CN104877964A - In vitro construction method for salivary glands organs and acinus - Google Patents
In vitro construction method for salivary glands organs and acinus Download PDFInfo
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Abstract
The invention discloses an in vitro construction method for biological engineering salivary glands organs and acinus based on human minor salivary gland adult stem cells belonging to the technical field of tissue and organ reconstruction. The in vitro construction method specifically is a method for obtaining organoids with salivary gland structures and functions by mixed cultivation of mesenchymal stem cells and epithelial stem/progenitor cells of the human minor salivary glands, or a method for obtaining acinar-like tissues with salivary gland structures and functions by individual cultivation of epithelial stem/progenitor cells of the human minor salivary glands. The method has the advantages of being wide in sampling source, easy to cut, and small in trauma; a donor site in a mouth is private; materials are drawn from adult tissues; and autoplastic transplantation can be carried out through amplification in vitro. The method has a wide application prospect in growth and development research of in vitro or in vivo salivary glands, research on a salivary gland disease model, research on physiological functions and pathological changes of the saliva and treatment of salivary gland diseases.
Description
Technical field
The invention belongs to tissue and Organ Reconstruction technical field, be specifically related to a kind of based on the Bioengineered sialisterium organoid of people's glandulae salivariae minores adult stem cell and the vitro construction method of class acinus.
Background technology
Existing research confirms that major salivary glands tissue is containing mescenchymal stem cell and epithelial stem cell.The sialisterium of people mainly contains three major salivary glandses and the many glandulae salivariae minoreses under being distributed in oral mucosa, and they are made up of essence (acinus, conduit) and interstitial (reticular tissue, blood vessel, lymphatic vessel and nerve etc.).Mainly concentrate on major salivary glands about the research of stem cell in sialisterium, there is mescenchymal stem cell in the existing experiment confirmer parotid gland and skeletonization, one-tenth fat and chondroblast can be induced to differentiate in vitro.This becomes the another source newly of mescenchymal stem cell.The multipotency progenitor cell (SGP) having bibliographical information to be separated from mouse submaxillary gland to obtain has cells and characteristic of stem and can be divided into entoderm system cell, implants liver by portal vein, finds that these cells incorporate hepatic trabjecula and can Albumin Secretion.Also have experiment to be separated salivary gland cell from murine submaxillary gland to cultivate by floating sphere culture system method in vitro, wherein comprise and express stem cell labeling Sca-1, the cell of c-Kit and Musashi-1, and confirm that these source of human stem cell are in ductal epithelium.In vitro these cytodifferentiation be salivary gland ductal cell and secreting mucus element and diastatic acinous cell.With c-Kit be mark selected by flow cytometry apoptosis stem cell enriched, these cytodifferentiation external are the acinous cell of secreting amylase.In body, in small number c-Kit+ cell gland, injection transplantation causes the long-term recovery of sialisterium morphology and function.Illustrate in sialisterium containing two kinds of stem cells and mescenchymal stem cell and epithelium ancestral cells.Although these researchs confirm there are this two kinds of stem cells in major salivary glands tissue, be difficult to be relatively easy to obtain appropriate separate tissue from major salivary glands clinically and cultivate these stem cells.
Under Mucosa of lip, glandulae salivariae minores and major salivary glands tissue have homology: just start to cut lip mucous gland in the eighties clinically and carry out biopsy to diagnose the disease of some major salivary glandses, which illustrate the homology of glandulae salivariae minores and major salivary glands.Under Mucosa of lip, the cell moiety of glandulae salivariae minores is identical with major salivary glands, and be all made up of acinous cell, ductal epithelial cell, myoepithelical cell and mesenchymal cell etc., theoretically, glandulae salivariae minores is organized and also should be contained above-mentioned two kinds of stem cells.
Under Mucosa of lip, glandulae salivariae minores is widely distributed, abundance, and draw materials easily, operation wound is small, hidden for district, does not substantially affect for district's function after cutting.We report the report obtaining adult stem cell from people's glandulae salivariae minores tissue the earliest.Therefore, study from people's glandulae salivariae minores separate tissue culture identification adult stem cell and carry out Induction of committed differentiation, can for stem-cell therapy or organizational project provide new seed cell source clinically.
