5.5 ﹪ CO
2, in the cell culture incubator of saturated humidity, hatch 3-6 minute, in the sterile test tube of a 50ml, add 4ml nutrient solution D standby simultaneously.
17) by step 16) described culture plate taking-up, use immediately 1ml transfer pipet gentle pressure-vaccum PBS-BSA solution 5 times in culture hole, make Laminin adherent cell from the de-wall of culture hole, the PBS-BSA solution that collection contains Laminin adherent cell, transfers to the PBS-BSA solution that contains Laminin adherent cell in the 50ml centrifuge tube that contains nutrient solution D;
18) by step 17) described centrifuge tube centrifugal 6min under 2000rpm rotating speed, remove supernatant liquor, reversing test tube, leniently removes remaining liquid;
19) in step 18) add 1ml nutrient solution D Eddy diffusion cell in described centrifuge tube, with cell counting count board, measure cell concn;
20) by CDH1 dyeing immunofluorescence cell method authentication step 19) cell in described cell suspending liquid is exactly sheep stem spermatogonium (Fig. 1).
Sheep stem spermatogonium purity 90% left and right of preparing by above-mentioned sheep stem spermatogonium separation purification method, is suitable for the long-term cultivation of sheep stem spermatogonium.
The long-period culture method that goes down to posterity to above-mentioned sheep stem spermatogonium, comprises the following steps:
The first step: obtain solution:
1) foetal calf serum that is 7% by volume percent adds makes STO cell culture fluid in DMEM/F12 nutrient solution;
2) foetal calf serum that is 20% by volume percent, 10% dimethyl sulfoxide (DMSO) add makes STO cells frozen storing liquid in DMEM/F12 nutrient solution;
3) in lmlSSCB solution, the anti-mycotic agent storage liquid 1 μ l of Streptomycin sulphate storage liquid 1 μ l, 2.5mg/ml of penicillin storage liquid 1 μ l, 100mg/ml of glial cellline-derived neurotrophic factor receptor alpha 1 sample protein antibodies storage liquid 1 μ l, 100kU/ml of Prostatropin storing solution 1 μ l, 100ng/ μ l that adds respectively glial cell line-derived neurotrophic factor storing solution 1 μ l, the 20ng/ μ l of 20ng/ μ l, mixes and makes SSCM nutrient solution;
The component that wherein said 1 liter of SSCB solution comprises is:
Second step: preparation trophocyte, successively operation as follows:
1) trophocyte's is prefabricated: with STO cell culture fluid, cultivate STO cell, when cell degree of converging reaches 70%-80%, by final concentration, 15 μ g/ml add mitomycin, put into cell culture incubator, at 37 ℃, 5%CO
2, saturated humidity condition under cultivate 2 hours, then remove the nutrient solution that contains mitomycin, and wash three times with 10mlPBS, with Trypsin/EDTA Digestive system described in 1ml claim 1, make cell take off wall again, and the digestion that stops Trypsin with nutrient solution C described in 5ml claim 1, collect postdigestive cell, the centrifugal 5min of 2000rpm, getting the cell of precipitation, is 6 * 10 by concentration
5the concentration of cell/mL adds STO cells frozen storing liquid, and liquid nitrogen is preserved;
2) prepare trophocyte: by step 1) the STO cell processed of described mitomycin, with 2 * 10
5the final concentration of cell/mL is seeded in cell in the 12 porocyte culture plates of being processed by gelatin in advance, at 37 ℃, and 5%CO
2, under the condition of saturated humidity, cultivate 12-24 hour;
The 3rd step: the long-term cultivation that goes down to posterity sheep stem spermatogonium, operation steps is as follows:
1) the former culture of stem spermatogonium: remove second step step 2) in described 12 porocyte culture plates in STO trophoderm containing serum free culture system liquid, and wash twice with PBS damping fluid, then use SSCM nutrient solution resuspended the good stem spermatogonium of separation and purification described in claim 2, with 3 * 10
4the concentration of cell/mL is inoculated into be completed in the trophoblastic 12 porocyte culture plates of STO, then culture plate is placed on to 37 ℃, 5%CO
2, saturated humidity cell culture incubator in, second day changes SSCM nutrient solution one time, changes one time SSCM nutrient solution every 2 days later;
2) long-term cultivation that goes down to posterity of stem spermatogonium: by step 1) stem spermatogonium of described former culture is cultivated 6-9 days, take out culture plate, remove SSCM nutrient solution, remove floating cell, the SSCM nutrient solution that adds the fresh configuration of 1ml in culture hole, and with 1mL pipette pressure-vaccum gently, stem spermatogonium is departed from from trophocyte, stem spermatogonium suspension is transferred to 50ml centrifuge tube, repetitive operation 1 time, to the centrifugal 5min of 2000rpm for centrifuge tube of stem spermatogonium suspension be housed, abandon supernatant, with SSCM nutrient solution re-suspended cell, be inoculated on new STO trophocyte, first three after former culture time goes down to posterity and carries out with the ratio of 1:1 or 1:1.5, go down to posterity afterwards and carry out with the ratio of 1:3, every 2 days, change one time SSCM nutrient solution, cultured stem spermatogonium carries out the frozen or cultivation that continues to go down to posterity as required.
