CN103083727A - Skin and soft tissue defect repair method by making use of own dermal fibroblast and umbilical cord mesenchymal stem cell - Google Patents
Skin and soft tissue defect repair method by making use of own dermal fibroblast and umbilical cord mesenchymal stem cell Download PDFInfo
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- CN103083727A CN103083727A CN2011103459256A CN201110345925A CN103083727A CN 103083727 A CN103083727 A CN 103083727A CN 2011103459256 A CN2011103459256 A CN 2011103459256A CN 201110345925 A CN201110345925 A CN 201110345925A CN 103083727 A CN103083727 A CN 103083727A
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Abstract
The present invention relates to a method for repairing subcutaneous tissue or dermal defects of patients. The method includes cultivating dermal fibroblasts from the patient living samples, cultivating umbilical cord mesenchymal stem cells of the same animal, mixing the two cells proportionally, and using the preparation essentially without immunogenic protein to repair subcutaneous or dermal tissues. The method can be used to repair wrinkles, skin expansion lines, pitting dense scar, and non-traumatic skin pit.
Description
Technical field
The present invention relates to a kind of method of utilizing self dermal fibroblast and umbilical cord mesenchymal stem cells repair of skin and soft tissue defects, belong to the bioengineered tissue field.
Background technology
A kind of material (being injection) is injected in vivo especially face to be obtained cosmetic result and can trace back to the 19th-century end.For example correcting the profile defective by injection paraffin accepts in " World War I " front short-term that obtains.But the existence of complication, long-term effect is undesirable is abandoned this way.The acquisition of the siloxanes of injection makes the recurrence from the sixties in this century of this process by a happy coincidence.Special medical siloxane solution.To part and the systemic reaction of siloxanes, the migration of injected material, the local organization fracture has limited the application of siloxanes injecting method.Although the initial proponent of this method still continues their planning of experiment with limited patient, this method can not be accepted by more surgery and medicine doctor.
The ill effect of injection non-biological material impels people to attempt utilizing foreign protein, and especially bovine collagen is used as injected material.Although unprocessed bovine collagen is too strong to human injection's immunogenicity, if but patient is through screening in advance, those immunoreactive patients have been got rid of, remove bovine collagen nitrogen end or carbon teminal with enzymatic degradation, the material of " without end collagen (atelocollagen) " of the generation use of still can limiting the quantity of.
Solution state has proved and can not be entirely satisfactory without end collagen, is not replaced by host material because this material can be absorbed from the injection site by the experimenter in the time of several thoughtful several months.Although someone considered with methods such as duplicate injections, these methods are because cattle produces immunoreation without end collagen, and its cost is higher, and patient resists etc. former thereby in fact uses limited.In order to overcome these restrictions, cattle can further be processed without end collagen: first use glutaraldehyde cross-linking, then filter and shear with dusting cover.Preparation and utilize the visible United States Patent (USP) of description 4,582, No. 640 and 4,642, No. 117 of these products.The crosslinked desired advantage that provides increases the Hangzhoupro of supporting to host's degraded, also supports to some extent the tip because of the increase of product viscosity.The increase of viscosity, especially viscosity irregular (agglomerate phenomenon) make this product more be difficult to use, and can't use even in some aspects.Give an example No. 5,366,498, visible United States Patent (USP).
The immunogenicity of bovine collagen product has limited the human body use, impels some to consider (the visible United States Patent (USP) 5,002 of giving an example from Placenta Hominis, (give an example No. 204,969,912, visible United States Patent (USP) and 5 in No. 071 and surgical samples, 332, No. 802) extraction people collagen.Because people's collagen and bovine collagen stand same degradation process, need further crosslinked and other chemical modification.
Show that without clear evidence injection people collagen is more lasting than bovine collagen produce an effect, and, utilize the collagen through processing of self also to be confined to the patient that esthetic surgery is performed the operation. because its preparation parent material is to take out in this operation process.Significantly, although the collagen through processing of self has been eliminated the immunogenicity of bovine collagen, it is the same with bovine collagen can not guarantee the long-term treatment effect.And it is confined to move the patient of operation.
Generally speaking, the existing abiotic injectable materials of neither one can completely satisfactorily increase neighbour's corium and soft tissue.