Organoid unit is the structure with each cell type in tissue that external dimensional culture is formed in extracellular matrix, and the method is widely used in the research of little intestinal stem cell and organizational project structure.There is bibliographical information, be separated the little intestinal stem cell of Lgr5+, organoid unit can be differentiated to form in Matrigel.Detect rear discovery to such organic unit, it has different cell types, comprises intestinal epithelial cell, mucous secreting cell and endocrine cell, and can be observed class enteric cavity structure.Based on this, investigators have carried out the research of organizational project small intestine and all kinds of stem cells hyperplasia and differentiation.Recently, scholar is had to obtain epithelial cell and mesenchymal cell from the mouse embryo submaxillary gland gland base tissue of 13.5 days to 14 days, two kinds of cells are put together and carries out external dimensional culture, within three days, form Bioengineered sialisterium bud afterwards, then orthotopic transplantation is in submaxillary gland or parotid gland position, transplants after 30 days and forms the ripe sialisterium with function in vivo.
Described mescenchymal stem cell substratum is MSCM substratum.
Described kerationcyte culture medium is KM substratum.
Still do not study and adopt people's glandulae salivariae minores adult mesenchymal stem cells and people's glandulae salivariae minores adult epithelium ancestral cells hybrid three-dimensional to cultivate acquisition people sialisterium organoid, or simple employment glandulae salivariae minores adult epithelium ancestral cells carries out dimensional culture, obtain the research report of people's sialisterium class acinar tissue.
Summary of the invention
The object of the present invention is to provide a kind of based on the Bioengineered sialisterium organoid of people's glandulae salivariae minores adult stem cell and the vitro construction method of class acinus.
A vitro construction method for sialisterium organoid and class acinus, carries out in accordance with the following steps:
(1) from people's glandulae salivariae minores tissue, glandulae salivariae minores adult mesenchymal stem cells and adult epithelium ancestral cells is obtained by tissue mass cell culture;
(2) separation and Culture glandulae salivariae minores mescenchymal stem cell and glandulae salivariae minores epithelium ancestral cells, carry out amplification in vitro; Or separation and Culture people glandulae salivariae minores adult epithelium ancestral cells, carries out amplification in vitro;
(3) in basement membrane matrix glue, hybrid three-dimensional cultivation is carried out to glandulae salivariae minores mescenchymal stem cell and glandulae salivariae minores epithelium ancestral cells, obtain people's sialisterium organoid; Or in basement membrane matrix glue, dimensional culture is carried out to people's glandulae salivariae minores epithelium ancestral cells, obtain people's sialisterium class acinar tissue.
Described basement membrane matrix glue is extracellular matrix Matrigel.
Further, describedly in basement membrane matrix glue to the concrete operation step that glandulae salivariae minores mescenchymal stem cell and glandulae salivariae minores epithelium ancestral cells carry out hybrid three-dimensional cultivation be: the glandulae salivariae minores mescenchymal stem cell and epithelium ancestral cells that are cultured to more than three generations or three generations are digested to unicellular, be inoculated in Matrigel after mixing by cell quantity 1: 1, the mescenchymal stem cell substratum of equal proportion mixing and kerationcyte culture medium is used to cultivate, concrete operations are trysinization mescenchymal stem cell and epithelium ancestral cells is single cell suspension, count rear substratum resuspended, and mix with equal-volume 4 DEG C of Matrigel thawed, be inoculated in 24 orifice plates, be placed in 37 DEG C of cell culture incubators, after 20 minutes, treat that Matrigel solidifies, add substratum, every hole inoculation 2.5 × 10
5glandulae salivariae minores mescenchymal stem cell and 2.5 × 10
5glandulae salivariae minores epithelium ancestral cells, inoculate after mixing with 50 μ LMatrigel, inoculate successfully, change liquid every other day, vitro culture is after 1 day, and cell aggregation becomes branch shape, and after 3 days, the organoid can seeing acinus shape and tubular branch structure is formed.Within 6-10 days, can grow to maximum, sialisterium substratum can be used after 6 days instead to promote its differentiation and maturation.
