CN106381281A - Culture method of embryonic stem cells - Google Patents
Culture method of embryonic stem cells Download PDFInfo
- Publication number
- CN106381281A CN106381281A CN201610940327.6A CN201610940327A CN106381281A CN 106381281 A CN106381281 A CN 106381281A CN 201610940327 A CN201610940327 A CN 201610940327A CN 106381281 A CN106381281 A CN 106381281A
- Authority
- CN
- China
- Prior art keywords
- culture
- cell line
- stem cell
- continuous cell
- umbilical cord
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a culture method of embryonic stem cells, and solves the problem to obtain a certain number of embryonic stem cells in a short time. The culture method has the main technical scheme that umbilical cord tissues are subjected to sterilization cleaning, slicing frozen storage and recovery air drying, and are finally cultured; in the culture process, a No.1 culture vessel is subjected to five times of operations of repeatedly taking out first-generation cell lines and supplementing a proper amount of mesenchymal stem cell culture medium for continuous culture; meanwhile, second-generation cell lines in the located culture vessel of the first-generation cell lines in the step S8 and later are subjected to centrifugal treatment to obtain second-generation cell line pure bodies. The culture method provided by the invention has the advantage that a great number of pure embryonic stem cells with few impurities can be cultured in a short time.
Description
Technical field
The present invention relates to technical field of cell culture, particularly to a kind of cultural method of embryonic stem cell.
Background technology
Embryonic stem cell is the class cell that body early embryo is separated, it have In vitro culture infinite multiplication, self more
The characteristic of new and Multidirectional Differentiation.No matter in vitro or internal milieu, embryonic stem cell can be induced to differentiate into body almost
All of cell type, stem cell culture is a continuous process, once leaving live body to start original cuiture, its various lifes
Thing characteristic all gradually changed and the change of the increase with passage number and vitro condition and constantly have new change
Change, and its required time is probabilistic, carries out frozen very necessary therefore in preparation process in time.Traditional mesenchyme is done
The preparation of cell includes collection, transport, separation, original cuiture and Secondary Culture, freezen protective and the recovery of umbilical cord.
The patent application of such as Publication No. CN101974484A discloses a kind of preparation of human umbilical cord mesenchymal stem cells
Method, the main collection including umbilical cord, the transport of umbilical cord, the handing-over of umbilical cord, the separation of umbilical cord, frozen, the piece of tissue of piece of tissue
Recovery, original cuiture and Secondary Culture, used frozen protection liquid includes basal liquid, permeability cryoprotective agent, non-oozes
Permeability cryoprotective agent, the basal liquid in described umbilical cord frozen protection liquid includes culture fluid DMEM/F12, phosphate buffer
PBS, normal saline, the culture fluid DMEM/F1 containing 10% hyclone.The culture side of another kind embryonic stem cell set forth below
Method.
Content of the invention
It is an object of the invention to provide a kind of cultural method of embryonic stem cell, turn out in the short time volume containing miscellaneous
Less, the more pure embryonic stem cell of matter.
