CN106566798A - Method for getting amniotic epithelial stem cells and kit - Google Patents
Method for getting amniotic epithelial stem cells and kit Download PDFInfo
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- CN106566798A CN106566798A CN201610983737.9A CN201610983737A CN106566798A CN 106566798 A CN106566798 A CN 106566798A CN 201610983737 A CN201610983737 A CN 201610983737A CN 106566798 A CN106566798 A CN 106566798A
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- digestion
- edta
- digestion product
- mixed liquor
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- 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]
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- 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 method for getting amniotic epithelial stem cells and a kit. The method comprises the following steps: using a mixed enzyme to digest an amniotic membrane to get a digestive product; and separating amniotic epithelial stem cells from the digestive product, wherein the mixed enzyme includes trypsin-EDTA mixture and dispase II. By using the method or the kit of the invention, a lot of high-purity and high-activity amniotic epithelial stem cells can be gotten.
Description
Technical field
The present invention relates to bioengineering field.In particular it relates to obtain method and the examination of amnioic epithelium stem cell
Agent box.
Background technology
Amniotic membrane is a part for fetal membrane, is made up of the amnioic epithelium and amnion mesenchymal of rising in blastocyst inner cell mass.Closely
Research over year has shown that human amnion membrane (human amniotic epithelial cells, hAECs) has part
Embryonic stem cell characteristic, can be divided into the different types of cell of three germinal layers, therefore, hAECs is provided as the cell of regenerative medicine
Source is of great interest.
However, the method for currently acquired amnioic epithelium stem cell still has much room for improvement.
The content of the invention
It is contemplated that at least solving at least one technical problem present in prior art to a certain extent.
It should be noted that the present invention is completed based on the following discovery of inventor：
Still lack the method effectively with regard to obtaining amnioic epithelium stem cell at present, traditional single enzyme digestion is detached
Amnioic epithelium stem cell yield is low, and activity is low, is unfavorable for further amplification and applies.
Inventor has found that during collagenase treatment, the factor appreciable impact enzyme such as species, consumption, digestion time of enzyme disappears
Change the species for the treatment of effect, especially enzyme, and then affect the yield and activity of amnioic epithelium stem cell.Inventor Jing is furtherd investigate
It was found that, the digestion effect of single enzyme is not good, it is difficult to obtain a large amount of and higher amnioic epithelium stem cell of purity, and mixed enzyme can
Effectively amniotic membrane is digested, so as to obtain a large amount of, the amnioic epithelium stem cell that purity is higher and activity is higher.Further
Ground, it is surprisingly found by the inventors that, the mixing of pancreas enzyme -EDTA mixed liquor (Trypsin-EDTA) and Bacillus polymyxa Neutral proteinase II (Dispase II)
The digestion effect of enzyme is optimal.
For this purpose, in one aspect of the invention, the present invention proposes a kind of method of acquisition amnioic epithelium stem cell.According to
Embodiments of the invention, methods described includes：Amniotic membrane is carried out into digestion process using mixing enzyme preparation, digestion product is obtained；With
And the amnioic epithelium stem cell is separated from the digestion product, wherein, the mixing enzyme preparation includes：Pancreas enzyme -EDTA is mixed
Close liquid；And Bacillus polymyxa Neutral proteinase II.
Using digestion process method, can be single so as to isolate by the protein degradation in amnion tissue between cell
Cell.Inventor has found that single enzyme is difficult amniotic membrane digestion completely, and resulting amnioic epithelium stem cell amount is less.Further, send out
A person of good sense has found that the effect digested using mixing enzyme preparation is preferable, to reduce the damage to cell.Inventor unexpectedly sends out
It is existing, using including that pancreas enzyme -EDTA mixed liquor and the mixing enzyme preparation of Bacillus polymyxa Neutral proteinase II are digested, it is obtained in that a large amount of, purity
The higher amnioic epithelium stem cell of higher and activity.However, using the effect on driving birds is not good of other kind of fermentoid.
