CN108728408A - Dog fetal membrane mescenchymal stem cell and preparation method and the culture medium used - Google Patents

Dog fetal membrane mescenchymal stem cell and preparation method and the culture medium used Download PDF

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CN108728408A
CN108728408A CN201810525818.3A CN201810525818A CN108728408A CN 108728408 A CN108728408 A CN 108728408A CN 201810525818 A CN201810525818 A CN 201810525818A CN 108728408 A CN108728408 A CN 108728408A
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cell
stem cell
mescenchymal stem
fetal membrane
dog
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CN108728408B (en
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王小武
迟大明
朱春颖
郭春明
许晓椿
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Tianjin ruibosi Biotechnology Co.,Ltd.
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Weihai North Boya Species Diversity Research Institute Co Ltd
Tianjin Boya Xiu Yan Biotechnology Co Ltd
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Abstract

The present invention relates to dog fetal membrane mescenchymal stem cell and preparation methods and the culture medium used.The dog fetal membrane mescenchymal stem cell is prepared by method comprising the following steps：Disinfection and cleaning, digestion process, cell culture, cell passage, cell cryopreservation.Excellent technique effect is presented in the method that the present invention prepares mescenchymal stem cell from dog fetal membrane, and culture medium used can greatly improve the efficiency for preparing stem cell.Obtained mescenchymal stem cell can be beneficial treatment canid arthritis, fracture, muscle damage, ligament injury, cartilage damage, joint injury, cognition dysfunction, immune-mediated disease, xerophthalmia, recurrent uveitis, liver diseases, heart disease, kidney trouble, diabetes, enterogastric diseases, thyroid disease, skin disease.

Description

Dog fetal membrane mescenchymal stem cell and preparation method and the culture medium used
Technical field
The invention belongs to the stem-cell therapy technical field of canid disease, it is related to preparing mesenchyma from the fetal membrane of dog The purposes of the method for stem cell and this mescenchymal stem cell in treating canid disease.The present invention is from dog fetal membrane system Excellent technique effect is presented in the method for standby mescenchymal stem cell.Obtained mescenchymal stem cell can be beneficial treatment Canidae It is the arthritis of animal, fracture, muscle damage, ligament injury, cartilage damage, joint injury, cognition dysfunction, immune mediating Disease, xerophthalmia, recurrent uveitis, liver diseases, heart disease, kidney trouble, diabetes, enterogastric diseases, thyroid gland Disease, skin disease.The present invention additionally relates to the culture medium used during preparing dog fetal membrane mescenchymal stem cell, this Culture medium provides possibility efficiently to obtain mescenchymal stem cell.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs or MSC) is present in body interstitial tissue Adult stem cell, can be obtained from a variety of sources such as placenta, umbilical cord, fetal membrane, marrow, adipose tissue.MSC have it is very strong from My proliferative capacity and multi-lineage potential.Under controlled condition, it can be divided into vivo or in vitro as nerve cell, cartilage are thin A plurality of types of cells such as born of the same parents, adipocyte, cardiac muscle cell and osteoblast.MSC also has immune compatibility, Bu Huizao simultaneously At immunological rejection, allograft can be carried out.In addition MSC does not have oncogenicity also, but can hematopoiesis support, and can secrete Multiple beneficial cell factor and immune factor, therefore regeneration and repairing and treating can be can be safely used for, in traditional medical means Huge treatment potential is shown in terms of helpless difficult and complicated cases.In veterinary applications, stem cell therapy can be carried effectively The quality of life of high animal helps them to break away from ailing puzzlement.Wherein dog is not only important companion animals, also can be trained As working dogs such as rescue dogs, seeing-eye dogs, in addition it is also the important animal model of new drug assessment and Preclinical Drug experiment. Currently, in terms of dog stem-cell therapy, had it is many successful treatment dog arthritis is injected by mescenchymal stem cell, fracture, The case of muscle, ligament or cartilage/joint injury.Other such as canine cognition dysfunctions, immune-mediated disease, dry eyes Disease, recurrent uveitis, all kinds of liver diseases, heart disease, kidney trouble, diabetes, enterogastric diseases, thyroid gland problem, The treatment of skin disease etc. is also being assessed.
Huge application prospect is there is an urgent need to establish efficient canid mescenchymal stem cell method for separating and preparing, with rule Isolate to modelling high quality MSC.The upper common MSC source of dog is fat and marrow at present, but obtains and do from both sources The process of cell inevitably brings wound and pain to donor animal, and although placenta/umbilical cord, fetal membrane belong to animal life Waste when production, but they are also abundant one of the sources MSC, therefore being used as sample separation cell by them can be more preferable Protect animal in ground.
At present from the organ-/ tissue of dog, especially from the fetal membrane of dog, the method that separation prepares mescenchymal stem cell is limited, And there is its limitation.Although detaching stem cell from mankind linked groups, organ has numerous method reports, such as the applicant The Chinese invention patent publication number CN102660501A (Chinese Patent Application No. 201210159918.1) of R&D team is disclosed A method of separation and amplification mesenchymal stem cell from fresh tissue of umbilical cord, however, it is possible to be due to species difference, originally Invention it was found by the inventors that the method for the above patent document is not appropriate for being directly used in canid.
Therefore, the method for having new method to prepare mescenchymal stem cell from the fetal membrane separation of dog is urgently expected in this field.
Invention content
The object of the present invention is to provide a kind of methods preparing mescenchymal stem cell from the fetal membrane of dog, unexpectedly It was found that preparing mescenchymal stem cell from the fetal membrane of dog using the method for the present invention, encouraging effect is presented, the present invention is based on this It was found that and being accomplished.
