US20190018000A1 - A method of non destructive monitoring of biological processes in microfluidic tissue culture systems - Google Patents
A method of non destructive monitoring of biological processes in microfluidic tissue culture systems Download PDFInfo
- Publication number
- US20190018000A1 US20190018000A1 US16/069,361 US201716069361A US2019018000A1 US 20190018000 A1 US20190018000 A1 US 20190018000A1 US 201716069361 A US201716069361 A US 201716069361A US 2019018000 A1 US2019018000 A1 US 2019018000A1
- Authority
- US
- United States
- Prior art keywords
- stem cells
- microfluidic device
- imaging
- cells
- detecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 77
- 230000001066 destructive effect Effects 0.000 title abstract description 9
- 230000031018 biological processes and functions Effects 0.000 title abstract description 6
- 238000012544 monitoring process Methods 0.000 title description 11
- 210000004027 cell Anatomy 0.000 claims description 59
- 239000003795 chemical substances by application Substances 0.000 claims description 47
- 238000002372 labelling Methods 0.000 claims description 47
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 36
- 238000001208 nuclear magnetic resonance pulse sequence Methods 0.000 claims description 28
- 210000004409 osteocyte Anatomy 0.000 claims description 27
- 210000000130 stem cell Anatomy 0.000 claims description 27
- 210000000963 osteoblast Anatomy 0.000 claims description 22
- 230000005415 magnetization Effects 0.000 claims description 20
- 229920002683 Glycosaminoglycan Polymers 0.000 claims description 19
- 230000033558 biomineral tissue development Effects 0.000 claims description 18
- 238000012546 transfer Methods 0.000 claims description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 229940122361 Bisphosphonate Drugs 0.000 claims description 14
- 150000004663 bisphosphonates Chemical class 0.000 claims description 13
- 230000001413 cellular effect Effects 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 13
- 239000000126 substance Substances 0.000 claims description 13
- 229920001436 collagen Polymers 0.000 claims description 12
- 239000012216 imaging agent Substances 0.000 claims description 11
- TYPGYRNAXWQZSC-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-10-[2-(diphosphonoamino)-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]acetic acid Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(=O)N(P(O)(O)=O)P(O)(O)=O)CCN(CC(O)=O)CC1 TYPGYRNAXWQZSC-UHFFFAOYSA-N 0.000 claims description 10
- 102000008186 Collagen Human genes 0.000 claims description 10
- 108010035532 Collagen Proteins 0.000 claims description 10
- 210000001612 chondrocyte Anatomy 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 10
- 210000002950 fibroblast Anatomy 0.000 claims description 10
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 10
- 229920002521 macromolecule Polymers 0.000 claims description 10
- 210000001074 muscle attachment cell Anatomy 0.000 claims description 10
- 238000001139 pH measurement Methods 0.000 claims description 10
- 239000007850 fluorescent dye Substances 0.000 claims description 9
- 239000013068 control sample Substances 0.000 claims description 8
- 238000002603 single-photon emission computed tomography Methods 0.000 claims description 6
- GKLVYJBZJHMRIY-OUBTZVSYSA-N Technetium-99 Chemical compound [99Tc] GKLVYJBZJHMRIY-OUBTZVSYSA-N 0.000 claims description 5
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 claims description 5
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims description 5
- KRHYYFGTRYWZRS-BJUDXGSMSA-M fluorine-18(1-) Chemical compound [18F-] KRHYYFGTRYWZRS-BJUDXGSMSA-M 0.000 claims description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 5
- WRUUGTRCQOWXEG-UHFFFAOYSA-N pamidronate Chemical group NCCC(O)(P(O)(O)=O)P(O)(O)=O WRUUGTRCQOWXEG-UHFFFAOYSA-N 0.000 claims description 5
- -1 phosphonomethyl Chemical group 0.000 claims description 5
- 229940056501 technetium 99m Drugs 0.000 claims description 5
- 238000012879 PET imaging Methods 0.000 claims description 4
- 210000001778 pluripotent stem cell Anatomy 0.000 claims description 4
- 238000003384 imaging method Methods 0.000 abstract description 52
- 108090000623 proteins and genes Proteins 0.000 abstract description 14
- 238000003306 harvesting Methods 0.000 abstract description 10
- 102000004169 proteins and genes Human genes 0.000 abstract description 10
- 230000014509 gene expression Effects 0.000 abstract description 8
- 238000000338 in vitro Methods 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 3
- 230000028327 secretion Effects 0.000 abstract description 3
- 230000009772 tissue formation Effects 0.000 abstract description 2
- 210000000988 bone and bone Anatomy 0.000 description 34
- 210000001519 tissue Anatomy 0.000 description 25
- 239000000523 sample Substances 0.000 description 20
- 238000013459 approach Methods 0.000 description 13
- 230000009818 osteogenic differentiation Effects 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 238000005259 measurement Methods 0.000 description 11
- 238000002591 computed tomography Methods 0.000 description 9
- 238000012632 fluorescent imaging Methods 0.000 description 9
- 210000004940 nucleus Anatomy 0.000 description 9
- 238000004624 confocal microscopy Methods 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 8
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 8
- 238000010859 live-cell imaging Methods 0.000 description 8
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 8
- 210000000845 cartilage Anatomy 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 230000008901 benefit Effects 0.000 description 6
- 238000010603 microCT Methods 0.000 description 6
- 230000002188 osteogenic effect Effects 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 210000002449 bone cell Anatomy 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 229910052500 inorganic mineral Inorganic materials 0.000 description 5
- 239000011707 mineral Substances 0.000 description 5
- 235000010755 mineral Nutrition 0.000 description 5
- 230000003068 static effect Effects 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 102000016921 Integrin-Binding Sialoprotein Human genes 0.000 description 4
- 108010028750 Integrin-Binding Sialoprotein Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 4
- 210000003041 ligament Anatomy 0.000 description 4
- 238000002595 magnetic resonance imaging Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000002123 temporal effect Effects 0.000 description 4
- 210000002435 tendon Anatomy 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 3
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000002600 positron emission tomography Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 2
- 208000004434 Calcinosis Diseases 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 101000762366 Homo sapiens Bone morphogenetic protein 2 Proteins 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 102000004067 Osteocalcin Human genes 0.000 description 2
- 108090000573 Osteocalcin Proteins 0.000 description 2
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 210000001789 adipocyte Anatomy 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 230000010072 bone remodeling Effects 0.000 description 2
- 230000024279 bone resorption Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 2
- 229960000367 inositol Drugs 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 229940102859 methylene diphosphonate Drugs 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000013425 morphometry Methods 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000000079 presaturation Methods 0.000 description 2
- 238000000163 radioactive labelling Methods 0.000 description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000016284 Aggrecans Human genes 0.000 description 1
- 108010067219 Aggrecans Proteins 0.000 description 1
- 241001436672 Bhatia Species 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- HWZUDASOMGNLSM-UHFFFAOYSA-N O=P1OCOP(=O)O1 Chemical compound O=P1OCOP(=O)O1 HWZUDASOMGNLSM-UHFFFAOYSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 108010009711 Phalloidine Proteins 0.000 description 1
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical class OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 1
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- 102000019307 Sclerostin Human genes 0.000 description 1
- 108050006698 Sclerostin Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001408 amides Chemical group 0.000 description 1
- 229910052586 apatite Inorganic materials 0.000 description 1
- 210000001188 articular cartilage Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000010256 biochemical assay Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000004221 bone function Effects 0.000 description 1
- 230000008468 bone growth Effects 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 238000007469 bone scintigraphy Methods 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000006364 cellular survival Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000003633 gene expression assay Methods 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000005067 joint tissue Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007762 localization of cell Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000011738 major mineral Substances 0.000 description 1
- 235000011963 major mineral Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 230000001089 mineralizing effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000003562 morphometric effect Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 238000002311 multiphoton fluorescence microscopy Methods 0.000 description 1
- 238000003333 near-infrared imaging Methods 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 238000012634 optical imaging Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 230000001582 osteoblastic effect Effects 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 230000001599 osteoclastic effect Effects 0.000 description 1
- 210000005009 osteogenic cell Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 description 1
- 231100000760 phototoxic Toxicity 0.000 description 1
- 208000007578 phototoxic dermatitis Diseases 0.000 description 1
- 231100000018 phototoxicity Toxicity 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000026447 protein localization Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000014493 regulation of gene expression Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000005469 synchrotron radiation Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- XRASPMIURGNCCH-UHFFFAOYSA-N zoledronic acid Chemical compound OP(=O)(O)C(P(O)(O)=O)(O)CN1C=CN=C1 XRASPMIURGNCCH-UHFFFAOYSA-N 0.000 description 1
- 229960004276 zoledronic acid Drugs 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0619—Neurons
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5038—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving detection of metabolites per se
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
- C12N5/0075—General culture methods using substrates using microcarriers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01T—MEASUREMENT OF NUCLEAR OR X-RADIATION
- G01T1/00—Measuring X-radiation, gamma radiation, corpuscular radiation, or cosmic radiation
- G01T1/16—Measuring radiation intensity
- G01T1/161—Applications in the field of nuclear medicine, e.g. in vivo counting
- G01T1/164—Scintigraphy
- G01T1/1641—Static instruments for imaging the distribution of radioactivity in one or two dimensions using one or several scintillating elements; Radio-isotope cameras
- G01T1/1644—Static instruments for imaging the distribution of radioactivity in one or two dimensions using one or several scintillating elements; Radio-isotope cameras using an array of optically separate scintillation elements permitting direct location of scintillations
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01T—MEASUREMENT OF NUCLEAR OR X-RADIATION
- G01T1/00—Measuring X-radiation, gamma radiation, corpuscular radiation, or cosmic radiation
- G01T1/29—Measurement performed on radiation beams, e.g. position or section of the beam; Measurement of spatial distribution of radiation
- G01T1/2914—Measurement of spatial distribution of radiation
- G01T1/2985—In depth localisation, e.g. using positron emitters; Tomographic imaging (longitudinal and transverse section imaging; apparatus for radiation diagnosis sequentially in different planes, steroscopic radiation diagnosis)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2205/00—General characteristics of the apparatus
- A61M2205/33—Controlling, regulating or measuring
- A61M2205/3331—Pressure; Flow
- A61M2205/3334—Measuring or controlling the flow rate
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
Definitions
- Described herein are methods and compositions related to imaging of stem cells and cells undergoing differentiation without sample manipulation.