Embodiments in accordance with the present invention, the method for the acquisition amnioic epithelium stem cell can also have following supplementary technology
Feature：
Embodiments in accordance with the present invention, the mixing enzyme preparation includes：The pancreatin of 0.05 volume %～0.25 volume %-
EDTA mixed liquors；And the Bacillus polymyxa Neutral proteinase II of 2U/mL～2.5U/mL.Inventor it was unexpectedly observed that pancreas enzyme -EDTA mixed liquor and point
The concentration appreciable impact digestive efficiency of scattered enzyme II, and then affect yield, purity and the activity of amniotic epithelial cells.In above-mentioned optimum
Under concentration, the yield of amnioic epithelium stem cell, purity and activity are higher.According to a particular embodiment of the invention, mixing enzyme preparation
Middle pancreas enzyme concentration can be 0.08 mass %, 0.10 mass %, 0.13 mass %, 0.18 mass % or 0.21 mass %.Work as pancreas
The excessive concentration of enzyme-EDTA mixed liquors and/or Bacillus polymyxa Neutral proteinase II, will cause to damage so as to activity decrease to amnioic epithelium stem cell,
If pancreas enzyme -EDTA mixed liquor and/or Bacillus polymyxa Neutral proteinase II concentration are too low, it is impossible to fully digestion, so as to make the amnioic epithelium for obtaining dry thin
The yield and purity of born of the same parents is relatively low.Thus, the method for acquisition amnioic epithelium stem cell according to embodiments of the present invention is obtained in that greatly
Amount, purity is higher and active preferable amnioic epithelium stem cell.
Embodiments in accordance with the present invention, the mixing enzyme preparation is 0.5～2 with the amount ratio of amniotic membrane:1, preferably 1:1.Send out
Person of good sense's discovery, the addition appreciable impact digestion process effect of mixing enzyme preparation, and then affect the yield of amnioic epithelium stem cell
And activity.Inventor is it was unexpectedly observed that when the amount ratio of mixing enzyme preparation and amniotic membrane is 0.5～2:1, preferably 1:When 1, can obtain
In a large number, the amnioic epithelium stem cell that purity is higher and activity is higher.If addition is excessive, cell injury is easily caused, activity
Decline；If addition is very few, enzymic digestion is insufficient, so as to cause amnioic epithelium stem cell yield relatively low.
Embodiments in accordance with the present invention, the digestion process includes：The amniotic membrane is carried out into the first digestion process, digestion is abandoned
Liquid, obtains the first digestion product；First digestion product is carried out into the second digestion process, Digestive system is abandoned, the second digestion is obtained
Product；Second digestion product is carried out into the 3rd digestion process, Digestive system is abandoned, the 3rd digestion product is obtained；And will be described
3rd digestion product carries out the 4th digestion process, abandons Digestive system, obtains the digestion product.
Inventor has found that the organizational structure of amniotic membrane is finer and close, and needs are digested for a long time.If disposably long
Time digests, and fragment of tissue, digestion product in Digestive system etc. can affect enzymatic activity, and then reduce digestive efficiency, so as to cause
Amnioic epithelium stem cell yield is reduced.Further, inventor has found, using multiple digestion process mode, will every time at digestion
Digestive system after reason is discarded, and changes to fresh mixing enzyme preparation.Thus, through 4 digestion process, can largely digest
Tissue so that amnioic epithelium stem cell yield is higher, and activity is higher.
According to a particular embodiment of the invention, after each digestion process, by adding tire Sanguis Bovis seu Bubali in digestion reaction system
Clearly (FBS), to terminate digestion reaction.