The above-mentioned technical purpose of the present invention technical scheme is that：A kind of culture of embryonic stem cell
Method, comprises the following steps：Step S1, is carried out disinfection to umbilical cord tissue using disinfectant solution, is carried out using buffer solution after sterilization
Cleaning；Step S2, the umbilical cord tissue of step S1 is cut into piece of tissue；Step S3, puts into frozen to the umbilical cord tissue block of step S2
In liquid under 1～7 DEG C of environment frozen 1～3d；Step S4, the umbilical cord tissue block of step S3 is carried out defrosting process, makes after taking-up
Cleaned with mescenchymal stem cell culture medium, realize umbilical cord tissue recovery；Step S5, the umbilical cord tissue block of step S4 is laid in training
10～25min is air-dried in foster ware one；Step S6, adds mescenchymal stem cell culture medium into the culture dish in S5 one and soaks
Do not had umbilical cord tissue block, and by culture dish No. one put into containing 5%CO2Incubator in the temperature inside the box be 37 DEG C at cultivated
The first continuous cell line is obtained after 5～7d；Step S7, the mescenchymal stem cell culture medium being mixed with the first continuous cell line is suctioned out, puts
Enter and obtain the second continuous cell line after adding appropriate mescenchymal stem cell culture medium culturing 2～3d in culture dish two；Simultaneously in umbilicuss
Fill into appropriate mescenchymal stem cell culture medium in the culture dish of band piece of tissue place and continue culture；Step S8, will cultivate in step S7
First continuous cell line of a number generation of ware is put into and adds mescenchymal stem cell culture medium to continue the culture second filial generation in culture dish three
Cell line, and fill into appropriate mescenchymal stem cell culture medium in culture dish one and continue to support；Produce in culture dish two simultaneously
The second raw continuous cell line carries out centrifugal treating and obtains the pure body of the second continuous cell line；Step S9, by culture dish one in step S8
Number carry out 5 times repeating to take out the first continuous cell line and fill into the operation that appropriate mescenchymal stem cell culture medium continues culture；With
When the second continuous cell line in step S8 and its first continuous cell line place culture dish afterwards carried out with centrifugal treating obtain the
The pure body of two continuous cell lines.
By technique scheme, in step S1, umbilical cord tissue is carried out removing minimizing interference culturing stem cells
Erythrocyte；It is cut into a certain size fast shape in step S2 to increase the culture area of umbilical cord tissue in unit mass, carry
Height turns out the quantity of the first continuous cell line；It is used for the preservation to umbilical cord tissue, to umbilical cord group in step S4 and step S5 in S3
Prepare culture after knitting recovery；In step S6, umbilical cord tissue, under 37 DEG C of environmental condition, is capable of cell division and goes out the first generation
Cell line, the first continuous cell line can observe by the naked eye；Can be by the first continuous cell line from umbilicuss using suction pipe in step S7
Isolate in band tissue, cultivated again and can obtain the second continuous cell line, the present invention fills in culture dish one simultaneously
Appropriate, realize former umbilical cord tissue and continue the first continuous cell line, and then the culture speed of stem cell is provided；In step S8, remove
The first continuous cell line that naked eyes are observed that, mixes to containing sightless second continuous cell line of naked eyes and mesenchyme culture medium
Conjunction liquid is put in centrifuge tube and is obtained in that second filial generation cell haplometrotic colony in centrifuge operation, second filial generation cell monomer here
Group refers to remove the second continuous cell line of the impurity such as mesenchyme culture medium；By the repetition the 5th to former umbilical cord tissue for step S9
Secondary use culture can quickly obtain the second continuous cell line.
The present invention is further：In described step S3, in frozen stock solution, comprise 70 weight portion albumin and 8 weight portions now joined
DMSO.
By technique scheme, under this concentration, DMSO is difficult to crack and discharges harmful substance and cause to damage umbilical cord group
Knit；Realize frozen speed in the unit interval low in frozen temperature-fall period, it is possible to increase the integrity of umbilical cord tissue, favorably simultaneously
The survival rate of umbilical cord tissue after thawing.
The present invention is further：In described step S3, the frozen ambient temperature of frozen stock solution is 1～4 DEG C.
By technique scheme, at this frozen temperature, DMSO is difficult to crack and discharges harmful substance and cause to damage umbilicuss
Band tissue, improves the survival rate of umbilical cord tissue after thawing.
The present invention is further：In described step S7, in culture dish two, it is provided with cell filtration film, the first continuous cell line sets
It is placed in the top of cell filtration film, the first continuous cell line is crossed in mescenchymal stem cell culture medium submergence.