- Bones consisting of mineralized bone tissue also consists of bone marrow, nerves and blood vessels. Development and homeostasis of bone relies heavily on communication between cells in the tissues as regulated by the bone environment. Bone is an active tissue maintained by bone cells such as osteoblasts that form bone and osteoclasts that resorb bone, and it is now understood that mesenchymal stem cells (MSCs) can differentiate to various skeletal cells including osteoblasts, chondrocytes, fibroblasts, adipocytes, tenocytes, nucleus pulposus cells and more. Additionally, within the collagen and mineral matrix osteocytes are also embedded and respond to the bone environment. The balance between these cells is necessary to maintain bone function.
- MSCs mesenchymal stem cells
- Paraffin tissue slices are a long standing conventional approach to evaluate microarchitecture and bone morphology.
- sample manipulation leads to changes in biochemical properties of antigenicity and mineral structure.
- Newer strategies to image cells within the bone such as MM, Micro-CT or Ultrasound can image bone structure and recently cells, however these techniques are limited by their low resolution at the cellular level given the surrounding physiological environment.
- Recent “organ-on-a-chip” technologies represent new and exciting opportunities for bone research.
- These devices include a microfluidic cell culture apparatus that is a more physiologically relevant in vitro model than cells cultured in dishes.
- providing for continuously perfused chambers inhabited by living cells arranged to simulate tissue- and organ-level physiology allow for the culturing of bone cells in a format mirroring their physiological environment. By studying bone cell function and response in this manner, a 3D environment can reveal completely different cellular dynamics compared to 2D cultures.
- the availability of cellular tissue material in this format further provides new avenues for apply imaging approaches of bone microarchitecture to identify features previously unavailable in tissue cultures or at insufficient resolution in vivo.
- Described herein is the use of non-destructive molecular imaging methods and systems in order to quantitatively monitor specific biological processes, over time, within the chip, without the need to harvest the tissue. Such methods can provide valuable data on developing tissues and their response to pharmaceutical, chemical and environmental agents.
- a method of detecting cellular mineralization in a microfluidic device including providing a microfluidic device including mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes, adding one or more labeling agents to the microfluidic device, and detecting the labeling agent, wherein the labeling agent is capable of binding to cellular mineralization.
- the microfluidic device further includes one or more channels for loading of a control sample.
- the one or more labeling agents comprise bisphosphonate imaging agents.
- the bisphosphonate imaging agent includes a pamidronate backbone with a fluorescent label.
- the one or more labeling agents comprise a radiolabel.
- the radiolabel includes technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled 4- ⁇ [(bis(phosphonomethyl))carbamoyl]methyl ⁇ -7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD).
- detecting the labeling agent includes Micro CT, Micro SPECT, and/or PET imaging.
- detecting the labeling agent further includes comparison of the quantity of detected labeling agent with one or more control samples. In other embodiments, further culturing of MSCs, osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells in the microfluidic device. In other embodiments, the method includes further detection of the labeling agent.
- the stem cells are mesenchymal stem cells (MSCs).
- the stem cells are pluripotent stem cells (pSCs).
- the stem cells are induced pluripotent stem cells (iPSCs).
- detecting the pulse sequence signal intensity includes chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX).
- CEST detects a quantity of glycosaminoglycans (GAGs).
- pH measurement of T1 rho detects a quantity of GAGs.
- MTC detects a quantity of collagen.
- MEX detects a quantity of collagen and/or osteoid.
- the microfluidic device further includes one or more channels for loading of a control sample.
- detecting the pulse sequence signal intensity further includes comparison of the quantity of detected pulse sequence signal intensity with one or more control samples.
- the method includes further culturing of stem cells in the microfluidic device.
- the method includes further detection of pulse sequence signal intensity.
- FIG. 1 Organ-on-chip dimensions and setting of the flow system. The flow was set to 30 ⁇ l/h and the media in the reservoirs was replaced or refilled twice a week.
- FIG. 2 Cell survival and proliferation on the organ-on-chip.
- FIG. 2A Micrographs of the cells on the chip grown for 3 weeks in osteogenic conditions.
- FIG. 2B bioluminescent imaging (BLI) images taken on Day 0.
- FIG. 3 Osteogenic differentiation at Week 3 measured with two probes (OsteoSense and BoneTag) using two different imaging systems: fluorescent imaging system (IVIS, Perkin Elmer) and Near Infrared imaging system (Odyssey® CLx, Li-Cor).
- FIG. 3A FLI images of chips incubated with BoneTag and in FIG. 3B the labeling was quantified
- FIG. 3C using fluorescent imaging IVIS Live staining where live cells arte stained with FITC (green) dye and OsteoSense is depicted in red and imaged using confocal microscopy, 10 ⁇ magnification.
- FIG. 4 Immunostaining of osteogenic differentiation of MSC-BMP2 in Organ-on-chip. The chips were sectioned using vibratome across the channels. The sections were stained using with immunofluorescent staining against the osteogenic markers osteocalcin and bone sialoprotein (BSP), and imaged using confocal microscopy.
- BSP bone sialoprotein
- microfluidic devices have been developed with the aim to replicate human tissues in vitro. These systems, also called microfluidic chips or “organ-on-a-chip”, have the potential to serve as an alternative for animal models that are used to test pharmaceutical, chemical and environmental agents.
- the microfluidic chips are attractive for biomedical research and drug discovery due to low cost and ethical considerations compared to animal models.
- Bhatia and Ingber “Microfluidic organs-on-chips.” Nat Biotechnol. 2014 August; 32(8):760-72, which is fully incorporated by reference herein.
- MSCs Mesenchymal stem cells
- skeletal cells including osteoblasts, chondrocytes, fibroblasts, adipocytes, tenocytes, nucleus pulposus cells and more.
- In situ imaging both in non-living and living specimens, have provided new insights, but for the above described reasons, quantitative experimental data requires destructive processing that may introduce bias, and lack temporal and spatial resolution.
- microfluidic organ-on-a-chip coupled with non-destructive labeling and imaging techniques may allow precise capture of MSC, osteoblast and osteocyte cell populations in micro and ultrastructure in 2D and 3D.
- Live cell imaging techniques which are able to track structural morphology and cellular differentiation in both space and time combined with the latest biochemical assays and microfluidic imaging techniques can provide further insight on the biological function of MSC, MSCs, osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells.
- fluorescent staining agents are used in conjunction with modern confocal laser scanning microscopy (CLSM), such as rhodamine and fluorescein, which can be incubated with undecalcified bone sections.
- CLSM confocal laser scanning microscopy
- More specific staining agents such as fluorescein isothiocyanate (FITC)-conjugated phalloidin and DAPI, label the actin skeleton and/or DNA of cell nuclei in such a way that the components cells can be directly imaged and separately displayed in 3D
- CLSM computed tomography
- image artifacts such as signal attenuation with increasing focal plane depth or aberrations due to refractive index mismatch.
- artifacts are absent in (conventional) X-ray absorption-based computed tomography (CT).
- CT computed tomography
- ⁇ CT Micro-computed CT
- 3D morphometric measures to quantify trabecular microarchitecture have laid the foundations for ⁇ CT to become a standard for bone morphometry.
- the standard application of desktop ⁇ CT systems with typical voxel sizes in the order of 5-100 ⁇ m is a core approach for quantitative characterization of whole bone geometry.
- Synchrotron radiation-based CT allows for imaging of bone microstructure, canal networks, as well as study of populations such as osteocytes within bone.
- optimized imaging protocol for SR CT provides spatial resolution closer to the diffraction limit of visible light at a few hundred nanometers.
- desktop ⁇ CT scanners with voxel sizes below 1 ⁇ m allow for new opportunities for imaging.
- GFPs green fluorescent proteins
- a large selection of fluorescent probes and reagents are commercially available to the researcher for investigating biological events in living cells, including fluorescent antibodies, kits for fluorescently labeling proteins of interest, dyes for cell and nuclear tracking, probes for labeling of membranes and organelles, fluorescence reagents for determining cell viability, probes for assessing pH and ion flux and probes for monitoring enzyme activity, etc.
- GFP-derived fluorescent protein vectors are available that can either be used as reporter constructs or to generate fusion constructs with a protein of interest. These enable the live monitoring of gene expression and protein localization in vivo, and in real time.
- fluorescent probes may perturb or alter the biology being examined. Validation studies are needed to make sure that the fusion protein still functions similarly to the wild type form. It is also advantageous to confirm findings with more than one type of imaging probe if possible.
