WO2015028900A1 - Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof - Google Patents
Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof Download PDFInfo
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- WO2015028900A1 WO2015028900A1 PCT/IB2014/062341 IB2014062341W WO2015028900A1 WO 2015028900 A1 WO2015028900 A1 WO 2015028900A1 IB 2014062341 W IB2014062341 W IB 2014062341W WO 2015028900 A1 WO2015028900 A1 WO 2015028900A1
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- Prior art keywords
- cells
- medium
- conditioned medium
- confluency
- mesenchymal
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Definitions
- the present disclosure relates to a method of obtaining conditioned medium (CM) rich in bioactive factors from pooled bone marrow derived mesenchymal stem cells (BM MSC).
- CM conditioned medium
- BM MSC bone marrow derived mesenchymal stem cells
- the instant method ensures a higher yield of desired growth factors and cytokines, by the use of cells pooled from multiple donors, which normalizes the inherent variability in the levels of the same secreted by cells from individual donors and by providing a cell feeding schedule at specified confluency.
- the disclosure further relates to the formulation of the conditioned medium for application in cosmetic and therapeutic areas.
- MSCs Mesenchymal stem cells have gained lot of interest in therapeutic/cosmetic field for various indications including skin conditions and wound healing.
- the mechanism through which MSCs participate in tissue repair is mainly through the secretion of trophic factors.
- MSCs provide the required microenvironment through the secretion of wide range of growth factors, cytokines and chemokines through their secretome.
- MSC secretome provides a new approach of treatment modalities in regenerative medicine and also in cosmetic applications.
- Growth factors, cytokines and chemokines serve as a tool for the cells to communicate and these molecules can be traced from the conditioned medium (CM) or spent medium harvested from cultured cells (Shohara et al., 2012).
- CM conditioned medium
- the present disclosure relates to a method of preparing a conditioned medium comprising bioactive factors secreted by mesenchymal stromal cells, said method comprising acts of : a. culturing the mesenchymal cells in a cell culture medium followed by expanding and harvesting of the cells; and b. subjecting the harvested cells to a process of: a. fed batch activation; b. fed batch activation followed by complete medium change; or c. complete medium change; or any combination thereof, to obtain the said conditioned medium; a method of preparing a conditioned medium comprising bioactive factors secreted by mesenchymal cells, said method comprising acts of: a.
- culturing the mesenchymal cells in a cell culture medium followed by expanding and harvesting of the cells and b. subjecting the harvested cells to a process of fed batch activation followed by complete medium change to obtain said conditioned medium; a conditioned medium comprising bioactive factors secreted by mesenchymal cells; a composition comprising conditioned medium along with pharmaceutically acceptable excipient and additive; and a method of managing skin related condition, said method comprising act of administering the conditioned medium or a formulation thereof, to a subject in need thereof.
- Figure 1 shows the variation in the VEGF secretion between three donors and it's normalization after pooling and culturing.
- Figure 2 shows the levels of expression of various cytokines and growth factors in the conditioned medium from pooled bone marrow derived mesenchymal stromal/stem cells, over and above the cell culture medium. Multiplex analysis using ELISA and Luminex® based assays is used for the estimation.
- Figure 3 shows the comparison of bioactive factors secreted from multipotent mesenchymal stromal/stem cells cultured by 3 different processes.
- PGE 2 PGE 2;
- Figure 4 shows the levels of (a) VEGF, (b) TGF beta 1 and (c) PGE-2 in conditioned medium generated by process 2 from three different batches.
- Figure 5 shows the enhanced bioactive factors retention in the conditioned medium as shown by the levels of VEGF after concentration by TFF technique using molecular cut-off >3kDa and >lkDa
- Figure 5 show the VEGF functionality by transwell migration assay using human umbilical vein endothelial cells.
- Figure 6 shows the anti- ageing effects of CM upon human foreskin fibroblast cells (FIFF) treated with Tert- butyl hydroperoxide (tbOH).
- FIFF human foreskin fibroblast cells
- tbOH Tert- butyl hydroperoxide
- Figure 7 shows the evaluation of protective effects of pre-treatment of 10X CM against collagen degradation caused by oxidative damage in dermal fibroblast cells.
- Figure 8 relates to a graph representing a comparative analysis in the proliferation potential of HFF cells in 5 dilutions of the conditioned media with control medium
- Figure 9a shows the significant fibroblast migration which is indicative of positive skin renewal effects upon the treatment of HFF- 1 cells with 10X CM.
- Figure 9b shows the effect of 10X control and 10X CM on HFF-1 cells migration which is indicative of positive skin renewal effects.
- the data points are indicated as mean ⁇ standard error of triplicate values.
- Figure 10 shows the cytoprotective/viability restorative effects of 10X CM against oxidative stress induced by tert-butyl hydroperoxide in human dermal fibroblast cells. The data points are indicated as mean ⁇ standard error of triplicate values.
- Figure 11 shows the anti- wrinkling effects of CM upon human foreskin fibroblast cells (HFF-1 cells) subjected to UV radiation and treated with 10X CM at doses of 10% and 50% compared to their respective control medium, (a) shows the restoration of elastin levels; (b) shows the decrease in caspase 3 activation; (c) shows the extent of protection against DNA lesions.
- Figure 12 shows the evaluation of anti-apoptotic effects of 10X CM against oxidative stress induced apoptosis in dermal fibroblast cells.
- Figure 13 shows a graphical representation of mean corneometer reading indicative of skin hydration levels with all the formulations.
- Figure 14 shows a graphical representation of mean mexameter reading indicative of erythema index with all the formulations
- Figure 15a shows photographic images of non-irradiated animals (control group- Gl) on day 1 in which the skin surface was smooth and even.
- Figure 15b shows photographic images of untreated animals (control group- Gl) on day 4, no visible macroscopic changes (redness, swelling, wrinkles) were observed.
- Figure 15c shows photographic images of untreated animals (control group-Gl) on day 6, no visible macroscopic changes (redness, swelling, wrinkles) were observed.
- Figure 15d shows photographic images of untreated animals (control group-Gl) on day 8, no visible macroscopic changes (redness, swelling, wrinkles) were observed.
- Figure 16a shows photographic images of UVB-irradiated animals (G2) on day 1 in which no visible macroscopic changes were observed.