By technique scheme, it is difficult to the less second filial generation of individuality observing by the naked eye after arranging cell filtration film
Cell line can pass through cell filtration film, and the larger first generation cell of the individuality that can observe by the naked eye will be by cell mistake
Membrane filtration above it is difficult to enter into below cell filtration film mesenchyme culture medium in, so being inhaled using suction pipe
After taking the mesenchyme culture medium below cell filtration film to carry out centrifugally operated, the second continuous cell line content of lower floor increases, can
Improve separating effect.
The present invention is further：In described step S7, new to filling into 2～3ml between cell filtration film and culture dish two
Fresh mesenchyme culture medium.
By technique scheme, realize induction the second continuous cell line and pass through the fresh mesenchyme training of cell filtration film picked-up
Foster base, reaches the second continuous cell line integrated distribution in the mesenchyme culture medium below cell filtration film, therefore can adopt suction pipe
The mesenchyme culture medium mixing liquid being mixed with the second continuous cell line is suctioned out and carries out centrifugally operated, improve the second continuous cell line and the
The separation degree of one continuous cell line.
The present invention is further：In described step S9, the 5th culture second of every generation first generation cell line of 5 taking-ups
After continuous cell line, using cell culture filter, the first continuous cell line is filtered, and use mesenchyme culture medium rinse cell mistake
Filter membrane, reuses centrifuge and carries out centrifugal treating to the mixing liquid of the mesenchyme culture medium containing the second continuous cell line.
By technique scheme, because the first continuous cell line is individual big, can be by cell culture filter by its point
Separate out, so carrying out after centrifugal treating using centrifuge to the mixing liquid of the mesenchyme culture medium containing the second continuous cell line,
Be difficult in second continuous cell line of lower floor contain the first continuous cell line again, improve the second continuous cell line by separation degree.
In sum, the present invention and prior art have the advantage that for：Umbilical cord cells in the present invention are turned out many successively
Organize the first continuous cell line, and the second continuous cell line is cultivated respectively by each group first continuous cell line it is achieved that can be in a short time
Obtain a number of second continuous cell line, the high purity of this second continuous cell line and survival rate are high simultaneously；By cell mistake
Filter membrane achieves being previously isolated from of the first continuous cell line and the second continuous cell line, and the first continuous cell line (is last the 5th time
Secondary) turn out the second continuous cell line after by using culture medium filter remove first continuous cell line collect filtrate carry out centrifugation behaviour
Improve the amount to obtain of the second continuous cell line, prevent from causing to obtain the second continuous cell line with the first casting out of continuous cell line
Loss.
Specific embodiment
Hereinafter the present invention is described in further detail.
Tested by formulating 6 embodiments and 2 comparative examples, and this 8 kinds of cultural methods are carried out repeating to test five
Secondary, carry out three parameter characterizations for cultural method, parameter characterization is as shown in Table 1；Embodiment 1～6 and comparative example 1～2
Concrete operation step is as follows.
Embodiment one, a kind of cultural method of embryonic stem cell, concrete incubation step is as follows：
S1, is carried out disinfection to umbilical cord tissue using ethanol, is carried out using PBS buffer solution after sterilization；
S2, the umbilical cord tissue of step S1 is cut into the umbilical cord tissue block of about 1.5mm*1.5mm size；
S3, some to the umbilical cord tissue block of step S2 puts into the jelly containing 70 weight portion albumin and 8 weight portion DMSO now joined
In liquid storage under 4 DEG C of environment frozen 2d；
S4, the umbilical cord tissue block of step S3 is carried out defrosting process, is cleaned using mescenchymal stem cell culture medium after taking-up, realizes
Umbilical cord tissue is recovered；
S5, the umbilical cord tissue block of step S4 is laid in the culture dish one of 10cm and air-dries 10～25min；
S6, adds mescenchymal stem cell culture medium not cross umbilical cord tissue block into the culture dish in S5 one, and by culture dish
Put into containing 5%CO for No. one2Incubator in the temperature inside the box be 37 DEG C at carry out cultivate 6d after obtain the first continuous cell line；S7,