Embodiments in accordance with the present invention, first digestion process is shaken under 37 DEG C of temperature, the rotating speed of 120r/min
Swing 10min～20min, preferred 10min；Second digestion product is shaken under 37 DEG C of temperature, the rotating speed of 150r/min
30min～40min, preferred 40min；3rd digestion product is shaken under 37 DEG C of temperature, the rotating speed of 150r/min
30min～40min, preferred 40min；3rd digestion product is shaken under 37 DEG C of temperature, the rotating speed of 150r/min
30min～40min, preferred 30min.Inventor obtains above-mentioned optimum digestion process condition through many experiments, with this understanding
Amnioic epithelium stem cell can be effectively obtained, and yield is higher, purity is larger and activity is stronger.
It should be noted that for the method that amnioic epithelium stem cell is separated from digestion product does not make considered critical, only
It is obtained in that amnioic epithelium stem cell.Embodiments in accordance with the present invention, do to obtain the higher amnioic epithelium of purity
Cell, the separation includes：The digestion product is filtered, filtrate is collected；The filtrate is centrifuged, is collected thin
Born of the same parents；And cleaned in the cell, to obtain the amnioic epithelium stem cell.Thereby, it is possible to effectively remove attachment removal
Digestive system on cell, and avoid that cell is caused to damage, do so as to obtain the amnioic epithelium that purity is higher and activity is stronger
Cell.
Embodiments in accordance with the present invention, methods described includes：It is using mixing enzyme preparation that size is described for 2cm × 2cm
Amniotic membrane carries out digestion process, obtains digestion product；By the digestion product with 300 mesh sieve net filtrations, filtrate is collected；By the filter
Liquid is centrifuged 5min under the rotating speed of 2000r/min, collects cell；And clean the cell with normal saline 2 times, to obtain
Obtain the amnioic epithelium stem cell.
Inventor has found that it is 2cm × 2cm that amniotic membrane is cut to into size so that the area for carrying out digestion reaction is larger, promotes
Digestion reaction fully occurs.Then, digestion product is sieved, to remove bulk tissue.Additionally, by filtrate under 2000r/min
Centrifugation 5min, can either sedimentation cell, be easy to collect, while smaller destroy cell structure and activity.Thus, it is possible to obtain
In a large number, the amnioic epithelium stem cell that purity is higher and activity is higher.
Embodiments in accordance with the present invention, methods described further includes that amplification is processed, and the amplification is processed to be included：Will be described
Amnioic epithelium stem cell is resuspended in culture medium, obtains mixed liquor；And the mixed liquor is inoculated in the culture medium, 37
Cultivate at DEG C to there is clone, it is long to passing on.According to a particular embodiment of the invention, the culture medium is alpha-MEM cultures
Base.Inventor has found that it is individual cells to separate the amnioic epithelium stem cell for obtaining, and is expanded to obtain substantial amounts of amniotic membrane
Epithelial stem cell.Under above-mentioned optimum amplification treatment conditions, while amplification its activity can also further improved.
In another aspect of this invention, the present invention proposes a kind of test kit.Embodiments in accordance with the present invention, the reagent
Box includes：Pancreas enzyme -EDTA mixed liquor；And Bacillus polymyxa Neutral proteinase II.Thus, it is obtained in that using test kit according to embodiments of the present invention
In a large number, the amnioic epithelium stem cell that purity is higher and activity is higher.
Embodiments in accordance with the present invention, the test kit is used to obtain amnioic epithelium stem cell.
Using digestion process method, can be single so as to isolate by the protein degradation in amnion tissue between cell
Cell.Inventor has found that single enzyme is difficult amniotic membrane digestion completely, and resulting amnioic epithelium stem cell amount is less.Further, send out
A person of good sense has found that the effect digested using mixing enzyme preparation is preferable, to reduce the damage to cell.Inventor unexpectedly sends out
It is existing, using including that pancreas enzyme -EDTA mixed liquor and the mixing enzyme preparation of Bacillus polymyxa Neutral proteinase II are digested, it is obtained in that a large amount of, purity
The higher amnioic epithelium stem cell of higher and activity.
Embodiments in accordance with the present invention, the test kit includes：The pancreas enzyme -EDTA of 0.05 volume %～0.25 volume % is mixed
Close liquid；And the Bacillus polymyxa Neutral proteinase II of 2U/mL～2.5U/mL.