- Figure 16b shows photographic images of UVB-irradiated animals (G2) on day 4 with noticeable induction of wrinkles and desquamation seen in animals of group that was UV-B exposed.
- Figure 16c shows photographic images of UVB-irradiated animals (G2) on day 6 with substantial wrinkles visible appearance of wrinkles, dullness, roughness and desquamation seen in animals of group that was UV-B exposed.
- Figure 16d shows photographic images of UVB-irradiated animals (G2) on day 8 with severe wrinkles and desquamation seen in animals of group that was UV-B exposed.
- Figure 17a shows photographic images of formulation 1 treated animals (G5) on day 1 in which no visible macroscopic changes were observed.
- Figure 17b shows photographic images of formulation 1 treated animals (G5) on day 4.
- Figure 17c shows photographic images of formulation 1 treated animals (G5) on day 6.
- Figure 17d shows photographic images of formulation 1 treated animals (G5) on day 8.
- Figure 18a shows photographic images of formulation 3 treated animals (G6) on day 1 in which no visible macroscopic changes were observed.
- Figure 18b shows photographic images of formulation 3 treated animals (G6) on day 4.
- Figure 18c shows photographic images of formulation 3 treated animals (G6) on day 6.
- Figure 18d shows photographic images of formulation 3 treated animals (-G6) on day 8.
- Figures 19a and 19b show the comparison of the VEGF and TGF- ⁇ in the conditioned medium generated from pooled bone marrow and Wharton's jelly derived mesenchymal stem cells, respectively.
- the present disclosure relates to a method of preparing a conditioned medium comprising bioactive factors secreted by mesenchymal stromal cells, said method comprising acts of: a. culturing the mesenchymal cells in a cell culture medium followed by expanding and harvesting of the cells; and
- the mesenchymal cells are mesenchymal stromal cells or mesenchymal stem cells or a combination thereof; and wherein the mesenchymal cells are bone marrow derived mesenchymal cells.
- the mesenchymal cells are isolated from individual donors and pooled to obtain pooled mesenchymal cells.
- the mesenchymal cells are seeded and expanded as passage 4 cultures at a seeding density of about 1000 cells/cm 2 to about 10000 cells/cm 2 preferably about 1000 cells/cm 2 ;
- the cells are expanded to a confluency of about 50% and subjected to media change and further cultured till about 80% to about 90% confluency.
- the stem cells are further seeded and expanded as passage 5 cultures at a seeding density of about 1000 cells/cm 2 to about 10000 cells/cm 2 preferably about 1000 cells/cm 2 till about 45% to about 50% confluency.
- the fed batch activation comprises acts of: a. adding about 500 ml of cell culture media to the passage 5 cells expanded till about 45% to about 50% confluency; and
- the fed batch activation followed by change of cell culture medium comprises acts of: a. adding about 500 ml of cell culture media to the passage 5 cells expanded till about 45% to about 50% confluency;
- the complete change of cell culture medium comprises acts of: a. subjecting the passage 5 cells expanded till about 45% to about 50% confluency to about 1.5L of complete change of cell culture media; and b. allowing culturing of the cells till about 80% to about 90% confluency and harvesting the conditioned medium.
- the conditioned medium is concentrated and enriched by ultrafiltration or tangential flow filtration using molecular weight cut- offs of about lkDa and above or about 3kDa and above; and wherein after the concentration, amino acid selected from a group comprising L- Arginine and L- Glutamic acid or a combination thereof is optionally added to the conditioned medium at a concentration ranging from about 50mM to about lOOmM.
- the method optionally comprises pooling of the conditioned media obtained from each of the three processes.
- the cell culture medium comprises components selected from a group comprising Dulbecco's Modified Eagle Medium-Knock Out, Dulbecco's Modified Eagle Medium - F12, Dulbecco's Modified Eagle Medium - Low Glucose, fetal bovine serum, L-alanine-L-glutamine, penicillin and streptomycin or any combination thereof.
- the cell culture medium comprises basic fibroblast growth factor at a concentration of about 1 ng/mL to about 10 ng/mL, preferably about 2 ng/mL to enhance secretion of the bioactive factors.
- the conditioned medium obtained by fed batch activation is enriched forAng-1; wherein the conditioned medium obtained by fed batch activation followed by change of cell culture medium is enriched for TGF- ⁇ ; and wherein the conditioned medium obtained by change of cell culture medium is enriched for VEGF and PGE-2.
- the present disclosure relates to a method of preparing a conditioned medium comprising bioactive factors secreted by mesenchymal cells, said method comprising acts of: a. culturing the mesenchymal cells in a cell culture medium followed by expanding and harvesting of the cells; and
- the mesenchymal cells are mesenchymal stromal cells or mesenchymal stem cells or a combination thereof; and wherein the mesenchymal cells are bone marrow derived mesenchymal cells.
- the mesenchymal cells are isolated from individual donors and pooled to obtain pooled mesenchymal cells.
- the fed batch activation followed by - complete medium change comprise acts of: a. adding about 500 ml of cell culture medium to the passage 5 cells expanded till about 45% to about 50% confluency;
- the present disclosure relates to a conditioned medium comprising bioactive factors secreted by mesenchymal cells.
- the bioactive factors comprises of growth factors, cytokines, chemokines, anti- oxidants and other factors that are known to function or mediate a biological process, wherein the biological process includes cell proliferation and cell migration.
- the bioactive factors are selected from a group comprising VEGF, TGF-b, PGE-2, PDGF, GD F, IGFBP, FGF, GCSF M-CSF, angiogenin, angiopoietin, KGF, FGF7, BMP6, IGF1, laminin, MMP1,MMP2,MMP9, TEVIP1 and TIMP2, HGF, SDFl and LIF, IL-10, IL-10 or any combination thereof; and wherein the conditioned medium, optionally contains amino acid selected from a group comprising L- Arginine and L- Glutamic acid or a combination thereof at a concentration ranging from about 50mM to about lOOmM.
- concentration of the VEGF ranges from about 2 to about 10 ng/mL. In yet another embodiment of the present disclosure, concentration of the TGF-b ranges from about 1 to about 5 ng/mL. In yet another embodiment of the present disclosure, the concentration of PGE-2 ranges from about 0.8 to about 2 ng/mL.