The mescenchymal stem cell culture medium being mixed with the first continuous cell line is suctioned out, puts into and in culture dish two, add appropriate mesenchyme to do carefully
The second continuous cell line is obtained after born of the same parents culture medium culturing 2.5d；Fill into appropriate mesenchyme in the culture dish of umbilical cord tissue block place simultaneously
Stem cell media continues culture；
S8, the first continuous cell line of a number generation of culture dish in step S7 is put into addition mescenchymal stem cell in culture dish three
Culture medium continues culture the second continuous cell line, and fills into appropriate mescenchymal stem cell culture medium in culture dish one and continue to support；
Centrifugal treating is carried out to the second continuous cell line producing in culture dish two simultaneously and obtain the pure body of the second continuous cell line；
S9, by culture dish in step S8, No. one carries out 5 times repeating to take out the first continuous cell line and fill into appropriate mesenchyme to do carefully
Born of the same parents' culture medium continues the operation of culture；Simultaneously to the second filial generation in step S8 and its first continuous cell line place culture dish afterwards
Cell line carries out 1600 revs/min of centrifugal treating on centrifuge, obtains and remove upper strata mescenchymal stem cell culture medium after 8 minutes
Layer, obtains the second continuous cell line pure body layer of lower floor, i.e. final product.
Comparative example one, using the cultural method of the application documents of background technology, cultivates embryonic stem cell within the 12d time.
Embodiment two, a kind of cultural method of embryonic stem cell, it is, frozen stock solution includes 50 with the difference implementing
Weight portion albumin and 10 weight portion DMSO, i.e. step S3, some to the umbilical cord tissue block of step S2 put into now join contain 50
In the frozen stock solution of weight portion albumin and 10 weight portion DMSO under 4 DEG C of environment frozen 2d.
Embodiment three, a kind of cultural method of embryonic stem cell, it is with the difference implementing, frozen in step S3
Environment is 9 DEG C, i.e. step S3, the umbilical cord tissue block of step S2 is put into now join containing 70 weight portion albumin and 8 weight portions
In the frozen stock solution of DMSO under 9 DEG C of environment frozen 2d；Remaining step all same.
Example IV, a kind of cultural method of embryonic stem cell, in step S3, frozen environment is 6 DEG C, i.e. step S3, to step
The umbilical cord tissue block of rapid S2 is put in the frozen stock solution containing 70 weight portion albumin and 8 weight portion DMSO now joined in 6 DEG C of environment
Under frozen 2d；Remaining step all same.
Embodiment five, a kind of cultural method of embryonic stem cell, it is provided with culture dish two in step S7 and containing aperture be
The cell filtration film of 0.22 micrometer Millipore, this cell filtration film is available for the second continuous cell line transmission but can control and prevent the first generation thin
Born of the same parents system passes through, i.e. step S7, the mescenchymal stem cell culture medium being mixed with the first continuous cell line is suctioned out, separately takes culture dish two simultaneously
It is placed with cell filtration film at it, the first continuous cell line suctioning out is put into and in culture dish two, is located at the upper of cell filtration film
Side, obtains the second continuous cell line after being simultaneously introduced appropriate mescenchymal stem cell culture medium culturing 2～3；Simultaneously in umbilical cord tissue block
Fill into appropriate mescenchymal stem cell culture medium in the culture dish of place and continue culture.
Comparative example two, a kind of cultural method of embryonic stem cell, it is with the difference implementing four, cultivate in step S7
Being provided with ware two containing aperture is 0.02 micrometer Millipore cell filtration film, and cell filtration membrane aperture is little, and to be difficult to the second filial generation thin
Born of the same parents system passes through, i.e. step S7, the mescenchymal stem cell culture medium being mixed with the first continuous cell line is suctioned out, separately takes culture dish two simultaneously
Place cell filtration film at it, the first continuous cell line suctioning out put into the top being located at cell filtration film in culture dish two,
The second continuous cell line is obtained after being simultaneously introduced appropriate mescenchymal stem cell culture medium culturing 2～3d；Simultaneously in umbilical cord tissue block institute
Fill into appropriate mescenchymal stem cell culture medium and continue culture in culture dish.