The present invention utilizes the living cells in self normal dermal tissue, increases to close on corium and subcutaneous tissue volume under damaged, reaches the cosmetic results such as repair of skin and soft tissue defects.Simultaneously, in self, can reduce the immunologic rejection effect due to cell derived.
Summary of the invention
The present invention partly is based on the recognition: the desirable material for increasing closing on corium and subcutaneous tissue volume under damaged should be the living cells that is present under normal circumstances in dermal tissue.
The present invention also is based on the recognition: by former weeks obtaining the live body sample from the patient in injection, then cultivating to provide a large amount of required type self cells.
The present invention further also is based on the recognition: through the amplification of such tissue culture, self cell can contain a certain amount of antigenic protein, but as long as carry out according to the application's guidance, this albumen can be removed before injection.
But the present invention can be undertaken by the undifferentiated mesoblastema of injecting any amplification culture.Because dermal fibroblast is easy to obtain and is easy to cultivate, and is that normal composition is closely organized in corium and its underpart, therefore in a preferred embodiment, inject (seeing Fig. 1 and Fig. 2) with it.
Description of drawings
Figure one: the SABC picture (200 *) of matched group
Figure two: the SABC picture of injection group (the SP staining, 200 *)
Draw from picture: in the skin biopsy of injection group, the obvious thickening of collagen fiber layer, cell is fine and close.Suffice to show that, this invention can significantly improve wrinkle, and has the effect that can repair skin scar.
The specific embodiment
The preparation of umbilical cord mesenchymal stem cells:
At once intercept umbilical cord by obstetrics' disconnected umbilicus of conventional ligation after delivery of baby, use the normal saline flushing umbilical cord, sterilize with medical alcohol again, umbilical cord is placed in umbilical cord preserving fluid 2-8 ℃ of constant temperature to be preserved, extract female blood and respectively be no less than 4ml as detecting sample from Placenta Hominis end extraction umbilical blood from parent, separately extract umbilical blood 4ml from the Placenta Hominis end and do collection serum use.
After umbilical blood, this detection of female blood examination test sample are qualified, the umbilical cord of laboratory is separated into 1-3mm
3Piece of tissue, the separating step of umbilical cord is to cut off along the ligature of the cord inboard, add appropriate normal saline to clean to the surface without after bloodstain, umbilical cord being cut into the segment of 2-3cm, cut off and peel off venous blood tube wall and arteries along umbilical cord vein blood vessel, after separating magnificent Tong Shi glue, magnificent Tong Shi glue is moved in centrifuge tube, with tissue shear, magnificent Tong Shi glue is shredded to 1-3mm
3The piece of tissue of size, umbilical cord can not be over 24 hours from being truncated to the front time of laboratory separation.
The culture bottle of inoculation piece of tissue is placed in 37 ℃, contains 5% CO
2, saturated humidity incubator in cultivate, changed a complete medium, and cultivated and obtain P0 for umbilical cord mesenchymal stem cells after 10-20 days in every 3 days;
The cultivation of going down to posterity
After in umbilical cord mesenchymal stem cells P0 generation, grown to cell fusion 80%-90%, obtain P1 for umbilical cord mesenchymal stem cells to go down to posterity at 1: 3 after 0.25% trypsinization, amplification can obtain the P5 mescenchymal stem cell in generation according to this.
Dermal fibroblast is cultivated since 2 * 5mm holostrome skin of living body sample.Because the known allotype rejection of spare-part surgery doctor and immunologist, the fibroblast of cultivation and host tissue are compatible very key.From waiting that the patient who revises its skin injury obtains a live body sample, and begin to cultivate fibroblast from it and can guarantee that histocompatibility is consistent.
Before beginning to cultivate, with the repeatedly rinsing in antibiotic and Hangzhoupro fungus reagent of live body sample, then 20 remove carefully epidermis and subcutaneus adipose tissue one so that the culture that obtains there is no non-fibroblast; Corium is cut with scalpel or operating scissors as far as possible.The sample section is placed on a dried glass culture dish with tweezers one by one.After its adherent 5-10 minute, slowly add a small amount of culture fluid, note not blowing afloat adherent piece of tissue.After hatching 24 hours, then add some culture medium.When beginning to cultivate with the T culture bottle, the initial amount of culture fluid is 1.5-2.0ml.Building 25 vertical cell lines from the live body sample needs 2-3 week usually, and cell just can take out amplification from initial culture bottle then.
At the commitment of cultivating, piece of tissue need to keep being attached to bottom culture dish.The piece of tissue that breaks away from need to be implanted in new culture bottle again.According to the technology that this respect professional knows, the of short duration EDTA-of the being exposed to trypsin of tissue culture can stimulate fibroblastic growth.Exposure should of short durationly be arrived deficiency so that fibroblast breaks away from the bottom of ware.Set up and reach soon when merging when culture, fibroblast can be sampled frozen.Frozen peaceful morning is evening not, because the passage number of normal fibroblast is limited.
Any dermal fibroblast cultural method that increases from biopsy samples, may be used to the present invention and come amplifying cells, can be referring to R.LFreshney chief editor's " animal cell culture: practical approach guide " and R.LFreshney chief editor's " animal cell culture: basic fundamental handbook.These books are as incorporating this application into list of references.
Anyly be fit to do the culture fluid that primary fibroblast cultivates and all can use.In most cases, the serum that adds 0.5%-20% (V/V) in culture fluid is accelerated fibroblastic growth.Serum-concentration is higher, and Growth of Cells is faster.In a preferred embodiment, serum is hyclone, and being added to final concentration is 10%.For example can use high glucose DMEM (GIBCO, 31600034) make culture fluid, add 2mM glutamine (Sigma, G-3126), the Sodium Pyruvate (Sigma, P2256) of 110mg/L, 10% (v/v) hyclone (Gibco, 16000-044) and antibiotic (Sima, P7539) namely form " complete medium ".
Cell go down to posterity at new culture bottle after can trypsinization, and for example one bottle of cell can divide and does three bottles and support.The three T-shaped culture bottles in the end have the culture area of 450 square centimeters, can be used for this invention, and the three T-shaped culture bottles in the end can inoculate 6 * 10
8Individual cell, covering with to have 1.8 * 10
9Individual cell.Usually after 5-7 days, cell can cover with, and at this moment growth-promoting media changes the serum-free complete medium into; Then cell be incubated in one namely remain on 30-40 ℃ at least 6 hours, better more than 12 hours.Preferably hatched 16-18 hour in 37 ℃ of protein-free mediums.Cell culture is cultivated in serum-free medium, basically can remove cell with the protein that is derived from bovine serum albumin.These albumen are immunogenic to human body, if be present in injected material, can produce allergy.
Serum-free culture digests cell get off with trypsin/EDTA from culture bottle, by centrifugal resuspended thorough washing after finishing.Finally be suspended from a saline such as equal-volume injection in order to injection.Six three T-shaped culture bottles in the end can cover with and can gather in the crops 10
5Cell, the most how much this can make about 1.0ml suspension.
Or as long as be injected in being mixed with 18 hours of suspension, these cells are transferred to 4 ℃.Cell suspension is used for transporting in isopyknic complete medium, except lacking phenol red pH indicator, replaces hyclone with experimenter's serum.Cell can be in sucking-off and injection in transporting culture medium.
The saline of re-suspended cell or transport volume with culture medium requires not strict.It depends on the cell quantity that the implementer plans to inject, damaged size to be treated and number, the factors such as urgency that the patient requires therapeutic effect.The implementer can allow the volume of cell suspension larger, and this 5 sample is in corresponding will the lacking of cell of each injection site injection.
Be used for obtaining the alternative of cell thickness suspension:
The invention provides a kind of alternative for preparing injection cell suspension, be applicable to the situation that dermis restoration needs lot of materials.Add thick lips, treatment muffle pleat and subcutaneously damagedly just can run into this situation.
Alternative and above-described method are in cell culture to 1 * 10
6The same in the past.In the culture dish of diameter 100mm, add self thrombin of the blood plasma of 2ml receptor and 50-100 unit, make its bottom form plasma clot, can form a cell culture face after processing.1 * 10 of 35ml will be suspended from
6The individual dermal fibroblast of cultivating is inoculated in the grumeleuse surface, continues to cultivate 7 days in complete medium.
During by 7 days, complete medium changes serum-free medium into.In certain operational approach, change two subcultures with the interval of a hour, then cell is continued to cultivate the effect that can obtain satisfaction in 1416 hours in serum-free medium.Hatch in serum-free medium complete after, grumeleuse can inhalation syringe, at any time injection.In flexible embodiment of the present invention, fibroblast is not made suspension, but with complete grumeleuse form or be cut into required form with scalpel and use.When corium abraded, this inoculation had fibroblastic grumeleuse surface as the covering of patient's corium.
Cell is used for laboratory animal:
Suspension of the present invention can be used for treating skin injury.Cell suspension can be used for substituting without end collagen, and has advantages of above drawing up.
A scheme of the present invention can be used for treating trickle wrinkle, and method is as follows: first process district to be treated with ethanol, then it is pulled into the plane; Get a syringe, fill cell suspension, set up the syringe needle of 30ga, the direction of as far as possible showing syringe needle to be thrust this district's one angle not crux one is pressurizeed shallowly gently, carries out the intradermal injection, until can see that the part slightly turns white, injects a plurality of places.
Implement example:
Adopt 7 all above nude mices, under uviol lamp, irradiation causes it to produce wrinkle.Irradiation time is 4 hours/day, Continuous irradiation 7 days.
It is 1 * 10 that cell mixing injection of the present invention, this injection contain the cell total amount
5, wherein the ratio of fibroblast and mescenchymal stem cell is 2: 3.Selecting the nude mice back is injected area, and selecting diameter is that the circular area of 2cm is as injected area.Get a syringe, fill cell suspension, set up syringe needle, carry out intradermal injection.In this circular scope, injection point is 8-10.
Be spaced apart a week inject time, inject 4 times in same skin scope altogether.After completing last injection, laboratory animal is put to death at interval 10 days.Situation of change with Using immunohistochemical collagen fibers of skin layer.Matched group is chosen (12 week) of the same age nude mice, normal saline injection, the same experimental group of injecting method.
Claims (12)
1.
OneKind can be used to the method for the subcutaneous or dermal tissue of in the mammal long-term patient of increasing, it comprises:
A) the auto derma fibroblast that provides the process that there is no immunogenic protein to go down to posterity;
B) provide the umbilical cord mesenchymal stem cells of the same race that there is no immunogenic protein;
C) confirm easily by closing on damaged that subcutaneous and dermal tissue improve under increasing;
D) suspension of effective volume is expelled to damaged lower adjacent tissue and makes its thickening.
2. the method for claim 1, fibroblast and mescenchymal stem cell according to the proportioning of various ratios all in the protection domain of patent of the present invention.
3. the method for claim 1, the proportioning ratio of fibroblast and mescenchymal stem cell is preferably 2: 3.
4. the process of claim 1 wherein damaged finger: skin stricture of vagina, skin expansion stricture of vagina, pitting disease trace, non-traumatic cutaneous or lip dysplasia.
5. the process of claim 1 wherein that suspension also comprises the animal fibrin of the effective dose that is enough to form injectable glue.
6. the method for claim 3, wherein injectable glue comprises the self blood plasma of thrombin activation.
7. the method for claim 1, also comprise the following steps:
A) gather laboratory animal corium live body sample;
B) will go down to posterity the culture medium that contains the 0.5-20% non-human serum from the dermal fibroblast that the live body sample obtains,
To provide essentially no adipose cell, the fibroblast of horn cell and extracellular matrix;
C) dermal fibroblast that goes down to posterity was hatched 6 hours in 30-40 ℃ in serum-free medium at least;
D) use proteolytic enzyme digest with the suspension fibroblast fibroblast that hatches.
8. according to claim 1 method, further comprising the steps of:
A) gather mammiferous umbilicus band of the same race;
B) former culture
The culture bottle of inoculation piece of tissue is placed in 37 ℃, contains 5% CO
2, saturated humidity incubator in cultivate, changed a complete medium, and cultivated and obtain P0 for umbilical cord mesenchymal stem cells after 10-20 days in every 3 days;
C) cultivation of going down to posterity
Go down to posterity for after growing to cell fusion 80%-90% until umbilical cord mesenchymal stem cells P0, amplification can obtain the P5 mescenchymal stem cell in generation according to this.
9. the preparation method of a kind of human umbilical cord mesenchymal stem cells according to claim 8 is characterized in that: described umbilical cord preserving fluid comprises one or more the combination in culture fluid DMEM/F12, penicillin, streptomycin, gentamycin, phosphate buffer PBS.
10. the device that comprises a kind of patient's of reparation Dermal defect of following content:
A) a kind of hypodermic syringe, it has an injecting cavity, and intracavity has a push rod, and the chamber has an outlet and the external world to communicate;
B) a kind of suspension comprises:
(1) fibroblast that obtains from the patient-this fibroblast there is no non-fibroblast, and cell also there is no has immunogenic albumen to this experimenter;
(2) from the umbilical cord mesenchymal stem cells in allogenic animal umbilical cord source, cell does not have immunogenic albumen to this laboratory animal substantially yet;
(3) a kind of this suspension of pharmaceutically acceptable carrier solution is placed in syringe cavity;
C) a kind of hypodermic needle that is positioned on syringe outlet.
11. the method for production of claim 7,8 equipment comprises following part:
A) take patient's corium live body sample;
B) dermal fibroblast in corium live body sample is gone down to posterity at the culture fluid that contains 0.5-20% non-human serum, there is no the fibroblast dermal fibroblast of cell in addition to provide;
C) dermal fibroblast that goes down to posterity was hatched minimum 6 hours with serum-free medium at 30-40 ℃;
D) cell that will hatch is exposed to proteolytic enzyme to have hanged fibroblast;
E) add in the fibroblast of hanging pharmaceutically that the acceptable carrier solution forms a kind of suspension, this suspension is placed in syringe cavity.
12. a method for the treatment of wrinkle, it comprises the following steps:
A) provide a kind of mixing suspension of inoculating dermal fibroblast and mescenchymal stem cell, this cell suspension does not contain has immunogenic albumen to animal subject;
B) product of the present invention is expelled to intradermal.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011103459256A CN103083727A (en) | 2011-11-07 | 2011-11-07 | Skin and soft tissue defect repair method by making use of own dermal fibroblast and umbilical cord mesenchymal stem cell |
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| CN2011103459256A CN103083727A (en) | 2011-11-07 | 2011-11-07 | Skin and soft tissue defect repair method by making use of own dermal fibroblast and umbilical cord mesenchymal stem cell |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881972A (en) * | 2013-08-28 | 2014-06-25 | 中国人民解放军南京军区福州总医院 | Method applicable to serum-free culture of mesenchymal stem cells |
| CN106995796A (en) * | 2016-01-26 | 2017-08-01 | 李建业 | Cellular processes, kit and freeze-dried powder based on cell factor |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1198090A (en) * | 1995-07-28 | 1998-11-04 | 埃索拉根技术有限公司 | Use of autologous dermal fibroblasts for repair of skin and soft tissue defects |
-
2011
- 2011-11-07 CN CN2011103459256A patent/CN103083727A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1198090A (en) * | 1995-07-28 | 1998-11-04 | 埃索拉根技术有限公司 | Use of autologous dermal fibroblasts for repair of skin and soft tissue defects |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881972A (en) * | 2013-08-28 | 2014-06-25 | 中国人民解放军南京军区福州总医院 | Method applicable to serum-free culture of mesenchymal stem cells |
| CN103881972B (en) * | 2013-08-28 | 2015-08-26 | 中国人民解放军南京军区福州总医院 | A kind of method being applicable to mesenchymal stem cell serum-free and cultivating |
| CN106995796A (en) * | 2016-01-26 | 2017-08-01 | 李建业 | Cellular processes, kit and freeze-dried powder based on cell factor |
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Addressee: Beijing QMLC Stem Cell Technology Co., Ltd. Document name: Notification of Publication of the Application for Invention |
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Address after: 100084, C building, Tsinghua Science and technology building, No. 1 Zhongguancun East Road, Beijing, Haidian District, 301 Applicant after: Beijing QMLC Stem Cell Technology Co., Ltd. Address before: 100097 Beijing city Haidian District minzhuang Road No. 3, Tsinghua Science Park building 204 Yuquan Huigu 7 Applicant before: Beijing QMLC Stem Cell Technology Co., Ltd. |
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Application publication date: 20130508 |