Further, describedly in basement membrane matrix glue to the concrete operation step that people's glandulae salivariae minores epithelium ancestral cells carries out dimensional culture be: the glandulae salivariae minores epithelium ancestral cells being cultured to more than three generations or three generations is digested to unicellular, be inoculated in Matrigel, kerationcyte culture medium is used to cultivate, concrete operations are trysinization epithelium ancestral cells is single cell suspension, count rear substratum resuspended, and mix with equal-volume 4 DEG C of Matrigel thawed, be inoculated in 24 orifice plates, be placed in 37 DEG C of cell culture incubators, after 20 minutes, treat that Matrigel solidifies, add substratum, every hole inoculation 5 × 10
4glandulae salivariae minores epithelium ancestral cells, inoculates after mixing, inoculate successfully, change liquid every other day, inoculate latter 4 hours, the ringwise polar alignment of cell with 50 μ L Matrigel, after 10 hours, forms the spheroplast of class acinus.After 6-12 days, class acinus spheroplast can grow to maximum.Sialisterium substratum can be used instead to promote its differentiation and maturation after 6 days.
Beneficial effect of the present invention: dimensional culture acquisition people sialisterium organoid is being carried out in method employment glandulae salivariae minores adult mesenchymal stem cells of the present invention and the combined inoculation of people's glandulae salivariae minores adult epithelium ancestral cells on extracellular matrix Matrigel, or simple employment glandulae salivariae minores adult epithelial stem cell/ancestral carries out dimensional culture on extracellular matrix Matrigel, obtain people's sialisterium class acinar tissue.Source of drawing material of the present invention is extensive, and be easy to cut, wound is small, and Kou Neigong district is hidden, draws materials from adult tissue, can be used for autotransplantation by amplification in vitro.The present invention is with a wide range of applications for the research of growing of sialisterium in external or body, the research of disease of salivary gland model, the physiological function of saliva and the research of pathological change, the treatment etc. of disease of salivary gland.
Accompanying drawing explanation
Fig. 1 is the present inventor's glandulae salivariae minores epithelium ancestral cells and the light microscopic figure of mescenchymal stem cell mixed culture on Matrigel.
Fig. 2 is that the present inventor's glandulae salivariae minores epithelial stem cell cultivates the light microscopic figure on Matrigel.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention will be further described.
Embodiment 1
The method used in culturing process and reagent:
(1) cultivation of primary cell: DMEM/F12 adds 10% foetal calf serum, 1% dual anti-and 1% glutamine;
(2) amplification in vitro:
1) people's glandulae salivariae minores mescenchymal stem cell is cultivated: mescenchymal stem cell substratum: DMEM/F12 adds 5% foetal calf serum, 1% is dual anti-, and BSA 10 μ g/mL, apo-transferrin 10 μ g/mL, insulin5mg/mL, EGF 2ng/mL, FGF-2 2ng/mL and hydrocortisone 1mg/mL.
2) people's glandulae salivariae minores epithelium ancestral cells is cultivated: kerationcyte culture medium
DMEM/F12 adds BSA (bovine serum albumin) 5 μ g/mL, transferrin (Transferrins,iron complexes) 5 μ g/mL, BPE (ox pituitary extract) 50 μ g/mL, insulin (Regular Insulin) 3.75g/mL, FGF-23ng/mL, EGF 1ng/mL, epinephrine (suprarenin) 500ng/mL, hydrocortisone (hydrocortisone) 0.5g/mL, Prostaglandin E2 (prostaglandin E2) 10
-8m and T330nM.
3) sialisterium organoid builds and acinus structure: extracellular matrix MatrigelTM (BD, US), sialisterium organoid (or class acinus) substratum:
(1) DMEM/F-12 (1: 1) adds 10%fetal bovine serum, 100Uml
-1penicillin, 100 μ g ml
-1streptomycin, 1% glutamine.
Or (2) DMEM/F12 1: 1,10%fetal bovine serum (FBS), 1%penicillin/streptomycin, 1%insulin-transferrin-seleniumsupplement, 10mM nicotinamide, 1 × 10
-7m dexamethasone, 1mMb-mercaptoethanol, 20mg/l epidermal growth factor (EGF) and 20mg/lhepatocyte growth factor (HGF).
