CN102763642B - Cryoprotectant and method for cryopreserving placenta amnion and chorion - Google Patents
Cryoprotectant and method for cryopreserving placenta amnion and chorion Download PDFInfo
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- CN102763642B CN102763642B CN201210288706.3A CN201210288706A CN102763642B CN 102763642 B CN102763642 B CN 102763642B CN 201210288706 A CN201210288706 A CN 201210288706A CN 102763642 B CN102763642 B CN 102763642B
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Abstract
The invention relates to the technical field of tissue engineering and discloses a cryoprotectant and a method for cryopreserving placenta amnion and chorion. The cryoprotectant is composed of fetal calf serum, dimethyl sulfoxide and dextran 40, and the volume ratio of the fetal calf serum, the dimethyl sulfoxide and the dextran 40 is 7-9:1:1. On the basis of deep research of placenta amnion and chorion structures, the dimethyl sulfoxide, the dextran 40 and the fetal calf serum suitable for protecting placenta amnion stem cells and chorion stem cells are selected to form the cryoprotectant. The fetal calf serum, the dimethyl sulfoxide and the dextran 40 coordinate to protect activity of cells, the cells are prevented from forming ice crystal and being damaged, and the activity of recovered stem cells and the activity of newly prepared amnion chorion stem cells have no difference.
Description
Technical field
The present invention relates to tissue engineering technique field, relate to specifically a kind of frozen solution and freezing depositary's placenta amnion and chorial method.
Background technology
Amnion and chorion are two chief components of people's placenta.Amnion is at the innermost layer of placenta, film body is smooth, without blood vessel, nerve and lymph, there is certain elasticity, it contains and abundant has multipotency, can not repel anti-multipotential stem cell by induction of immunity after transplanting, and amniotic cell of human placenta also can be expressed neurocyte specific mark, secretory nerve nutritional factor, for nervous system disorders, in addition people's placenta amnion can also be as the basilar membrane of ophthalmologic operation; And chorion is placenta main part, adjacent with amnion, on chorion, contain equally the abundant stem cell with many differentiation potentials, in the injuries of tissues and organs reparation and transplanting that can be applicable to cause due to old and feeble and pathology.
Utopian engineering tissue, except needs possess very high biological activity, also needs to meet at any time and transplants the needs that wait operation, and therefore, the engineering tissue that contains multipotential stem cell as people's placenta amnion and chorion is preserved with regard to needs are long-term.It is the common method that biological tissue is preserved that cryogenic freezing is preserved, and it is generally to preserve at 0～-196 ℃.Although cryogenic freezing technology can make engineering tissue preserve for a long time, also brought the problem of freezing injury.In order to reduce the problem of freezing injury, when preserving cell and tissue, need to add frozen solution, thereby alleviate or avoid the damage to cell of ice crystal in cooling, recovery process.
The patent of Chinese Patent Application No. 201010528721.1 provides a kind of " people's umbilical cord China Tong Shi glue tissue block freezing protection liquid "; it is according to the feature of claimed mescenchymal stem cell; disclose by perviousness cryoprotectant, impermeability cryoprotectant and basal liquid etc. Multiple components and formed protection liquid, the degree of impairment that reply mescenchymal stem cell may occur in freezing preservation.But, not yet have at present for people's placenta amnion and chorial cryogenic freezing Techniques of preserving report.Therefore, provide a kind of freezing depositary's placenta amnion and chorial technology, can improve undoubtedly amnion and chorial preservation quality, guarantee the effect of amnion and the clinical application of chorion stem cell.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of frozen solution and freezing depositary's placenta amnion and chorial method, make described method can effectively protect people's placenta amnion and chorion, the Stem Cell Activity after its recovery is reached more than 90%.
To achieve these goals, the invention provides following technical scheme:
A frozen solution, is comprised of foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40, and the volume ratio of described foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40 is 7-9:1:1.