- a GFP fusion protein can be used for in vivo localization of a specific protein and key data can be confirmed using a fluorescence-conjugated antibody against the same protein.
- microfluidic imaging approaches which allow for spatial and temporal mapping in three dimensions and quantitative measurement of gene expression cells in an organized “organ-on-a-chip” niche.
- Examples of a “microfluidic imaging” approach can be briefly described by the following workflow: bone formation and/or resorption are spatially mapped and quantified in technologies such as in vivo ⁇ CT and 3D image registration techniques; labeling (e.g., fluorescence, radio labeling) or other techniques, (e.g., chemical exchange saturation transfer (CEST), pH measurement T1rho, magnetization transfer contrast, magnetization exchange or other technologies.
- labeling e.g., fluorescence, radio labeling
- CEST chemical exchange saturation transfer
- pH measurement T1rho pH measurement T1rho
- magnetization transfer contrast magnetization exchange or other technologies.
- the microfluidic device includes mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes.
- the microfluidic device includes cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells among others. It is emphasized that the described methods and techniques find wide applicability to biological tissues.
- the microfluidic device includes stem cells.
- the stem cells are mesenchymal stem cells (MSCs).
- the stem cells are induced pluripotent stem cells (iPSCs).
- the microfluidic device further includes one or more channels for loading of a control sample.
- the properties are biochemical properties of the one or more cells in a microfluidic device.
- the method includes providing a microfluidic device, adding one or more labeling agents to the microfluidic device, and detecting the labeling agent, wherein the labeling agent is capable of binding to one or more biochemical properties of one or more cells in the microfluidic device.
- one or more labeling agents comprise bisphosphonate imaging agents.
- the bisphosphonate imaging agent includes a pamidronate backbone with a fluorescent label.
- the one or more labeling agents comprise a radiolabel.
- the radiolabel includes technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled (4- ⁇ [(bis(phosphonomethyl))carbamoyl]methyl ⁇ -7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD).
- detecting the labeling agent includes Micro CT, Micro SPECT, and/or PET imaging.
- detecting the labeling agent further includes comparison of the quantity of detected labeling agent with one or more control samples.
- the method includes further culturing of MSCs, osteoblasts and/or osteocytes in the microfluidic device. In other embodiments, the method includes further detection of the labeling agent.
- the method includes applying one or more pulse sequences to the microfluidic device, and detecting the pulse sequence signal intensity, wherein the pulse sequence signal intensity is capable of measuring one or more biochemical properties.
- detecting the pulse sequence signal intensity includes chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX).
- CEST detects a quantity of glycosaminoglycans (GAGs).
- pH measurement of T1 rho detects a quantity of GAGs.
- MTC detects a quantity of collagen.
- MEX detects a quantity of collagen and/or osteoid.
- the microfluidic device further includes one or more channels for loading of a control sample.
- detecting the pulse sequence signal intensity further includes comparison of the quantity of detected labeling agent with one or more control samples.
- the method includes further culturing of stem cells in the microfluidic device. In other embodiments, the method includes further culturing of cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells among others. In other embodiments, the method includes further detection of pulse sequence signal intensity. In various embodiments, the method includes detecting cellular mineralization. In other embodiments, the method includes detecting secreted extracellular macromolecules. In various embodiments, the method includes detecting cellular survival, differentiation and/or proliferation.
- a method of detecting cellular mineralization in a microfluidic device including providing a microfluidic device including mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes, adding one or more labeling agents to the microfluidic device, and detecting the labeling agent, wherein the labeling agent is capable of binding to cellular mineralization.
- the microfluidic device further includes one or more channels for loading of a control sample.
- the one or more labeling agents comprise bisphosphonate imaging agents.
- the bisphosphonate imaging agent includes a pamidronate backbone with a fluorescent label.
- the one or more labeling agents comprise a radiolabel.
- the radiolabel includes technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled (4- ⁇ [(bis(phosphonomethyl))carbamoyl]methyl ⁇ -7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD).
- detecting the labeling agent includes Micro CT, Micro SPECT, and/or PET imaging.
- detecting the labeling agent further includes comparison of the quantity of detected labeling agent with one or more control samples.
- the method includes further culturing of MSCs, osteoblasts and/or osteocytes in the microfluidic device.
- the method includes further culturing cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells in the microfluidic device.
- the method includes further detection of the labeling agent.
- detecting secreted extracellular macromolecules in a microfluidic device including providing a microfluidic device including stem cells, applying one or more pulse sequences to the microfluidic device, and detecting the pulse sequence signal intensity, wherein the pulse sequence signal intensity is capable of measuring one or more macromolecules secreted by the stem cells.
- the stem cells are mesenchymal stem cells (MSCs).
- the stem cells are induced pluripotent stem cells (iPSCs).
- detecting the pulse sequence signal intensity includes chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX).
- CEST detects a quantity of glycosaminoglycans (GAGs).
- pH measurement of T1 rho detects a quantity of GAGs.
- MTC detects a quantity of collagen.
- MEX detects a quantity of collagen and/or osteoid.
- the microfluidic device further includes one or more channels for loading of a control sample.
- detecting the pulse sequence signal intensity further includes comparison of the quantity of detected pulse sequence signal intensity with one or more control samples.
- the method includes further culturing of stem cells in the microfluidic device.
- the method includes further culturing cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells in the microfluidic device. In other embodiments, the method includes further detection of pulse sequence signal intensity.
- MSCs Mesenchymal stem cells
- osteoblasts A common assay of MSC differentiation to osteogenic cells includes measurements of mineralization within the culture. Several methods can be used to monitor mineralization over time in chips
- Hydroxyapatite (HA) is a mineral form of calcium apatite and is the major mineral product of osteoblasts. Therefore, HA levels are a good biomarker for osteoblast activity. In addition, abnormal accumulation of HA can be indicative of a disease state.
- OsteoSenseTM imaging agents bind with high affinity to HA. Since hydroxyapatite (HA) is known to bind pyrophosphonates and phosphonates as well as synthetic bisphosphonates with high affinity, OsteoSenseTM agents were designed as bisphosphonate imaging agents.
- These probes consist of a pamidronate backbone functionalized with near-infrared fluorophore off the amino terminus of the R2 side chain.
- OsteoSenseTM imaging agents can be used to image areas of microcalcifications, bone remodeling and enables imaging of bone growth and resorption.
- the bisphosphonate probe attaches to micro calcifications and the fluorescent readout provides quantification of mineralization.
- BPs Bisphosphonates
- MDP methylene diphosphonate
- zoledronic acid can be labeled with technetium-99m ([99mTc]-BPs) for use in bone scintigraphy as has been used to detect osteoporosis and other skeletal-related events (SREs).
- SREs skeletal-related events
- [18F]-Fluoride is another nuclide that is commonly used for bone imaging, and positron emission tomography (PET) and is believed to be superior to [99mTc]-BPs for the diagnosis of SREs.
- Micro SPECT/PET imaging-99mTc-Methyl diphosphonate can be added to the chip at different time points, washed and then the chip is imaged using a micro SPECT scanner.
- Alternative probes are [′8F)-Fluoride or 68Ga-Labeled (4- ⁇ [(bis(phosphonomethyl))carbamoyl]methyl ⁇ -7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) [68Ga]BPAMD that can be imaged using a micro PET scanner.
- BPAMD 68Ga]BPAMD that can be imaged using a micro PET scanner.
- Micro CT high-resolution micro CT scanners can detect mineral particles as small as 500 nm.
- a non-destructive scan of the chip can provide an accurate measurement of mineralization generated by the developing tissues.
- MSCs and induced pluripotent stem cells have been shown to differentiate to joint tissue cells such as osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells cells. While differentiating, the cells secret characteristic extracellular molecules such as aggrecan, glycosaminoglycans (GAGs), collagens and more.
- iPSCs induced pluripotent stem cells
- a way to monitor the secretion of these molecules in a chip will include the use of micro MRI using different pulse sequences, including but not limited to: chemical exchange saturation transfer (CEST)—GAGs measurement; pH measurement T1 rho—GAGs measurement, magnetization transfer contrast (MTC)—collagen measurement, magnetization exchange (MEX)—collagen and osteoid measurement.
- CEST chemical exchange saturation transfer
- MTC magnetization transfer contrast
- MEX magnetization exchange
- CEST Chemical exchange saturation transfer
- the most common method for acquisition of a CEST data set is to acquire multiple image data sets with presaturation at different offset frequencies around the water resonance and one reference data set without saturation or with saturation at a very large offset frequency.
- the normalized signal as a function of the presaturation offset (termed the z-spectrum) can then be used to determine and quantify CEST effects, which are asymmetric with respect to the water resonance (ie, a CEST effect appears either up- or down-field from water and therefore can be extracted from the z-spectrum via analysis of its asymmetry with respect to the water resonance).
- Chemical exchange saturation transfer is a magnetic resonance imaging (MRI) contrast enhancement technique that enables indirect detection of metabolites with exchangeable protons.
- Endogenous metabolites with exchangeable protons including many endogenous proteins with amide protons, glycosaminoglycans (GAG), glycogen, myo-inositol (MI), glutamate (Glu), creatine (Cr) and several others have been identified as potential in vivo endogenous CEST agents. These endogenous CEST agents can be exploited as non-invasive and non-ionizing biomarkers of disease diagnosis and treatment monitoring.