Inventor it was unexpectedly observed that the concentration appreciable impact digestive efficiency of pancreas enzyme -EDTA mixed liquor and Bacillus polymyxa Neutral proteinase II, and then
Affect yield, purity and the activity of amniotic epithelial cells.According to a particular embodiment of the invention, pancreas enzyme concentration in mixing enzyme preparation
Can be 0.08 mass %, 0.10 mass %, 0.13 mass %, 0.18 mass % or 0.21 mass %.When pancreas enzyme -EDTA mixing
The excessive concentration of liquid and/or Bacillus polymyxa Neutral proteinase II, will cause to damage so as to activity decrease, if pancreas enzyme -EDTA to amnioic epithelium stem cell
Mixed liquor and/or Bacillus polymyxa Neutral proteinase II concentration are too low, it is impossible to fully digestion, so as to make the yield of amnioic epithelium stem cell that obtains and pure
Degree is relatively low.Thus, test kit according to embodiments of the present invention be obtained in that in a large number, purity is higher and active preferable amnioic epithelium
Stem cell.
Embodiments in accordance with the present invention, pancreatin and the amount ratio of EDTA are 2.5 in the pancreas enzyme -EDTA mixed liquor:1, root
According to the preferred embodiments of the present invention, the pancreas enzyme -EDTA mixed liquor contains 0.5mg/mL pancreatin and 0.2mg/mL EDTA.Thus,
Test kit according to embodiments of the present invention be obtained in that in a large number, purity is higher and active preferable amnioic epithelium stem cell.
The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become from the description with reference to accompanying drawings below to embodiment
It is substantially and easy to understand, wherein：
Fig. 1 shows cell viability analysis chart according to an embodiment of the invention.
Specific embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this
It is bright, and be not considered as limiting the invention.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carry out according to the technology or condition described by document in the art or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, be can pass through city available from conventional products.
Embodiment
In this embodiment, amnioic epithelium stem cell is obtained in following manner：
(1) amniotic membrane peeled off from fresh human placenta, cuts to 2cm × 2cm sizes, and physiology salt is washed three times, will killed in a large scale as far as possible
Only；
(2) amniotic membrane that 5g is shredded is fitted in 50ml centrifuge tubes, adds the pancreas enzyme -EDTA mixed liquors of 5mL 0.25% (to contain
0.5mg/mL pancreatin and 0.2mg/mL EDTA) and 2.5U/mL Bacillus polymyxa Neutral proteinase II, shake under 37 DEG C of temperature, the rotating speed of 120r/min
10min, adds FBS to terminate Trypsin digestion, and the first resulting digestion product is centrifuged under the rotating speed of 2000r/min
5min, the Digestive system of acquisition is discarded；
(3) 5mL 0.25% pancreas enzyme -EDTA mixed liquor (pancreatin containing 0.5mg/mL and 0.2mg/mL are added in centrifuge tube
EDTA) with 2.5U/mL Bacillus polymyxa Neutral proteinase II, 40min is shaken under 37 DEG C of temperature, the rotating speed of 150r/min, adds FBS to terminate
Trypsin digests, and the second resulting digestion product is centrifuged into 5min, the Digestive system of acquisition under the rotating speed of 2000r/min
Discard；
(4) 5mL 0.25% pancreas enzyme -EDTA mixed liquor (pancreatin containing 0.5mg/mL and 0.2mg/mL are added in centrifuge tube
EDTA) with 2.5U/mL Bacillus polymyxa Neutral proteinase II, 40min is shaken under 37 DEG C of temperature, the rotating speed of 150r/min, adds FBS to terminate
Trypsin digests, and the 3rd resulting digestion product is centrifuged into 5min, the Digestive system of acquisition under the rotating speed of 2000r/min
Discard；
(5) 5mL 0.25% pancreas enzyme -EDTA mixed liquor (pancreatin containing 0.5mg/mL and 0.2mg/mL are added in centrifuge tube
EDTA) with 2.5U/mL Bacillus polymyxa Neutral proteinase II, 30min is shaken under 37 DEG C of temperature, the rotating speed of 150r/min, adds FBS to terminate
Trypsin digests, and the 4th resulting digestion product is collected into filtrate with 300 mesh sieve net filtrations；
(6) filtrate is centrifuged into 5min under the rotating speed of 2000r/min, collects cell, cleaned with normal saline 2 times, so as to
Obtain amnioic epithelium stem cell；
(7) by the resuspended rear inoculation of 2ml alpha-MEM culture medium of amnioic epithelium stem cell, cultivate at 37 DEG C, every 48h
Change liquid once, long to passing on until clone occurs, the amnioic epithelium stem cell after being expanded.