- the concentration of Angiopoietin- 1 ranges from about 10 to about 12 ng/mL.
- the concentration of HGF ranges from about 20 to about 200 ng/ml. In yet another embodiment of the present disclosure, the concentration of SDF1 ranges from about 0.4 to about 3ng/ml.
- the concentration of IL-10 ranges from about 10 to about 50 ng/ml.
- the present disclosure relates to a composition
- a composition comprising conditioned medium along with pharmaceutically acceptable excipient or cosmetically acceptable carriers.
- the excipient is selected from a group comprising additive, carrier, granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, glidants, anti-adherents, anti-static agents, surfactants, anti-oxidants, gums, coating agents, coloring agents, flavouring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents, plant cellulosic material and spheronization agents or any combination thereof.
- the excipient is selected from a group comprising Hydroxy ethyl cellulose, Hydroxy propyl cellulose, Hydroxy propyl methyl cellulose, Carbopol, EDTA, Methyl paraben, Propyl paraben, Deionised water, Glycerin, DL- Panthenol, D-Panthenol, Phenoxyethanol, Allantoin, PEG, Purified water, Xanthan gum, Sodium PCA, Gluconolactone, Sodium Benzoate, Sodium Hydroxide, Phenoxyethanol, Ethylhexylglycerin, Sodium Polyacrylate, Caprylic or Capric Triglyceride, Mineral oil, Tri (PPG-3 Myristyl ether) Citrate, SorbitanLaurate, Trideceth-6, PEG-75 Lanolin, Beta Glucan, Sodium Hyaluronate, Phosphatidylcholine, Cholesterol, Essential Oil,
- the composition is formulated into dosage forms selected from a group comprising, oily suspensions, hydrogel, nanogel, wet tissues, ointment, patch, gel, lotion, serum, emulsion, creams, spray, drops, or any combination thereof.
- the present disclosure relates to a method of managing a skin related condition, said method comprising act of administering the conditioned medium or a formulation thereof, to a subject in need thereof.
- mesenchymal stromal cells refer to multipotent mesenchymal stem cells and the said terms can be used interchangeably.
- the term 'skin' throughout this disclosure is used to mean skin as an organ as a whole, or any components or variants thereof, such as outer covering, outer coating, cutis, derma, dermis, epidermis, integument, peel, pelt, sheath and surface.
- the term 'skin conditions' includes cosmetic skin conditions such as wrinkles, frown lines, scarring, folds, sagging, age spots, uneven pigmentation, thinning, elasticity, scarring, skin roughness and dryness, open pores and other skin conditions,
- 'complete medium change' refers to removal of spent media and feeding the cells with fresh cell culture medium.
- the term multipotent stem cells includes mesenchymal stromal cells, mesenchymal stem cells derived from bone marrow, adipose tissue, Wharton's jelly dental pulp and other known sources.
- the term carriers includes pharmaceutical carrier, cosmetically acceptable carries and dermatologically acceptable carriers etc.
- the term carrier as referred in this application means an excipient or an vehicle, used for administration of the active ingredient (CM) topically to skin.
- Conditioned medium(CM) or spent medium refers to the medium rich in various different types of biological active factor secreted by the stem cells, which are hereinafter collectively referred to as the bioactive factors.
- bioactive factors' comprises of biological active growth factors, cytokines, chemokines, antioxidants, small molecules, ECM and other factors that are known to function or mediate a biological process, for example cell proliferation, cell adhesion, differentiation, inflammatory response, cell migration and other biological functions etc.
- Conditioned medium is obtained from the cell culture collected at 80-90% confluency of the culture (10- 14 days) which is the confluency at which the cells are in the mitotic phase and there is maximum production of bioactive factors.
- the present disclosure provides an efficient method for enhancing the final availability of bioactive factors within the conditioned/spent media derived from bone marrow derived multipotent mesenchymal stem/stromal cells (BM-MSCs) and also to reduce any biological variability.
- the method involves pooling the said BM-MSCs from multiple donors to reduce the biological variability.
- the pooling of mesenchymal cells maximize the probability of generating - conditioned medium representing large number of bioactive factors with specific biological function/property and also to compensate biological factors not secreted/or secreted on lower levels by one of the selected individuals being pooled.
- the pooled cells with reduced biological variability are then cultured in cell culture medium and subjected to selective cell feeding schedule/media change processes at specified confluency to enhance the cell secretion and collection of bioactive factors by separating the conditioned medium from the cultured mesenchymal stromal cells.
- the mesenchymal stromal cells are pooled to reduce the biological variability and overall heterogeneity general observed in CM obtained from individual donors.
- the pooling leads to producing bioactive factors which can be used to improve the skin health for cosmetic applications.
- a conditioned medium produced in a serum and xeno-free conditions also includes serum and xeno-free medium used for culturing and maintaining the stem cells.
- the pooled MSCs are subjected to a specific feeding cycle at specific confluency to obtain maximum amount of CM rich in all the bioactive factors.
- the CM rich in bioactive factor was collected and concentrated using known technology.
- the concentration process involves using a specific molecular weight cutoff to retain all the required bioactive factors which benefit the skin health.
- the CM is purified and sterilized by generally known methods in the art.
- the CM is formulated for topical administration to skin /for various cosmetic applications.
- the CM is used for the improving the skin conditions such as wrinkles, frown lines, scarring, folds, sagging, age spots, uneven pigmentation, thinning, elasticity, scarring, skin roughness and dryness, open pores and other skin conditions,
- the method of culturing and pooling the BM- MSCs from multiple donors is previously known and is briefly as follows: a) Cryopreserved cells at passage 1, from individual donors, are thawed at 37 °C in a water bath.
- the cells in the freezing medium are revived with 9 part cell culture medium in (1 :9 ratio), after which the sample is centrifuged at about 1200 to 1500 rpm for 10- 20 minutes at room temperature.
- the cells from all the donors are mixed in equal proportion, after which the cells are counted, the viability is checked and the viable cells are plated for passage 2 at a seeding density of about 1000 cells/cm 2 in cell culture vessel including and not limiting to culture flask, cell stack etc.
- the culture vessels are transferred to a 5% C0 2 incubator at about 37°C.
- the instant example compares the secretion of bioactive factors between individual and pooled bone marrow derived mesenchymal stem cells.
- Human bone marrow was obtained from healthy donors at KMC Manipal hospital, Manipal, India.
- Human bone marrow derived mesenchymal stromal/stem cells isolated from multiple donors preferably from at least 2, or more preferably from three healthy donors are individually cultured using a cell culture medium composition as described in table 1 for five passages.
- the cell culture medium is not limited to the composition given in table 1 but can include any known cell culture medium including serum-free and xeno-free culture medium or a combination thereof known in the art.
- the cells are cultured in 25 cm 2 tissue culture flasks, complete change of cell culture medium is given every 3 rd day till cultures reach 80- 90% confluency.
- the conditioned medium is collected from the confluent (day 9-10) cultures at passage 5 and preserved at -80°C for further analysis.
- Dulbecco's Modified Eagle Medium - Knock out (DMEM- About 85 to about 95% KO) or Dulbecco's Modified Eagle Medium - F12 (DMEM- preferably about 88.5% F12) or Dulbecco's Modified Eagle Medium - Low Glucose
- DMEM-LG Dulbecco's Modified Eagle Medium
- Glucose (DMEM-HG) or any other basal medium or serum
- FBS Fetal Bovine Serum
- L-alanine-L-glutamine(GLUTAMAX) About 0.5 to about 2% preferably about 1% Penicillin Streptomycin(PENSTREP) About 0.1 to about 1% preferably about 0.5%
- bFGF Basic Fibroblast growth factor
- the basic fibroblast growth factor is an optional ng/mL preferably about 2 component which enhances the secretion of bioactive factors.
- BMMSC bone marrow derived mesenchymal stem/stromal cells
- Table 2 indicates the relative levels of the various growth factors in conditioned medium from individual BMMSC samples, over and above the medium control. Values represent arbitrary fluorescence units. Analysis of bioactive factor secretion from pooled cells:
- Human bone marrow derived mesenchymal stem cells isolated from at least 2, or preferably three healthy donors are pooled and cultured using the cell culture medium as described in table 1 for five passages.
- the cell culture medium is not limited to the composition given in table 1 but can include any known cell culture medium including serum-free and xeno-free culture medium or a combination thereof known in the art.
- the cells are cultured in 25 cm 2 tissue culture flasks, complete change of cell culture medium is given every 3 rd day till cultures reach 80- 90% confluency.
- the conditioned medium is collected from the confluent (day 9-10) cultures at passage 5 and preserved at -80°C for further analysis.
- the method of culturing the pooled cells in a large scale involves expanding he pooled cells at passage 3 as passage 4 cultures in one cell stacks (CS- 1) at a seeding density of 1000 cells/cm 2 , subjected to a change of the medium at 50% confluency and further cultured till 80-90%> confluency is achieved.
- the pooled cells are then harvested and expanded as passage 5 cultures at a seeding density of 1000 cells/cm 2 in ten cell stacks (CS- 10) till 45- 50%) confluency is achieved. Thereafter 500 mL of cell culture medium is added to each cell stack and the conditioned medium is collected from the cell stacks at 80- 90% confluency of the cultures.
- the table 3 represents the bioactive factor rich conditioned medium from pooled culture derived from three donors, where significant levels of growth factors important in therapeutic processes for skin conditions are observed. This data suggests that pooling contributes to averaging out/compensates the inherent individual variability among donors and therefore increases the probability of representation of the majority of important GFs in the conditioned medium.
- Table 3 indicates the relative levels of the various growth factors in conditioned medium from pooled BMMSC samples, over and above the medium control. Values represent arbitrary fluorescence units.
- the sample of cells derived after pooling of various cells from multiple donors are less in some bioactive factors there would also be cases where the levels of growth factors/cytokines are improved significantly from that of the individual samples, as the pooling would have made the levels of growth factors/cytokines more homogeneous. It further maximizes the probability of generating mesenchymal stromal/stem cells derived conditioned medium representing large number of bioactive factors and others molecules with specific biological function/property and also compensates for biological factor not secreted by one of the selected individual being pooled.
- the aforementioned observation establishes the importance of pooling for reduction of variability and heterogeneity and ensures that when a final product derived from the pooled cells is obtained for specific therapeutic and or cosmetic applications, the overall levels of bioactive factors- are maintained.
- Figure 1 shows the variation in the VEGF secretion between three donors and it's normalization after pooling and culturing.
- Figure 2 shows the levels of expression of various -bioactive factors in the conditioned medium from pooled bone marrow derived mesenchymal stem cells, over and above the cell culture medium.
- Figure 19a and 19b shows the comparison of the VEGF and TGF in the conditioned medium generated from pooled bone marrow and Wharton's jelly derived MSCs respectively. It is observed that the Wharton's jelly derived conditioned medium has less of both the factors when compared to the bone marrow derived conditioned medium.
- the cryopreserved pooled MSCs [obtained as per the process of isolation and pooling provided above] at passage 3 (P3) are thawed and expanded as P4 cultures in one Cell Stacks (CS-1) at a seeding density of 1000 cells/cm 2 .
- a complete change of cell culture medium is given at around 50% of confluency (about 6-8 day cultures) and then cells are cultured until they become 80-90% confluent.
- Cells are then harvested and expanded as P5 cultures at a seeding density of 1000 cells/cm 2 in ten cell stacks (CS-10); and each CS-10 is filled with 1.5 L of the cell culture medium.
- One of the following 3 cell culture medium change processes (or also referred as feeding schedule processes) is performed once cells achieve 45-50% confluency (around day 7 cultures):
- the medium change/ cell feeding schedule comprises one partial medium change referred to as fed-batch activation or media top-up.
- fed-batch activation fresh medium is added to the cell culture without removing the spent medium.
- the fed batch activation is performed with a volume of 500ml per CS-10 on Day 06/07 (or when the cell are about 45- 50% confluency) of culture to all the CS-10 cultures. Once the cultures reach 80-90% confluency, conditioned medium is collected and preserved from all CS-10.
- Media Change Process 2 :
- the media change/cell feeding schedule comprises one partial and one complete medium change at a specific confluency.