1, cell primary is cultivated
Tissue mass cell culture is adopted to carry out uterus tissue pieces to people's glandulae salivariae minores that biopsy or other modes (in art in discarded tissue) obtain.Detailed process is: fully rinse glandulae salivariae minores tissue with cold PBS, careful coating or other reticular tissue removing remnants.Fully tissue is shredded, to 0.5mm with eye scissors
3the little block organization of size, is affixed on bottom T25 culturing bottle with tweezers respectively, eachly organizes interblock gap 5mm, substratum is slightly moistening be placed on incubator (37 DEG C, 5%CO
2) in make tissue block fully adherent bottom culturing bottle.After 6-8 hour, add perfect medium (DMEM/F12, adds 10% foetal calf serum, 1% mycillin and 1% glutamine).Being cultured to the 4th day has cell to climb out of around visible tissue block, within every three days subsequently, changes liquid, about 10-14 days passages.
During original cuiture, be fine and close epithelioid cell around tissue block, for spindle sample becomes fiber-like cell around epithelioid cell.For separation and Culture people glandulae salivariae minores mescenchymal stem cell and epithelium ancestral cells, be separated two kinds of cells with cloning ring method.1 concrete operations, for determining a kind of cell type with clone's ring, after clone smears a small amount of Vaseline bottom ring, with 0.25% trysinization ring inner cell, are transferred in six orifice plates and are cultivated.
2, glandulae salivariae minores derived mesenchymal stem cells in vitro is cultivated
Be separated obtain glandulae salivariae minores mescenchymal stem cell cultivate with mescenchymal stem cell substratum MSCM, when cytogamy is to 80-90%, can 1: 3 stablize be passaged to 20 generations or more.
3, glandulae salivariae minores epithelium hematopoietic stem/progenitor cells in vitro is cultivated
Be separated the epithelium ancestral cells obtained to cultivate in kerationcyte culture medium, when cytogamy is to 80-90%, also use 0.25% trysinization, go down to posterity according to cell growth state 1: 3 or 1: 4.Epithelium ancestral cells also Absorbable organic halogens is passaged to 20 generations or more.
4, sialisterium organoid external structure
The glandulae salivariae minores mescenchymal stem cell and epithelium ancestral cells that are cultured to more than three generations or three generations are digested to unicellular, be inoculated in Matrigel after mixing by cell quantity 1: 1, use the mescenchymal stem cell substratum of equal proportion mixing and kerationcyte culture medium to cultivate.Concrete operations are trysinization mesenchymal cell and epithelial stem cell is single cell suspension, counts rear substratum resuspended, and mixes with equal-volume 4 DEG C of Matrigel thawed, be inoculated in 24 orifice plates, be placed in 37 DEG C of cell culture incubators, after 20 minutes, treat that Matrigel solidifies, add substratum.Every hole inoculation 2.5 × 10
5glandulae salivariae minores mescenchymal stem cell and 2.5 × 10
5glandulae salivariae minores epithelium ancestral cells, inoculates after mixing with 50 μ L Matrigel.Inoculate successfully, change liquid every other day.Vitro culture is after 1 day, and cell aggregation becomes branch shape, and by 3 days, the organoid can seeing obvious acinus shape and tubular branch structure was formed.After 5 days, these structures will seem more obvious, and organoid can obviously be grown up, and within 6-10 days, can grow to maximum.Sialisterium substratum can be used instead to promote its differentiation and maturation after 6 days.Result as shown in Figure 1.
Above-mentioned Matrigel can also be replaced by other basement membrane matrix glue, basement membrane matrix as containing following portions by weight: LN1 0 part, Herba Cirsii extract 1 part, nidogen 20 parts, 20 parts, heparin sulfate glycoprotein, epidermal growth factor 0.1 part, fibroblastic growth factor 0.1 part, thrombocyte source reproduction factor 0.1 part, somatostatin 0.2 part, hydrocortisone 0.01 part.Above-mentioned Herba Cirsii extract is adopted and is prepared with the following method: pulverized by field thistle, loads to decoct in bag to soak 10min-11h, moves in Decocting pot, decocts 10min-7h, finally by decoction liquor spray pulverization, obtain Herba Cirsii extract under temperature 80-120 degree condition.Adopt above-mentioned basement membrane matrix to repeat above-mentioned experiment vitro culture after 1 day, cell aggregation becomes branch shape, and by 3 days, the organoid can seeing obvious acinus shape and tubular branch structure was formed.After 5 days, these structures will seem more obvious, and organoid can obviously be grown up.Having arrived 6-10 days can grow to maximum.Sialisterium substratum can be used instead after 6 days.
Or available type i collagen, hydrogel etc. replace Matrigel.
5, the structure of sialisterium class acinar tissue
The glandulae salivariae minores epithelium ancestral cells being cultured to three generations is digested to unicellular, is inoculated in Matrigel, use KM substratum to cultivate.Concrete operations are trysinization epithelium ancestral cells is single cell suspension, counts rear substratum resuspended, and mixes with equal-volume 4 DEG C of Matrigel thawed, be inoculated in 24 orifice plates, be placed in 37 DEG C of cell culture incubators, after 20 minutes, treat that Matrigel solidifies, add substratum.Every hole inoculation 5 × 10
4salivary gland epithelia ancestral cells, inoculates after mixing with 50 μ L Matrigel.Inoculate successfully, change liquid every other day.Inoculate latter 4 hours, cell just ringwise polar alignment, after 10 hours, just defines the spheroplast of class acinus, and along with the prolongation of incubation time, volume is disconnected grows up for acinus, and acinus quantity is on the increase, and after 6-12 days, volume can grow to maximum.Sialisterium substratum can be used instead to promote its differentiation and maturation after 6 days.Result as shown in Figure 2.
Above-mentioned Matrigel can also be replaced by other basement membrane matrixs, basement membrane matrix as containing following portions by weight: LN1 0 part, Herba seu Radix Cirsii Japonici 1 part, nidogen 20 parts, 20 parts, heparin sulfate glycoprotein, epidermal growth factor 0.1 part, fibroblastic growth factor 0.1 part, thrombocyte source reproduction factor 0.1 part, somatostatin 0.2 part, hydrocortisone 0.01 part.Above-mentioned Herba seu Radix Cirsii Japonici is adopted and is prepared with the following method: pulverized by field thistle, loads to decoct in bag to soak 10min-11h, moves in Decocting pot, decocts 10min-7h, finally by decoction liquor spray pulverization, obtain Herba seu Radix Cirsii Japonici under temperature 80-120 degree condition.Above-mentioned basement membrane matrix is adopted to repeat above-mentioned experiment, inoculate latter 4 hours, cell is ringwise polar alignment just, after 10 hours, just define the spheroplast of class acinus, along with the prolongation of incubation time, volume is disconnected grows up for acinus, acinus quantity is on the increase, and has arrived 6-12 days volumes and just can grow to maximum.Sialisterium substratum can be used instead to promote its differentiation and maturation after 6 days.
Or available type i collagen, hydrogel etc. replace Matrigel.
Claims (6)
1. a vitro construction method for sialisterium organoid and class acinus, is characterized in that, carries out in accordance with the following steps:
(1) from people's adult glandulae salivariae minores tissue, adult glandulae salivariae minores mescenchymal stem cell and adult glandulae salivariae minores epithelium ancestral cells is obtained by tissue mass cell culture;
(2) separation and Culture glandulae salivariae minores mescenchymal stem cell and glandulae salivariae minores epithelium ancestral cells, carry out amplification in vitro; Or separation and Culture people glandulae salivariae minores epithelium ancestral cells, carries out amplification in vitro;
(3) in basement membrane matrix glue, hybrid three-dimensional Dual culture is carried out to glandulae salivariae minores mescenchymal stem cell and glandulae salivariae minores epithelium ancestral cells, obtain people's sialisterium organoid; Or in basement membrane matrix glue, dimensional culture is carried out to people's glandulae salivariae minores epithelium ancestral cells, obtain people's sialisterium class acinar tissue.
2. the vitro construction method of a kind of sialisterium organoid and class acinus according to claim 1, it is characterized in that, described basement membrane matrix glue is extracellular matrix Matrigel.
3. the vitro construction method of a kind of sialisterium organoid and class acinus according to claim 1, it is characterized in that, describedly in basement membrane matrix glue to the concrete operation step that glandulae salivariae minores mescenchymal stem cell and glandulae salivariae minores epithelium ancestral cells carry out hybrid three-dimensional Dual culture be: the glandulae salivariae minores mescenchymal stem cell and epithelial stem cell that are cultured to more than three generations or three generations are digested to unicellular, be inoculated in Matrigel after mixing by cell quantity 1: 1, the mescenchymal stem cell substratum of equal proportion mixing and kerationcyte culture medium is used to cultivate, sialisterium organoid substratum is used instead after 2 days, concrete operations are trysinization mescenchymal stem cell and epithelium ancestral cells is single cell suspension, count rear substratum resuspended, and mix with equal-volume 4 DEG C of Matrigel thawed, be inoculated in 24 orifice plates, be placed in 37 DEG C of cell culture incubators, after 20 minutes, treat that Matrigel solidifies, add substratum, every hole inoculation 2.5 × 10
5sialisterium mescenchymal stem cell and 2.5 × 10
5salivary gland epithelia ancestral cells, inoculate after mixing with 50 μ L Matrigel, inoculate successfully, change liquid every other day, vitro culture is after 1 day, and cell aggregation becomes branch shape, after 3 days, the organoid can seeing acinus shape and tubular branch structure is formed, and after 6-10 days, the organoid of Tubuli-vesicular structure can grow to maximum, can use sialisterium substratum instead to promote its differentiation and maturation after 6 days.
4. the vitro construction method of a kind of sialisterium organoid and class acinus according to claim 1, it is characterized in that, describedly in basement membrane matrix glue to the concrete operation step that people's glandulae salivariae minores epithelium ancestral cells carries out dimensional culture be: the glandulae salivariae minores epithelium ancestral cells being cultured to more than three generations or three generations is digested to unicellular, be inoculated in Matrigel, kerationcyte culture medium is used to cultivate, concrete operations are trysinization epithelial stem cell is single cell suspension, count rear substratum resuspended, and mix with equal-volume 4 DEG C of Matrigel thawed, be inoculated in 24 orifice plates, be placed in 37 DEG C of cell culture incubators, after 20 minutes, treat that Matrigel solidifies, add substratum, every hole inoculation 5 × 10
4salivary gland epithelia stem cell, inoculate after mixing with 50 μ L Matrigel, inoculate successfully, change liquid every other day, sialisterium organoid substratum can be used after 6 days instead to promote its differentiation and maturation, inoculate latter 4 hours, the ringwise polar alignment of cell, after 10 hours, form the spheroplast of class acinus, within 6-12 days, class acinus spheroplast can grow to maximum.
5. the vitro construction method of a kind of sialisterium organoid and class acinus according to claim 1, it is characterized in that, described mescenchymal stem cell substratum is MSCM substratum.
6. the vitro construction method of a kind of sialisterium organoid and class acinus according to claim 1, it is characterized in that, described kerationcyte culture medium is KM substratum.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105695394A (en) * | 2016-04-07 | 2016-06-22 | 中山大学 | In vitro culture method of mouse salivary gland organ |
| CN106318901A (en) * | 2016-05-06 | 2017-01-11 | 天津市海河医院 | Method for culturing in vitro three-dimensional respiratory tract stem/ancestral cells |
| KR20190053960A (en) * | 2016-10-06 | 2019-05-20 | 피로고프 러시안 내셔널 러서치 메디컬 유니버시티 (알엔알엠유) | Culture method of human salivary gland cells |
| KR102218549B1 (en) * | 2016-10-06 | 2021-02-22 | 피로고프 러시안 내셔널 러서치 메디컬 유니버시티 (알엔알엠유) | Human salivary gland cell culture method |
| CN111197024A (en) * | 2018-11-16 | 2020-05-26 | 杭州捷诺飞生物科技股份有限公司 | Pancreas-like structure and construction method and application thereof |
| CN111197024B (en) * | 2018-11-16 | 2023-08-18 | 杭州捷诺飞生物科技股份有限公司 | Pancreatic-like structure, construction method and application thereof |
| CN113544258A (en) * | 2019-03-29 | 2021-10-22 | 国立大学法人长崎大学 | Cultured tissue and method for producing same |
| CN114599380A (en) * | 2019-09-06 | 2022-06-07 | 美德邈医药公司 | Composition for preventing or treating salivary gland diseases using cell-derived vesicle |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2016168950A9 (en) | 2016-12-22 |
| WO2016168950A1 (en) | 2016-10-27 |
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