- Magnetization Transfer Contrast is an imaging method that evolved from NMR spectroscopy.
- MTC Magnetization Transfer Contrast
- tissue imaging MTC relies upon the interaction of less mobile protons associated with macromolecules such as proteins and their interactions with protons freely associated with water.
- the premise is that in a system where molecules move and exchange position, whether it be a change in spatial position in asymmetrical molecules or an exchangeable proton between a molecule and water, the magnetization state will also move and be transferred.
- a two pool model can be utilized to illustrate the theory behind MTC MM.
- Conventional MRI detects only the free water pool while the macromolecular pool remains mostly undetected. Both the macromolecular and free water pools are centered around the same frequency but the macromolecular pool is shallower and wider. Saturation is achieved by applying an off-resonance radio frequency (RF) pulse specific to a peak in the macromolecular pool before excitation at the center frequency. The RF pulse saturates the signal from the section leading to ideally no signal at the off-resonance frequency. Since both pools interact this saturation is transferred to the free water pool. While it is not possible to detect the changes in the macromolecular pool directly, it can be assumed that the loss in signal intensity of the free water pool corresponds to the changes in the macromolecular pool.
- RF radio frequency
- MTR Magnetization Transfer Ratio
- Quantitative magnetization transfer (qMT) imaging is MR technique which utilizes a two-pool model of magnetization exchange to acquire information regarding the cartilage macromolecular matrix.
- qMT imaging techniques typically require multiple MT-contrast images with different magnetization preparatory pulses resulting in long scan times which have limited cartilage assessment to ex-vivo specimens.
- Cross-relaxation imaging is a qMT method which can create three-dimensional parametric maps of articular cartilage measuring the fraction of macromolecular bound protons (f), the exchange rate constant between macromolecular bound protons and free water protons (k), and the T2 relaxation time of macromolecular bound protons (T2B) with high resolution and relatively short scan time based upon a limited number of MT-contrast images.
- the parameter f provides an indirect measure of macromolecular content
- the parameters k, and T2B reflect the efficiency of magnetization exchange between macromolecular bound protons and free water protons and the spin diffusion between proton sites in macromolecules respectively which may be influenced by macromolecular organization and ultra-structure
- Microfluidic culture devices are attractive systems to model physiological and pathological conditions of tissues and organs. Although these devices allow fluorescent and light microcopy imaging of cultured cells, one of its current limitations is that various types of analyses require sacrificing of the culture. The Inventors have previously utilized micro imaging systems to monitor stem cell differentiation in ex-vivo 3D tissue constructs.
- the organ-on-chip was coated with ECM crosslinked with UV prior to cell seeding. Then mesenchymal stem cell line overexpressing BMP2 and Luciferase reporter genes were seeded on the coated organ-on-a-chip (see dimensions and the set up for microfluidic studies in FIG. 1 and supplemented with osteogenic media.
- the static cultures were performed using 200 ⁇ l media reservoirs that were changed every other day.
- the flow studies were performed using 30 ⁇ l/h flow of media pulled through using specialized pump ( FIG. 1 ). Micrographs were taken twice a week and survival of the cells was monitored using bioluminescent imaging. The media was changed to media with Luciferin and imagined using IVIS (Perkin Elmer).
- osteogenic differentiation after 3 weeks of culture in osteogenic media was monitored using florescent probes OsteoSense650 and BoneTag800 that were introduced 24 hours before the imaging and were imaged using fluorescent imaging (FLI) and near infrared (NIR) imaging, confocal microscopy and immunostaining
- FLI fluorescent imaging
- NIR near infrared
- FIG. 1A A comparison of chips grown in static culture condition to chips grown under constant flow of media (30 ⁇ l/h) was performed along with evaluation of the effect of the flow on cell survival/proliferation of cells and the extent of osteogenic differentiation.
- the microscopic images ( FIG. 1A ) show proliferation of the cells under the flow conditions, however it is difficult to quantify the extent of proliferation using this method without disrupting the cultures. Therefore, the Inventors used cell that express Luciferase reporter gene and the cell proliferation was quantified using bioluminescent imaging (BLI) twice a week ( FIG. 2B , C). This imaging method allowed monitor the proliferation of the cells without the need to harvest or disrupt the culture and significant advantage to the flow system was observed. Also microfluidic environment had positive effect on osteogenic differentiation, when compared with static cultures.
- the harvested chips were sectioned using vibratome creating transvers sections across the channels. Then these sections were subjected to immunofluorescent staining using primary antibody against Osteocalcin and Bone Sialoprotein (BSP) osteogenic markers.
- BSP Bone Sialoprotein
- Organ-on-chip system allows monitoring of the cell survival and proliferation in vitro using BLI imaging system and monitor the osteogenic differentiation of the cell on the chip in real time, without the need of harvesting the cells and disrupting the culture conditions.
- the Inventors demonstrate that the flow conditions affect both proliferation and the differentiation of the MSCs that overexpress BMP2.
- the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
Abstract
Description
- Described herein are methods and compositions related to imaging of stem cells and cells undergoing differentiation without sample manipulation.
- Bones consisting of mineralized bone tissue also consists of bone marrow, nerves and blood vessels. Development and homeostasis of bone relies heavily on communication between cells in the tissues as regulated by the bone environment. Bone is an active tissue maintained by bone cells such as osteoblasts that form bone and osteoclasts that resorb bone, and it is now understood that mesenchymal stem cells (MSCs) can differentiate to various skeletal cells including osteoblasts, chondrocytes, fibroblasts, adipocytes, tenocytes, nucleus pulposus cells and more. Additionally, within the collagen and mineral matrix osteocytes are also embedded and respond to the bone environment. The balance between these cells is necessary to maintain bone function. Studying bone is a challenging field due to microarchitecture defining the bone environment, which involve the intricately dense structural composition of the bone morphology. Unlike other tissues that can be processed and prepared for experiments, including cultured cell lines, working with bone is difficult. Studying intracellular dynamics of the bone cells embedded within the mineralized tissue has proven to be a challenging task.
- Compounding these challenges related to underlying properties of the cellular material, imaging cells at subcellular level within the bone environment is very difficult. Paraffin tissue slices are a long standing conventional approach to evaluate microarchitecture and bone morphology. However, sample manipulation leads to changes in biochemical properties of antigenicity and mineral structure. Newer strategies to image cells within the bone such as MM, Micro-CT or Ultrasound can image bone structure and recently cells, however these techniques are limited by their low resolution at the cellular level given the surrounding physiological environment.
- Recent “organ-on-a-chip” technologies represent new and exciting opportunities for bone research. These devices include a microfluidic cell culture apparatus that is a more physiologically relevant in vitro model than cells cultured in dishes. Importantly, providing for continuously perfused chambers inhabited by living cells arranged to simulate tissue- and organ-level physiology allow for the culturing of bone cells in a format mirroring their physiological environment. By studying bone cell function and response in this manner, a 3D environment can reveal completely different cellular dynamics compared to 2D cultures. The availability of cellular tissue material in this format further provides new avenues for apply imaging approaches of bone microarchitecture to identify features previously unavailable in tissue cultures or at insufficient resolution in vivo. Real time imaging of the bone marrow niche within bone and fluorescent imaging of cells within the bone marrow niche has reportedly been achieved. Recent advancements in imaging techniques allows for the identification of osteocytes embedded in the bone matrix. However, determining the localization of cell types and protein expression dynamics of single cells within the bone is still very difficult. And more research is needed to identify intracellular protein activities of the cell bodies embedded within mineralized matrix.
- Described herein is the use of non-destructive molecular imaging methods and systems in order to quantitatively monitor specific biological processes, over time, within the chip, without the need to harvest the tissue. Such methods can provide valuable data on developing tissues and their response to pharmaceutical, chemical and environmental agents.
- Described herein is a method of detecting cellular mineralization in a microfluidic device including providing a microfluidic device including mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes, adding one or more labeling agents to the microfluidic device, and detecting the labeling agent, wherein the labeling agent is capable of binding to cellular mineralization. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, the one or more labeling agents comprise bisphosphonate imaging agents. In other embodiments, the bisphosphonate imaging agent includes a pamidronate backbone with a fluorescent label. In other embodiments, the one or more labeling agents comprise a radiolabel. In other embodiments, the radiolabel includes technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled 4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD). In other embodiments, detecting the labeling agent includes Micro CT, Micro SPECT, and/or PET imaging. In other embodiments, detecting the labeling agent further includes comparison of the quantity of detected labeling agent with one or more control samples. In other embodiments, further culturing of MSCs, osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells in the microfluidic device. In other embodiments, the method includes further detection of the labeling agent.
- Also described herein is a method of detecting secreted extracellular macromolecules in a microfluidic device including providing a microfluidic device including stem cells, applying one or more pulse sequences to the microfluidic device; and, detecting the pulse sequence signal intensity, wherein the pulse sequence signal intensity is capable of measuring one or more macromolecules secreted by the stem cells. In other embodiments, the stem cells are mesenchymal stem cells (MSCs). In other embodiments, the stem cells are pluripotent stem cells (pSCs). In other embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In other embodiments, detecting the pulse sequence signal intensity includes chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX). In other embodiments, CEST detects a quantity of glycosaminoglycans (GAGs). In other embodiments, pH measurement of T1 rho detects a quantity of GAGs. In other embodiments, MTC detects a quantity of collagen. In other embodiments, MEX detects a quantity of collagen and/or osteoid. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, detecting the pulse sequence signal intensity further includes comparison of the quantity of detected pulse sequence signal intensity with one or more control samples. In other embodiments, the method includes further culturing of stem cells in the microfluidic device. In other embodiments, the method includes further detection of pulse sequence signal intensity.