Embodiment six, a kind of cultural method of embryonic stem cell, it is with the difference of example IV, described step S9
In, after the 5th culture the second continuous cell line of every generation first generation cell line of 5 taking-ups, all will using cell culture filter
First continuous cell line filtration, and use mesenchyme culture medium rinse cell filtration film, reuse centrifuge to thin containing the second filial generation
The mixing liquid of the mesenchyme culture medium of born of the same parents system carries out centrifugal treating, i.e. step S9, and by culture dish in step S8, No. one carries out 5 times
Repeat to take out the first continuous cell line, and fill into the operation that appropriate mescenchymal stem cell culture medium continues culture, wherein front 4 cultures
After end, centrifugal treating is carried out to the mixing liquid below cell filtration film, and after the 5th culture the second continuous cell line, first
Filtration process is carried out to the first continuous cell line using cell culture filter；Filtrate is carried out with the centrifugal treating acquisition second filial generation again thin
Born of the same parents are pure body, remaining step all same.
Cultural method characterizes one：After embodiment and comparative example carry out cultivating five times, collect each embodiment and contrast respectively
Example is to prepare the second continuous cell line or embryonic stem cell in the test tube of 100m in 12 days at the appointed time, measures its body
Long-pending, record the meansigma methodss of the volume of five times, and seek the standard deviation of its five volumes, result is as shown in Table 1.
Cultural method characterizes two：Cell counting, will be clean with wiping to blood counting chamber and cover plate, and cover plate is covered counting
On plate；Cell suspension is suctioned out a little, Deca, at cover plate edge, makes suspension be full of between cover plate and counting chamber；Standing 3 minutes；
Second filial generation cell after Microscopic observation, the big lattice total cellular score of count plate four, testing example and five cultures of comparative example
The meansigma methodss of number and standard deviation, result is as shown in Table 1.
Cultural method characterizes three：Cell viability, cell suspension is added in test tube with 0.5ml；Add 0.5ml 0.4%
Expect blue dye liquor, dye 2～3 minutes；Draw a little suspension to be applied on microscope slide, add cover plate；Several any visuals field are taken to divide under mirror
Not Ji dead cell and viable count, meter living cells account for the percentage ratio of total cellular score, after testing example and five cultures of comparative example
Meansigma methodss and standard deviation, result is as shown in Table 1.
Form 1：The characterization parameter of cultural method
By above table 1, the data according to volumetric quantities meansigma methodss can show, the second filial generation turned out using the present invention is thin
Born of the same parents system content is more, can draw the calculated second filial generation on unit count plate according to the meansigma methodss of second filial generation cell amount
Cell is many, and the impurity containing is few, and can draw there is that survival rate is high according to the data that cell viability test obtains；
The uniformity of every sign performance is good simultaneously.
This specific embodiment is only explanation of the invention, and it is not limitation of the present invention, people in the art
Member can make to the present embodiment after reading this specification as needed does not have the modification of creative contribution, but as long as at this
All protected by Patent Law in the right of invention.