The sheep stem spermatogonium of the long-term cultivation that goes down to posterity is carried out to immunofluorescence double labelled staining with the tagged molecule OCT4/CDH1 of stem spermatogonium, visible two kinds of tagged molecule coloration results overlapping (Fig. 2), prove that adopting the go down to posterity cell of long-term cultivation of the technical program is exactly sheep stem spermatogonium.
The sheep stem spermatogonium of the long-term cultivation that goes down to posterity is carried out to immunofluorescence double labelled staining with the tagged molecule PLZF/Gfra1 of stem spermatogonium, visible two kinds of tagged molecule coloration results overlapping (Fig. 3), prove that adopting the go down to posterity cell of long-term cultivation of the technical program is exactly sheep stem spermatogonium.。
Sheep stem spermatogonium to the above-mentioned long-term cultivation that goes down to posterity carries out frozen: by the stem spermatogonium of the above-mentioned long-term cultivation that goes down to posterity, with the leniently piping and druming gently of 1mL pipette, stem spermatogonium is departed from from STO trophocyte, collect stem spermatogonium to 50ml centrifuge tube, carry out cell counting, then the centrifugal 5min of 2000rpm, abandons supernatant, with serum-free cell frozen storing liquid re-suspended cell and adjust concentration to 5 * 10
5cell/ml, divides and installs in cryopreservation tube, places 30min for 4 ℃, places 24h, proceeds to afterwards the medium-term and long-term preservation of liquid nitrogen container for-80 ℃.
Above-mentioned frozen sheep stem spermatogonium is recovered: above-mentioned cryopreservation tube is taken out from liquid nitrogen container, in 37 ℃ of water-baths, melt 3 minutes fast, cell is proceeded in the 50ml centrifuge tube that contains 10mlSSCM nutrient solution in Bechtop, centrifugal 6 minutes of 2000rpm, abandons supernatant liquor, and throw out is resuspended in 3mlSSCM nutrient solution, transfer to 12 well culture plates, every hole 1ml, is placed in 37 ℃, 5%CO
2, in the cell culture incubator of saturated humidity, every 2 days, change one time SSCM nutrient solution, cultivate 2 days.
Sheep stem spermatogonium after recovery was entered to vitro differentiation inducing culture, cellular form changes, obviously increase, and express the special labelled protein Scp3 (Fig. 4) of spermatocyte, illustrate that adopting the technical program to cultivate sheep stem spermatogonium frozen and recovery still has physiological potential.
The method of sheep stem spermatogonium provided by the invention separation and purification, the long-term cultivation that goes down to posterity, freezing and thawing, has formed a set of complete sheep stem spermatogonium long-term cultivation technology.Because sheep stem spermatogonium has remarkable stability and proliferation potential in long-term cultivation process, gene or abnormal epigenetic proterties that can prevent from having damaged are propagated to offspring, make sheep spermatogonial stem cell transplantation technology become attractive Transgenic Sheep manufacturing technology, and in the cross-breeding of sheep, external or wild good character is utilized.Sheep stem spermatogonium has the ability that is divided into reproduction multipotential stem cell of moving back, sheep stem spermatogonium long-term cultivation technology is reproduction multipotential stem cell by reincarnation, can be used for the research of sheep regenerative medicine model, thereby regenerative medicine and clinical tissue rehabilitation are produced to active influence.
Above a kind of sheep stem spermatogonium provided by the present invention separation and purification, the long-term cultivation that goes down to posterity, method frozen and recovery are described in detail.By embodiment, principle of the present invention and embodiment are set forth herein, above explanation is just for helping to understand method of the present invention and core concept thereof.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of the claims in the present invention.
Claims (6)
1. a method for sheep stem spermatogonium separation and purification, is characterized in that, comprises the following steps:
The first step: obtain solution:
1) antibacterial-anti-mycotic agent that is 0.1% by volume percent adds in DMEM/F12 nutrient solution makes nutrient solution A, in two weeks, uses;
2) IV Collagenase Type is added and in DMEM low sugar nutrient solution, makes IV Collagenase Type storing solution ,-20 ℃ of preservations with the concentration of 20mg/ml;
3) Unidasa is added in DMEM low sugar nutrient solution and makes Unidasa storing solution ,-20 ℃ of preservations with the concentration of 10mg/ml;
4) DnaseI is added to DMEM low sugar nutrient solution with the concentration of 5mg/ml, then to add volume percent be 20% glycerine, make DNaseI storing solution ,-20 ℃ of preservations;
5) Trypsin that is 0.5% by volume percent and concentration are that 2mM EDTA is dissolved in and in PBS damping fluid, makes Trypsin/EDTA storing solution ,-20 ℃ of preservations;
6) foetal calf serum that is 10% by volume percent and 0.1% antibacterial-anti-mycotic agent adds and in DMEM/F12 nutrient solution, makes nutrient solution C, in two weeks, use;
7) horse serum that is 5.5% by volume percent, 2.4% foetal calf serum and 0.1% antibacterial-anti-mycotic agent adds in DMEM/F12 nutrient solution and makes nutrient solution B, in two weeks, use;
8) β-thioglycol of 30 μ Μ is added in nutrient solution C, filtration sterilization is made nutrient solution D, now with the current;
9) bovine serum albumin that is 0.52% by volume percent is dissolved in PBS damping fluid, and filtration sterilization is made PBS-BSA solution, now with the current;
Second step: application two step enzymolysis processs are prepared sheep testicle and organized single-cell suspension liquid, and under aseptic condition, operation steps is as follows successively:
1) from the sheep testicle at 3-4 monthly age, get 2-3g convoluted seminiferous tubule tissue block, put into the 100ml culture dish that is loaded with 2.5ml nutrient solution A, with tweezers, remove albuginea testis in convoluted seminiferous tubule tissue block and larger blood vessel;
2) last tissue block is put into 20ml bottle, with ophthalmology scissors straight, shred, continue to cut 3 minutes;
3) tissue block shredding is transferred in 100ml jar with cover, added nutrient solution A to 7.5ml;
4) carry out the first step enzymic digestion, concrete operations are as follows: in above-mentioned 100ml jar with cover, add the IV Collagenase Type storing solution of 0.63ml, be placed in 37 ℃ of shaking baths, with the rotating speed of 90rpm, sway 40min; Then add the Unidasa storing solution of 0.825ml and the DNaseI storing solution of 0.125ml, continue to be placed in 37 ℃ of shaking baths, with the rotating speed of 90rpm, sway 20min.
5) from shaking bath, take out 100ml jar with cover, with 10ml transfer pipet, leniently inhale and blow suspended substance 5 times up and down;
6) by step 5) suspension in described 100ml jar with cover proceeds to 50ml centrifuge tube, and the centrifugal 6min of rotating speed with 2000rpm, removes supernatant liquor;
7) in step 6) add the PBS damping fluid of 12ml in described centrifuge tube, shake up and make throw out Eddy diffusion, the centrifugal 6min of rotating speed with 2000rpm, removes supernatant liquor;
8) operation of repeating step 7 once;
9) carry out second step enzymolysis, in step 8) add the nutrient solution A Eddy diffusion throw out of 0.75ml in described centrifuge tube, add again the Trypsin/EDTA storing solution of 1ml and the DNaseI storing solution of 0.25ml, be placed in 37 ℃ of shaking baths, rotating speed with 90rpm sways 10min, with 10ml transfer pipet, leniently inhales and blows suspended substance 5 times up and down;
10) in step 9) add the nutrient solution C of 10ml in described centrifuge tube, with 10ml transfer pipet leniently inhale up and down blow suspended substance until significantly block disappear;
11) by step 10) described centrifuge tube is with the centrifugal 6min of rotating speed of 2000rpm, removes supernatant liquor, then uses 2ml nutrient solution C re-suspended cell;
12) first with 200 order nylon filters, then with 300 order nylon filters, distinguish filtration steps 11) described cell suspending liquid;
13) by step 12) cell suspending liquid after described filtration leniently inhales and blows 5-10 time with 10ml transfer pipet, carries out cell counting, then adds nutrient solution C adjustment cell density to 1.25x10
6cell/ml, makes single-cell suspension liquid;
The 3rd step: purifying stem spermatogonium from single-cell suspension liquid described in second step, operation steps is as follows successively:
1) by 10ml second step step 13) described single-cell suspension liquid is laid on the coated culture dish of gelatin that diameter is 100mm, is placed on 32.5 ℃, 5.5%CO
2, in the cell culture incubator of saturated humidity, cultivate 2 days half to 3 day half;
2) with 5ml transfer pipet from step 1) described in leniently remove all nutrient solutions culture dish;
3) first with nutrient solution A, wash cell: from the edge of culture dish, slowly add the nutrient solution A of 4ml preheating, leniently rock back and forth culture dish 1 time, then with transfer pipet, remove nutrient solution;
4) with PBS damping fluid, wash cell twice again: from the edge of culture dish, slowly add the PBS damping fluid of 4ml preheating, leniently rock back and forth culture dish 1 time, with transfer pipet, remove supernatant liquor immediately, then repetitive operation once;
5) add 4ml nutrient solution A to culture dish, with transfer pipet suction, blow nutrient solution A and rinse gently the whole surface of culture dish about 2 times;
6) by step 5) washing fluid in described culture dish transfers in aseptic 50ml centrifuge tube;
7) by step 6) described centrifuge tube is in the centrifugal 6min of 2000rpm, removes supernatant liquor, retains cell precipitation;
8) in step 7) add the nutrient solution B re-suspended cell of 4ml in described centrifuge tube;
9) in the coated culture dish of collagen I, add 4ml nutrient solution B, repave step 8) described cell suspending liquid, then culture dish is put into 32.5 ℃, 5.5 ﹪ CO
2, saturated humidity cell culture incubator in hatch 2 hours;
10) from incubator, take out culture dish, with 5ml transfer pipet, leniently on culture dish surface, repeatedly inhale and blow 5-10 time, make cytomixis, then culture dish is put back in incubator and continued to cultivate 2 hours;
11) from incubator, take out after culture dish, with 5ml transfer pipet, in culture dish, leniently inhale and blow cell suspending liquid, until rinsed whole culture dish surface, then from culture dish, collect the non-adsorbable cell suspending liquid of collagen I with 5ml transfer pipet, all transfer in 50ml centrifuge tube;
12) by step 11) described centrifuge tube centrifugal 6min under 2000rpm, sedimentation cell, removes supernatant liquor, with 4ml nutrient solution D suspension cell, then draws cell suspending liquid by 200 order nylon filters, with 50ml test tube, collects filtrate;
13) promptly by step 12) filtrate that obtains blows 4-5 time with the suction of 5ml transfer pipet gentleness, then the filtrate mixing is spread to the 12 coated well culture plates into Laminin by every hole 1ml, wherein said culture plate is prepared in advance, facing the used time first removes the supernatant liquor in each hole, add again 1ml nutrient solution A to clean, just spread into above-mentioned filtrate after removing all nutrient solution A;
14) by step 13) described culture plate is placed in 32.5 ℃, 5.5 ﹪ CO
2in the cell culture incubator of saturated humidity, hatch 20-40 minute, then take out, with the suction as mild as a dove of 1ml micropipet, blow surperficial one time of the culture hole of described culture plate, cell suspending liquid levels in hole is mixed, then put back to rapidly and in incubator, continue to hatch 10-30 minute;
15) by step 14) described culture plate taking-up, with 1ml micropipet, inhale and blow once, cell suspending liquid in hole is mixed, now the cell in suspension is not adherent cell of Laminin, these cell suspending liquids is removed from culture hole, then, in culture hole, add immediately the freshly prepared nutrient solution D of 1ml, with 1ml micropipet, inhale and blow culture hole surface one time again, then remove the nutrient solution D in culture hole, again in hole, add the freshly prepared nutrient solution D of 1ml immediately.Nutrient solution D wants gentle and drips equably the whole surface in hole, and the cell adhering in hole is Laminin adherent cell;
16) with 1ml micropipet from step 15) remove nutrient solution D described culture hole, then in culture hole, add the freshly prepared PBS-BSA solution of 1ml immediately, then culture plate is put into 32.5 ℃, 5.5 ﹪ CO
2, in the cell culture incubator of saturated humidity, hatch 3-6 minute, in the sterile test tube of a 50ml, add 4ml nutrient solution D standby simultaneously.
17) by step 16) described culture plate taking-up, use immediately 1ml transfer pipet gentle pressure-vaccum PBS-BSA solution 5-10 time in culture hole, make Laminin adherent cell from the de-wall of culture hole, the PBS-BSA solution that collection contains Laminin adherent cell, transfers to the PBS-BSA solution that contains Laminin adherent cell in the 50ml centrifuge tube that contains nutrient solution D;
18) by step 17) described centrifuge tube centrifugal 6min under 2000rpm rotating speed, remove supernatant liquor, reversing test tube, leniently removes remaining liquid;
19) in step 18) add 1ml nutrient solution D Eddy diffusion cell in described centrifuge tube, with cell counting count board, measure cell density;
20) by dyeing immunofluorescence cell method authentication step 19) cell in described cell suspending liquid is exactly sheep stem spermatogonium.
2. by the sheep stem spermatogonium that described in claim 1 prepared by sheep stem spermatogonium separation purification method.
3. the long-period culture method that goes down to posterity of sheep stem spermatogonium described in pair claim 2, comprises the following steps:
The first step: obtain solution:
1) foetal calf serum that is 7% by volume percent adds makes STO cell culture fluid in DMEM/F12 nutrient solution;
2) foetal calf serum that is 20% by volume percent, 10% dimethyl sulfoxide (DMSO) add makes STO cells frozen storing liquid in DMEM/F12 nutrient solution;
3) in lml SSCB solution, the anti-mycotic agent storage liquid 1 μ l of Streptomycin sulphate storage liquid 1 μ l, 2.5mg/ml of penicillin storage liquid 1 μ l, 100mg/ml of glial cellline-derived neurotrophic factor receptor alpha 1 sample protein antibodies storage liquid 1 μ l, 100kU/ml of Prostatropin storing solution 1 μ l, 100ng/ μ l that adds respectively glial cell line-derived neurotrophic factor storing solution 1 μ l-2 μ l, the 20ng/ μ l of 20ng/ μ l, mixes and makes SSCM nutrient solution;
Second step: preparation trophocyte, successively operation as follows:
1) trophocyte's is prefabricated: with STO cell culture fluid, cultivate STO cell, when cell degree of converging reaches 70%-80%, by final concentration, 15 μ g/ml add mitomycin, put into cell culture incubator, at 37 ℃, 5%CO
2, saturated humidity condition under cultivate 2 hours, then remove the nutrient solution that contains mitomycin, and wash three times with 10mlPBS, with Trypsin/EDTA Digestive system described in 1ml claim 1, make cell take off wall again, and the digestion that stops Trypsin with nutrient solution C described in 5ml claim 1, collect postdigestive cell, the centrifugal 5min of 2000rpm, getting the cell of precipitation, is 6 * 10 by concentration
5the concentration of cell/mL adds STO cells frozen storing liquid, and liquid nitrogen is preserved;
2) prepare trophocyte: by step 1) the STO cell processed of described mitomycin, with 2 * 10
5the final concentration of cell/mL is seeded in cell in the 12 porocyte culture plates of being processed by gelatin in advance, at 37 ℃, and 5%CO
2, under the condition of saturated humidity, cultivate 12-24 hour;
The 3rd step: the long-term cultivation that goes down to posterity sheep stem spermatogonium, operation steps is as follows:
1) the former culture of stem spermatogonium: remove second step step 2) in described 12 porocyte culture plates in STO trophoderm containing serum free culture system liquid, and wash twice with PBS damping fluid, then use SSCM nutrient solution resuspended the good stem spermatogonium of separation and purification described in claim 2, with 2-5 * 10
4the density of cell/mL is inoculated into be completed in the trophoblastic 12 porocyte culture plates of STO, then culture plate is placed on to 37 ℃, 5%CO
2, saturated humidity cell culture incubator in, second day changes SSCM nutrient solution one time, changes one time SSCM nutrient solution every 2 days later;
2) long-term cultivation that goes down to posterity of stem spermatogonium: by step 1) stem spermatogonium of described former culture is cultivated 6-9 days, take out culture plate, remove SSCM nutrient solution, remove floating cell, the SSCM nutrient solution that adds the fresh configuration of 1ml in culture hole, and with 1mL transfer pipet pressure-vaccum gently, stem spermatogonium is departed from from trophocyte, stem spermatogonium suspension is transferred to 50ml centrifuge tube, repetitive operation 1-2 time, to the centrifugal 5min of 2000rpm for centrifuge tube of stem spermatogonium suspension be housed, abandon supernatant, with SSCM nutrient solution re-suspended cell, be inoculated on new STO trophocyte, first three after former culture time goes down to posterity and carries out with the ratio of 1:1 or 1:1.5, go down to posterity afterwards and carry out with the ratio of 1:2-1:3, every 2 days, change one time SSCM nutrient solution, cultured stem spermatogonium carries out frozen as required, utilize or the cultivation that continues to go down to posterity.
4. the sheep stem spermatogonium of the long-term cultivation that goes down to posterity described in pair claim 3 carries out frozen method, comprise the following steps: by the stem spermatogonium of the long-term cultivation that goes down to posterity described in claim 3, with the leniently piping and druming gently of 1mL pipette, stem spermatogonium is departed from from STO trophocyte, collect stem spermatogonium to 50ml centrifuge tube, carry out cell counting, then the centrifugal 5min of 2000rpm, abandon supernatant, with serum-free cell frozen storing liquid re-suspended cell and adjust concentration to 5 * 10
4-2 * 10
5cell/ml, divides and installs in cryopreservation tube, places 30min for 4 ℃, places 24h, proceeds to afterwards the medium-term and long-term preservation of liquid nitrogen container for-80 ℃.
5. the method that described in pair claim 4, frozen sheep stem spermatogonium is recovered, comprise the following steps: cryopreservation tube described in claim 4 is taken out from liquid nitrogen container, in 37 ℃ of water-baths, melt 3 minutes fast, cell is proceeded in the 50ml centrifuge tube that contains 10ml SSCM nutrient solution to centrifugal 6 minutes of 2000rpm in Bechtop, abandon supernatant liquor, throw out is resuspended in 1-3mlSSCM nutrient solution, transfers to 12 well culture plates, every hole 1ml, be placed in 37 ℃, 5%CO
2, in the cell culture incubator of saturated humidity, every 2 days, change one time SSCM nutrient solution, cultivate 2-3 days.
6. the long-period culture method that goes down to posterity of sheep stem spermatogonium according to claim 3, is characterized in that, the component that wherein said 1 liter of SSCB solution comprises is:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410265999.2A CN104004708B (en) | 2014-06-16 | 2014-06-16 | The separation and purification of a kind of sheep stem spermatogonium, the long-term cultivation that goes down to posterity, frozen and recovery method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105331582A (en) * | 2015-11-16 | 2016-02-17 | 黄林海 | Stem cell recovery method of cryopreserved umbilical cord blood with high recovery rate |
| CN105385655A (en) * | 2015-11-16 | 2016-03-09 | 黄林海 | Resuscitation fluid applied to vitrified cryopreserved mesenchymal stem cells |
| CN105400736A (en) * | 2016-01-18 | 2016-03-16 | 黄林海 | High-recovery-rate cryopreserved mesenchymal stem cell recovery method |
| CN105969723A (en) * | 2016-06-01 | 2016-09-28 | 内蒙古大学 | Method for efficiently separating mouse spermatogonial stem cells |
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| CN106754724A (en) * | 2016-12-09 | 2017-05-31 | 西北农林科技大学 | A kind of ox stem spermatogonium system of immortalization and its construction method |
| CN108378019A (en) * | 2018-03-13 | 2018-08-10 | 洛阳轩智生物科技有限公司 | A kind of frozen stock solution of people's stem spermatogonium |
| CN109371111A (en) * | 2018-12-07 | 2019-02-22 | 卢克焕 | The identification method of cell type in a kind of buffalo stem spermatogonium like cell purification process |
| CN109385395A (en) * | 2018-10-15 | 2019-02-26 | 卢克焕 | A kind of purification process of the buffalo stem spermatogonium like cell in vitro culture |
| CN109402045A (en) * | 2018-10-15 | 2019-03-01 | 卢克焕 | A kind of in vitro culture and propagating method of buffalo stem spermatogonium like cell |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638633A (en) * | 2009-09-04 | 2010-02-03 | 西北农林科技大学 | In-vitro separation and culture method of goat male germ stem cells |
| CN101818127A (en) * | 2010-03-31 | 2010-09-01 | 安徽农业大学 | Method for separating and culturing mouse primitive spermatogonia |
| CN102344909A (en) * | 2011-10-20 | 2012-02-08 | 上海交通大学医学院附属仁济医院 | Method for separating human spermatogonial stem cells |
| CN102382799A (en) * | 2010-08-31 | 2012-03-21 | 吴际 | In-vitro isolation and culture method of mouse spermatogonial stem cells and special culture medium thereof |
| CN103074297A (en) * | 2012-12-07 | 2013-05-01 | 内蒙古大学 | Livestock spermatogonial stem cell medium |
-
2014
- 2014-06-16 CN CN201410265999.2A patent/CN104004708B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638633A (en) * | 2009-09-04 | 2010-02-03 | 西北农林科技大学 | In-vitro separation and culture method of goat male germ stem cells |
| CN101818127A (en) * | 2010-03-31 | 2010-09-01 | 安徽农业大学 | Method for separating and culturing mouse primitive spermatogonia |
| CN102382799A (en) * | 2010-08-31 | 2012-03-21 | 吴际 | In-vitro isolation and culture method of mouse spermatogonial stem cells and special culture medium thereof |
| CN102344909A (en) * | 2011-10-20 | 2012-02-08 | 上海交通大学医学院附属仁济医院 | Method for separating human spermatogonial stem cells |
| CN103074297A (en) * | 2012-12-07 | 2013-05-01 | 内蒙古大学 | Livestock spermatogonial stem cell medium |
Non-Patent Citations (4)
| Title |
|---|
| 乌云毕力格: "绵羊精原干细胞的分离富集与体外培养研究", 《中国博士学位论文全文数据库》 * |
| 乌云毕力格: "绵羊精原干细胞的分离富集与体外培养研究", 《中国博士学位论文全文数据库》, no. 11, 15 November 2012 (2012-11-15), pages 3 * |
| 刘代艳: "海藻酸微囊在鼠胚成纤维细胞及绵羊精原干细胞冷冻保存中的应用", 《中国优秀硕士学位论文全文数据库》 * |
| 唐红: "绵羊精原干细胞非分化扩增的影响因素研究", 《中国优秀硕士学位论文全文数据库》 * |
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| CN108378019A (en) * | 2018-03-13 | 2018-08-10 | 洛阳轩智生物科技有限公司 | A kind of frozen stock solution of people's stem spermatogonium |
| CN108378019B (en) * | 2018-03-13 | 2021-03-02 | 诺赛联合(北京)生物医学科技有限公司 | Cryopreservation liquid for human spermatogonial stem cells |
| CN109385395A (en) * | 2018-10-15 | 2019-02-26 | 卢克焕 | A kind of purification process of the buffalo stem spermatogonium like cell in vitro culture |
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| CN110241074A (en) * | 2019-05-09 | 2019-09-17 | 青岛农业大学 | A kind of preparation method of Novel goat sperm in vitro agonist |
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