-
FIG. 1 . Organ-on-chip dimensions and setting of the flow system. The flow was set to 30 μl/h and the media in the reservoirs was replaced or refilled twice a week. -
FIG. 2 . Cell survival and proliferation on the organ-on-chip.FIG. 2A . Micrographs of the cells on the chip grown for 3 weeks in osteogenic conditions.FIG. 2B . bioluminescent imaging (BLI) images taken onDay 0.FIG. 2C . Quantitative analysis BLI that was done twice a week for 3 weeks. Bars indicate standard deviation, n=5, *p<0.05; ***P<0.001. -
FIG. 3 . Osteogenic differentiation atWeek 3 measured with two probes (OsteoSense and BoneTag) using two different imaging systems: fluorescent imaging system (IVIS, Perkin Elmer) and Near Infrared imaging system (Odyssey® CLx, Li-Cor).FIG. 3A . FLI images of chips incubated with BoneTag and inFIG. 3B the labeling was quantifiedFIG. 3C . using fluorescent imaging IVIS Live staining where live cells arte stained with FITC (green) dye and OsteoSense is depicted in red and imaged using confocal microscopy, 10× magnification. InFIG. 3D , labeling was quantified, bars indicate standard deviation, n=5, *p<0.05; ***P<0.001. -
FIG. 4 . Immunostaining of osteogenic differentiation of MSC-BMP2 in Organ-on-chip. The chips were sectioned using vibratome across the channels. The sections were stained using with immunofluorescent staining against the osteogenic markers osteocalcin and bone sialoprotein (BSP), and imaged using confocal microscopy. - All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and
Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7th ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton, Dictionary of DNA andGenome Technology 3rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies ALaboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089 (1996 December); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7. - One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.
- As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
- In recent years, microfluidic devices have been developed with the aim to replicate human tissues in vitro. These systems, also called microfluidic chips or “organ-on-a-chip”, have the potential to serve as an alternative for animal models that are used to test pharmaceutical, chemical and environmental agents. The microfluidic chips are attractive for biomedical research and drug discovery due to low cost and ethical considerations compared to animal models. A variety of examples are described in Bhatia and Ingber, “Microfluidic organs-on-chips.” Nat Biotechnol. 2014 August; 32(8):760-72, which is fully incorporated by reference herein.
- An important caveat of the “‘chips” is that currently there is no option to quantitatively monitor biological processes that take place within the chip, over time. To date, researchers are using destructive methods in order to analyze tissue formation, gene expression, protein secretion etc. These methods include histology, immunofluorescence or PCR and require the harvest of the “tissue” at a certain time point. The use of non-destructive molecular imaging methods and systems in order to quantitatively monitor specific biological processes, over time, within the chip, without the need to harvest the tissue would be a significant improvement in the field. Such methods can provide valuable data on developing tissues and their response to pharmaceutical, chemical and environmental agents.
- Mesenchymal stem cells (MSCs) can differentiate to various skeletal cells including osteoblasts, chondrocytes, fibroblasts, adipocytes, tenocytes, nucleus pulposus cells and more. In situ imaging, both in non-living and living specimens, have provided new insights, but for the above described reasons, quantitative experimental data requires destructive processing that may introduce bias, and lack temporal and spatial resolution. In this regard, microfluidic organ-on-a-chip coupled with non-destructive labeling and imaging techniques may allow precise capture of MSC, osteoblast and osteocyte cell populations in micro and ultrastructure in 2D and 3D. Live cell imaging techniques which are able to track structural morphology and cellular differentiation in both space and time combined with the latest biochemical assays and microfluidic imaging techniques can provide further insight on the biological function of MSC, MSCs, osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells.
- Existing techniques for imaging of cells in skeletal and other tissues has proved challenging due to the need to develop methodologies for sectioning specimens, labeling or imaging of specimens or to develop protocols for decalcifying specimens to enable conventional sectioning and imaging techniques to be used. Current imaging approaches rely mainly on histological stains combined with conventional light microscopy. Confocal imaging approaches allows for three-dimensional (3D) imaging in situ within the bone environment. In contrast to inherently two-dimensional (2D) imaging techniques such as light microscopy, confocal microscopy stacks optical sections at different focal planes to generate a three-dimensional (3D) representation of the sample. Endogenous (auto)fluorescence of the bone tissue can be used to provide contrast for confocal microscopy measurements. More often, various fluorescent staining agents are used in conjunction with modern confocal laser scanning microscopy (CLSM), such as rhodamine and fluorescein, which can be incubated with undecalcified bone sections. More specific staining agents, such as fluorescein isothiocyanate (FITC)-conjugated phalloidin and DAPI, label the actin skeleton and/or DNA of cell nuclei in such a way that the components cells can be directly imaged and separately displayed in 3D
- However, a major drawback with CLSM is the limited maximum focal plane depth of around 100-150 μm. Additionally, CLSM is tainted with image artifacts, such as signal attenuation with increasing focal plane depth or aberrations due to refractive index mismatch. Such artifacts are absent in (conventional) X-ray absorption-based computed tomography (CT). Micro-computed CT (μCT) and 3D morphometric measures to quantify trabecular microarchitecture have laid the foundations for μCT to become a standard for bone morphometry. In bone research, the standard application of desktop μCT systems with typical voxel sizes in the order of 5-100 μm is a core approach for quantitative characterization of whole bone geometry. Synchrotron radiation-based CT allows for imaging of bone microstructure, canal networks, as well as study of populations such as osteocytes within bone. Most recently, optimized imaging protocol for SR CT provides spatial resolution closer to the diffraction limit of visible light at a few hundred nanometers. The recent availability of desktop μCT scanners with voxel sizes below 1 μm allow for new opportunities for imaging.
- Over the past two decades or so, technologies for imaging of living cells using light and confocal microscopy have advanced at a rapid rate. Coupled with enhanced green fluorescent proteins (GFPs) and a seemingly limitless array of fluorescent imaging probes has made it possible to image almost any intracellular or extracellular structure or protein in living cells and tissues. A large selection of fluorescent probes and reagents are commercially available to the researcher for investigating biological events in living cells, including fluorescent antibodies, kits for fluorescently labeling proteins of interest, dyes for cell and nuclear tracking, probes for labeling of membranes and organelles, fluorescence reagents for determining cell viability, probes for assessing pH and ion flux and probes for monitoring enzyme activity, etc. In addition, a variety of GFP-derived fluorescent protein vectors are available that can either be used as reporter constructs or to generate fusion constructs with a protein of interest. These enable the live monitoring of gene expression and protein localization in vivo, and in real time.
- The traditional approach of collecting “static” images of fixed or post mortem cells and tissues provides a snapshot view of events at a single fixed point in time. However, this inherently overlooks the dynamic aspects of the biology being examined. In contrast, live cell imaging enables the visualization of temporal changes in living specimens and can reveal novel aspects of the biology that may not otherwise have been appreciated. Additionally, the datasets generated from time-lapse imaging are information rich and can be interrogated quantitatively to enable measurement of cellular, subcellular and tissue dynamic events as a function of time
- Although these approaches are leading to exciting discoveries that are advancing our understanding of biological systems, there are several limitations that need to be acknowledged. Firstly, fluorescent probes may perturb or alter the biology being examined. Validation studies are needed to make sure that the fusion protein still functions similarly to the wild type form. It is also advantageous to confirm findings with more than one type of imaging probe if possible. For example, a GFP fusion protein can be used for in vivo localization of a specific protein and key data can be confirmed using a fluorescence-conjugated antibody against the same protein. When developing live cell imaging protocols, there is always a compromise between obtaining a high enough signal-to-noise ratio to enable quantitative measurements and to obtain sufficient image resolution, while at the same time avoiding phototoxic effects to the cells. Therefore, to ensure cell viability, the researcher may have to accept a lower image quality and resolution than would be acceptable for equivalent images of fixed specimens. Nevertheless, technologies such as multiphoton fluorescence microscopy can increase the depth of tissue penetration for live cell imaging applications and reduce phototoxicity by using a longer wavelength light to excite fluorophores. These instruments are becoming more widely used for live imaging applications due to their advantages over conventional widefield and confocal microscopy systems.
- Recently, live cell imaging approaches have been applied to the study of MSCs, osteoblasts and osteocytes. Organ cultures of neonatal calvaria from mice have provided a useful model for imaging the dynamic properties of osteocytes. Another way in which this model can be used for imaging osteocyte dynamics is by using long term cultures of MSCs and osteoblasts. These cells differentiate when cultured under mineralizing conditions to form mineralized nodules in which the transition to the osteocyte-like phenotype can be monitored by fluorescent labeling or radiolabeling. To gain maximum information, imaging of these can be combined with other fluorescent probes, such as alizarin red to monitor mineral deposition. Live cell imaging studies as applied to investigating osteocyte biology are still in their infancy. In addition to revealing the dynamic properties of MSCs, osteoblasts and osteocytes, identifying the underlying intracellular signaling pathways, such as calcium oscillations, monitoring the temporal integration of osteocyte differentiation and mineralization, live imaging studies have considerable potential to address many as yet unresolved questions in osteocyte biology.