Claims (6)
1. a kind of cultural method of embryonic stem cell is it is characterised in that comprise the following steps：Step S1, using disinfectant solution to umbilicuss
Band tissue carries out disinfection, and is carried out using buffer solution after sterilization；Step S2, the umbilical cord tissue of step S1 is cut into tissue
Block；Step S3, puts in frozen stock solution frozen 1 ~ 3d under 1 ~ 7 DEG C of environment to the umbilical cord tissue block of step S2；Step S4, by step
The umbilical cord tissue block of S3 carries out defrosting process, is cleaned using mescenchymal stem cell culture medium after taking-up, realizes umbilical cord tissue recovery；
Step S5, the umbilical cord tissue block of step S4 is laid in culture dish one and air-dries 10 ~ 25min；Step S6, to the culture in S5
Add mescenchymal stem cell culture medium umbilical cord tissue block is crossed in submergence in ware one, and by culture dish No. one put into containing 5%CO2's
The first continuous cell line is obtained after carrying out cultivating 5 ~ 7d in incubator at the temperature inside the box is 37 DEG C；Step S7, will be mixed with the first generation
The mescenchymal stem cell culture medium of cell line suctions out, and puts into and adds appropriate mescenchymal stem cell culture medium culturing in culture dish two
The second continuous cell line is obtained after 2 ~ 3d；Fill into appropriate mescenchymal stem cell culture medium in the culture dish of umbilical cord tissue block place simultaneously
Continue culture；Step S8, the first continuous cell line of a number generation of culture dish in step S7 is put between adding in culture dish three
Mesenchymal stem cell media continues culture the second continuous cell line, and fills into appropriate mescenchymal stem cell culture in culture dish one
Base continues to support；The second continuous cell line producing in culture dish two is carried out with centrifugal treating simultaneously, and to obtain the second continuous cell line pure
Body；Step S9, by culture dish in step S8, No. one carries out 5 times repeating to take out the first continuous cell line and fill into appropriate mesenchyme to do
Cell culture medium continues the operation of culture；Simultaneously to second in step S8 and its first continuous cell line place culture dish afterwards
Continuous cell line carries out centrifugal treating and obtains the pure body of the second continuous cell line.
2. a kind of cultural method of embryonic stem cell according to claim 1 is it is characterised in that in described step S3, freeze
70 weight portion albumin and the 8 weight portion DMSO now joining are comprised in liquid storage.
3. a kind of cultural method of embryonic stem cell according to claim 2 is it is characterised in that in described step S3, freeze
The frozen ambient temperature of liquid storage is 1 ~ 4 DEG C.
4. a kind of cultural method of embryonic stem cell according to claim 3 is it is characterised in that in described step S7, train
It is provided with cell filtration film, the first continuous cell line is arranged at the top of cell filtration film, mescenchymal stem cell is cultivated in foster ware two
The first continuous cell line is crossed in base submergence.
5. a kind of cultural method of embryonic stem cell according to claim 4 is it is characterised in that in described step S7, to
2 ~ 3ml fresh mesenchyme culture medium is filled between cell filtration film and culture dish two.
6. a kind of cultural method of embryonic stem cell according to claim 5 is it is characterised in that in described step S9,5 times
After the 5th culture the second continuous cell line of the every generation first generation cell line taken out, using cell culture filter, the first generation is thin
The filtration of born of the same parents system, and use mesenchyme culture medium rinse cell filtration film, reuse centrifuge containing the second continuous cell line
The mixing liquid of mesenchymal culture medium carries out centrifugal treating.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610940327.6A CN106381281A (en) | 2016-11-01 | 2016-11-01 | Culture method of embryonic stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610940327.6A CN106381281A (en) | 2016-11-01 | 2016-11-01 | Culture method of embryonic stem cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106381281A true CN106381281A (en) | 2017-02-08 |
Family
ID=57958153
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610940327.6A Pending CN106381281A (en) | 2016-11-01 | 2016-11-01 | Culture method of embryonic stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106381281A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191217A (en) * | 2010-03-12 | 2011-09-21 | 上海市第一人民医院 | Method for inducing differentiation from human umbilical cord mesenchymai stem cells (hucMSCs) into neural cells |