Comparative example
Method according to embodiment obtains amnioic epithelium stem cell, and difference is, in step (2), (3), (4), (5), profit
Digested with 0.25% pancreas enzyme -EDTA mixed liquor.
Vitality test is carried out to the amnioic epithelium stem cell after embodiment amplification
The vigor of the amnioic epithelium stem cell obtained by the step of comparing embodiment and comparative example (6), comprises the following steps that：
The amnioic epithelium stem cell obtained by (6) the step of embodiment and comparative example is washed 2 times with physiology salt respectively, plus
Enter the digestion about 30s of the TrypLE Express without animal component, visible short fusiformis attached cell is rounded and suspends under mirror
Afterwards, add normal saline piping and druming cell and cell is transferred to into centrifuge tube, 1200rpm centrifugation 5min discard supernatant, use 2ml
Normal saline it is resuspended, by cell viability analyser detect cell vigor.
Amnioic epithelium stem cell vigor obtained by embodiment step (6) is 92.1%.Illustrate to utilize the method for the present invention
Obtain amnioic epithelium stem cell during, digestion process mild condition is less to amnioic epithelium stem cell injuries, so as to get sheep
Film epithelial stem cell activity is stronger.The vigor of the amnioic epithelium stem cell obtained by comparative example is only 70.7% (as shown in Figure 1),
And the yield of the amnioic epithelium stem cell of embodiment step (6) is 3 times of comparative example.Illustrate the sheep obtained by the method for the present invention
The yield and activity of film epithelial stem cell is higher.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.And, the specific features of description, structure, material or feature can be with office
Combine in an appropriate manner in one or more embodiments or example.Additionally, in the case of not conflicting, the skill of this area
Art personnel can be tied the feature of the different embodiments or example described in this specification and different embodiments or example
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (10)
1. it is a kind of obtain amnioic epithelium stem cell method, it is characterised in that include：
Amniotic membrane is carried out into digestion process using mixing enzyme preparation, digestion product is obtained；And
The amnioic epithelium stem cell is separated from the digestion product,
Wherein, the mixing enzyme preparation includes：
Pancreas enzyme -EDTA mixed liquor；And
Bacillus polymyxa Neutral proteinase II,
Preferably, the mixing enzyme preparation includes：
The pancreas enzyme -EDTA mixed liquor of 0.05 volume %～0.25 volume %；And
The Bacillus polymyxa Neutral proteinase II of 2U/mL～2.5U/mL.
2. method according to claim 1, it is characterised in that the use of pancreatin and EDTA in the pancreas enzyme -EDTA mixed liquor
Amount is than being 2.5:1,
Preferably, the pancreas enzyme -EDTA mixed liquor contains 0.5mg/mL pancreatin and 0.2mg/mL EDTA.
3. method according to claim 1, it is characterised in that the digestion process includes：
The amniotic membrane is carried out into the first digestion process, Digestive system is abandoned, the first digestion product is obtained；
First digestion product is carried out into the second digestion process, Digestive system is abandoned, the second digestion product is obtained；
Second digestion product is carried out into the 3rd digestion process, Digestive system is abandoned, the 3rd digestion product is obtained；And
3rd digestion product is carried out into the 4th digestion process, Digestive system is abandoned, the digestion product is obtained.