- fed batch activation is performed with a volume of 500 mL per Ten Cell Stack (TCS) is on Day 06/07 (or when the cells reach about 50% confluency) of culture in all the CS-10.
- TCS Ten Cell Stack
- a complete medium change is carried out, wherein the spent medium is removed from the culture and fresh 2L of cell culture medium is added per CS-10. Once the cultures reach 80-90% confluency, conditioned media is collected and preserved from all CS-10.
- the media change/cell feeding schedule comprises only one complete medium change schedule.
- the spent medium is removed completely from all TCS and replaced with 1.5 L/TCS of freshly prepared cell culture medium.
- the cultures reach 80-90% confluency, conditioned media is collected and preserved from all CS-10.
- CM conditioned medium
- CM enriched for Angiopoietin-1 can be obtained by following Process 1
- CM enriched for TGF -beta can be obtained by following Process 2
- CM enriched for VEGF and PGE-2 can be obtained by following Process 3.
- a specific production processes can therefore be used to obtain tailor-made CM for a specific therapeutic application or set of applications demanding large amounts of the relevant GFs/Cytokines or other bioactive factors.
- Process 2 can be employed for applications requiring cartilage regeneration, due to the large amounts of TGF-beta in the CM.
- Process 3 can be employed for immune modulation due to the large amounts of PGE-2 in the CM.
- the secretion of specific bioactive factors in optimum levels during the processes 1, 2, and 3, by the pooled BM-MSCs is correlated to the rate of secretion, in vitro half-lives and stability of the growth factors/ cytokines.
- different bioactive factors are secreted in the cell culture medium by the cells at different point of time, depending on the stage at which the conditioned medium is collected.
- the pooled BM-MSCs are subjected to Process 1 that involves fed- batch activation on the 6 th or 7 th day of culturing wherein the cultures are 50% confluent, as a result of which the cell density increases and the secreted growth factor gets accumulated in the media.
- VEGF is relatively secreted faster at desired confluency
- the pooled BM-MSCs are subjected to Process 3 which involves administering complete media change at 45- 50% confluency on the 6 th to 8 th day of culturing, thereby inducing more production of the growth factor by the cells, which would ensure its optimum level in the media.
- specific conditioned medium can be arrived at, which comprises optimum levels of specific bioactive factors-, along with other growth factors/cytokines which are non-process specific.
- CM from different individual processes can also be pooled selectively to obtain CM containing maximum quantities of relevant GFs/Cytokines for the given application.
- CM from process 1 & 3 can be pooled to obtain maximum enrichment of the angiogenic growth factors like Ang-1 and VEGF.
- Table 4 indicates the absolute levels of the growth factors and cytokines in the CM generated from the three different processes.
- VEGF vascular endothelial growth factor (ng/mL) 2.6 ⁇ 0.24 3.7 ⁇ 0.36 6.3 ⁇ 0.16
- TGF-b ng/mL 1.15 ⁇ 0.05 3.0 ⁇ 0.1 2.4 ⁇ 0.01
- Angiopoietin- 1 about 11.3 to about about 6.3 to about about 2.6 to about 4 (ng/mL) 11.6 6.4
- Figure 4(a,b,c) provides the graph for VEGF, TGF beta and PGE-2 levels in the conditioned medium generated by process 2 from 3 from different batches.
- the batch comparison was performed after the TFF concentration, which resulted in a conditioned medium which is 10 times concentrated when compared to the original conditioned medium prior to the TFF.
- the conditioned medium obtained is a 10X concentrate.
- the graph shows the
- process 2 generated conditioned medium from 3 different batches with the average concentration of 0.440 ⁇ 0.125 ng/ml and with variations which are statistically insignificant.
- the CM collected from the above methods is optionally further concentrated and enriched by ultra-filtration/Tangential flow filtration (TFF) using defined molecular weight cut offs and the concentrated conditioned media was used for analytical estimation and formulation.
- the CM can also be concentrated by other known and established methods.
- the concentration and enrichment by ultra-filtration/Tangential flow filtration (TFF) is performed by using molecular weight cutoffs of 3KDa and above. While, in another embodiment the CM is concentrated using a molecular cutoff of lKDa and above.
- Figures 5a and 5b show the enhanced growth factor retention in the conditioned medium as shown by the levels of VEGF, after concentration by TFF concentration technique using molecular cutoff of lkDa, which is further proven to be functional by transwell migration assay using human umbilical vein endothelial cells (HUVEC) as described below.
- the migration of the HUVEC cells in response to the factors in CM that is concentrated using two different molecular cutoffs of 1 kDa and 3 kDa is compared to the migration of the HUVEC cells with response to the factors in Plain KO, KO ⁇ 10%FBS and M199 ⁇ 20%FBS.
- TFF Tangential Flow Filtration
- the fluid (feed) is pumped tangentially along the surface of the membrane.
- An applied pressure serves to force a portion of the fluid through the membrane to the filtrate side.
- a pump is used to generate pressure on the feed flow.
- the TFF unit for the purpose of concentrating human bone marrow mesenchymal stem cell derived conditioned media (CM) utilizes a filter membrane with a cut-off of 1 kDa as this cut- off value is substantially lower than the molecular weight of the protein molecules that are desired to be retained. Particles which are of greater size are swept along with the tangential flow of the fluid. Thus, the tangential flow of the fluid prevents accumulation of particles on the flow path of the filtrate thereby enabling continuous operation at relatively high solids loads.
- Diafiltration (DF) process can be performed in combination with the ultrafiltration run during protein concentration to enhance either product yield or purity. During DF (continuous diafiltration), buffer is introduced into the feed tank while filtrate is removed from the unit operation. This washes components out of the product pool into the filtrate, thereby exchanging buffers and reducing the concentration of undesirable species.
- the membrane is installed and the TFF system is initialized (typically flushed with water and tested for water filtrate flow rate and integrity).
- About 1- 2 litres of the CM is added to the feed tank) and the cross flow is established by maintaining the feed inlet pressure of 30 psi (PI) and a retentate pressure of 25 psi (P2). This run is continued till the CM is reduced to ten times its original volume (100-200 ml; depending on the feed quantity).
- the TFF concentration thus resulted in a conditioned medium which is 10 times concentrated when compared to the original conditioned medium prior to the TFF.