| CN102258809A (en) * | 2011-07-15 | 2011-11-30 | 中国人民解放军海军总医院 | Application of mesenchymal stem cells from human umbilical cord Wharton's jelly for preparation of cell transplantation materials |
| CN102660497A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells |
| CN103146642A (en) * | 2013-02-07 | 2013-06-12 | 中国人民解放军总医院第一附属医院 | Method for directional induction of differentiation of mesenchymal stem cells (MSCs) |
| CN103266081A (en) * | 2012-01-21 | 2013-08-28 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| CN106701669A (en) * | 2015-07-29 | 2017-05-24 | 华南生物医药研究院 | Mesenchymal stem cell for clinical treatment as well as preparation method and application thereof |
-
2016
- 2016-11-01 CN CN201610940327.6A patent/CN106381281A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191217A (en) * | 2010-03-12 | 2011-09-21 | 上海市第一人民医院 | Method for inducing differentiation from human umbilical cord mesenchymai stem cells (hucMSCs) into neural cells |
| CN102258809A (en) * | 2011-07-15 | 2011-11-30 | 中国人民解放军海军总医院 | Application of mesenchymal stem cells from human umbilical cord Wharton's jelly for preparation of cell transplantation materials |
| CN103266081A (en) * | 2012-01-21 | 2013-08-28 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| CN102660497A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells |
| CN103146642A (en) * | 2013-02-07 | 2013-06-12 | 中国人民解放军总医院第一附属医院 | Method for directional induction of differentiation of mesenchymal stem cells (MSCs) |
| CN106701669A (en) * | 2015-07-29 | 2017-05-24 | 华南生物医药研究院 | Mesenchymal stem cell for clinical treatment as well as preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Spiliotis et al. | Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generationof primary cell cultures | |
| CN103667176B (en) | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof | |
| CN104450611B (en) | A kind of primary isolation and culture method of human amnion mesenchymal stem cell | |
| CN103667187A (en) | Isolated culture method of human adipose-derived stem cells and construction method of stem cell bank | |
| CN101591638A (en) | The construction process of kidney cell line of scophthalmus maximus | |
| CN109082402A (en) | A kind of purposes of the external efficient infection organoid of AAV-DJ type adeno-associated virus | |
| CN109294974A (en) | A kind of hybridized prussian carp myeloid tissue cell line and its construction method are applied with it | |
| EP3339427B1 (en) | Preparation method for olfactory ensheathing cells | |
| CN104818241B (en) | Epinephelus lanceolatus fish-skin skin tissue cell line and its construction method | |
| CN106702499A (en) | Canine mesenchymal stem cell bank and construction method thereof | |
| CN108728408A (en) | Dog fetal membrane mescenchymal stem cell and preparation method and the culture medium used | |
| CN103484425B (en) | Pseudosciaena crocea head kidney cell line and construction method thereof | |
| CN107227296A (en) | A kind of method of myelomonocyte separation, identification and induction differentiation | |
| CN114807008B (en) | Preparation method and application of tomato leaf protoplast single-cell suspension | |
| CN106381281A (en) | Culture method of embryonic stem cells | |
| CN107641614A (en) | Purposes of the microcapsule bubble in induction stem cell macronucleus differentiation | |
| CN104419676A (en) | Construction of down's syndrome cell model and cell bank of down's syndrome cell by employing hTERT gene recombination | |
| CN108795853A (en) | Prepare the method and dog fetal membrane mescenchymal stem cell of dog fetal membrane mescenchymal stem cell | |
| CN107641612A (en) | Triploid cruciancarp caudal fin cell system 3nFC and its construction method and application | |
| TWI671399B (en) | Cell culture device and cell culture system | |
| CN104419684A (en) | Construction of 18-trisomy syndrome cell model and cell bank of 18-trisomy syndrome cell by SV40LT gene transfection | |
| CN104419677A (en) | Construction of trisomy 18 syndrome cell model and cell bank thereof by hTERT transgenosis | |
| CN104419727A (en) | Construction of neural tube defect cell model and cell bank thereof under induction of hTERT recombinant | |
| CN104419682A (en) | Construction of trisomy 21 syndrome cell model and cell bank thereof by using recombined SV40LT and hTERT genes | |
| CN104480067A (en) | Method for researching isolation, culture and ultrastructure features of human umbilical cord mesenchymal stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170208 |
|
| RJ01 | Rejection of invention patent application after publication |