4. method according to claim 3, it is characterised in that
First digestion process is concussion 10min～20min under 37 DEG C of temperature, the rotating speed of 120r/min, preferably
10min；
Second digestion product is concussion 30min～40min under 37 DEG C of temperature, the rotating speed of 150r/min, preferably
40min；
3rd digestion product is concussion 30min～40min under 37 DEG C of temperature, the rotating speed of 150r/min, preferably
40min；
3rd digestion product is concussion 30min～40min under 37 DEG C of temperature, the rotating speed of 150r/min, preferably
30min。
5. method according to claim 1, it is characterised in that the separation includes：
The digestion product is filtered, filtrate is collected；
The filtrate is centrifuged, cell is collected；And
The cell is cleaned, to obtain the amnioic epithelium stem cell.
6. method according to claim 1, it is characterised in that include：
The amniotic membrane using mixing enzyme preparation by size for 2cm × 2cm carries out digestion process, obtains digestion product；
By the digestion product with 300 mesh sieve net filtrations, filtrate is collected；
The filtrate is centrifuged into 5min under the rotating speed of 2000r/min, cell is collected；And
The cell is cleaned 2 times with normal saline, to obtain the amnioic epithelium stem cell,
Preferably, methods described further includes that amplification is processed,
The amplification is processed to be included：
The amnioic epithelium stem cell is resuspended in culture medium, mixed liquor is obtained；And
The mixed liquor is inoculated in the culture medium, is cultivated at 37 DEG C to there is clone, it is long to passing on.
7. method according to claim 1, it is characterised in that the mixing enzyme preparation and the amount ratio of amniotic membrane be 0.5～
2:1, preferably 1:1.
8. a kind of test kit, it is characterised in that include：
Pancreas enzyme -EDTA mixed liquor；And
Bacillus polymyxa Neutral proteinase II,
Preferably, including：
The pancreas enzyme -EDTA mixed liquor of 0.05 volume %～0.25 volume %；And
The Bacillus polymyxa Neutral proteinase II of 2U/mL～2.5U/mL.
9. test kit according to claim 8, it is characterised in that pancreatin is with EDTA's in the pancreas enzyme -EDTA mixed liquor
Amount ratio is 2.5:1,
Preferably, the pancreas enzyme -EDTA mixed liquor contains 0.5mg/mL pancreatin and 0.2mg/mL EDTA.
10. test kit according to claim 8 or claim 9, it is characterised in that the test kit is used to obtaining amnioic epithelium dry thin
Born of the same parents.
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| CN201610983737.9A CN106566798A (en) | 2016-11-08 | 2016-11-08 | Method for getting amniotic epithelial stem cells and kit |
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|---|---|---|---|
| CN201610983737.9A CN106566798A (en) | 2016-11-08 | 2016-11-08 | Method for getting amniotic epithelial stem cells and kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108707577A (en) * | 2018-05-25 | 2018-10-26 | 苏州博福生物医药科技有限公司 | The elution process and catching method of a kind of cell or biomolecule |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000073421A2 (en) * | 1999-06-02 | 2000-12-07 | Lifebank Services, L.L.C. | Methods of isolation, cryopreservation, and therapeutic use of human amniotic epithelial cells |
-
2016
- 2016-11-08 CN CN201610983737.9A patent/CN106566798A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000073421A2 (en) * | 1999-06-02 | 2000-12-07 | Lifebank Services, L.L.C. | Methods of isolation, cryopreservation, and therapeutic use of human amniotic epithelial cells |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108707577A (en) * | 2018-05-25 | 2018-10-26 | 苏州博福生物医药科技有限公司 | The elution process and catching method of a kind of cell or biomolecule |
| CN108707577B (en) * | 2018-05-25 | 2023-11-10 | 江苏汇先医药技术有限公司 | Elution method and capture method of cells or biomolecules |
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Application publication date: 20170419 |