- Most importantly, biochemical data characterizing the precise role of MSCs, osteoblasts and osteocytes in bone remodeling remains severely limited. A number of in vivo models have been developed to study their function. Existing technologies typically harvest large osteocyte populations and employ technologies which provide a comprehensive assessment of a large number of genes which are both up-regulated and down-regulated in response mechanical stimulation. For example, to comprehensively assess osteocyte gene expression in a mouse model for load induced bone adaptation, current state-of-the-art approaches extract large populations of osteocytes from loaded bone and perform micro-array-analysis to quantify the expression levels of tens of thousands of different genes. Global gene expression assays derived from in vivo models for bone adaptation have identified a number of candidate genes and revealed potential load regulated pathways. Nevertheless, there are significant limitations when interpreting these data. The harvesting and analysis of large populations of osteocytes reports gene expression averaged over tens of thousands of cells, each of which reside in different micro-environments characterized by different levels of mechanical strain and local osteoblastic/osteoclastic activity. It is therefore possible that key genes and networks are being concealed. Emerging studies investigate local regulation of gene expression in osteocytes by comparing 2D histology sections from loaded bone stained for specific molecular targets (sclerostin) with micro finite element (μFE) models. Whilst informative, these approaches are still very much qualitative and only permit the analysis of one specific molecular target at a time.
- Addressing these limitations are microfluidic imaging approaches which allow for spatial and temporal mapping in three dimensions and quantitative measurement of gene expression cells in an organized “organ-on-a-chip” niche. Examples of a “microfluidic imaging” approach can be briefly described by the following workflow: bone formation and/or resorption are spatially mapped and quantified in technologies such as in vivo μCT and 3D image registration techniques; labeling (e.g., fluorescence, radio labeling) or other techniques, (e.g., chemical exchange saturation transfer (CEST), pH measurement T1rho, magnetization transfer contrast, magnetization exchange or other technologies. The vast amount of data generated using these approaches can be used to build, feed and validate computational models of various skeletal and other tissues, which incorporate all of the different length scales, from the organ-level to the cellular-level. Further examples include those described in Trussel et al., “Toward mechanical systems biology in bone.” Ann Biomed Eng. 2012 November; 40(11):2475-87.
- Described herein is a method of detecting properties of one of more cells in a microfluidic device. In other embodiments, the microfluidic device includes mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes. In other embodiments, the microfluidic device includes cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells among others. It is emphasized that the described methods and techniques find wide applicability to biological tissues. In other embodiments, the microfluidic device includes stem cells. In other embodiments, the stem cells are mesenchymal stem cells (MSCs). In other embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In various embodiments, the properties are biochemical properties of the one or more cells in a microfluidic device.
- In various embodiments, the method includes providing a microfluidic device, adding one or more labeling agents to the microfluidic device, and detecting the labeling agent, wherein the labeling agent is capable of binding to one or more biochemical properties of one or more cells in the microfluidic device. In other embodiments, one or more labeling agents comprise bisphosphonate imaging agents. In other embodiments, the bisphosphonate imaging agent includes a pamidronate backbone with a fluorescent label. In other embodiments, the one or more labeling agents comprise a radiolabel. In other embodiments, the radiolabel includes technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD). In other embodiments, detecting the labeling agent includes Micro CT, Micro SPECT, and/or PET imaging. In other embodiments, detecting the labeling agent further includes comparison of the quantity of detected labeling agent with one or more control samples. In other embodiments, the method includes further culturing of MSCs, osteoblasts and/or osteocytes in the microfluidic device. In other embodiments, the method includes further detection of the labeling agent.
- In various embodiments, the method includes applying one or more pulse sequences to the microfluidic device, and detecting the pulse sequence signal intensity, wherein the pulse sequence signal intensity is capable of measuring one or more biochemical properties.
- In other embodiments, detecting the pulse sequence signal intensity includes chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX). In other embodiments, CEST detects a quantity of glycosaminoglycans (GAGs). In other embodiments, pH measurement of T1 rho detects a quantity of GAGs. In other embodiments, MTC detects a quantity of collagen. In other embodiments, MEX detects a quantity of collagen and/or osteoid. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, detecting the pulse sequence signal intensity further includes comparison of the quantity of detected labeling agent with one or more control samples. In other embodiments, the method includes further culturing of stem cells in the microfluidic device. In other embodiments, the method includes further culturing of cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells among others. In other embodiments, the method includes further detection of pulse sequence signal intensity. In various embodiments, the method includes detecting cellular mineralization. In other embodiments, the method includes detecting secreted extracellular macromolecules. In various embodiments, the method includes detecting cellular survival, differentiation and/or proliferation.
- Described herein is a method of detecting cellular mineralization in a microfluidic device including providing a microfluidic device including mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes, adding one or more labeling agents to the microfluidic device, and detecting the labeling agent, wherein the labeling agent is capable of binding to cellular mineralization. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, the one or more labeling agents comprise bisphosphonate imaging agents. In other embodiments, the bisphosphonate imaging agent includes a pamidronate backbone with a fluorescent label. In other embodiments, the one or more labeling agents comprise a radiolabel. In other embodiments, the radiolabel includes technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD). In other embodiments, detecting the labeling agent includes Micro CT, Micro SPECT, and/or PET imaging. In other embodiments, detecting the labeling agent further includes comparison of the quantity of detected labeling agent with one or more control samples. In other embodiments, the method includes further culturing of MSCs, osteoblasts and/or osteocytes in the microfluidic device. In other embodiments, the method includes further culturing cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells in the microfluidic device. In other embodiments, the method includes further detection of the labeling agent.
- Also described herein is method of detecting secreted extracellular macromolecules in a microfluidic device including providing a microfluidic device including stem cells, applying one or more pulse sequences to the microfluidic device, and detecting the pulse sequence signal intensity, wherein the pulse sequence signal intensity is capable of measuring one or more macromolecules secreted by the stem cells. In other embodiments, the stem cells are mesenchymal stem cells (MSCs). In other embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In other embodiments, detecting the pulse sequence signal intensity includes chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX). In other embodiments, CEST detects a quantity of glycosaminoglycans (GAGs). In other embodiments, pH measurement of T1 rho detects a quantity of GAGs. In other embodiments, MTC detects a quantity of collagen. In other embodiments, MEX detects a quantity of collagen and/or osteoid. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, detecting the pulse sequence signal intensity further includes comparison of the quantity of detected pulse sequence signal intensity with one or more control samples. In other embodiments, the method includes further culturing of stem cells in the microfluidic device. In other embodiments, the method includes further culturing cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells in the microfluidic device. In other embodiments, the method includes further detection of pulse sequence signal intensity.
- Mesenchymal stem cells (MSCs) can differentiate to various skeletal cells including osteoblasts. A common assay of MSC differentiation to osteogenic cells includes measurements of mineralization within the culture. Several methods can be used to monitor mineralization over time in chips
- Fluorescence imaging—bisphosphonate imaging probes such as OsteoSense™ (Perkin Elmer) can be added to the chip at different time points, washed and then the chip is imaged in an optical scanner. Hydroxyapatite (HA) is a mineral form of calcium apatite and is the major mineral product of osteoblasts. Therefore, HA levels are a good biomarker for osteoblast activity. In addition, abnormal accumulation of HA can be indicative of a disease state. OsteoSense™ imaging agents bind with high affinity to HA. Since hydroxyapatite (HA) is known to bind pyrophosphonates and phosphonates as well as synthetic bisphosphonates with high affinity, OsteoSense™ agents were designed as bisphosphonate imaging agents. These probes consist of a pamidronate backbone functionalized with near-infrared fluorophore off the amino terminus of the R2 side chain. Specifically, OsteoSense™ imaging agents can be used to image areas of microcalcifications, bone remodeling and enables imaging of bone growth and resorption. The bisphosphonate probe attaches to micro calcifications and the fluorescent readout provides quantification of mineralization.
- Bisphosphonates (BPs; also known as diphosphonates), such as methylene diphosphonate (MDP) and zoledronic acid, can be labeled with technetium-99m ([99mTc]-BPs) for use in bone scintigraphy as has been used to detect osteoporosis and other skeletal-related events (SREs). These chemicals bind hydroxyapatite, which allow for imaging of bisphosphonates as described above. [18F]-Fluoride is another nuclide that is commonly used for bone imaging, and positron emission tomography (PET) and is believed to be superior to [99mTc]-BPs for the diagnosis of SREs.
- Micro SPECT/PET imaging-99mTc-Methyl diphosphonate (Tc-MDP) can be added to the chip at different time points, washed and then the chip is imaged using a micro SPECT scanner. Alternative probes are [′8F)-Fluoride or 68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) [68Ga]BPAMD that can be imaged using a micro PET scanner. These probes also attach to mineralization foci and the uptake readouts can provide quantitative data of mineralization.
- Micro CT—high-resolution micro CT scanners can detect mineral particles as small as 500 nm. A non-destructive scan of the chip can provide an accurate measurement of mineralization generated by the developing tissues.
- Different types of stem cells including MSCs and induced pluripotent stem cells (iPSCs) have been shown to differentiate to joint tissue cells such as osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells cells. While differentiating, the cells secret characteristic extracellular molecules such as aggrecan, glycosaminoglycans (GAGs), collagens and more.