- the conditioned medium obtained is a 1 OX concentrate.
- the amino acids L- Arginine and L- Glutamic acid are added to the TFF concentrate, in their purified form solubilized in minimum quantity of Dulbecco's phosphate buffered saline, at a concentration of about 50 mM to about 100 mM. These amino acids function as protein stabilizers.
- the Migration assay is performed using a transwell two-chamber cell culture method and Transwell inserts (3422; Corning, Cambridge, MA) with an 8 ⁇ pore polycarbonate membrane.
- Human umbilical vein endothelial cells (HUVEC) are serum starved for 24 hours and 1X10 5 cells/well are plated into the upper chamber of the transwell insert.
- the cell culture media plain media or conditioned media is added.
- 20 ⁇ g/mL of VEGF blocking antibody R&D Systems-MAB293 is added as one of the condition. Cells are allowed to migrate for about 16-18 hours.
- the non-migrated cells are carefully removed using a pre-wetted cotton swab and the membrane is fixed with 4% paraformaldehyde and stained with haematoxylin for 10 minutes. After washing, the membrane is mounted with DPX and the cells that had migrated from the upper to the lower side of the membrane are counted under bright field microscope at 100X objective. (100X magnifications- Nikon 90i microscope). All the assays are done in triplicates. The data is represented as mean ⁇ SEM. The assay shows the concentrated conditioned medium is functional and the bioactive factors are not destroyed during the processing.
- Oxidative stress plays a central role in initiating and driving events that contribute to skin ageing at the cellular level by degrading vital building blocks of the skin including elastin and hyaluronic acid and even alters cellular renewal cycles.
- Elastin protein fibres combine with collagen to give the skin elasticity while hyaluronic acid is found at the base of the dermis and forms a "cementing and gelling" base that binds to water molecules, allows nutrients and oxygen into the tissue and protects the dermal structural layer.
- cyclin B l is a regulatory protein involved in mitosis whose levels are known to decrease in living cells during senescence.
- CM at 10X concentration
- tbOH Tert-butyl hydroperoxide
- conditioned medium is obtained from culturing of MSCs derived from three donors, when pooled together and cultured by following Process 1.
- Human foreskin fibroblast (HFF-1 ATCC® SCRC-1041TM- source: human foreskin) cells are treated with 300 ⁇ tbOH for 2 hours, followed by exposure of different doses of 10X CM and control medium for 48hours.
- Elastin and hyaluronic acid levels are estimated in culture supernatant by ELISA method.
- Cyclin Bl cells are lysed in lysis buffer to obtain cell lysates and protein levels are estimated by ELISA method.
- Bone marrow mesenchymal stem cell conditioned media possess the potential to regain structural framework to treat damaged skin, to restore elasticity and retain moisture in damaged / aged skin, prevent loss of skin structure and reverse senescence by increasing cyclin B l levels caused by oxidative stress.
- the protective effects of 10X CM generated by process- 2 against collagen degradation caused by oxidative damage in dermal fibroblast cells upon treatment with 600 ⁇ of H 2 O 2 for 2 h is evaluated by measuring levels of collagen 48 h after H 2 O 2 damage.
- HFF-1 cells are treated with different doses of 10X CM and 10X control medium for 24 h prior to H2O2 damage.
- 10X CM possess the ability to protect collagen degradation induced by oxidative stress and has the potential to prevent loss of skin structure caused by oxidative stress.
- HFF cells About 2000 HFF cells are seeded per well in a 96-well micro-titre plate in IX DMEM + 15% FBS. On the next day, the cells are serum starved in IX DMEM + 0.1% FBS for 24 hours. Post serum starvation, the cells are treated with different doses of IX / 10X of control medium / CM generated by process 2. MTT assay is performed 6 days post- addition of the control/ conditioned medium. The assay employs a dye that is used for measuring the viability and proliferation of the cells colorimetrically.
- This assay indicates the proliferation potential of Human fibroblast cells in the presence of conditioned media (Figure 8).
- the proliferation potential of HFF is found to be the highest when 25% of IX CM is used. It represents the ability of skin cells to grow new cells contributing to youthful skin.
- the invitro scratch assay is an easy, low-cost and well-developed method to measure cell migration in vitro.
- the assay is particularly suitable for studies on the effects of cell-matrix and cell- cell interactions on cell migration, it mimics cell migration and enables imaging of live cells during migration to monitor intracellular events if desired.
- the basic steps involve creating a "scratch" in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to quantify the migration rate of the cells.
- Cytoprotective viability restorative effects of 10X CM generated by process 2 against oxidative damage in dermal fibroblast cells upon treatment with 300 uM of tert-butyl hydroperoxide (tbOH) for 2 h and post treatment of damaged fibroblast with 10X CM for 48 h is evaluated by MTT assay.
- Treatment of HFF-1 cells with 300 uM of tbOH leads to 35% decrease in cellular viability in SFM treated as compared to untreated cells.
- the cytoprotection obtained with 10X CM is 11%, 18% and 12% higher at the doses of 5%, 10% and 50% respectively as compared to respective 10X control medium treated cells against tbOH damage ( Figure 10).
- serum free medium with ascorbic acid SFM+AA
- Treatment of HFF-1 cells with 100 uM ascorbic acid(AA) also leads to 12% cytoprotection against tbOH damage.
- Treatment of damaged HFF-1 cells with 10X CM demonstrates cytoprotective / cellular viability restorative abilities at all the doses tested.
- 10X CM has shown a trend in the ability to restore the viability of oxidative stressed fibroblast cells at the concentrations 5 %, 10 % and 50 %. Maximum activity is observed at 10 % 10X CM. Hence, 10X CM has the ability to restore the viability of oxidative stressed fibroblasts cells and hence possesses the potential to restore skin health.
- UV radiation damages elastin, a protein in the skin that normally maintains the springiness and strength of tissue beneath the skin.
- elastin a protein in the skin that normally maintains the springiness and strength of tissue beneath the skin.
- large amounts of metalloproteinase are generated which may result in collagen damage. This results in the formation of disorganized collagen fibers known as solar scars.