For this purpose, first aspect present invention provides the method for preparing mescenchymal stem cell from the fetal membrane of dog, this method includes Following steps：
(1) it sterilizes and cleans：Placenta is taken out from Placenta samples collecting cassette, is placed in stainless steel cup, uses alcohol successively (such as 75% alcohol, alcohol soaking disinfection time are 10~60s, preferably 30s) and physiological saline rinsed surfaces repeatedly, to tire Disk carries out disinfection processing；Then it is slowly torn with surgical forceps and takes fetal membrane, be placed in glass dish, it is slow using phosphoric acid after rejecting blood vessel Fliud flushing (such as 0.025M phosphate sodium dihydrogen buffer solutions of pH6.5)) it rinses again, removal surface crimson blood, impurity；(this step is logical It is often to be handled in Biohazard Safety Equipment)
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block (in another cell culture plate) (for example, the size of tissue block is about 0.05-0.5 cubic centimetres, preferably about 0.05-0.2 cubic centimetres, preferably from about 0.1 cube Centimetre cube it is blocky), by tissue block be put into digestion enzyme solutions (such as it includes collagenase type I, DMEM-F12, for example, according to Following method is prepared：The collagenase type I of 0.1g is added to the DMEM-F12 of 100ml, is then obtained by filtration with 0.45 μm of filter Digestive juice) in, digestion process 0.5-3 hours (for example, 37 DEG C of digestion process 1-2 hours, such as 1.5 hours), filter removing group After knitting block (for example, being carried out by strainer, the strainer is 50-150 μm of strainer, preferably from about 100 μm of strainers), filled between addition Then matter stem cell media carries out cell cleaning to the cell that digestion obtains, finally obtains cell suspension to terminate digestion； (if not otherwise indicated, the present invention used in mescenchymal stem cell culture medium in the FBS containing 15 parts by weight, 1 parts by weight L- The DMEM-F12 of Glutamine, the Gentamicin of 0.05 parts by weight and 84 parts by weight)
(3) cell culture：By the cell suspension that step (2) obtains be put into culture vessel (for example, with density 0.2-2 × 104/cm2It is added in culture vessel, preferably with density about 1 × 104/cm2It is added), then culture vessel is put into incubator and is carried out Culture, whens culture to the 2-7 days (such as the 3-6 days, such as the 4th day, such as the 5th day), take culture vessel from incubator Go out, adds appropriate (such as 3ml) mescenchymal stem cell culture medium, continue to cultivate；It will culture at the 8-11 days (such as the 9th day) Container takes out from incubator, carries out changing liquid entirely for the first time, continues to cultivate；It carries out once changing entirely per 1-3 days (such as 2 days) backward Liquid；
(4) cell passes on：After the attached cell fusion rate in culture vessel reaches 40%-70% (such as 60%), Using digestive ferment (for example, in the present invention, if not otherwise specified, using TrypLETMExpress) by attached cell detachment vessel Supernatant is taken in bottom, centrifugation away, and mesenchymal stem cell media suspension cell again is added, is inoculated in culture vessel and is trained It supports, is hereafter changed the liquid once per 1-3 days (such as every 2 days), to get P1 generations after fusion rate reaches 70-90% (such as 80%) Fetal membrane mescenchymal stem cell；Then necessary passage is carried out (for example, used in the present invention according to above-mentioned cultural method TrypLETMExpress, consisting of：Potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, The rProtease of seven water disodium hydrogen phosphate 2160.0mg/L, EDTA 457.6mg/L, commercialization amount；The TrypLETMExpress Can be by commercial sources purchased from match Mo Feishier companies, relevant technical information is for example, see http:// www.thermofisher.com/cn/zh/home/technical-resources/media- formulation.346.html)；Optional
(5) it freezes：Will addition cells frozen storing liquid in fetal membrane mescenchymal stem cell obtained by step (4) (such as with volume ratio 1:1 Amount be added) freezed in liquid nitrogen, it is spare.(for example, the cells frozen storing liquid includes DMEM-F12, dimethyl sulfoxide (DMSO) and people's blood Albumin.In one embodiment, the cells frozen storing liquid include about 65 parts of DMEM-F12, about 10 parts of dimethyl sulfoxide (DMSO), About 15 parts of human serum albumin.In one embodiment, which includes 50% low sugar DMEM culture solutions, 40% FBS, 10% dimethyl sulfoxide (DMSO).)
According to method of the first aspect of the present invention, PBS buffer solution described in step (1) is the sodium salt and/or sylvite of phosphoric acid It prepares, pH 5.0-8.0, preferably pH are 5.5-76, and preferably pH is 6.0-7.0.In one embodiment, the PBS A concentration of 0.01-0.5M of phosphate radical, preferably 0.02-0.1M in buffer solution.In following experiments of the present invention, do not say especially such as Bright, PBS buffer solution used is sodium ascorbyl phosphate, wherein a concentration of 0.025M of phosphate radical, pH 6.5.It should be noted that this hair A person of good sense has found that PBS buffer solution concentration and pH value within the above range is little for the influential effect of the method for the present invention.
According to method of the first aspect of the present invention, it is that collagenase type I is added enzyme solutions to be digested described in step (2) DMEM-F12 is obtained by filtration by filter, and digestive ferment 0.05g-0.5g, preferably digestive ferment are 0.08g-0.2g, excellent It is 0.1g to select digestive ferment, and DMEM-F12 50-500ml, preferably DMEM-F12 are 80-200ml, and preferably DMEM-F12 is 100ml, filter are 0.45 μm of filter.In one embodiment, the digestion enzyme solutions are by the type i collagen of 0.1g Enzyme is added in the DMEM-F12 of 100ml, mixing, and filtering (such as being filtered with 0.45um filters) obtains.
According to method of the first aspect of the present invention, wherein in step (2), the size of tissue block is about 0.05-0.5 cubes li Rice, preferably about 0.05-0.2 cubic centimetres, preferably from about 0.1 cubic centimetre of cube is blocky.
According to method of the first aspect of the present invention, wherein in step (2), organize block size at 0.05-0.5 cubic centimetres, It is preferred that being highly preferred when about 0.1 cubic centimetre of 0.05-0.2 cubic centimetres, especially size.While it is contemplated that tissue pieces are small Be conducive to the realization of the method for the present invention, however the present inventor in experiments it is found that 0.05 cubic centimetre, 0.1 cubic centimetre, 0.5 Under three kinds of states of cubic centimetre, they are almost the same to the digestion process effect of digestive ferment, and volume is more than after 1 cubic centimetre There is notable adverse effect to the digestion effect of digestive ferment, which can be weak to a certain extent by extending digestion time Change.
According to method of the first aspect of the present invention, wherein in step (2), the time of digestion process is 0.5-3 hours, preferably 1-2.5 hours, preferably 1.5-2 hours.The inventors discovered that within 1-2.5 hours digestion process time, disappear to tissue block It is best to change treatment effect, both can guarantee that tissue block obtains sufficient digestion process, was also avoided that cell is destroyed.
According to method of the first aspect of the present invention, wherein in step (2), digestion process is the temperature near body temperature It is carried out in range, preferably 34-40 DEG C, preferably 36-38 DEG C, preferably 37 DEG C.
According to method of the first aspect of the present invention, wherein in step (2), digestion process carries out in constant-temperature table.
According to method of the first aspect of the present invention, wherein in step (2), tissue block is removed in filtering to be carried out by strainer , the strainer is 50-150 μm of strainer, preferably from about 100 μm of strainers.
According to method of the first aspect of the present invention, wherein in step (2), terminating the mescenchymal stem cell culture medium of digestion is According to 2:1~1:2 ratio is added, and preferably 1:1 ratio, the ratio are volume ratio.
According to method of the first aspect of the present invention, wherein in step (2), cell cleaning comprises the concrete steps that 5-15 points of centrifugation Clock removes supernatant, PBS buffer solution is added, cell is resuspended, then centrifuge 5-15 minutes, remove supernatant, and it is dry thin that mesenchyma is added Born of the same parents' culture medium extracts a small amount of samples and carries out cell count.Centrifugal rotational speed is 800-2000rpm, preferably 1250rpm, centrifugation time It is preferably 10 minutes.
According to method of the first aspect of the present invention, wherein including FBS, L- in the mescenchymal stem cell culture medium Glutamine (Pidolidone), Gentamicin (gentamicin) and DMEM-F12.In one embodiment, it is filled between described FBS containing 10-20% in matter stem cell media.In one embodiment, contain in the mescenchymal stem cell culture medium There is about 15% FBS.In one embodiment, the L- containing 0.5-2% in the mescenchymal stem cell culture medium Glutamine.In one embodiment, about 1% L-Glutamine is contained in the mescenchymal stem cell culture medium.? In one embodiment, the Gentamicin of about 0.01-0.1% is contained in the mescenchymal stem cell culture medium.In a reality It applies in scheme, about 0.05% Gentamicin is contained in the mescenchymal stem cell culture medium.In one embodiment, institute State the DMEM-F12 containing 80-90% in mescenchymal stem cell culture medium.In one embodiment, the mescenchymal stem cell Contain about 84% DMEM-F12 in culture medium.In one embodiment, containing about in the mescenchymal stem cell culture medium The FBS of 15 parts by weight, the L-Glutamine of about 1 parts by weight, the Gentamicin of about 0.05 parts by weight and about 84 parts by weight DMEM-F12。
According to method of the first aspect of the present invention, wherein the mescenchymal stem cell culture medium, which is also augmented, glycine.Example Such as, the FBS containing 15 parts by weight, the L-Glutamine of 1 parts by weight, 0.05 parts by weight in the mescenchymal stem cell culture medium Gentamicin, 84 parts by weight DMEM-F12,0.025%w/v glycine.
According to method of the first aspect of the present invention, wherein the TrypLETMIt is also additionally added to 1 in Express digestive ferments, 2- propylene glycol.For example, the TrypLETMThe 1,2- propylene glycol of 0.05%w/v is also additionally added in Express digestive ferments.? Supplement propylene glycol in glycine and digestive ferment is augmented through having now surprisingly been found that, in mescenchymal stem cell culture medium to be helped to carry The yield of high mescenchymal stem cell.
According to method of the first aspect of the present invention, CO wherein in step (3) described incubator2A concentration of 3-7% is preferably dense Degree is 5%, and incubator temperature controls in body temperature environs, preferably 34-40 DEG C, preferably 36-38 DEG C, preferably 37 DEG C. Such CO2Concentration and temperature are usually also condition of culture commonly used in the art.
In addition, in the first aspect of the present invention, it is dry thin to provide separation and amplification mesenchyma from dog fetal membrane flesh tissue The method of born of the same parents.Therefore, second aspect of the present invention provides a kind of dog fetal membrane mescenchymal stem cell.
Dog fetal membrane mescenchymal stem cell according to a second aspect of the present invention is any implementation according to a first aspect of the present invention What scheme the method obtained.
Dog fetal membrane mescenchymal stem cell according to a second aspect of the present invention, cell purity are more than 85%, are greater than 90%.In one embodiment, the fetal membrane mescenchymal stem cell is after 3 more than generation pass on, and cell purity is more than 85%, example Such as larger than 90%.
Further, third aspect present invention is related to dog fetal membrane mescenchymal stem cell produced by the present invention in preparation for controlling Treat and/or prevent the purposes in the preparation selected from following canid disease：Arthritis, fracture, muscle damage, ligament injury, Cartilage damage, joint injury, cognition dysfunction, immune-mediated disease, xerophthalmia, recurrent uveitis, liver diseases, Heart disease, kidney trouble, diabetes, enterogastric diseases, thyroid disease, skin disease.
Further, fourth aspect present invention is related to a kind of mescenchymal stem cell culture medium, wherein containing about 15 parts by weight FBS, the L-Glutamine of about 1 parts by weight, about 0.05 parts by weight Gentamicin and about 84 parts by weight DMEM-F12.
Mescenchymal stem cell culture medium according to a fourth aspect of the present invention, wherein also supplement has glycine.For example, between described The L-Glutamine of FBS, 1 parts by weight containing 15 parts by weight in mesenchymal stem cell media, 0.05 parts by weight DMEM-F12,0.025%w/v glycine of Gentamicin, 84 parts by weight.
The present invention is further illustrated below.Cited text in document and the document cited in the present invention It offers, their full content is incorporated herein by reference.
In the present invention, in any technical solution of either side of the present invention, any technical characteristic is equally applicable to this Any embodiment of the either side of invention, as long as they will not cause contradiction, and this be mutually applicable in if necessary may be used To be suitably modified.
In the present invention, term " fetal membrane mescenchymal stem cell " refers to the mescenchymal stem cell from fetal membrane.Therefore exist In the present invention, more particularly to the present invention context in, term " fetal membrane mescenchymal stem cell " can with " fetal membrane stem cell ", " stem cell ", " mescenchymal stem cell " are used interchangeably, unless otherwise clearly indicating.
In the present invention, term " PBS buffer solution " or " PBS " refer to phosphate buffer.Those skilled in the art are ripe Know the general formula and preparation method and their general aspects such as pH value or pH of the PBS used under situation of the present invention Range.
In the present invention, term " fetal membrane " refers to the fetal membrane of dog, particularly relates to the fetal membrane within 4 hours postpartum.
It is filled between mescenchymal stem cell (mesenchymal stem cell, MSC) such as the mammal such as mankind or dog Matter stem cell is separated from marrow earliest, from mesoblastic a kind of with multi-lineage potential and self-renewing The tissue stem cell of ability has under external specified conditions to osteoblast, cartilage cell, adipocyte, endothelium in vivo Ability (the Caplan AI.Mesenchymal of a variety of adult cell differentiation such as cell, nerve cell, myocyte, liver cell stem cells.J Orthop Res.1991,9:641-650.Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999； 284:143-147).It is newest research shows that mescenchymal stem cell has immunological regulation and Hematopoiesis Support affect, and be easy to outer Source channel genes expression.Therefore mescenchymal stem cell not still tissue-engineered bone, cartilage and cardiac muscle structure in seed cell, Important carrier cell in gene therapy, and since mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host of inhibition of transplant anti- Function is answered, is with a wide range of applications in hematopoietic stem cell transplantation and organ transplant.Mescenchymal stem cell has external patch The characteristic of wall growth, using this characteristic, people have succeeded from liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood It is separately cultured out mescenchymal stem cell in equal Various Tissues.
The method of the invention discloses a kind of from fetal membrane a large amount of separating mesenchymal stem cells, and protected using this method It deposits fetal membrane mescenchymal stem cell and establishes fetal membrane stem cell bank.The present inventor was separately cultured mesenchyma in summary and did in the past On the basis of cell, using tissue digestion enzymic digestion fetal membranes block, in conjunction with stationary culture, success is isolated from fetal membrane A large amount of mescenchymal stem cells.Mescenchymal stem cell purity that the method for the present invention obtains is high, quantity is more, has dry with medulla mesenchyma The identical biological characteristics of cell, can be to differentiation such as osteoblast, cartilage cell, adipocyte, endothelial cell, nerve cells. Since stem cell is clinically with a wide range of applications compared with adult stem cell naivety, rich content in fetal membrane, we use Conventional cell freezing method freezes mescenchymal stem cell as bleeding of the umbilicus, establishes fetal membrane stem cell bank, is later dry The further investigation of cell and clinical treatment lay the foundation.
Due to containing abundant candidate stem cell in bleeding of the umbilicus, people are this important biology of the fetal membrane candidate stem cell of dog Resource stores, and a kind for the treatment of means are provided for a variety of diseases in the blood system and disease of immune system.Same fetal membrane mesenchyma As a kind of more importantly stem cell resource, we are frozen in -196 with conventional cell freezing method and are taken the photograph stem cell It is preserved for a long time in the profound hypothermia liquid nitrogen of family name's degree, establishes fetal membrane stem cell bank, be stem cell in the future or application for the treatment of preservation seed.
According to the method for the present invention, in-between mesenchymal stem cell media, which is matched, to succeed and effectively to fetal membrane mesenchyma Stem cell carries out amplification in vitro.According to the method for the present invention, wherein changing liquid and to organize the setting of checkout time to shorten adherent thin Born of the same parents reach the time of specified fusion rate.According to the method for the present invention, the formula of digestive ferment and the digestion time of fetal membranes and side Method can succeed and effectively the full cell in tissue is separated.
The present invention is easy to operate, convenient and practical, can obtain a large amount of mescenchymal stem cell, and differentiation performance is good, have at The ability of the cell differentiations such as osteocyte, adipocyte, cartilage cell, endothelial cell, nerve cell.Tire of the present invention success from dog Separation obtains a large amount of higher mescenchymal stem cells of purity in film, and can establish the fetal membrane stem cell bank of dog with this method to store up The stem cell of standby this great application prospect.The method is simple and easy to do, and since fetal membrane is as bleeding of the umbilicus, Cell Component is inmatureer, It derives from a wealth of sources, is conveniently easy to get, therefore the method for the present invention will above have extensive foreground in the canid application of stem cell.
Specific implementation mode
The present invention can be further described by the following examples, however, the scope of the present invention and unlimited In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out the material and test method that are arrived used in experiment general And/or specific description.Although to realize the present invention many materials and operating method used in purpose be it is known in the art that But the present invention still makees description as detailed as possible herein.
Embodiment 1 prepares mescenchymal stem cell from the fetal membrane of dog
(1) it sterilizes and cleans：In Biohazard Safety Equipment, placenta is taken out from Placenta samples collecting cassette, is placed in stainless steel cup In, alcohol (75% alcohol) and physiological saline rinsed surfaces (alcohol soaking disinfection time 30s) repeatedly are used successively), to tire Disk carries out disinfection processing；Then it is slowly torn with surgical forceps and takes fetal membrane, be placed in glass dish, it is slow using phosphoric acid after rejecting blood vessel Fliud flushing (the 0.025M phosphate sodium dihydrogen buffer solutions of pH6.5)) it rinses again, removal surface crimson blood, impurity；
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block in another cell culture plate Tissue block is put into digestion enzyme solutions (preparation method by (about 0.1 cubic centimetre of cube is blocky)：By the collagenase type I of 0.1g The DMEM-F12 of 100ml is added, digestive juice then is obtained by filtration with 0.45 μm of filter) in, 0.5-3 hours (these of digestion process In example, 37 DEG C of digestion process 1.5 hours), after tissue block (this example passes through 100 μm of strainers and carries out) is removed in filtering, mesenchyma is added Stem cell media is (with volume ratio 1:1 ratio addition；In this example, 15 parts by weight are contained in mescenchymal stem cell culture medium The DMEM-F12 of FBS, the L-Glutamine of 1 parts by weight, the Gentamicin of 0.05 parts by weight and 84 parts by weight) disappeared with terminating Change, then carrying out cell cleaning to the cell that digestion obtains, (in this example, 1250rpm centrifuges 10 minutes removal supernatants, is added PBS buffer solution is resuspended after cell again 1250rpm and centrifuges 10 minutes removal supernatants, and mescenchymal stem cell culture medium is added and (extracts A small amount of samples carry out cell count)), finally obtain cell suspension；
(3) cell culture：By the cell suspension that step (2) obtains be put into culture vessel (in this example, with density about 1 × 104/cm2It is added), then put culture vessel into incubator (CO2A concentration of 5%, 37 DEG C of temperature) in cultivated, culture to the Culture vessel is taken out from incubator when 2-7 days (this example, general culture was to the 4th day), it is dry thin to add appropriate (3ml) mesenchyma Born of the same parents' culture medium continues to cultivate；Culture vessel is taken out from incubator at the 8-11 days (this example, the 9th day), is carried out for the first time Liquid is changed entirely, continues to cultivate；Per 1-3 days (this example, 2 days), progress once changed liquid entirely backward；
(4) cell passes on：After the attached cell fusion rate in culture vessel reaches 40%-70% (this example, 60%), Using digestive ferment, (this example uses TrypLETMExpress) by attached cell detachment vessel bottom, centrifugation is taken supernatant away, is added Enter mesenchymal stem cell media suspension cell again, is inoculated in culture vessel and is cultivated, hereafter 1-3 days every (this example, every 2 It) it changes the liquid once, until fusion rate reaches after 70-90% (this example up to 80%) to get P1 for fetal membrane mescenchymal stem cell；Then Necessary passage, which is carried out, according to above-mentioned cultural method (in this example, P15 generations is passaged to, wherein cell purity is more than after 3 generations passed on 95%；This example, commercial prod TrypLE usedTMExpress consisting of：Potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, seven water disodium hydrogen phosphate 2160.0mg/L, EDTA457.6mg/L, commercialization amount rProtease；The TrypLETMExpress can be by commercial sources purchased from match Mo Feishier companies, relevant technical information ginseng See http://www.thermofisher.com/cn/zh/home/technical-resources/media- formulation.346.html)；
(5) it freezes：Cells frozen storing liquid (1 will be added in fetal membrane mescenchymal stem cell obtained by step (4):1), cold in liquid nitrogen Freeze, it is spare (this example, cells frozen storing liquid used include 65 parts of DMEM-F12,10 parts of dimethyl sulfoxide (DMSO)s, 15 parts of human serum albumins).? Before freezing, by the details of mescenchymal stem cell (including dog age, kind of dog, hereditary information, immunization campaign information, viral diagnosis Information etc.) input computer data, it is for future reference to establish data archival.
Embodiment 2 prepares mescenchymal stem cell from the fetal membrane of dog
(1) it sterilizes and cleans：In Biohazard Safety Equipment, placenta is taken out from Placenta samples collecting cassette, is placed in stainless steel cup In, alcohol (75% alcohol) and physiological saline rinsed surfaces (alcohol soaking disinfection time 30s) repeatedly are used successively), to tire Disk carries out disinfection processing；Then it is slowly torn with surgical forceps and takes fetal membrane, be placed in glass dish, it is slow using phosphoric acid after rejecting blood vessel Fliud flushing (the 0.025M phosphate sodium dihydrogen buffer solutions of pH6.5)) it rinses again, removal surface crimson blood, impurity；
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block in another cell culture plate Tissue block is put into digestion enzyme solutions (preparation method by (about 0.1 cubic centimetre of cube is blocky)：By the collagenase type I of 0.1g The DMEM-F12 of 100ml is added, digestive juice then is obtained by filtration with 0.45 μm of filter) in, 0.5-3 hours (these of digestion process In example, 37 DEG C of digestion process 1.5 hours), after tissue block (this example passes through 100 μm of strainers and carries out) is removed in filtering, mesenchyma is added Stem cell media is (with volume ratio 1:1 ratio addition；In this example, 15 parts by weight are contained in mescenchymal stem cell culture medium DMEM-F12,0.025%w/v of FBS, the L-Glutamine of 1 parts by weight, the Gentamicin of 0.05 parts by weight, 84 parts by weight Glycine) to terminate digestion, then carrying out cell cleaning to the cell that digestion obtains, (in this example, 1250rpm is centrifuged 10 minutes and is gone Except supernatant, PBS buffer solution is added, 10 minutes removal supernatants of 1250rpm centrifugations again are resuspended after cell, it is dry thin that mesenchyma is added Born of the same parents' culture medium (extract a small amount of samples and carry out cell count)), finally obtain cell suspension；
(3) cell culture：By the cell suspension that step (2) obtains be put into culture vessel (in this example, with density about 1 × 104/cm2It is added), then put culture vessel into incubator (CO2A concentration of 5%, 37 DEG C of temperature) in cultivated, culture to the Culture vessel is taken out from incubator when 2-7 days (this example, general culture was to the 4th day), it is dry thin to add appropriate (3ml) mesenchyma Born of the same parents' culture medium continues to cultivate；Culture vessel is taken out from incubator at the 8-11 days (this example, the 9th day), is carried out for the first time Liquid is changed entirely, continues to cultivate；Per 1-3 days (this example, 2 days), progress once changed liquid entirely backward；
(4) cell passes on：After the attached cell fusion rate in culture vessel reaches 40%-70% (this example, 60%), Using digestive ferment, (this example uses TrypLETMExpress is also additionally added to 1, the 2- the third two of 0.05%w/v in the digestive ferment Alcohol) by attached cell detachment vessel bottom, supernatant is taken in centrifugation away, and mesenchymal stem cell media suspension cell again is added, then It is inoculated in culture vessel to be cultivated, hereafter be changed the liquid once per 1-3 days (this example, every 2 days), until fusion rate reaches 70-90% To get P1 for fetal membrane mescenchymal stem cell after (this example up to 80%)；Then necessary passage (this example is carried out according to above-mentioned cultural method In, P15 generations are passaged to, wherein cell purity is more than 95% after 3 generations passed on；This example, TrypLE usedTMIts composition of Express For：Potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, seven water disodium hydrogen phosphates The rProtease of 2160.0mg/L, EDTA 457.6mg/L, commercialization amount；The TrypLETMExpress can pass through business way Diameter is purchased from match Mo Feishier companies, and relevant technical information is referring to http://www.thermofisher.com/cn/zh/ home/technical-resources/media-formulation.346.html)；
(5) it freezes：Cells frozen storing liquid (1 will be added in fetal membrane mescenchymal stem cell obtained by step (4):1), cold in liquid nitrogen Freeze, it is spare (this example, cells frozen storing liquid used include 65 parts of DMEM-F12,10 parts of dimethyl sulfoxide (DMSO)s, 15 parts of human serum albumins).? Before freezing, by the details of mescenchymal stem cell (including dog age, kind of dog, hereditary information, immunization campaign information, viral diagnosis Information etc.) input computer data, it is for future reference to establish data archival.
In the present embodiment 2, from step (1) to step (4), gained P1 is for dog fetal membrane mescenchymal stem cell, every gram of fetal membrane The average yield of tissue is 4~7 × 107In cell context (average result tested through 5 times, similarly hereinafter)；In the present embodiment 2, If do not augment glycine in mescenchymal stem cell culture medium, every gram fetal membranes of the P1 for dog fetal membrane mescenchymal stem cell Average yield is 6~8 × 105Cell context；In the present embodiment 2, if digestive ferment TrypLETM1 is not augmented in Express, 2- propylene glycol, then P1 for the average yield of every gram of fetal membranes of dog fetal membrane mescenchymal stem cell 5~6 × 105Cell context； This shows to augment glycine only in mescenchymal stem cell culture medium while in digestive ferment TrypLETM1 is augmented in Express, When 2- propylene glycol, the yield of dog fetal membrane mescenchymal stem cell can be just significantly improved.Similar, in embodiment 1, from step (1) it is tested to P1 obtained by step (4) through 5 times, the average yield of every gram of fetal membranes is 5 for dog fetal membrane mescenchymal stem cell ~8 × 105In cell context (when being handled people's fetal membranes in 1 same method of the embodiment of the present invention, every gram of fetal membrane group The average yield knitted can reach 7~8 × 107Cell context, big two orders of magnitude, this may be caused by due to species variation). The present inventor has found that the fetal membrane mesenchyma with reference to the method preparation dog of CN 102660501A embodiments 1 is dry thin in complementary testing When born of the same parents, P1 for every gram of fetal membranes of stem cell average yield 3~5 × 105In cell context；With reference to CN 106702499A In embodiment 1 to 5 method of embodiment when various scheme combinations, P1 is respectively less than 7 for the average yield of every gram of fetal membranes of stem cell ×105Cell.
The Identification of Biological Characteristics of embodiment 3, fetal membrane MSC
Following tests, the experiment of two kinds of stem cells are carried out to embodiment 1 and 2 gained dog fetal membrane mescenchymal stem cell of embodiment As a result indifference.
1, cell growth and its Morphological Characteristics
By being separately cultured for embodiment 1 and embodiment 2, aobvious after gained dog fetal membrane mescenchymal stem cell culture 48 hours Fusiformis attached cell can be obviously seen under micro mirror, can form within 8 days or so turbine-like cell clone, 75% can be formed after had digestive transfer culture The leapfrog structure of left and right fusion.In incubation, it is found that this cellular morphology is relatively uniform, growth rate is fast, and adherent speed is fast, easily It is digested by pancreatin, is passaged to 3-15 generations, form and growth characteristic are also without substantially changeing.
2, flow cytometry identification of M SC surface markers
The 1st, 3,5,10,15 generation cells, Flow cytometry cell surface marker, dynamic observation incubation are taken respectively The variation of middle cell surface marker.Cell phenotype testing result show the distinctive mark CD44 of cell statement mescenchymal stem cell, CD90, CD105 do not express CD11, CD19, CD34.
3, the measurement of the drafting of fetal membrane MSC growth curves and exponential phase doubling time
Logarithmic growth phase cell, digestion count, with the LG-DMEM culture mediums of 10%FBS be made cell suspension (2 × 104/ ml), it is inoculated with 0.5ml in 24 orifice plates per hole, 37 DEG C, 5%CO2, cultivate under saturated humidity.3 multiple holes, trypan blue dye are taken daily Living cell counting number after color calculates average value, is observed continuously 7 days.Using incubation time as horizontal axis, cell number is the longitudinal axis, is drawn thin Intracellular growth curve.Cell is calculated in the doubling time of exponential phase, i.e. Td=Tlg2/Lg (Nt/ with Patterson formula No), Td：Doubling time (h), T：Cell increases to the time (h) used in Nt, N by No：Cell number.
Cell growth curve is drawn by the result of daily cell count, calculates the doubling time.It can by cell growth curve To find out, cell was in exponential phase of growth at the 3-5 days.According to formula calculate the 5th generation cell exponential phase of growth multiplication Time is within the scope of 24~33 hours.
4, the identification of fetal membrane MSC multi-lineage potentials
(1) osteogenic induction：The 3 generations above MSC, by 5 × 104/ hole is inoculated with six orifice plates, is put in 37 DEG C, 5%CO2, saturated humidity Under, after being cultivated for 24 hours in MSC culture mediums, use instead containing 10% through screening the DMEM-HG of FBS and 0.1 μM of dexamethasone, anti-bad being added 50 μM of hematic acid phosphate, β-phosphoglycerol 5mM are put in 37 DEG C, 5%CO2, cultivate under saturated humidity, every 3 days half amounts change liquid, Coinduction 2-4 weeks.Alkaline phosphatase staining identification osteoblast is formed, and Von Kossa dyeing identification bone tubercles are formed.Containing 0.1 μM of dexamethasone, 50 μM of ascorbyl phosphate, β-phosphoglycerol 5mM cultures are added in 10% DMEM-HG through screening FBS 1 week, apparent change occurred for cellular morphology, becomes polygonal from fusiform fibroblast sample, is similar to neuronal cell Sample, there is long filiform and protrudes in cell periphery, and can extend to surrounding.After continuing culture 2 weeks or more, occurs calcification in cellular matrix Spot, mineralizer gradually appear, and initially form the small junction structure of multilayer, until after cultivating 4 weeks, it is seen that apparent calcium scoring.At 2 weeks Alkaline phosphatase staining is in strong positive reaction, reaches 94% or more, and the control group not induced is largely then feminine gender, only Have and be shown as weakly positive less than 5%, shows that cell is converted to osteoblast.Von Kossa dyeing can will deposit in bone tubercle Calcium dye black, the visible a large amount of black bone tubercle of induction group has an apparent stereochemical structure, and control group is at any time all There is no positive reaction.
(2) Adipogenic induction：The 3 generations above MSC, by 5 × 104/ hole is inoculated in six orifice plates, is put in 37 DEG C, 5%CO2, saturation Under humidity, after being cultivated for 24 hours in MSC culture mediums, use instead containing 10% through screening the DMEM in high glucose of FBS, and dexamethasone 0.5 is added μM, 25 μM of indocin, IBMX 0.5mM, 2 μ g/ml of insulin, be put in 37 DEG C, 5%CO2, cultivate under saturated humidity, every 3 days half Amount changes liquid, coinduction 2 weeks, and oil red dyeing identification fat drips are formed.Containing 10% DMEM-HG through screening FBS, dexamethasone is added 0.5 μM, 50 μM of indocin, IBMX 0.5mM, 5 μ g/ml of insulin cultivate 3 days, morphologic change occurs for cell, by fusiform Fibroblast sample, which gradually tapers up, to shorten, and 90% or more cell becomes cube or polygonal；Continuous culture 7 days, it is visible under mirror There are small fat drips to occur into the cell, with the extension of incubation time, fat drips gradually increase and merge, until when cultivating 2 weeks, it is seen that melt It closes pockets of fat drips and is full of entire cell.The fat generated in oil red O stain visible cell dyes red by specificity.
By the detection of above series of data target, show dry using the isolated dog mesenchyma of the method for the present invention Cell has the ability to differentiation such as osteoblast, adipocytes, it was demonstrated that the dog mescenchymal stem cell tool that the method for the present invention obtains There are stem cell properties.
This method is easy to operate, stablizes, and the fetal membrane mescenchymal stem cell activity being thus prepared is high, can be used for dog routine The treatment of means refractory disease.

Claims (10)

1. a kind of dog fetal membrane mescenchymal stem cell, cell purity is more than 85%；The dog fetal membrane mescenchymal stem cell is to shine to include What the method for following steps was prepared：
(1) it sterilizes and cleans：Placenta is used to alcohol and physiological saline rinsed surfaces repeatedly successively, is carried out disinfection place to placenta Reason；Then it tears and takes fetal membrane, after rejecting blood vessel, rinsed again using phosphate buffer, removal surface crimson blood, impurity；
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block, tissue block is put into digestion enzyme solutions, is disappeared Change processing 0.5-3 hours, after tissue block is removed in filtering, mescenchymal stem cell culture medium is added to terminate digestion, then to digestion Obtained cell carries out cell cleaning, finally obtains cell suspension；
(3) cell culture：The cell suspension that step (2) obtains is put into culture vessel, then culture vessel is put into incubator It is cultivated, culture takes out culture vessel at the 2-7 days from incubator, adds appropriate mescenchymal stem cell culture medium, Continue to cultivate；Culture vessel is taken out from incubator at the 8-11 days, carries out changing liquid entirely for the first time, continues to cultivate；Backward It carried out once changing liquid entirely per 1-3 days；
(4) cell passes on：After the attached cell fusion rate in culture vessel reaches 40%-70%, it will be pasted using digestive ferment Supernatant is taken in parietal cell detachment vessel bottom, centrifugation away, and mesenchymal stem cell media suspension cell again is added, is inoculated in Culture vessel is cultivated, hereafter per changing the liquid once within 1-3 days, until fusion rate is filled to get P1 between fetal membrane after reaching 70-90% Matter stem cell；Then necessary passage is carried out according to above-mentioned cultural method；Optional
(5) it freezes：Addition cells frozen storing liquid in fetal membrane mescenchymal stem cell obtained by step (4) is freezed in liquid nitrogen, it is spare.
2. the method for preparing mescenchymal stem cell from the fetal membrane of dog, this approach includes the following steps：
(1) it sterilizes and cleans：Placenta is used to alcohol and physiological saline rinsed surfaces repeatedly successively, is carried out disinfection place to placenta Reason；Then it tears and takes fetal membrane, after rejecting blood vessel, rinsed again using phosphate buffer, removal surface crimson blood, impurity；
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block, tissue block is put into digestion enzyme solutions, is disappeared Change processing 0.5-3 hours, after tissue block is removed in filtering, mescenchymal stem cell culture medium is added to terminate digestion, then to digestion Obtained cell carries out cell cleaning, finally obtains cell suspension；
(3) cell culture：The cell suspension that step (2) obtains is put into culture vessel, then culture vessel is put into incubator It is cultivated, culture takes out culture vessel at the 2-7 days from incubator, adds appropriate mescenchymal stem cell culture medium, Continue to cultivate；Culture vessel is taken out from incubator at the 8-11 days, carries out changing liquid entirely for the first time, continues to cultivate；Backward It carried out once changing liquid entirely per 1-3 days；
(4) cell passes on：After the attached cell fusion rate in culture vessel reaches 40%-70%, it will be pasted using digestive ferment Supernatant is taken in parietal cell detachment vessel bottom, centrifugation away, and mesenchymal stem cell media suspension cell again is added, is inoculated in Culture vessel is cultivated, hereafter per changing the liquid once within 1-3 days, until fusion rate is filled to get P1 between fetal membrane after reaching 70-90% Matter stem cell；Then necessary passage is carried out according to above-mentioned cultural method；Optional
(5) it freezes：Addition cells frozen storing liquid in fetal membrane mescenchymal stem cell obtained by step (4) is freezed in liquid nitrogen, it is spare.
3. method according to claim 2, PBS buffer solution described in step (1) is that the sodium salt of phosphoric acid and/or sylvite are prepared, Its pH is 5.0-8.0, and preferably pH is 5.5-76, and preferably pH is 6.0-7.0；For example, PBS buffer solution used is sodium ascorbyl phosphate, A concentration of 0.025M of middle phosphate radical, pH 6.5.
4. method according to claim 2, the cells frozen storing liquid includes DMEM-F12, dimethyl sulfoxide (DMSO) and human serum albumin.
5. method according to claim 2, it is that DMEM-F12 is added in collagenase type I that enzyme solutions are digested described in step (2), is led to Cross what filter was obtained by filtration, digestive ferment 0.05g-0.5g, preferably digestive ferment are 0.08g-0.2g, and preferably digestive ferment is 0.1g, DMEM-F12 50-500ml, preferably DMEM-F12 are 80-200ml, and preferably DMEM-F12 is 100ml, filter For 0.45 μm of filter；For example, the digestion enzyme solutions are that the collagenase type I of 0.1g is added in the DMEM-F12 of 100ml, Mixing, filtering (such as being filtered with 0.45um filters) obtain.
6. in method according to claim 2, wherein step (2), the time of digestion process is 0.5-3 hours, and digestion process is It is carried out in 36-38 DEG C of temperature range.
7. method according to claim 2, the FBS containing 15 parts by weight in the mescenchymal stem cell culture medium, 1 parts by weight The DMEM-F12 of L-Glutamine, the Gentamicin of 0.05 parts by weight and 84 parts by weight.
8. method according to claim 7, the mescenchymal stem cell culture medium, which is also augmented, glycine；For example, being filled between described Propylene glycol is also additionally added in matter stem cell media；Further, CO in step (3) described incubator2A concentration of 3- 7%, preferred concentration 5%, incubator temperature controls within the scope of 36-38 DEG C.
9. a kind of mescenchymal stem cell culture medium, wherein the L-Glutamine of FBS, about 1 parts by weight containing about 15 parts by weight, The DMEM-F12 and optional glycine of the Gentamicin of about 0.05 parts by weight and about 84 parts by weight.
10. the dog tire that the dog fetal membrane mescenchymal stem cell of claim 1 or the method for any one of claim 2-8 are prepared Film mescenchymal stem cell is being prepared for treating and/or preventing the purposes in the preparation selected from following canid disease：Joint Inflammation, fracture, muscle damage, ligament injury, cartilage damage, joint injury, cognition dysfunction, immune-mediated disease, dry eyes Disease, recurrent uveitis, liver diseases, heart disease, kidney trouble, diabetes, enterogastric diseases, thyroid disease, skin Disease.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109370986A (en) * 2018-12-11 2019-02-22 南京农业大学 The extracting method and its preparation of a kind of dog fat stem cell and application
CN112080464A (en) * 2020-09-16 2020-12-15 中国科学院昆明动物研究所 Canine umbilical cord-derived mesenchymal stem cell culture medium and culture method
CN114736856A (en) * 2022-06-15 2022-07-12 天津百恩生物科技有限公司 Preparation method and application of canine placenta mesenchymal stem cells

Citations (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1229405A (en) * 1996-09-06 1999-09-22 日本化药株式会社 Novel acetamide derivatives and protease inhibitors
EP1876233A1 (en) * 2005-01-24 2008-01-09 Japan Health Sciences Foundation Cells capable of differentiating into cardiac muscle cells
WO2009067601A1 (en) * 2007-11-20 2009-05-28 Pioneer Surgical Orthobiologics, Inc. Cryopreservation of cells using cross-linked bioactive hydrogel matrix particles
US20120094380A1 (en) * 2010-10-18 2012-04-19 Sunshine Biotech Inc. Human multipotent embryonic stem cell-like progenitor cells
CN102586184A (en) * 2011-09-05 2012-07-18 博雅干细胞科技有限公司 Method for establishing placental mesenchyme stem cell library
CN102660501A (en) * 2012-05-21 2012-09-12 博雅干细胞科技有限公司 Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord
CN102676451A (en) * 2011-09-05 2012-09-19 博雅干细胞科技有限公司 Method for separating mesenchymal stem cells from placenta
CN103422176A (en) * 2013-07-29 2013-12-04 庞希宁 Construction method of human amniotic mesenchymal stem cell bank
CN103469309A (en) * 2013-09-12 2013-12-25 博雅干细胞科技有限公司 Method for separating living cell and constructing cell bank by means of tissue homogenate method
CN103857789A (en) * 2011-08-31 2014-06-11 世元世龙技术株式会社 Method for preparing a basic culture medium for mesenchymal stem cells, basic culture medium for mesenchymal stem cells, and cell therapeutic agent cultured and differentiated using same
US20140322182A1 (en) * 2011-12-06 2014-10-30 Kang Stem Biotech, Co., LTD. Canine Amniotic Membrane-Derived Multipotent Stem Cells
CN105521523A (en) * 2015-11-20 2016-04-27 金艺鹏 Preparation and preservation method of canine and amniotic membrane
CN105769910A (en) * 2016-04-15 2016-07-20 遵义医学院附属医院 Application of human amniotic mesenchymal stem cells
WO2016187308A1 (en) * 2015-05-20 2016-11-24 Amgen Inc. Triazole agonists of the apj receptor
WO2017001783A1 (en) * 2015-06-30 2017-01-05 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Method for the cryopreservation of cells for therapeutic purposes
CN106434557A (en) * 2016-11-25 2017-02-22 博雅干细胞科技有限公司 Method for preparing CD34 positive cells from umbilical cord mesenchymal stem cells
CN106520676A (en) * 2016-12-09 2017-03-22 博雅干细胞科技有限公司 Method for preparing human amniotic membrane epithelial cells from human placenta amnion and application thereof
CN106702499A (en) * 2017-01-13 2017-05-24 天津百恩生物科技有限公司 Canine mesenchymal stem cell bank and construction method thereof
CN106727704A (en) * 2016-12-26 2017-05-31 云南农业大学 Dog stem cell secretion factor injection
CN106754674A (en) * 2016-12-09 2017-05-31 博雅干细胞科技有限公司 Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion
CN106890193A (en) * 2017-04-21 2017-06-27 云南农业大学 A kind of dog, cat stem cell eye-drops preparations and its application
CN107022521A (en) * 2017-02-13 2017-08-08 广东唯泰生物科技有限公司 Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell
CN107164319A (en) * 2017-05-25 2017-09-15 佛山科学技术学院 A kind of method of the mescenchymal stem cell in original cuiture dog umbilical cord source
CN107236704A (en) * 2017-06-15 2017-10-10 博雅干细胞科技有限公司 From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used
CN107299082A (en) * 2017-08-02 2017-10-27 广州中科博雅干细胞科技有限公司 Placenta interstitial cell and the method for being trained mescenchymal stem cell are separated from tissue
CN107375329A (en) * 2017-06-12 2017-11-24 佛山科学技术学院 One kind treats arthritic stem cell medicine of dog and preparation method thereof
CN107384857A (en) * 2017-08-02 2017-11-24 广州中科博雅干细胞科技有限公司 The cultural method of autologous fat mescenchymal stem cell and the culture medium used
CN107635567A (en) * 2015-04-17 2018-01-26 加利福利亚大学董事会 People's amnion for the acellular of cell delivery, cell culture and inflammation prevention
CN107746829A (en) * 2017-10-19 2018-03-02 佛山科学技术学院 A kind of method separated with the mescenchymal stem cell in original cuiture dog placenta source
CA3036391A1 (en) * 2016-10-04 2018-04-12 Transwell Biotech Co., Ltd. Compositions and methods for cell cryopreservation
WO2018083700A1 (en) * 2016-11-03 2018-05-11 Exostem Biotec Ltd. Mesenchymal stem cells populations, their products, and use thereof
WO2018108859A1 (en) * 2016-12-12 2018-06-21 Health And Biotech France (H & B France) Perinatal tissue derived mesenchymal stem cells: method of preparation and uses thereof
CN108456657A (en) * 2018-03-31 2018-08-28 无锡博雅基因科技有限公司 Dog umbilical cord mesenchymal stem cells and preparation method thereof and cryopreservation methods
CN108486050A (en) * 2018-03-31 2018-09-04 无锡博雅基因科技有限公司 The method for preparing mescenchymal stem cell from the umbilical cord of dog
CN108795853A (en) * 2018-05-28 2018-11-13 天津博雅秀岩生物技术有限公司 Prepare the method and dog fetal membrane mescenchymal stem cell of dog fetal membrane mescenchymal stem cell
CN109674819A (en) * 2018-12-21 2019-04-26 博雅干细胞科技有限公司 Placenta mesenchyma stem cell preparation and its purposes for treating hardening illness
CN110075124A (en) * 2019-02-19 2019-08-02 浙江大学 AMSC-MALAT1-Exo is used to prepare application in the drug for the treatment of liver diseases and preparation method thereof
CN110317781A (en) * 2019-07-18 2019-10-11 天津博雅秀岩生物技术有限公司 The method of mesenchymal stem cell cryopreserving and recovery
CN110317782A (en) * 2019-07-18 2019-10-11 天津博雅秀岩生物技术有限公司 Improve the method survived after mescenchymal stem cell recovery and frozen stock solution used
CN112029717A (en) * 2020-09-17 2020-12-04 英科博雅基因科技（天津）有限公司 Serum-free in vitro domestication culture of human mesenchymal stem cells

Patent Citations (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1229405A (en) * 1996-09-06 1999-09-22 日本化药株式会社 Novel acetamide derivatives and protease inhibitors