Wherein, as preferably, the volume ratio of described foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40 is 8:1:1.
Dimethyl sulfoxide (DMSO) (DMSO) is a kind of important osmosis type cytoprotective.When preserving cell, profound hypothermia (subzero 196 degree) in frozen process, can prevent the damage that the formation of intracellular fluid ice crystal, osmotic pressure change, cellularstructure disorder etc. cause.DMSO can enter in cell by quick penetration cytolemma, reduces freezing point, delays frozen process, improves intracellular ion concentration simultaneously, reduces the formation of intracellular ice crystal, thereby reduces cell injury.And Dextran 40 belongs to impermeability antifreezing agent, can be water-soluble, but can not enter cell, and make solution be supercooled state, can under specified temp, reduce solute concentration, thereby play a protective role.Because its molecular weight is large, molecular conecentration is low, very little to the active function of solvent, can reduce low molecule solute (ionogen) concentration in solution, alleviates salt damage, and slow freezing effect is better.Freeze-stored cell is a process slowly, and foetal calf serum provides necessary nutrition to freeze-stored cell, and in serum, contains rich in protein cell is also had to provide protection.
Foetal calf serum, Dextran 40 and DMSO mix protection liquid when frozen as people's placenta amnion and chorion according to ratio of the present invention; Dextran 40 is extracellular protective agent; DMSO has hypertonicity; it is protective material in cell; foetal calf serum is nutritive substance, and three is adapted to the characteristic of amnion stem cell and chorion stem cell, the activity of coordinating protection cell; prevent that cell from forming ice crystal in freezing and thawing, and then affect the activity of cell.Therefore, institute of the present invention book frozen solution can be applied in cryogenic freezing depositary placenta amnion and chorion.
In addition; the present invention also provides a kind of freezing depositary's placenta amnion and chorial method; take foetal calf serum: dimethyl sulfoxide (DMSO): Dextran 40 is 7-9:1: 1 volume ratio weighs foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40; be mixed with frozen solution; people's placenta amnion and chorion to be stored are immersed in respectively in frozen solution; then be cooled to-100 ℃, be finally transferred in liquid nitrogen and preserve.
As preferably, the volume ratio of described foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40 is 8:1:1,
Described cooling operation is preferably:
First with the speed of 1 ℃/min, be down to-40 ℃, and then lower the temperature with the speed of 5 ℃/min.
People's placenta amnion and chorion according to the frozen mistake of the method for the invention are recovered; then separated, culturing stem cells; with the stem cell that utilizes the new people's placenta amnion gathering and chorion to prepare, carry out activity contrast; both activities is all 90%; no significant difference; shown that frozen solution of the present invention and method can extremely effectively protect amnion and chorionic cells active, cell is not almost subject to freezing injury, with freshly prepd amnion and chorion stem cell indifference.
From above technical scheme; the present invention is furtheing investigate on the structural basis of people's placenta amnion and chorion; selection is suitable for protecting dimethyl sulfoxide (DMSO), Dextran 40 and the foetal calf serum of people's placenta amnion stem cell and chorion stem cell to form protection liquid; the activity of three's coordinating protection cell; prevent that cell from forming ice crystal and sustaining damage, guaranteed Stem Cell Activity and freshly prepd amnion and the chorion Stem Cell Activity indifference after recovery.
Embodiment
The embodiment of the invention discloses a kind of frozen solution and freezing depositary's placenta amnion and chorial method.Those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Product of the present invention and method are described by preferred embodiment, related personnel obviously can be within not departing from content of the present invention, spirit and scope to product as herein described and method is changed or suitably change and combination, realize and apply the technology of the present invention.
In order further to understand the present invention, below in conjunction with embodiment, a kind of frozen solution provided by the invention and freezing depositary's placenta amnion and chorial method are elaborated.
Embodiment 1: utilize the method for the invention cryopreserved human placenta amnion and chorion
1, cryopreservation methods
Choosing the transmissible disease such as hepatitis, syphilis, acquired immune deficiency syndrome (AIDS) detects negative and without the placenta of obstetric complication, preoperative warp obtains puerpera's informed consent and signs Informed Consent Form.The aseptic polystyrene liquid storage bottle of preoperative preparation is as transportation of bottles, and in-built commercially available cell culture fluid gathers rear 4 ℃ of conditions and delivers to frozen place in lower 12 hours.
Under gnotobasis, with pincers gently separate amnion and chorion, operating scissors clip amnion and chorion.With physiological saline, rinse respectively amnion and chorion 2-3 time, remove after the dirts such as surperficial clot, amnion and chorion are cut into respectively to about 1cm in culture dish
2size.
Take foetal calf serum: dimethyl sulfoxide (DMSO): the volume ratio that Dextran 40 is 8:1:1 weighs foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40; be mixed with frozen solution; people's placenta amnion and chorion to be stored are immersed in frozen solution respectively, then are distributed in cryopreservation tube, tighten pipe lid.Cryopreservation tube is put into programmed cooling instrument, first with the speed of 1 ℃/min, be down to-40 ℃, and then be cooled to-100 ℃ with the speed of 5 ℃/min, finally cryopreservation tube is proceeded in liquid nitrogen, preserve for-196 ℃ long-term.
2, active contrast
According to collection standard in 1, again get fresh human placenta separated amnion and chorion, with physiological saline, rinse respectively amnion and chorion 2-3 time, remove after the dirts such as surperficial clot, amnion and chorion are cut into respectively to about 1cm in culture dish
2size is placed in centrifuge tube, adds 0.2% II Collagenase Type, 37 ℃ of water-bath digestion 40min.Toward centrifuge tube, add physiological saline, 300 eye mesh screens filter; The centrifugal 5min of filtrate 300G, washs 2-3 time.Then perfect medium re-suspended cell, is inoculated in culturing bottle, in 37 ℃, 5%CO
2cultivate.Approximately, after 12-24 hour, observation of cell is adherent can change liquid for the first time, after this every 3 days, changes liquid, until Growth of Cells, after merging, obtains stem cell, detects Stem Cell Activity and reaches more than 90%.
Amnion frozen in 1 and chorion are taken out, put into immediately in 37 ℃ of water-baths, and rock rapidly, after frozen storing liquid dissolves, take out immediately cryopreservation tube.After cryopreservation tube surface sterilization, in Bechtop, open, amnion and chorion suspension are transferred in centrifuge tube, add pre-cold saline, the centrifugal 5min of 300G, washs 2-3 time.According to the amnion of aforementioned new collection placenta and chorion, prepare the method for stem cell afterwards, amnion stem cell and chorion stem cell after preparation recovery, detect Stem Cell Activity and reach more than 90% equally.
Result shows, amnion and chorion stem cell after frozen are not almost subject to freezing injury, and with freshly prepd amnion and chorion stem cell indifference, cytoactive is all more than 90%, and both Growth of Cells are also identical to merging the time used.
Embodiment 2: utilize the method for the invention cryopreserved human placenta amnion and chorion
1, cryopreservation methods
Choosing the transmissible disease such as hepatitis, syphilis, acquired immune deficiency syndrome (AIDS) detects negative and without the placenta of obstetric complication, preoperative warp obtains puerpera's informed consent and signs Informed Consent Form.The aseptic polystyrene liquid storage bottle of preoperative preparation is as transportation of bottles, and in-built commercially available cell culture fluid gathers rear 4 ℃ of conditions and delivers to frozen place in lower 12 hours.
Under gnotobasis, with pincers gently separate amnion and chorion, operating scissors clip amnion and chorion.With physiological saline, rinse respectively amnion and chorion 2-3 time, remove after the dirts such as surperficial clot, amnion and chorion are cut into respectively to about 1cm in culture dish
2size.
Take foetal calf serum: dimethyl sulfoxide (DMSO): the volume ratio that Dextran 40 is 7:1:1 weighs foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40; be mixed with frozen solution; people's placenta amnion and chorion to be stored are immersed in frozen solution respectively, then are distributed in cryopreservation tube, tighten pipe lid.Cryopreservation tube is put into programmed cooling instrument, first with the speed of 1 ℃/min, be down to-40 ℃, and then be cooled to-100 ℃ with the speed of 5 ℃/min, finally cryopreservation tube is proceeded in liquid nitrogen, preserve for-196 ℃ long-term.
2, active contrast
According to collection standard in 1, again get fresh human placenta separated amnion and chorion, with physiological saline, rinse respectively amnion and chorion 2-3 time, remove after the dirts such as surperficial clot, amnion and chorion are cut into respectively to about 1cm in culture dish
2size is placed in centrifuge tube, adds 0.2% II Collagenase Type, 37 ℃ of water-bath digestion 40min.Toward centrifuge tube, add physiological saline, 300 eye mesh screens filter; The centrifugal 5min of filtrate 300G, washs 2-3 time.Then perfect medium re-suspended cell, is inoculated in culturing bottle, in 37 ℃, 5%CO
2cultivate.Approximately, after 12-24 hour, observation of cell is adherent can change liquid for the first time, after this every 3 days, changes liquid, until Growth of Cells, after merging, obtains stem cell, detects Stem Cell Activity and reaches more than 90%.
Amnion frozen in 1 and chorion are taken out, put into immediately in 37 ℃ of water-baths, and rock rapidly, after frozen storing liquid dissolves, take out immediately cryopreservation tube.After cryopreservation tube surface sterilization, in Bechtop, open, amnion and chorion suspension are transferred in centrifuge tube, add pre-cold saline, the centrifugal 5min of 300G, washs 2-3 time.According to the amnion of aforementioned new collection placenta and chorion, prepare the method for stem cell afterwards, amnion stem cell and chorion stem cell after preparation recovery, detect Stem Cell Activity and reach more than 90% equally.
Result shows, amnion and chorion stem cell after frozen are not almost subject to freezing injury, and with freshly prepd amnion and chorion stem cell indifference, cytoactive is all more than 90%, and both Growth of Cells are also identical to merging the time used.
Embodiment 3: utilize the method for the invention cryopreserved human placenta amnion and chorion
1, cryopreservation methods
Choosing the transmissible disease such as hepatitis, syphilis, acquired immune deficiency syndrome (AIDS) detects negative and without the placenta of obstetric complication, preoperative warp obtains puerpera's informed consent and signs Informed Consent Form.The aseptic polystyrene liquid storage bottle of preoperative preparation is as transportation of bottles, and in-built commercially available cell culture fluid gathers rear 4 ℃ of conditions and delivers to frozen place in lower 12 hours.
Under gnotobasis, with pincers gently separate amnion and chorion, operating scissors clip amnion and chorion.With physiological saline, rinse respectively amnion and chorion 2-3 time, remove after the dirts such as surperficial clot, amnion and chorion are cut into respectively to about 1cm in culture dish
2size.
Take foetal calf serum: dimethyl sulfoxide (DMSO): the volume ratio that Dextran 40 is 9:1:1 weighs foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40; be mixed with frozen solution; people's placenta amnion and chorion to be stored are immersed in frozen solution respectively, then are distributed in cryopreservation tube, tighten pipe lid.Cryopreservation tube is put into programmed cooling instrument, first with the speed of 1 ℃/min, be down to-40 ℃, and then be cooled to-100 ℃ with the speed of 5 ℃/min, finally cryopreservation tube is proceeded in liquid nitrogen, preserve for-196 ℃ long-term.
2, active contrast
According to collection standard in 1, again get fresh human placenta separated amnion and chorion, with physiological saline, rinse respectively amnion and chorion 2-3 time, remove after the dirts such as surperficial clot, amnion and chorion are cut into respectively to about 1cm in culture dish
2size is placed in centrifuge tube, adds 0.2% II Collagenase Type, 37 ℃ of water-bath digestion 40min.Toward centrifuge tube, add physiological saline, 300 eye mesh screens filter; The centrifugal 5min of filtrate 300G, washs 2-3 time.Then perfect medium re-suspended cell, is inoculated in culturing bottle, in 37 ℃, 5%CO
2cultivate.Approximately, after 12-24 hour, observation of cell is adherent can change liquid for the first time, after this every 3 days, changes liquid, until Growth of Cells, after merging, obtains stem cell, detects Stem Cell Activity and reaches more than 90%.
Amnion frozen in 1 and chorion are taken out, put into immediately in 37 ℃ of water-baths, and rock rapidly, after frozen storing liquid dissolves, take out immediately cryopreservation tube.After cryopreservation tube surface sterilization, in Bechtop, open, amnion and chorion suspension are transferred in centrifuge tube, add pre-cold saline, the centrifugal 5min of 300G, washs 2-3 time.According to the amnion of aforementioned new collection placenta and chorion, prepare the method for stem cell afterwards, amnion stem cell and chorion stem cell after preparation recovery, detect Stem Cell Activity and reach more than 90% equally.
Result shows, amnion and chorion stem cell after frozen are not almost subject to freezing injury, and with freshly prepd amnion and chorion stem cell indifference, cytoactive is all more than 90%, and both Growth of Cells are also identical to merging the time used.
The explanation of above embodiment 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 (5)
1. a frozen solution, is characterized in that, foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40, consists of, and the volume ratio of described foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40 is 7-9:1:1.
2. frozen solution according to claim 1, is characterized in that, the volume ratio of described foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40 is 8:1:1.
3. the application of frozen solution in cryogenic freezing depositary placenta amnion and chorion described in claim 1.
4. freezing depositary's placenta amnion and chorial method; it is characterized in that; take foetal calf serum: dimethyl sulfoxide (DMSO): the volume ratio that Dextran 40 is 7-9:1:1 weighs foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40; be mixed with frozen solution; people's placenta amnion and chorion to be stored are immersed in respectively in frozen solution; then first with the speed of 1 ℃/min, be down to-40 ℃, and then be cooled to-100 ℃ with the speed of 5 ℃/min, be finally transferred in liquid nitrogen and preserve.
5. method according to claim 4, is characterized in that, the volume ratio of described foetal calf serum, dimethyl sulfoxide (DMSO) and Dextran 40 is 8:1:1.
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| CN105165803B (en) * | 2015-10-28 | 2017-12-08 | 广州赛莱拉干细胞科技股份有限公司 | A kind of placenta amnion mescenchymal stem cell freezes protection liquid and cryopreservation methods |
| CN108077243A (en) * | 2018-01-24 | 2018-05-29 | 北京臻溪谷医学研究中心（有限合伙） | A kind of freezen protective Human plactnta amnion and chorial protection liquid and preparation method thereof and application method |
| CN108029679B (en) * | 2018-01-29 | 2020-09-22 | 山东省齐鲁细胞治疗工程技术有限公司 | Freezing medium for freezing mononuclear cells |
| CN108812643B (en) * | 2018-07-18 | 2021-09-21 | 银丰生物工程集团有限公司 | Preparation and cryopreservation method and application of human placental chorionic villus tissue |
| CN108812640B (en) * | 2018-07-18 | 2021-11-26 | 银丰生物工程集团有限公司 | Preparation and cryopreservation method and application of human placental amniotic membrane and decidua tissue |
| CN109392891B (en) * | 2018-10-26 | 2020-10-16 | 银丰生物工程集团有限公司 | Method for cryopreserving human umbilical cord tissues according to structural hierarchy system and application |
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