- A way to monitor the secretion of these molecules in a chip will include the use of micro MRI using different pulse sequences, including but not limited to: chemical exchange saturation transfer (CEST)—GAGs measurement; pH measurement T1 rho—GAGs measurement, magnetization transfer contrast (MTC)—collagen measurement, magnetization exchange (MEX)—collagen and osteoid measurement.
- Chemical exchange saturation transfer (CEST) also provides the ability to analyze the GAG content in cartilage. The most common method for acquisition of a CEST data set is to acquire multiple image data sets with presaturation at different offset frequencies around the water resonance and one reference data set without saturation or with saturation at a very large offset frequency. The normalized signal as a function of the presaturation offset (termed the z-spectrum) can then be used to determine and quantify CEST effects, which are asymmetric with respect to the water resonance (ie, a CEST effect appears either up- or down-field from water and therefore can be extracted from the z-spectrum via analysis of its asymmetry with respect to the water resonance).
- Chemical exchange saturation transfer (CEST) is a magnetic resonance imaging (MRI) contrast enhancement technique that enables indirect detection of metabolites with exchangeable protons. Endogenous metabolites with exchangeable protons including many endogenous proteins with amide protons, glycosaminoglycans (GAG), glycogen, myo-inositol (MI), glutamate (Glu), creatine (Cr) and several others have been identified as potential in vivo endogenous CEST agents. These endogenous CEST agents can be exploited as non-invasive and non-ionizing biomarkers of disease diagnosis and treatment monitoring.
- Magnetization Transfer Contrast (MTC) MRI is an imaging method that evolved from NMR spectroscopy. In tissue imaging, MTC relies upon the interaction of less mobile protons associated with macromolecules such as proteins and their interactions with protons freely associated with water. The premise is that in a system where molecules move and exchange position, whether it be a change in spatial position in asymmetrical molecules or an exchangeable proton between a molecule and water, the magnetization state will also move and be transferred.
- A two pool model can be utilized to illustrate the theory behind MTC MM. Conventional MRI detects only the free water pool while the macromolecular pool remains mostly undetected. Both the macromolecular and free water pools are centered around the same frequency but the macromolecular pool is shallower and wider. Saturation is achieved by applying an off-resonance radio frequency (RF) pulse specific to a peak in the macromolecular pool before excitation at the center frequency. The RF pulse saturates the signal from the section leading to ideally no signal at the off-resonance frequency. Since both pools interact this saturation is transferred to the free water pool. While it is not possible to detect the changes in the macromolecular pool directly, it can be assumed that the loss in signal intensity of the free water pool corresponds to the changes in the macromolecular pool.
- Ideally, an increase is preferable to a decrease in signal intensity since it is easier to visualize changes in brightness over changes in darkness. To achieve this type of image, a Magnetization Transfer Ratio (MTR) is calculated using a base image without saturation to measure the relative loss of signal intensity in a pixel by pixel basis: MTR=Nonsaturated−Saturated/Nonsaturated. MTC is very similar in function to CEST. CEST focuses on a limited part of magnetization transfer by linking it to chemical exchange systems.
- Quantitative magnetization transfer (qMT) imaging is MR technique which utilizes a two-pool model of magnetization exchange to acquire information regarding the cartilage macromolecular matrix. qMT imaging techniques typically require multiple MT-contrast images with different magnetization preparatory pulses resulting in long scan times which have limited cartilage assessment to ex-vivo specimens. Cross-relaxation imaging (CRI) is a qMT method which can create three-dimensional parametric maps of articular cartilage measuring the fraction of macromolecular bound protons (f), the exchange rate constant between macromolecular bound protons and free water protons (k), and the T2 relaxation time of macromolecular bound protons (T2B) with high resolution and relatively short scan time based upon a limited number of MT-contrast images. The parameter f provides an indirect measure of macromolecular content, while the parameters k, and T2B reflect the efficiency of magnetization exchange between macromolecular bound protons and free water protons and the spin diffusion between proton sites in macromolecules respectively which may be influenced by macromolecular organization and ultra-structure
- Microfluidic culture devices are attractive systems to model physiological and pathological conditions of tissues and organs. Although these devices allow fluorescent and light microcopy imaging of cultured cells, one of its current limitations is that various types of analyses require sacrificing of the culture. The Inventors have previously utilized micro imaging systems to monitor stem cell differentiation in ex-vivo 3D tissue constructs.
- Of interest is utilizing optical imaging to non-invasively monitor stem cell survival and differentiation while cultured in an “organ-on-chip” device. Stiffer membrane and microfluidic environment will promote more efficient osteogenic differentiation.
- To explore this possibility, the organ-on-chip was coated with ECM crosslinked with UV prior to cell seeding. Then mesenchymal stem cell line overexpressing BMP2 and Luciferase reporter genes were seeded on the coated organ-on-a-chip (see dimensions and the set up for microfluidic studies in
FIG. 1 and supplemented with osteogenic media. The static cultures were performed using 200 μl media reservoirs that were changed every other day. The flow studies were performed using 30 μl/h flow of media pulled through using specialized pump (FIG. 1 ). Micrographs were taken twice a week and survival of the cells was monitored using bioluminescent imaging. The media was changed to media with Luciferin and imagined using IVIS (Perkin Elmer). The osteogenic differentiation after 3 weeks of culture in osteogenic media was monitored using florescent probes OsteoSense650 and BoneTag800 that were introduced 24 hours before the imaging and were imaged using fluorescent imaging (FLI) and near infrared (NIR) imaging, confocal microscopy and immunostaining - A comparison of chips grown in static culture condition to chips grown under constant flow of media (30 μl/h) was performed along with evaluation of the effect of the flow on cell survival/proliferation of cells and the extent of osteogenic differentiation. The microscopic images (
FIG. 1A ) show proliferation of the cells under the flow conditions, however it is difficult to quantify the extent of proliferation using this method without disrupting the cultures. Therefore, the Inventors used cell that express Luciferase reporter gene and the cell proliferation was quantified using bioluminescent imaging (BLI) twice a week (FIG. 2B , C). This imaging method allowed monitor the proliferation of the cells without the need to harvest or disrupt the culture and significant advantage to the flow system was observed. Also microfluidic environment had positive effect on osteogenic differentiation, when compared with static cultures. - This effect was observed in fluorescent imaging of osteogenic differentiation probes using two different systems—FLI and Near Infrared (
FIG. 3 ). The probes can be detected using different wavelengths of fluorescence, therefore both probes can be added simultaneously and imaged separately. The quantification of FLI (FIG. 3A , B) of BoneTag showed higher osteogenic differentiation of the cell under the flow conditions. OsteoSense was also imaged using confocal microscopy in conjugation with Live/Dead staining (FIG. 3C ) showing that most of the live cells absorbed OsteoSense probe and again the flow chips were stained in more efficiently than the static cultures. The NIR system is considered more sensitive and the quantification of the image more accurate. Here, the Inventors demonstrate that they system is capable of detecting the same trend using both probes (FIG. 3D ). - In order to confirm osteogenic differentiation of the MSSC-BMP2 cells, the harvested chips were sectioned using vibratome creating transvers sections across the channels. Then these sections were subjected to immunofluorescent staining using primary antibody against Osteocalcin and Bone Sialoprotein (BSP) osteogenic markers. The staining shows cells on both sides of the membrane in both conditions, but mainly in the top channel. In both conditions there was positive staining for both marker, indicating osteogenic differentiation, however the staining looks more prominent in the chips that were cultured in flow (
FIG. 4 bottom panel). - Organ-on-chip system allows monitoring of the cell survival and proliferation in vitro using BLI imaging system and monitor the osteogenic differentiation of the cell on the chip in real time, without the need of harvesting the cells and disrupting the culture conditions. Here, the Inventors demonstrate that the flow conditions affect both proliferation and the differentiation of the MSCs that overexpress BMP2.
- The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.
- Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.
- Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.
- Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are sources of mesenchymal stem cells, osteoblasts, bone cells or stem cells, seeding and culturing on a microfluidic device, imaging methods, including labeling and detection, and the particular use of the products created through the teachings of the invention. Various embodiments of the invention can specifically include or exclude any of these variations or elements.
- In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
- In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
- Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
- Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
- Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.
- In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.