- UVB exposure induces cell death by activating apoptotic cell death pathways such as increase in caspase 3 whose activation indicates mitochondrial mediated apoptotic pathway activation.
- DNA damage also results from UVB exposure, with prominent UV- induced lesions on DNA resulting in CPD (cyclobutane pyrimidine dimer) formation. Further, the activation of caspase 3 and increase in cellular DNA damage results in malignant changes in the epidermis and disorganization in the dermal matrix leading to the appearance of wrinkled skin.
- CPD cyclobutane pyrimidine dimer
- caspase 3 activation cells are lysed in cell lysis buffer to make cell lysates to estimate protein levels using BCA (biocinchoninic acid assay) protein estimation.
- the caspase 3 activity is measured using ELISA.
- the level of CPD formation was estimated by ELISA.
- Bone marrow mesenchymal stem cell derived conditioned media aids in skin structure building /strengthening effects, skin tightening, retention of elasticity, protects against UVB induced apoptosis and possess DNA repairing properties against UVB induced DNA lesions.
- the percentage of cells in sub-GO/Gl phase of cell cycle indicates apoptotic population of cells.
- the anti-apoptotic effect of 10X CM obtained by Process- 1 against UVB induced apoptosis in dermal fibroblast cells upon treatment with 230 mJ/crm UVB is evaluated by analyzing the percentage of cells in sub-GO/Gl phase of cell cycle 48 h after irradiation.
- the treatment of human dermal fibroblast cells with 300 mJ/crm UVB leads to 55% increase in number of cells in sub-GO/Gl phase of cells cycle.
- CM protects human dermal fibroblast cells against UVB induced apoptosis at higher doses (10% and 50%).
- the conditioned medium produced from the above process is formulated for topical administration.
- the CM is formulated into but not limited to oily suspensions, hydrogel, nanogel, liposome, wet tissues, ointment, patch, gel, lotion, serum, emulsion, creams, spray, drops, and other known formulation in art can be used or any combination thereof.
- Conditioned media (Drug) is added into the solution and stirred until it is dispersed uniformly. Gel forming polymer is added slowly with continuous stirring to the above solution until it forms a viscous gel.
- the anti-wrinkle formulations are evaluated for physicochemical properties and in-vitro screening studies. Based on the feasibility, the initial in-vitro screening studies are conducted in Franz diffusion cell using rat skin or dialysis membrane. For the final optimized formula cadaver skin is used. During the in-vitro screening studies receptor sample is collected at specified time intervals (0,0.10,0.20,0.30,0.45, 1,2,4,6,8,10, 12 and 24 hours) and it is analyzed by UV spectroscopy at 280 and 200 nm.
- the proteins (growth factors) present in the receptor sample 1 absorb ultraviolet light with absorbance maxima at 280 and 200 nm. These parameters is compared with marketed and placebo formulations.
- the final formulation is packed and labeled in a suitable container meant for marketing and kept for stability under various conditions as per suitable ICH guidelines. At certain decided interval referred as stability time point samples are withdrawn and all the physicochemical evaluation (PCE) is performed.
- PCE physicochemical evaluation
- a gel formulation with appropriate viscosity and spreadability is selected for animal studies. Further the gels are placed in stability chambers and evaluated for the above physical parameters at specified time points as per the proposal.
- Conditioned Medium for skin health including anti-ageing and skin rejuvenation
- Concentrated conditioned medium is formulated into a serum for cosmetic application.
- the cosmetic application includes use the serum formulation for general skin health including, anti-ageing, anti-wrinkling, anti-scaring and for skin revival and skin rejuvenations.
- BM-MSC conditioned media (CM) based anti-wrinkle serum is prepared by suitable preparation method with compatible ingredients to stabilize and to protect varies bioactive factors present in the conditioned media.
- Each batch formula is prepared with IX, 3X and 7X concentration of the BMMSC conditioned media.
- the table below illustrates the 4 different type of serum formulation.
- the anti-aging liposomal serum formulation comprises of conditioned medium rich in bioactive factor secreted by mesenchymal stem/stromal cell which can promote youthful skin.
- the anti-wrinkle formulations are evaluated for physicochemical properties and in-vitro screening studies. Based on the feasibility, the initial in-vitro screening studies are conducted in Franz diffusion cell using rat skin or dialysis membrane. For the final optimized formula cadaver skin is used. During the in-vitro screening studies receptor sample is collected at specified time intervals (0,0.10,0.20,0.30,0.45, 1,2,4,6,8,10, 12 and 24 hours) and it is analyzed by UV spectroscopy at 280 and 200 nm.
- the proteins (growth factors) present in the receptor sample 1 absorb ultraviolet light with absorbance maxima at 280 and 200 nm. These parameters is compared with marketed and placebo formulations. Table 17: Physicochemical evaluation of formulations
- the final formulation is packed and labeled in a suitable container meant for marketing and kept for stability under various conditions as per suitable ICH guidelines. At certain decided interval referred as stability time point samples are withdrawn and all the physicochemical evaluation (PCE) is performed.
- PCE physicochemical evaluation
- Emulsifier, Solubiliser and Wetting agent Emulsifier, Solubiliser and Wetting agent
- Viscosity is the measure of fluid friction which can be considered as the internal friction resulting when a layer of fluid is made to move in relationship to another layer. It is the measure of the ratio of shearing stress to rate of shear.
- the viscosity of the given formulation 1 and 3 was tested using a Brookfield Viscometer. The Brookfield Viscometer measures viscosity by measuring the force required to rotate a spindle in a fluid. Spreadability of the given formulation was tested against standard weight applied on the sample glass plate and measuring the area of sample traversed.
- the present study is conducted to evaluate the anti-wrinkling efficacy of the test formulations against pathophysiological changes induced by UVB irradiations in nude mice.
- 6 to 8 weeks old nude mice are selected. All animals except non-irradiated group (Gl) are irradiated with UVB up to 7 days. The dose of irradiation is maintained at 150 mJ/cm 2 .
- test formulations [as described below] and vehicles (placebo serum formula 1 and placebo serum formula 2) are topically administered to the entire dorsal back of each animal in respective groups.
- UVB exposure leads to pathophysiological changes in animal skin, all the animals are closely observed for appearance of any wrinkles; roughness of skin; loss of water (moisture) and erythema (redness).
- Hydration of the skin and erythema index (E.I.) are measured using corneometer and mexameter respectively each day before UVB exposure. A comparison is made between day 1 and 8 for the mean corneometer and mexameter units of test formulation and it is revealed that there is low extent of decrease in skin moisture in groups G5 (21.80 %) and G6 (23.86 %) as compared to day 1.
- Formulation 1 applied at dose of IX concentration (i.e. 10% of the 10X CM) in G5 and formulation 3 applied at dose of 3X (i.e. 30% of the 10X CM) concentration in G6 could substantially prevent UVB induced moisture loss from the skin.
- formulation 1 IX concentration treated group G5 (2.26 %) and formulation 3 (3X concentration) treated group G6 (2.73 %) as compared to day 1.
- Formulation 1 applied at dose of IX concentration in G5 and formulation 3 applied at dose of 3X concentration in G6 can substantially prevent UVB induced erythema in the skin.
- test formulations were evaluated in nude mice. The degree of protection was assessed using macroscopic and microscopic parameters. Photographic images were recorded to capture the effect of UV-B and the anti -wrinkling potential of the formulations 1 and 3. The camera was clamped on a stand at a fixed distance from the animals and photography was done under aseptic conditions. In this study, the anti-wrinkling efficacy of the formulations was evaluated against pathophysiological changes induced by UV-B irradiations in nude mice. Six to eight weeks old nude mice were selected. All animals except non-irradiated group (control group) were irradiated with UV-B up to 7 days.
- Table 22 Mean Corneometer reading indicative (% change) of skin hydration levels.
- the comeometer reading indicative of skin hydration levels is considered at the end of 8th day which is after 7 days of UVB irradiation according to the experimental design.
- the graphical representation it is observed that there is a gradual decrease in skin hydration levels in all the samples including UVB treated, and placebo samples whereas the skin hydration level is retained to 76-78% at the end of 8th day.
- Table 23 Mean mexameter readings indicative of erythema index.
- the mexameter reading indicative of erythema index is considered at the end of 8th day which is after 7 days of UVB irradiation according to the experimental design.
- the erythema index obtained upon treatment with formulations remains constant to the order of 270 similar to the untreated control.
- the invention addresses the variation in the levels of bioactive factors produced/secreted by the mesenchymal stem cells from individual donors as compared to that of pooled donors. Certain bioactive factors that are not produced or produced at a very low amount by individual donors are secreted at higher levels in conditioned media obtained from by pooling of mesenchymal stem cells from multiple donors. Hence cytokines and growth factors secreted by pooled donors minimize the heterogeneity seen in conditioned media obtained from individual donors.
- the pooling reduced the individual variability of the BMMSC samples.
- TGF-beta is produced by only one donor (donor 1), and not produced by donors 2 and 3. Therefore TGF- beta is absent in the conditioned media from donors 2 or 3.
- pooled condition medium contains TFG-beta. Hence, pooling minimizes or eliminates individual variation.
- conditioned medium having functional VEGF, at a concentration to carry out the required angiogenesis.
- pooling is done to compensate biological factors not secreted by one of the selected individual being pooled. The present method of obtaining conditioned medium is found to show higher levels of cytokines and growth factor expression necessary for biological/medicinal applications.
- the CM based formulation can be used for various cosmetic and therapeutic purposes depending on the expression and secretion of cytokines/GF by screening. • The expression and secretion of specific type of cytokines and GF are used for specific indications. For example, FGF and HGF are important in anti-scarring properties. ⁇ The conditioned medium of the present disclosure can also be employed in the form of wet wipes, cosmetic patches and hydrogels.
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Abstract
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Priority Applications (12)
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EP14752380.7A EP3039124A1 (en) | 2013-08-29 | 2014-06-18 | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
MX2016002084A MX2016002084A (en) | 2013-08-29 | 2014-06-18 | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof. |
BR112016002040A BR112016002040A2 (en) | 2013-08-29 | 2014-06-18 | stromal cell derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
AU2014313874A AU2014313874A1 (en) | 2013-08-29 | 2014-06-18 | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
CN201480046745.XA CN105473709A (en) | 2013-08-29 | 2014-06-18 | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
SG11201600219TA SG11201600219TA (en) | 2013-08-29 | 2014-06-18 | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
MYPI2016000340A MY186324A (en) | 2013-08-29 | 2014-06-18 | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
JP2016537403A JP2016528911A (en) | 2013-08-29 | 2014-06-18 | Conditioned medium derived from stromal cells, method, preparation and application for obtaining said conditioned medium composition |
US14/912,306 US20160206550A1 (en) | 2013-08-29 | 2014-06-18 | Stromal dells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
IL243530A IL243530A0 (en) | 2013-08-29 | 2016-01-10 | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
PH12016500252A PH12016500252A1 (en) | 2013-08-29 | 2016-02-05 | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
HK16105530.8A HK1217514A1 (en) | 2013-08-29 | 2016-05-13 | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
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US (1) | US20160206550A1 (en) |
EP (1) | EP3039124A1 (en) |
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CN (1) | CN105473709A (en) |
AU (1) | AU2014313874A1 (en) |
BR (1) | BR112016002040A2 (en) |
HK (1) | HK1217514A1 (en) |
IL (1) | IL243530A0 (en) |
MX (1) | MX2016002084A (en) |
MY (1) | MY186324A (en) |
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CN116410921A (en) * | 2023-02-09 | 2023-07-11 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
CN116410921B (en) * | 2023-02-09 | 2024-01-23 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
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HK1217514A1 (en) | 2017-01-13 |
AU2014313874A1 (en) | 2016-02-25 |
BR112016002040A2 (en) | 2017-08-01 |
PH12016500252B1 (en) | 2016-05-16 |
JP2016528911A (en) | 2016-09-23 |
MY186324A (en) | 2021-07-09 |
PH12016500252A1 (en) | 2016-05-16 |
SG11201600219TA (en) | 2016-02-26 |
MX2016002084A (en) | 2016-06-23 |
US20160206550A1 (en) | 2016-07-21 |
CN105473709A (en) | 2016-04-06 |
IL243530A0 (en) | 2016-03-31 |
EP3039124A1 (en) | 2016-07-06 |
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