EP1876233A1 (en) * 2005-01-24 2008-01-09 Japan Health Sciences Foundation Cells capable of differentiating into cardiac muscle cells
WO2009067601A1 (en) * 2007-11-20 2009-05-28 Pioneer Surgical Orthobiologics, Inc. Cryopreservation of cells using cross-linked bioactive hydrogel matrix particles
EP2222159A1 (en) * 2007-11-20 2010-09-01 Pioneer Surgical Orthobiologics, Inc. Cryopreservation of cells using cross-linked bioactive hydrogel matrix particles
US20120094380A1 (en) * 2010-10-18 2012-04-19 Sunshine Biotech Inc. Human multipotent embryonic stem cell-like progenitor cells
CN103857789A (en) * 2011-08-31 2014-06-11 世元世龙技术株式会社 Method for preparing a basic culture medium for mesenchymal stem cells, basic culture medium for mesenchymal stem cells, and cell therapeutic agent cultured and differentiated using same
CN102586184A (en) * 2011-09-05 2012-07-18 博雅干细胞科技有限公司 Method for establishing placental mesenchyme stem cell library
CN102676451A (en) * 2011-09-05 2012-09-19 博雅干细胞科技有限公司 Method for separating mesenchymal stem cells from placenta
US9771555B2 (en) * 2011-12-06 2017-09-26 Kang Stem Biotech, Co., LTD. Canine amniotic membrane-derived multipotent stem cells
US20140322182A1 (en) * 2011-12-06 2014-10-30 Kang Stem Biotech, Co., LTD. Canine Amniotic Membrane-Derived Multipotent Stem Cells
CN102660501A (en) * 2012-05-21 2012-09-12 博雅干细胞科技有限公司 Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord
CN103422176A (en) * 2013-07-29 2013-12-04 庞希宁 Construction method of human amniotic mesenchymal stem cell bank
CN103469309A (en) * 2013-09-12 2013-12-25 博雅干细胞科技有限公司 Method for separating living cell and constructing cell bank by means of tissue homogenate method
CN107635567A (en) * 2015-04-17 2018-01-26 加利福利亚大学董事会 People's amnion for the acellular of cell delivery, cell culture and inflammation prevention
CA2985542A1 (en) * 2015-05-20 2016-11-24 Amgen Inc. Triazole agonists of the apj receptor
WO2016187308A1 (en) * 2015-05-20 2016-11-24 Amgen Inc. Triazole agonists of the apj receptor
WO2017001783A1 (en) * 2015-06-30 2017-01-05 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Method for the cryopreservation of cells for therapeutic purposes
CN105521523A (en) * 2015-11-20 2016-04-27 金艺鹏 Preparation and preservation method of canine and amniotic membrane
CN105769910A (en) * 2016-04-15 2016-07-20 遵义医学院附属医院 Application of human amniotic mesenchymal stem cells
CA3036391A1 (en) * 2016-10-04 2018-04-12 Transwell Biotech Co., Ltd. Compositions and methods for cell cryopreservation
WO2018083700A1 (en) * 2016-11-03 2018-05-11 Exostem Biotec Ltd. Mesenchymal stem cells populations, their products, and use thereof
CN106434557A (en) * 2016-11-25 2017-02-22 博雅干细胞科技有限公司 Method for preparing CD34 positive cells from umbilical cord mesenchymal stem cells
CN106520676A (en) * 2016-12-09 2017-03-22 博雅干细胞科技有限公司 Method for preparing human amniotic membrane epithelial cells from human placenta amnion and application thereof
CN106754674A (en) * 2016-12-09 2017-05-31 博雅干细胞科技有限公司 Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion
WO2018108859A1 (en) * 2016-12-12 2018-06-21 Health And Biotech France (H & B France) Perinatal tissue derived mesenchymal stem cells: method of preparation and uses thereof
CN106727704A (en) * 2016-12-26 2017-05-31 云南农业大学 Dog stem cell secretion factor injection
CN106702499A (en) * 2017-01-13 2017-05-24 天津百恩生物科技有限公司 Canine mesenchymal stem cell bank and construction method thereof
CN107022521A (en) * 2017-02-13 2017-08-08 广东唯泰生物科技有限公司 Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell
CN106890193A (en) * 2017-04-21 2017-06-27 云南农业大学 A kind of dog, cat stem cell eye-drops preparations and its application
CN107164319A (en) * 2017-05-25 2017-09-15 佛山科学技术学院 A kind of method of the mescenchymal stem cell in original cuiture dog umbilical cord source
CN107375329A (en) * 2017-06-12 2017-11-24 佛山科学技术学院 One kind treats arthritic stem cell medicine of dog and preparation method thereof
CN107236704A (en) * 2017-06-15 2017-10-10 博雅干细胞科技有限公司 From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used
CN107384857A (en) * 2017-08-02 2017-11-24 广州中科博雅干细胞科技有限公司 The cultural method of autologous fat mescenchymal stem cell and the culture medium used
CN107299082A (en) * 2017-08-02 2017-10-27 广州中科博雅干细胞科技有限公司 Placenta interstitial cell and the method for being trained mescenchymal stem cell are separated from tissue
CN107746829A (en) * 2017-10-19 2018-03-02 佛山科学技术学院 A kind of method separated with the mescenchymal stem cell in original cuiture dog placenta source
CN108456657A (en) * 2018-03-31 2018-08-28 无锡博雅基因科技有限公司 Dog umbilical cord mesenchymal stem cells and preparation method thereof and cryopreservation methods
CN108486050A (en) * 2018-03-31 2018-09-04 无锡博雅基因科技有限公司 The method for preparing mescenchymal stem cell from the umbilical cord of dog
CN108795853A (en) * 2018-05-28 2018-11-13 天津博雅秀岩生物技术有限公司 Prepare the method and dog fetal membrane mescenchymal stem cell of dog fetal membrane mescenchymal stem cell
CN109674819A (en) * 2018-12-21 2019-04-26 博雅干细胞科技有限公司 Placenta mesenchyma stem cell preparation and its purposes for treating hardening illness
CN110075124A (en) * 2019-02-19 2019-08-02 浙江大学 AMSC-MALAT1-Exo is used to prepare application in the drug for the treatment of liver diseases and preparation method thereof
CN110317781A (en) * 2019-07-18 2019-10-11 天津博雅秀岩生物技术有限公司 The method of mesenchymal stem cell cryopreserving and recovery
CN110317782A (en) * 2019-07-18 2019-10-11 天津博雅秀岩生物技术有限公司 Improve the method survived after mescenchymal stem cell recovery and frozen stock solution used
CN112029717A (en) * 2020-09-17 2020-12-04 英科博雅基因科技（天津）有限公司 Serum-free in vitro domestication culture of human mesenchymal stem cells

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"2017年《现代化工》总目次(按栏目索引)", 《现代化工》 *
MANUEL FILIOLI URANIO 等: "Characterization and In Vitro Differentiation Potency of Early-Passage Canine Amnion- and Umbilical Cord-Derived Mesenchymal Stem Cells as Related to Gestational Age", 《MOLECULAR REPRODUCTION & DEVELOPMENT》 *
刘雄昊 等: "胎膜来源的间充质干细胞促进烧伤愈合的研究", 《现代生物医学进展》 *
李春雨 等: "不同消化酶对体外培养的原代乳鼠雪旺细胞的影响", 《中国实验诊断学》 *
章守琴 等: "胎膜干细胞的分离方法和特性", 《医学综述》 *
陈亭 等: "间充质干细胞在低氧环境下的增殖、代谢与成骨分化：胎盘羊膜及骨髓来源间充质干细胞的对比", 《中国组织工程研究与临床康复》 *
马海英 等: "组织块贴壁法提取胎盘、脐带和胎膜间充质干细胞效果观察", 《山东医药》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109370986A (en) * 2018-12-11 2019-02-22 南京农业大学 The extracting method and its preparation of a kind of dog fat stem cell and application
CN112080464A (en) * 2020-09-16 2020-12-15 中国科学院昆明动物研究所 Canine umbilical cord-derived mesenchymal stem cell culture medium and culture method
CN114736856A (en) * 2022-06-15 2022-07-12 天津百恩生物科技有限公司 Preparation method and application of canine placenta mesenchymal stem cells

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