Claims (23)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/069,361 US20190018000A1 (en) | 2016-01-12 | 2017-01-12 | A method of non destructive monitoring of biological processes in microfluidic tissue culture systems |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662277857P | 2016-01-12 | 2016-01-12 | |
PCT/US2017/013250 WO2017123806A1 (en) | 2016-01-12 | 2017-01-12 | A method of non destructive monitoring of biological processes in microfluidic tissue culture systems |
US16/069,361 US20190018000A1 (en) | 2016-01-12 | 2017-01-12 | A method of non destructive monitoring of biological processes in microfluidic tissue culture systems |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2017/013250 A-371-Of-International WO2017123806A1 (en) | 2016-01-12 | 2017-01-12 | A method of non destructive monitoring of biological processes in microfluidic tissue culture systems |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/130,498 Continuation US20190009270A1 (en) | 2016-01-12 | 2018-09-13 | Method of osteogenic differentiation in microfluidic tissue culture systems |
Publications (1)
Publication Number | Publication Date |
---|---|
US20190018000A1 true US20190018000A1 (en) | 2019-01-17 |
Family
ID=59311493
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/069,361 Abandoned US20190018000A1 (en) | 2016-01-12 | 2017-01-12 | A method of non destructive monitoring of biological processes in microfluidic tissue culture systems |
US15/958,142 Pending US20180305668A1 (en) | 2016-01-12 | 2018-04-20 | Method of osteogenic differentiation in microfluidic tissue culture systems |
US15/958,976 Pending US20180237741A1 (en) | 2016-01-12 | 2018-04-20 | Method of osteogenic differentiation in microfluidic tissue culture systems |
US16/130,498 Pending US20190009270A1 (en) | 2016-01-12 | 2018-09-13 | Method of osteogenic differentiation in microfluidic tissue culture systems |
Family Applications After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/958,142 Pending US20180305668A1 (en) | 2016-01-12 | 2018-04-20 | Method of osteogenic differentiation in microfluidic tissue culture systems |
US15/958,976 Pending US20180237741A1 (en) | 2016-01-12 | 2018-04-20 | Method of osteogenic differentiation in microfluidic tissue culture systems |
US16/130,498 Pending US20190009270A1 (en) | 2016-01-12 | 2018-09-13 | Method of osteogenic differentiation in microfluidic tissue culture systems |
Country Status (3)
Country | Link |
---|---|
US (4) | US20190018000A1 (en) |
GB (2) | GB2562406B (en) |
WO (1) | WO2017123806A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11414648B2 (en) | 2017-03-24 | 2022-08-16 | Cedars-Sinai Medical Center | Methods and compositions for production of fallopian tube epithelium |
US11473061B2 (en) | 2016-02-01 | 2022-10-18 | Cedars-Sinai Medical Center | Systems and methods for growth of intestinal cells in microfluidic devices |
US11767513B2 (en) | 2017-03-14 | 2023-09-26 | Cedars-Sinai Medical Center | Neuromuscular junction |
US11913022B2 (en) | 2017-01-25 | 2024-02-27 | Cedars-Sinai Medical Center | In vitro induction of mammary-like differentiation from human pluripotent stem cells |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10605877B2 (en) * | 2017-01-20 | 2020-03-31 | The General Hospital Corporation | System and method for chemical exchange saturation transfer (CEST) magnetic resonance fingerprinting |
US20210062131A1 (en) * | 2018-04-11 | 2021-03-04 | Georgia Tech Research Corporation | In Vitro, Multi-Niche, Bone Marrow-on-a-Chip |
US10859656B2 (en) * | 2018-04-23 | 2020-12-08 | Cedars-Sinai Medical Center | Methods and systems for chemical exchange saturation transfer signal matching |
WO2019226780A1 (en) * | 2018-05-22 | 2019-11-28 | Nirrin Bioprocess Analytics, Inc. | Near infrared spectroscopy of culture media in micro-physiological systems |
CN109337813B (en) * | 2018-10-19 | 2023-07-11 | 杭州捷诺飞生物科技股份有限公司 | System and method suitable for biological tissue culture and real-time monitoring |
CN112098362B (en) * | 2020-09-15 | 2024-02-27 | 南京工程学院 | Cancellous bone in-vitro time assessment method and system based on near infrared spectrum characteristics |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2002236683A1 (en) * | 2000-10-27 | 2002-05-21 | Beth Israel Deaconess Medical Center | Non-isotopic detection of osteoblastic activity in vivo using modified bisphosphonates |
US9381273B2 (en) * | 2008-01-31 | 2016-07-05 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Scaffolds with oxygen carriers, and their use in tissue regeneration |
EP3778858A1 (en) * | 2008-07-16 | 2021-02-17 | Children's Medical Center Corporation | Device with microchannels and method of use |
AU2015204375A1 (en) * | 2008-07-16 | 2015-08-06 | Children's Medical Center Corporation | Organ mimic device with microchannels and methods of use and manufacturing thereof |
US9149806B2 (en) * | 2012-01-10 | 2015-10-06 | Biopico Systems Inc | Microfluidic devices and methods for cell sorting, cell culture and cells based diagnostics and therapeutics |
KR101200049B1 (en) * | 2012-05-14 | 2012-11-13 | 한국원자력연구원 | Preparation of Technetium-99m tricarbonyl labeled glycine monomer or oligomer containing probes that have biomolecules and its application as imaging complex-composition |
EP2986210B1 (en) * | 2013-04-19 | 2019-03-27 | Cedars-Sinai Medical Center | Imaging biomarkers for diagnosis of back pain |
US10047344B2 (en) * | 2014-02-18 | 2018-08-14 | National University Of Singapore | Biophysically sorted osteoprogenitors from culture expanded bone marrow derived mesenchymal stromal cells (MSCs) |
AU2015241133B2 (en) * | 2014-03-31 | 2019-11-28 | Brigham And Women's Hospital, Inc. | Systems and methods for biomimetic fluid processing |
GB201415804D0 (en) * | 2014-09-08 | 2014-10-22 | Univ Singapore | Assay Device |
-
2017
- 2017-01-12 GB GB1811716.8A patent/GB2562406B/en active Active
- 2017-01-12 GB GB1903007.1A patent/GB2569058B/en active Active
- 2017-01-12 WO PCT/US2017/013250 patent/WO2017123806A1/en active Application Filing
- 2017-01-12 US US16/069,361 patent/US20190018000A1/en not_active Abandoned
-
2018
- 2018-04-20 US US15/958,142 patent/US20180305668A1/en active Pending
- 2018-04-20 US US15/958,976 patent/US20180237741A1/en active Pending
- 2018-09-13 US US16/130,498 patent/US20190009270A1/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11473061B2 (en) | 2016-02-01 | 2022-10-18 | Cedars-Sinai Medical Center | Systems and methods for growth of intestinal cells in microfluidic devices |
US11913022B2 (en) | 2017-01-25 | 2024-02-27 | Cedars-Sinai Medical Center | In vitro induction of mammary-like differentiation from human pluripotent stem cells |
US11767513B2 (en) | 2017-03-14 | 2023-09-26 | Cedars-Sinai Medical Center | Neuromuscular junction |
US11414648B2 (en) | 2017-03-24 | 2022-08-16 | Cedars-Sinai Medical Center | Methods and compositions for production of fallopian tube epithelium |
Also Published As
Publication number | Publication date |
---|---|
GB2569058B (en) | 2021-04-14 |
US20180305668A1 (en) | 2018-10-25 |
GB201811716D0 (en) | 2018-08-29 |
GB2562406A (en) | 2018-11-14 |
US20190009270A1 (en) | 2019-01-10 |
WO2017123806A1 (en) | 2017-07-20 |
GB201903007D0 (en) | 2019-04-17 |
GB2562406B (en) | 2020-09-02 |
US20180237741A1 (en) | 2018-08-23 |
GB2569058A (en) | 2019-06-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20190009270A1 (en) | Method of osteogenic differentiation in microfluidic tissue culture systems | |
JP6755975B2 (en) | System for brightfield simulation | |
Di Rienzo et al. | Probing short-range protein Brownian motion in the cytoplasm of living cells | |
Webster et al. | Studying osteocytes within their environment | |
US10222335B2 (en) | Phasor method to fluorescence lifetime microscopy to discriminate metabolic state of cells in living tissue | |
Dmitriev et al. | Luminescence lifetime imaging of three-dimensional biological objects | |
JP2023505265A (en) | Systems and methods for high-throughput drug screening | |
JP5953293B2 (en) | Long-term monitoring and analysis methods using photoproteins | |
Scalfi-Happ et al. | Investigation of lipid bodies in a colon carcinoma cell line by confocal Raman microscopy | |
Pinkert et al. | Imaging the cardiac extracellular matrix | |
Zarychta-Wiśniewska et al. | In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models | |
Tubbesing et al. | Raman microspectroscopy fingerprinting of organoid differentiation state | |
Jenkins et al. | Imaging cell and tissue O 2 by TCSPC-PLIM | |
Croix et al. | Intravital fluorescence microscopy in pulmonary research | |
Slaughter et al. | Fluorescence fluctuation spectroscopy and imaging methods for examination of dynamic protein interactions in yeast | |
Sarfraz et al. | Establishing Riboglow-FLIM to visualize noncoding RNAs inside live zebrafish embryos | |
Habibalahi et al. | Non-invasive real-time imaging of reactive oxygen species (ROS) using multispectral auto-fluorescence imaging technique: a novel tool for redox biology | |
Dittmar et al. | In situ label‐free cell viability assessment of nucleus pulposus tissue | |
Kolanowski et al. | Fluorescent probes for the analysis of labile metals in brain cells | |
Lawrence et al. | Intracellular targeting of Cyclotides for therapeutic applications | |
Pérez Parets | The use of advanced imaging techniques for studying neuromuscular disorders | |
Haridoss | In vivo assessment of focal adhesion kinase (FAK) activity in breast cancer cells using fluorescence resonance energy transfer (FRET) sensor and confocal laser scanning microscope (CLSM) | |
WO2020049642A1 (en) | Cell analysis apparatus and cell analysis method | |
Miao | Stimulated Raman scattering: a biophysical perspective for imaging cells and tissues | |
Yun et al. | Bioimaging Probes Development by DOFLA (Diversity Oriented Fluorescence Library Approach) for in Vitro, in Vivo and Clinical Applications |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: CEDARS-SINAI MEDICAL CENTER, CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GAZIT, DAN;PELLED, GADI;GAZIT, ZULMA;AND OTHERS;SIGNING DATES FROM 20170113 TO 20170116;REEL/FRAME:046321/0214 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: APPLICATION DISPATCHED FROM PREEXAM, NOT YET DOCKETED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |