WO2012131733A2 - Media compositions, method of initiating and enriching cultures of stem cells and/or cancer stem-like cells - Google Patents
Media compositions, method of initiating and enriching cultures of stem cells and/or cancer stem-like cells Download PDFInfo
- Publication number
- WO2012131733A2 WO2012131733A2 PCT/IN2012/000234 IN2012000234W WO2012131733A2 WO 2012131733 A2 WO2012131733 A2 WO 2012131733A2 IN 2012000234 W IN2012000234 W IN 2012000234W WO 2012131733 A2 WO2012131733 A2 WO 2012131733A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- medium
- cancer
- present
- stem
- Prior art date
Links
- 210000004027 cell Anatomy 0.000 title claims abstract description 199
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 119
- 201000011510 cancer Diseases 0.000 title claims abstract description 110
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 107
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000000203 mixture Substances 0.000 title claims description 40
- 230000000977 initiatory effect Effects 0.000 title description 7
- 239000002609 medium Substances 0.000 claims abstract description 70
- 239000001963 growth medium Substances 0.000 claims abstract description 63
- 239000003102 growth factor Substances 0.000 claims abstract description 32
- 235000015097 nutrients Nutrition 0.000 claims abstract description 30
- 238000004113 cell culture Methods 0.000 claims abstract description 29
- 229920000609 methyl cellulose Polymers 0.000 claims abstract description 26
- 235000010981 methylcellulose Nutrition 0.000 claims abstract description 26
- 239000001923 methylcellulose Substances 0.000 claims abstract description 26
- 108010010803 Gelatin Proteins 0.000 claims abstract description 23
- 239000008273 gelatin Substances 0.000 claims abstract description 23
- 229920000159 gelatin Polymers 0.000 claims abstract description 23
- 235000019322 gelatine Nutrition 0.000 claims abstract description 23
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 23
- 210000002966 serum Anatomy 0.000 claims abstract description 16
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 32
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 27
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 19
- 238000012258 culturing Methods 0.000 claims description 18
- 230000012010 growth Effects 0.000 claims description 18
- 102000004877 Insulin Human genes 0.000 claims description 17
- 108090001061 Insulin Proteins 0.000 claims description 17
- 229940125396 insulin Drugs 0.000 claims description 16
- 229960000890 hydrocortisone Drugs 0.000 claims description 14
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 13
- 239000006285 cell suspension Substances 0.000 claims description 13
- 229960002897 heparin Drugs 0.000 claims description 13
- 229920000669 heparin Polymers 0.000 claims description 13
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 13
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 12
- 229940116977 epidermal growth factor Drugs 0.000 claims description 12
- 239000012583 B-27 Supplement Substances 0.000 claims description 9
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 8
- 210000002950 fibroblast Anatomy 0.000 claims description 8
- 101150021185 FGF gene Proteins 0.000 claims description 7
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 7
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims description 5
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 4
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 4
- 239000006147 Glasgow's Minimal Essential Medium Substances 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 4
- 239000007640 basal medium Substances 0.000 claims description 3
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 claims description 2
- 125000000017 cortisol group Chemical group 0.000 claims 1
- 239000002299 complementary DNA Substances 0.000 description 21
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 12
- 206010006187 Breast cancer Diseases 0.000 description 12
- 208000026310 Breast neoplasm Diseases 0.000 description 12
- 230000001464 adherent effect Effects 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 11
- 102000004142 Trypsin Human genes 0.000 description 10
- 108090000631 Trypsin Proteins 0.000 description 10
- 239000012091 fetal bovine serum Substances 0.000 description 10
- 239000013642 negative control Substances 0.000 description 10
- 239000012588 trypsin Substances 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 102400001368 Epidermal growth factor Human genes 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 239000000499 gel Substances 0.000 description 9
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 108010088751 Albumins Proteins 0.000 description 4
- 102000009027 Albumins Human genes 0.000 description 4
- 206010025323 Lymphomas Diseases 0.000 description 4
- 206010060862 Prostate cancer Diseases 0.000 description 4
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 4
- -1 artificial analogs Proteins 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 102100021083 Forkhead box protein C2 Human genes 0.000 description 3
- 101000818305 Homo sapiens Forkhead box protein C2 Proteins 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 206010039491 Sarcoma Diseases 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000009401 metastasis Effects 0.000 description 3
- 201000005962 mycosis fungoides Diseases 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000001172 regenerating effect Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 239000004017 serum-free culture medium Substances 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- 235000001809 DL-alpha-tocopherylacetate Nutrition 0.000 description 2
- 239000011626 DL-alpha-tocopherylacetate Substances 0.000 description 2
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 2
- 208000021309 Germ cell tumor Diseases 0.000 description 2
- 108010009202 Growth Factor Receptors Proteins 0.000 description 2
- 102000009465 Growth Factor Receptors Human genes 0.000 description 2
- 208000017604 Hodgkin disease Diseases 0.000 description 2
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 2
- 102000003746 Insulin Receptor Human genes 0.000 description 2
- 108010001127 Insulin Receptor Proteins 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 208000034176 Neoplasms, Germ Cell and Embryonal Diseases 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- 238000011530 RNeasy Mini Kit Methods 0.000 description 2
- 238000010240 RT-PCR analysis Methods 0.000 description 2
- 208000000453 Skin Neoplasms Diseases 0.000 description 2
- 208000021712 Soft tissue sarcoma Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 210000000013 bile duct Anatomy 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 210000001608 connective tissue cell Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000007783 downstream signaling Effects 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 206010061289 metastatic neoplasm Diseases 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 201000002530 pancreatic endocrine carcinoma Diseases 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 208000010626 plasma cell neoplasm Diseases 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 201000000849 skin cancer Diseases 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 210000000278 spinal cord Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229940042585 tocopherol acetate Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 206010061424 Anal cancer Diseases 0.000 description 1
- 208000007860 Anus Neoplasms Diseases 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 206010060971 Astrocytoma malignant Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000006734 Beta-Globulins Human genes 0.000 description 1
- 108010087504 Beta-Globulins Proteins 0.000 description 1
- 206010004593 Bile duct cancer Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006143 Brain stem glioma Diseases 0.000 description 1
- 206010007275 Carcinoid tumour Diseases 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 102000009016 Cholera Toxin Human genes 0.000 description 1
- 108010049048 Cholera Toxin Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 102000001045 Connexin 43 Human genes 0.000 description 1
- 108010069241 Connexin 43 Proteins 0.000 description 1
- OMFXVFTZEKFJBZ-UHFFFAOYSA-N Corticosterone Natural products O=C1CCC2(C)C3C(O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 OMFXVFTZEKFJBZ-UHFFFAOYSA-N 0.000 description 1
- ZAKOWWREFLAJOT-CEFNRUSXSA-N D-alpha-tocopherylacetate Chemical compound CC(=O)OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-CEFNRUSXSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 101100335722 Drosophila melanogaster Gapdh1 gene Proteins 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 206010014967 Ependymoma Diseases 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 208000012468 Ewing sarcoma/peripheral primitive neuroectodermal tumor Diseases 0.000 description 1
- 102100028072 Fibroblast growth factor 4 Human genes 0.000 description 1
- 208000022072 Gallbladder Neoplasms Diseases 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 101001060274 Homo sapiens Fibroblast growth factor 4 Proteins 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 206010021042 Hypopharyngeal cancer Diseases 0.000 description 1
- 206010056305 Hypopharyngeal neoplasm Diseases 0.000 description 1
- 206010061252 Intraocular melanoma Diseases 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 206010023825 Laryngeal cancer Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 208000030070 Malignant epithelial tumor of ovary Diseases 0.000 description 1
- 208000032271 Malignant tumor of penis Diseases 0.000 description 1
- 208000000172 Medulloblastoma Diseases 0.000 description 1
- 208000002030 Merkel cell carcinoma Diseases 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 1
- 208000014767 Myeloproliferative disease Diseases 0.000 description 1
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 206010029266 Neuroendocrine carcinoma of the skin Diseases 0.000 description 1
- 102000008763 Neurofilament Proteins Human genes 0.000 description 1
- 108010088373 Neurofilament Proteins Proteins 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 206010031096 Oropharyngeal cancer Diseases 0.000 description 1
- 206010057444 Oropharyngeal neoplasm Diseases 0.000 description 1
- 208000007571 Ovarian Epithelial Carcinoma Diseases 0.000 description 1
- 206010061328 Ovarian epithelial cancer Diseases 0.000 description 1
- 206010033268 Ovarian low malignant potential tumour Diseases 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 208000000821 Parathyroid Neoplasms Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 208000002471 Penile Neoplasms Diseases 0.000 description 1
- 206010034299 Penile cancer Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 208000007641 Pinealoma Diseases 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 101150085390 RPM1 gene Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 208000004337 Salivary Gland Neoplasms Diseases 0.000 description 1
- 206010061934 Salivary gland cancer Diseases 0.000 description 1
- 208000009359 Sezary Syndrome Diseases 0.000 description 1
- 208000021388 Sezary disease Diseases 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 208000031673 T-Cell Cutaneous Lymphoma Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- 206010057644 Testis cancer Diseases 0.000 description 1
- 201000009365 Thymic carcinoma Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 206010046431 Urethral cancer Diseases 0.000 description 1
- 206010046458 Urethral neoplasms Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 201000005969 Uveal melanoma Diseases 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 206010047741 Vulval cancer Diseases 0.000 description 1
- 208000004354 Vulvar Neoplasms Diseases 0.000 description 1
- 208000008383 Wilms tumor Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 101000818331 Xenopus tropicalis Forkhead box protein C2 Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000020990 adrenal cortex carcinoma Diseases 0.000 description 1
- 208000007128 adrenocortical carcinoma Diseases 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 201000011165 anus cancer Diseases 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 208000012172 borderline epithelial tumor of ovary Diseases 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 208000002458 carcinoid tumor Diseases 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 201000007335 cerebellar astrocytoma Diseases 0.000 description 1
- 208000030239 cerebral astrocytoma Diseases 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 201000007241 cutaneous T cell lymphoma Diseases 0.000 description 1
- 208000017763 cutaneous neuroendocrine carcinoma Diseases 0.000 description 1
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 1
- 229940068840 d-biotin Drugs 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- HSXUHWZMNJHFRV-UHFFFAOYSA-L disodium;6-oxido-5-phenyldiazenyl-4-sulfonaphthalene-2-sulfonate Chemical compound [Na+].[Na+].OC1=CC=C2C=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C2=C1N=NC1=CC=CC=C1 HSXUHWZMNJHFRV-UHFFFAOYSA-L 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 210000002514 epidermal stem cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 201000008819 extrahepatic bile duct carcinoma Diseases 0.000 description 1
- 208000024519 eye neoplasm Diseases 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 102000003977 fibroblast growth factor 18 Human genes 0.000 description 1
- 108090000370 fibroblast growth factor 18 Proteins 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 201000010175 gallbladder cancer Diseases 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 201000007116 gestational trophoblastic neoplasm Diseases 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 210000000442 hair follicle cell Anatomy 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 210000003897 hepatic stem cell Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 201000006866 hypopharynx cancer Diseases 0.000 description 1
- 230000002267 hypothalamic effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 206010023841 laryngeal neoplasm Diseases 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 208000030883 malignant astrocytoma Diseases 0.000 description 1
- 208000006178 malignant mesothelioma Diseases 0.000 description 1
- 208000026045 malignant tumor of parathyroid gland Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 208000018795 nasal cavity and paranasal sinus carcinoma Diseases 0.000 description 1
- 210000001178 neural stem cell Anatomy 0.000 description 1
- 210000005044 neurofilament Anatomy 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 201000008106 ocular cancer Diseases 0.000 description 1
- 201000002575 ocular melanoma Diseases 0.000 description 1
- 201000005443 oral cavity cancer Diseases 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 201000006958 oropharynx cancer Diseases 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 208000021284 ovarian germ cell tumor Diseases 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 208000028591 pheochromocytoma Diseases 0.000 description 1
- 230000008288 physiological mechanism Effects 0.000 description 1
- 201000002511 pituitary cancer Diseases 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 208000025638 primary cutaneous T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 208000015347 renal cell adenocarcinoma Diseases 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000002363 skeletal muscle cell Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 201000002314 small intestine cancer Diseases 0.000 description 1
- 210000001057 smooth muscle myoblast Anatomy 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 206010046885 vaginal cancer Diseases 0.000 description 1
- 208000013139 vaginal neoplasm Diseases 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 201000005102 vulva cancer Diseases 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
Classifications
-
- 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
- C12N5/0693—Tumour cells; Cancer 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
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
-
- 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
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- 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
- C12N2501/90—Polysaccharides
- C12N2501/91—Heparin
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Definitions
- the present invention relates to media compositions and method for initiating, enriching and maintaining culture of stem cells or cancer stem-like cells.
- the present invention further relates to culture media of the defined composition and cell cultures comprising the same.
- Stem cells are characterized by their ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialized cells types. Stem cells can be divided into two basic categories i.e. embryonic stem cells that are isolated from embryos and adult stem cells that are found in adult tissues.
- stem cells differentiate to produce the various tissues during growth whereas in adult organisms stem cells and progenitor cells act as repair system for the body replenishing cells and maintain the normal regeneration of regenerative organs for example, oval cells (hepatic stem cells) that produce hepatocytes and bile duct cells, cardiac stem cells that produce cardiomyocytes, "satellite cells” and “myoblasts” produce skeletal muscle cells, neural stem cells that are found in neural tissue such as the brain and spinal cord and produce neurons and glial cells, epidermal stem cells that produce epidermal cells and hair follicle cells, progenitor cells derived from stem cells known as "hematopoietic stem cells", produce blood cells such as erythrocytes, lymphocytes and megakaryocytes.
- oval cells hepatic stem cells
- cardiac stem cells that produce cardiomyocytes
- satellite cells and “myoblasts”
- neural stem cells that are found in neural tissue such as the brain and spinal cord and produce neurons and glial cells
- Stem cells are therefore important in regenerative therapy to cures various diseases and heal wounds.
- protocols and media compositions contemplated for culturing stem cells are specific and vary as per the origin of stem cells.
- necessitating the use and investing in unreasonable inventory for different media and protocols for culturing stem cells or cancer stem cells This makes it challenging and an uneconomical proposition for researchers as well as those in the industrial set up equally causing hindrances in expanding the scope of research or utility of different types of stem cells and/or cancer stem-like cells.
- stem-like cells are found within cancerous tumors, which possesses characteristics of stem cells and are considered to be the cause of tumor formation and recurrence. Such cells are proposed to persist in tumors as a distinct population with the ability to give rise to all cell types found in cancerous tumor as well as replenish them from time to time and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at Cancer stem cells or cancer stem-like cells holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. It is considered difficult to separate CSCs from tumors cells. Therefore, it is desirable to develop culture medium, method for obtaining cultures with enriched cancer stem cells or cancer stem-like cells and cultures comprising the same for further use.
- cancer stem-like cells differ based on their nature of origin. Cancer stem-like cells derived from breast cancer differs substantially in growth requirements for ln-vitro culturing as opposed to cancer stem-like cell derived from epithelial tissue or prostrate tissue. Besides, most culturing media available in the market are specific to the different types of cancer cells and require addition of fetal bovine serum (FBS) which is considered as crucial component. Addition of FBS poses additional problems of contamination with pathogens and bacteria. Thus, problems with existing media are that they are suitable for only selective stem cells proliferation and cannot be used for all cells lines for proliferation of stem cells/ stem like-cells.
- FBS fetal bovine serum
- the present invention in one aspect provides compositions for media for culturing, selectively enriching and maintaining stem cells or cancer stem-like cells which is essentially serum free.
- the present invention provides a composition for a culture medium for selectively enriching and maintaining stem cells or cancer stemlike cells comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount, wherein the composition is essentially serum free.
- the present invention provides a composition for feeding media for selectively enriching and maintaining stem cells or cancer stem-like cells.
- the present invention provides a composition for a feeding media for selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, and optionally growth factors or other supplementary elements in an effective amount wherein the composition is essentially serum free.
- the present invention provides a culture media for selectively enriching and maintaining stem cells or cancer stem-like cells.
- the present invention provides a culture medium for selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount wherein the culture medium is essentially serum free.
- the present invention provides a feeding media for selectively enriching and maintaining stem cells or cancer stem-like cells.
- the present invention provides a feeding medium for selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, and optionally growth factors or other supplementary elements in an effective amount wherein the feeding medium is essentially serum free.
- a cell culture which comprises the stem cells or cancer stem-like cells and any of the culture media of the present invention.
- the present invention provides a cell culture comprising cancer stem-like cells in essentially serum free culture medium comprised of basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an amount sufficient for maintaining stem cells or cancer stem-like cells.
- the present invention provides a cell culture comprising stem cells in essentially serum free culture medium comprised of basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an amount sufficient for maintaining stem cells or cancer stem-like cells.
- the present invention provides a method for isolation and proliferation of stem cells or cancer stem-like cells.
- the present invention provides a method comprising steps of culturing the single cell suspension of cancer cells or normal cells in the culture medium of the present invention comprising the basic nutrients, heparin, gelatin and methyl cellulose at 37°C in a 5% C0 2 atmosphere and adding feeding medium to the growing culture intermittently to obtain spheres of stem cells or cancer stem-like cells.
- the present invention provides a kit for use in the method of the present invention.
- the present invention provides a kit comprising the culture medium or components thereof, feeding medium or components thereof, the cell culture system comprising the stem cells or cancer stem-like cells, cell culture containers.
- the kit may also further comprise written instructions for how to perform the cell culture method.
- Figure 2 Shows photomicrograph of spheroids formed from the cultured LNCAP prostate cancer cells as per Example 2.
- Figure 3 Shows photomicrograph of spheroids formed from the cultured MCF-IOA breast cancer cells as per Example 3.
- Figure 4 Shows photomicrograph of spheroids formed from the cultured MDA-MB- 231 breast cancer cells as per Example 4.
- Figure 5 Shows photomicrograph of spheroids formed from the cultured L 929 mouse connective tissue cells as per Example 5.
- Figures 6A - 6C shows RT-PCR - gene expression analysis of spheroids of cells obtained in Example 3-5.
- FIG 6A is a diagrammatic representation of FIG 6A.
- Lane 1 Marker 1- lOObp
- Lane 2 Marker 2- 1Kb
- Lane 3-4 primer quantification of primers 1 & 2 FOXC2 I
- Lane 5-6 Lane(3-6) primer quantification of primers 3 & 4 GADPH 1.
- Lane 1 Marker 1 lOObp
- Lane 2 Marker 2 - 1Kb
- Lane 3 cDNA : negative control : 1 ul :no band
- Lane 4 Enzyme negative control: no band
- Lane 5 Enzyme + water negative control :no band
- Lane 6 cDNA :MCF 10 A - faint band seen near the periphery of well
- Lane 7 cDIMA : L929 - faint band seen near the periphery of well
- Lane 8 cDNA: MDA MB 231 : faint band seen near the periphery of well.
- FIG 6B is a diagrammatic representation of FIG 6B.
- Lane 1 Ladder - indicating 494bps
- Lane 2 Control 1: good prominent band
- Lane 3 Control 2: good prominent band
- Lane 4 Negative control : enzyme negative
- Lane 5 MCF 10 A: good prominent band
- Lane 6 L929 : good prominent band
- Lane 7 MDA MB 231: good prominent band
- Lane 1 Ladder - indicating 494bps
- Lane 2 Blank Lane
- Lane 3 Control : good prominent band
- Lane 4 Negative control * : enzyme negative
- Lane 5 MCF 10 A: good prominent band
- Lane 6 L929 : good prominent band
- Lane 7 MDA MB 231: good prominent band
- Lane 8 Negative: negative .
- FIG 6C is a diagrammatic representation of FIG 6C.
- Lane 1 Ladder - indicating 494bps
- Lane 2 Control 2: good prominent band
- Lane 3 Negative control : enzyme negative: no band seen
- Lane 4 MCF 10 A: with P1/P2 2ul cDNA .-Faint band
- Lane 5 L929 : with P1/P2 : 2ul cDNA No band
- Lane 6 MDA MB 231: with P1/P2: 2ul cDNA faint band
- Lane 7 Control 2: 5ul cDNA good prominent band
- Lane 8 Negative control : enzyme negative: no band seen
- Lane 9 MCF 10 A: with P1/P2 5ul cDNA:Faint band
- Lane 10 L929 : with P1/P2 5ul cDNA: No band
- Lane 11 MDA MB 231: with P1/P2 5ul cDNA: faint band.
- Lane 1 Ladder - indicating 494bps
- Lane 2 Control 2: good prominent band
- Lane 3 Negative control : enzyme negative: no band seen
- Lane 4 MCF 10 A: with P3/P4 2ul cDNA : prominent band
- Lane 5 L929 : with P3/P4 : 2ul cDNA prominent band
- Lane 6 MDA MB 231: with P3/P4: 2ul cDNA : prominent band
- Lane 7 Control 2: 5ul cDNA good prominent band
- Lane 8 Negative control : enzyme negative: no band seen
- Lane 9 MCF 10 A: with P3/P4 5ul cDNA: prominent band
- Lane 10 L929 : with P3/P4 5ul cDNA:Faint band
- Lane 11 MDA MB 231: with P3/P4 5ul cDNA: prominent band.
- the present invention is directed towards obviating the problems associated with initiation, proliferation and maintenance of cultures of stem cells or cancer stem-like cells. More particularly the present invention is directed to selectively enriching and maintaining the population of stem cells or cancer stem-like cells and to make them accessible for further research, regenerative therapies, clinical investigations or drug screening for anticancer drug therapeutics.
- the present invention is directed towards providing compositions for media for example culture medium and feeding medium, for culturing, selectively enriching and maintaining stem cells or cancer stem-like cells.
- the present invention provides a composition of a medium for selectively enriching and maintaining stem cells or cancer stem-like cells originating from cancerous and non-cancerous tissue comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount wherein the composition is essentially serum free.
- serum free refers to being devoid of a human or an animal serum.
- the serum free media compositions do not comprise of serum or portions thereof.
- the basic nutrients refers to a mixture comprising of salts, amino acids, vitamins that provide cells with water and certain bulk inorganic ions essential for normal cell metabolism, maintain intra- and extra-cellular osmotic balance, provide a carbohydrate as an energy source, and provide a buffering system to maintain the medium within the physiological pH range.
- the basic nutrients may be formulated into a base media by mixing the above mentioned nutrient components. Alternately, basic nutrients may be incorporated based on the known basal media as such, or after modification.
- Non-limiting examples of basic nutrients to be incorporated based on the known basal media include Dulbecco's Modified Eagle's Medium (DMEM), DMEM/F-12, or KO-DMEM, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), BGJb Medium, RPM1 1640, Ham's F-10, Ham's F-12, ex- Minimal Essential Medium (ctMEM), Brinster's BMOC-3 Medium, C02-lndependent Medium, CMRL Medium, Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium, Waymouth's MB 752/1 Media, Williams Media E, Medium NCTC-109, neuroplasma medium, Leibovitz's L-15 Media, McCoy's 5A Media (modified), MCDB 131 Mediumor the likes and/or mixtures thereof.
- DMEM Dulbecco's Modified Eagle's Medium
- BME Basal Medium Eagle
- BME Bas
- gelatin is present typically from about 0.001% to about 0.01% w/v, for example from about 0.001% to about 0.5% w/v, for example from about 0.001% to about 0.05% w/v, or for example from about 0.001% to about 0.01% w/v.
- gelatin is incorporated in an amount from about 0.001% to about 0.01% w/v.
- methyl cellulose is present typically from about 0.01% to about 10% w/v, for example from about 0.01% to about 5% w/v, for example from about 0.05 % to about 2.5% w/v, or for example from about 0.05 % to about 1.5% w/v.
- the composition further comprises heparin.
- the concentration of said heparin is from about 0 ng/ml to about 100 ng/ml, for example from about 2 ng/ml to about 50 ng/ml, for example from about 2.5 ng/ml to about 25 ng/ml, for example from about 5 ng/ml to about 15 ng/ml, for example from about 4 ng/ml to about 10 ng/ml, for example from about 4 ng/ml to about 8 ng/ml.
- composition of a medium for selectively enriching and maintaining stem cells or cancer stem-like cells of the present invention further comprises growth factors selected from the group consisting of but not limited to, members of the epidermal growth factor family (EGFs), members of the fibroblast growth factor family (FGFs), members of the hydrocortisone growth factor family, B27 supplement, insulin and/or other suitable growth factors.
- EGFs epidermal growth factor family
- FGFs fibroblast growth factor family
- B27 supplement insulin and/or other suitable growth factors.
- one or more growth factors and insulin to be incorporated in the composition are recombinantly produced molecules. Alternately, they may be isolated from natural sources. With regard to the growth factors and insulin proteins, the invention also contemplates the use of homologs, or proteins having sequence identity of at least about 70% and the receptor activating activity of the respective naturally occurring protein that is growth factors or insulin, artificial analogs, polypeptide fragments that activate the respective growth factors or insulin receptor and/or downstream signaling, and other molecules that activate one or more growth factors or insulin receptors and/or their downstream signaling.
- the composition comprises Basic fibroblast growth factor (also known as bFGF, FGF2 or FGF- ⁇ ) a member of the fibroblast growth factor family.
- bFGF Basic fibroblast growth factor
- the concentration of said bFGF is from about 5 ng/ml to about 100 ng/ml, for example from about 10 ng/ml to about 50 ng/ml, for example from about 10 ng/ml to about 25 ng/ml, for example from about 15 ng/ml to about 25 ng/ml.
- the composition comprises Epidermal growth factor (also known as EGF).
- EGF Epidermal growth factor
- the concentration of said EGF is from about 1 ng/ml to about 100 ng/ml, for example from about 2.5 ng/ml to about 50 ng/ml, for example from about 2.5 ng/ml to about 25 ng/ml, for example from about 5 ng/ml to about 15 ng/ml.
- the composition comprises insulin.
- the concentration of said insulin is from about 1 ⁇ / ⁇ to about 100 ⁇ g/ml, for example from about 2.5 ⁇ g/ml to about 50 ⁇ g/ml, for example from about 2.5 ⁇ g/ml to about 25 ⁇ g/ml, for example from about 5 ⁇ g/ml to about 15 ⁇ g/ml.
- the composition comprises hydrocortisone.
- concentration of said hydrocortisone is at least about 0.1 ⁇ g/ml, for example at least about 2.5 ⁇ g/ml, for example at least about 5 ⁇ g/ml, for example at least about 7.5 ⁇ g/ml, for example at least about 1 g ml.
- the composition comprises B27 supplement.
- B27 supplement is without vitamin A which is available from Gibco-lnvitrogen, Corporation, Grand Island, NY USA, Catalogue No. 12587-010.
- the B27 supplement is a serum-free formulation which includes d-biotin, fatty acid free fraction V bovine serum albumin (BSA), catalase, L- carnitine HC1, corticosterone, ethanolamine HC1, D-galactose (Anhyd.), glutathione (reduced), recombinant human insulin, linoleic acid, linolenic acid, progesterone, putrescine-2-HCI, sodium selenite, superoxide dismutase, T- 3/albumin complex, DL alpha-tocopherol and DL alpha tocopherol acetate.
- BSA bovine serum albumin
- catalase L- carnitine HC1, corticosterone
- ethanolamine HC1 D
- composition may further comprise of supplementary nutrients for example L- glutamine or stable glutamine.
- supplementary nutrients for example L- glutamine or stable glutamine.
- the composition of a medium according to the invention can also be supplemented with any compound(s) that will not interfere with, and preferably supports the enrichment and/or maintenance of stem cells or cancer stem-like cells over time.
- Preferred examples of such compounds include non-essential amino acids, anti-oxidants, reducing agents, vitamins, organic compounds, inorganic salts, transferring, and albumins.
- the present invention provides a culture medium.
- the culture medium for culturing, selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount wherein the culture medium is essentially serum free.
- culture medium refers to a solid or a liquid substance used to support the growth of stem cells and selectively enriching and maintaining stem cells or cancer stem-like cells.
- culture medium refers to a liquid substance.
- the present invention provides a medium for initiating, selectively enriching and maintaining stem cells or cancer stem-like cells originating from cancerous and non-cancerous cells comprising gelatin, heparin, the basic nutrients comprising of an approximately 50:50 mixture of DMEM and Ham's F12 nutrients, L- glutamine or stable glutamine, basic Fibroblast Growth Factor- FGF, Epidermal Growth Factor -EGF, recombinant growth factor Insulin, B27 supplement, and hydrocortisone.
- the medium when used as a culture medium is supplemented with methyl cellulose prior to use.
- the medium may be used as such, as a feeding medium.
- the present invention provides a medium for initiating, selectively enriching and maintaining stem cells or cancer stem-like cells originating from cancerous and non-cancerous cells comprising gelatin present at concentration from about 0.001% to about 0.01% w/v, methyl cellulose present at concentration from about 0.05 % to about 1% w/v, heparin present at the concentration from about 4 ng/ml to about 8 ng/ml, the basic nutrients comprising of an approximately 50:50 mixture of DMEM and Ham's F12 nutrients, basic Fibroblast Growth Factor- bFGF present at concentration from about 15 ng/ml to about 25 ng/ml, Epidermal Growth Factor -EGF present at concentration from about 5 ng/ml to about 15 ng/ml, Insulin present at concentration from about 5 g/ml to about 15 ⁇ g/ml, B27 supplement IX to 5X, and hydrocortisone present at concentration of at least about 1 g/ml.
- the medium when used as
- stem cells or cancer stem-like cells of the present invention are derived from a cell line. In another embodiment, stem cells or cancer stem-like cells of the present invention are derived from a primary cell culture.
- stem cells of the present invention are derived using a well known protocol from blood, or from tissues or from various organs comprising the stem cells such tissues or organs may be selected from but not limiting to liver, bile duct, cardiac tissue, skeletal muscles, neural tissue from brain and spinal cord, epidermal tissue, skin, hair follicle or any other organ or tissue comprising the stem cells.
- the primary cell culture comprising cancer stem-like cells is derived from a tumor or cell metastasis.
- tumors and cell metastasis are derived from but not limited to: prostate cancer, breast cancer, carcinoid tumor, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewings family of tumors (pnet), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, laryngeal cancer, adrenocortical carcinoma/ anal cancer, bladder cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroecto
- a cell culture which comprises the stem cell or cancer stem-like cells and any of the culture media described hereinabove.
- the stem cell or cancer stem-like cells or cell cultures comprising the same are capable of keeping their self-renewal potential during 1-100 passages of in-vitro cultivation.
- stem cell or cancer stem-like cells are capable of keeping their self-renewal potential during 1-90 passages of in-vitro cultivation.
- stem cell or cancer stem-like cells are capable of keeping their self-renewal potential during 20-60 passages of in-vitro cultivation.
- a method for isolation and proliferation of stem cells or cancer stem-like cells comprises steps of cuituring the single cell suspension of cancer cells or normal cells in culture medium as per the present invention comprising the basic nutrients, heparin, gelatin and methyl cellulose at 37°C in a 5% C0 2 atmosphere and adding feeding medium to the growing culture intermittently to obtain spheres of stem cells or cancer stem-like cells.
- the propagation time for generation of spheroids of stem cells or cancer stem-like cells by the culture method and using the culture medium with methyl cellulose as well as feeding medium of the present invention is shorter compared to the other known protocols.
- Spheres or spheroids of stem cells or cancer stem-like cells may be obtained by the method and media of the present invention in 5-10 days depending upon the type of cells / cell line and cell density plated. Unless stated otherwise the terms “spheroid” and “spheres” are used interchangeably herein and refer to a certain "ball-shaped" globular structure, consisting of more than a single cell that has initially developed from a single or from multiple cells.
- the single cell suspension of the normal cells or cancer cells is cultured as per the present invention method at an appropriate cell density.
- the cell density may range from about 1X10 4 cells /ml to 1X10 6 cells/ml.
- normal cells or cancer cells are expanded by incubating them in a complete minimal essential medium supplemented with antibiotic, vitamins and serum for period of 24-48 hours at about 37 degree centigrade in a 5% C0 2 atmosphere.
- cells are washed twice with DPBS and trypsinised using 0.25% trypsin-EDTA at 37°C in a 5% C0 2 atmosphere for 2-5 minutes and cells are dissociated into a single cell suspension, which are initially cultured in a 96 well non-treated plate with the medium as per the present invention.
- small clusters may also be used. While using the small clusters, enzymatic digestion (such as with type IV collagenase) utilized for cluster disruption is terminated before stem cells become completely dispersed and the cells are triturated with a pipette such that clumps (i.e., 10-200 cells) are formed. However, measures are taken to avoid large clusters which may cause cell differentiation.
- the spheres or spheroids comprising stem cells or cancer stem-like cells are isolated from the primary growing sphere cultures and passaged in the culture medium of the present invention, further supplemented periodically with the feeding medium to obtain large spheres comprising of stem cells or cancer stem-like cells.
- the methods of the present invention provide that stem cells or cancer stem-like cells can be passaged without loosing their respective phenotype for at least 5 passages. In other embodiments, the methods of the present invention provide that stem cells or cancer stem-like cells can be passaged without loosing their respective phenotype for at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 passages.
- the growth of the stem cells or cancer stem-like cells is monitored.
- Monitoring the formation of spheres or spheroids comprising stem cells or cancer stem-like cells is within the capabilities of those skilled in the art and can be effected by morphological evaluations and determination of expression of differentiation-specific markers [e.g., using immunological techniques or RNA- based analysis (e.g., RT-PCR, cDIMA microarray)].
- the growth of the stem cells or cancer stem-like cells is monitored to determine their differentiation state.
- the differentiation state can be determined using various approaches including, for example, morphological evaluation (e.g., as shown in Figures 1-5) and/or detection of the expression pattern of typical markers of the undifferentiated state using immunological techniques such as flow cytometry for membrane-bound markers, immunohistochemistry or immunofluorescence for extracellular and intracellular markers and enzymatic immunoassay, for secreted molecular markers.
- the level of transcripts of specific markers e.g., FOXC2 or undifferentiation markers e.g., Oct 4, IManog, Sox2, Rexl, Cx43, FGF4 or differentiation markers e.g., albumin, glucagons, -cardiac actin, ⁇ -globulin, Flkl, AC133 and neurofilament can be detected using RNA-based techniques such as RT-PCR analysis and/or cDNA microarray analysis.
- the present invention provides a kit comprising the culture medium or components thereof, feeding medium or components thereof, the cell culture system comprising the normal cells or stem cells or cancer cells or cancer stem-like cells, cell culture containers.
- the kit also comprises written instructions for how to perform the cell culture method.
- compositions of the culture and feeding media, the use of the culture and feeding media of the present invention and the method of culturing as per the present invention allows selectively enriching and maintaining stem cells or cancer stem-like cells in liquid suspension as spheroids and thereby provides ease in separating said stem cells or cancer stem-like cells from differentiated cells.
- the present invention employs passaging suspended cells in liquid serum-free medium to enrich the cells with non-differentiated cells.
- the system is an ideal in vivo mimicking study model for stem cells or cancer stemlike cells.
- compositions of media and methods of culturing of the present invention provides advantages of a well-defined culture system for obtaining spheroids of stem cells or cancer stem-like cells of different origin; shorter culture cycle; reduced exposure to pathogens and ability for the isolation and proliferation of stem cells or cancer stem-like cells. Due to the serum-free nature of the media as per the present invention toxic effects of serum are avoided. Further, sensitive proteins are not degraded by serum proteases. Also, downstream processing of products from such cultures of stem cells or stem-like cells is easier.
- the selectively enriched stem cells or cancer stem-like cells or cell cultures comprising the same as obtained by the present invention can provide a means of exploring basic mechanisms in cancer cell biology and disease, the same can be used advantageously in cancer stem cell research, clinical research and drug screening of anticancer therapeutics.
- the selective enrichment and maintains of stem cells or cancer stem-like cells from a particular tumor or metastatic lesion is useful, for example, in diagnosing a pathology and/or developing a rational therapeutic treatment that targets a developing pathology.
- enrichment and maintains of stem cells or cancer stem-like cells is desirable for further in-vitro studies exploring physiological and molecular mechanisms, wherein in other instances, these cells can be used to inoculate a test animal for further studies of cancer progression or therapy.
- Effects of the test compound on cancer stem cells can also be measured for example by determining the number of cancer stem cells that persist in culture or in the tumors in vivo after treatment with the test compound. In addition to determining the number of cancer stem cells, the effects of the test compound on cancer stem cells, cell cycle status and marker expression can also be determined by flow-cytometry.
- Cells from breast cancer cell line MCF 7 were expanded in Minimal essential Medium MEM containing IX antibiotic, L - glutamine and 10 % FBS in a cell culture plate and incubated at 37°C in a 5% C02 atmosphere. After attaining 90% confluency the cells were washed twice with PBS and trypsinised with 0.25% trypsin -EDTA.
- a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 8ng/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 20ng/ml, Epidermal Growth Factor -EGF lOng/ml, recombinant Insulin lOug/ml, growth factor B27- IX and hydrocortisone lug/ml, and incubated at 37°C in a 5% C0 2 atmosphere. Feeding medium comprising of all the constituents of the culture medium except methyl cellulose was added at three days intervals.
- LNCaP prostate cancer cell line were firstly expanded in Rose Parker Memorial Institute 1640 (RPMI 1640) containing 2mM sodium Pyruvate, 0.075% sodium bicarbonate, IX antibiotic, 2 mM L-glutamine and 10 % FBS in a cell culture plate 100mm and incubated for 24-48 hrs at 37°C in a 5% C02 atmosphere. After attaining 90% confluency, the cells were washed twice with PBS and trypsinised with 0.25% trypsin - EDTA.
- RPMI 1640 Rose Parker Memorial Institute 1640
- the cells were dissected into single cells using a trypsin solution and lOOul of cell suspension containing 2.5xl0 4 /ml cells were seeded into 96 well low attachment plates with the medium as per the present invention comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 4 ng/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 15 ng/ml, Epidermal Growth Factor -EGF 12 ng/ml, recombinant Insulin 15 ug/ml, growth factor B27- 0.5X and hydrocortisone 2 ug/ml, and incubated at 37°C in a 5% C0 2 atmosphere. Feeding medium comprising of all the constituents of the culture medium except methyl cellulose was added at three days intervals.
- Spheres were extracted, passaged three times and newly seeded into the culture medium initially and further supplemented with feeding medium periodically, wherein the composition of culture medium and feeding medium was the same as disclosed above for culturing and feeding. It was observed that after such passaging again new spheres were formed.
- the obtained spheres were dissociated enzymatically with 0.25% trypsin- EDTA and seeded onto a 96 well plate to a density of an average of 1000 cells per well and incubated at 37°C in a 5% C02 atmosphere. This gave large spheroids comprising of prostate cancer/ stem cells or cancer stem-like cells as seen in FIG. 2.
- Cells from breast cancer cell line MCF-IOA were expanded in DMEM/F12 500 ml, supplemented with FBS 10%, ⁇ EGF (100 mg/ml), 250 ⁇ Hydrocortizone (lmg/ml), 50 ⁇ CholeraToxin (lmg/ml), 500 ⁇ Insulin (lOmg/ml and 5.0ml Pen/Strep in a cell culture plate and incubated at 37°C in a 5% C02 atmosphere. After attaining 90% confluency the cells were washed twice with PBS and trypsinised with 0.25% trypsin -EDTA.
- the cells were dissociated into single cells using a trypsin solution and lOOul of cell suspension containing a pre-defined number of (1000) cells were seeded into 96 well low attachment plates in a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 3 ng/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 16 ng/ml, Epidermal Growth Factor -EGF 8 ng/ml, recombinant Insulin 12 g/ml, growth factor B27- IX and hydrocortisone lug/ml, and incubated at 37°C in a 5% C02 atmosphere.
- a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 3 ng/ml, and other growth
- Cells from breast cancer cell line MDA-MB-231 were expanded in DMEM/F12 500 ml, supplemented with containing IX antibiotic, L - glutamine and 10 % FBS in a cell culture plate and incubated at 37°C in a 5% C02 atmosphere. After attaining 90% confluency the cells were washed twice with PBS and trypsinised with 0.25% trypsin -EDTA.
- the cells were dissociated into single cells using a trypsin solution and lOOul of cell suspension containing a pre-defined number of (1000) cells were seeded into 96 well low attachment plates in a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 5 ng/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 20 ng/ml, Epidermal Growth Factor -EGF 10 ng/ml, recombinant Insulin 14 ug/ml, growth factor B27- IX and hydrocortisone lug/ml, and incubated at 37oC in a 5% C02 atmosphere.
- a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 5 ng/ml, and other
- the obtained spheres were dissociated enzymatically with 0.25% trypsin-EDTA and seeded onto a 96 well plate to a density of an average of 1000 cells per well and incubated at 37°C in a 5% C02 atmosphere. This gave large spheroids comprising of breast cancer stem cells or cancer stem-like cells as seen in FIG. 4.
- Example 5 Cells from mouse connective tissue cell line L929 were expanded in DMEM supplemented with 1% Penicillin Streptomycin / L-glutamine and 10% heat inactivated Fetal Bovine Serum at 56°C for 30 min. and 7.5% C02 atmosphere. After attaining 90% confluency the cells were washed twice with PBS and trypsinised with 0.25% trypsin -EDTA.
- a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 6 g/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 18 ng/ml, Epidermal Growth Factor - EGF 8ng/ml, recombinant Insulin 13 ug/ml, growth factor B27- IX and hydrocortisone lug/ml, and incubated at 37°C in a 5% C02 atmosphere. C0 2 atmosphere. Feeding medium comprising of all the constituents of the culture medium except methyl cellulose was added at three days intervals.
- the obtained spheres were dissociated enzymatically with 0.25% trypsin-EDTA and seeded onto a 96 well plate to a density of an average of 1000 cells per well and incubated at 37°C in a 5% C02 atmosphere. This gave large spheroids comprising of breast cancer stem cells or cancer stem-like cells as seen in FIG. 5.
- Primer 1 hFOXC2 (Forward) : lOOmM stock- GCCTAAGGACCTGGTGAAGC
- Primer 2 hFOXC2 (Reverse) : lOOmM stock-TTG ACG AAG CACTCGTTG AG
- Primer 3 hGADPH (Forward): lOOmM stock- ACCCAGAAGACTGTGGATGG
- Primer 4 hGADPH ⁇ (Reverse) : lOOmM stock-TCTAGACGGCAGGTCAGGTC
- PCR products were size- fractionated using 1 % agarose gel electrophoresis using ethidium bromide and 10X orange G dye. DNA markers were used to confirm the size of the resultant fragments.
- Q-PCR quantitative PCR
- FOXC2 results show that FoxC2 expression which is prominent in cancer stem-like cells is highly expressed in -human breast cell line MDA MB 231 and non transformed breast cell line MCF 10 A cultured using the media and the method of the present invention.
Abstract
The present invention provides a culture medium for selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount wherein the culture medium is essentially serum free. The present invention further provides feeding medium comprising components similar to the culture medium except the feeding medium is devoid of methyl cellulose. Present invention still further provides cell cultures comprising stem cells or cancer stem-like cells and the culture medium of the present invention. Also provides are methods for selectively enriching and maintaining stem cells or cancer stem-like cells using the culture medium of the present invention and kits for the same.
Description
Title: Media Compositions, method of initiating and enriching cultures of stem cells and/or cancer stem-like cells
Field of Invention
The present invention relates to media compositions and method for initiating, enriching and maintaining culture of stem cells or cancer stem-like cells. The present invention further relates to culture media of the defined composition and cell cultures comprising the same. Background of the Invention
Stem cells are characterized by their ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialized cells types. Stem cells can be divided into two basic categories i.e. embryonic stem cells that are isolated from embryos and adult stem cells that are found in adult tissues. In embryos, stem cells differentiate to produce the various tissues during growth whereas in adult organisms stem cells and progenitor cells act as repair system for the body replenishing cells and maintain the normal regeneration of regenerative organs for example, oval cells (hepatic stem cells) that produce hepatocytes and bile duct cells, cardiac stem cells that produce cardiomyocytes, "satellite cells" and "myoblasts" produce skeletal muscle cells, neural stem cells that are found in neural tissue such as the brain and spinal cord and produce neurons and glial cells, epidermal stem cells that produce epidermal cells and hair follicle cells, progenitor cells derived from stem cells known as "hematopoietic stem cells", produce blood cells such as erythrocytes, lymphocytes and megakaryocytes. Stem cells are therefore important in regenerative therapy to cures various diseases and heal wounds. There are protocols available to isolate and grow such stem cells for further studies and research. However, such protocols and media compositions contemplated for culturing stem cells are specific and vary as per the origin of stem
cells. Thus, necessitating the use and investing in unreasonable inventory for different media and protocols for culturing stem cells or cancer stem cells. This makes it challenging and an uneconomical proposition for researchers as well as those in the industrial set up equally causing hindrances in expanding the scope of research or utility of different types of stem cells and/or cancer stem-like cells.
Further, recently, it has been proposed that stem-like cells are found within cancerous tumors, which possesses characteristics of stem cells and are considered to be the cause of tumor formation and recurrence. Such cells are proposed to persist in tumors as a distinct population with the ability to give rise to all cell types found in cancerous tumor as well as replenish them from time to time and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at Cancer stem cells or cancer stem-like cells holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. It is considered difficult to separate CSCs from tumors cells. Therefore, it is desirable to develop culture medium, method for obtaining cultures with enriched cancer stem cells or cancer stem-like cells and cultures comprising the same for further use. However, it is challenging to isolate, culture and enrich various types of cancer stem cells or cancer stem-like cells. This may be primarily because cancer stem-like cells differ based on their nature of origin. Cancer stem-like cells derived from breast cancer differs substantially in growth requirements for ln-vitro culturing as opposed to cancer stem-like cell derived from epithelial tissue or prostrate tissue. Besides, most culturing media available in the market are specific to the different types of cancer cells and require addition of fetal bovine serum (FBS) which is considered as crucial component. Addition of FBS poses additional problems of contamination with pathogens and bacteria.
Thus, problems with existing media are that they are suitable for only selective stem cells proliferation and cannot be used for all cells lines for proliferation of stem cells/ stem like-cells. This creates a unique challenge in devising appropriate culture media and techniques for growth and proliferation of stem cells or cancer stem-like cells. Thus, there is a need in the art of media composition free of fetal bovine serum (FBS), culturing protocols, which can be used mostly universally without much deviation for initiating, enriching and maintaining cultures of different types of stem cells or cancer stem-like cells as well as cell cultures comprising the same. Summary of the Invention
The present invention in one aspect provides compositions for media for culturing, selectively enriching and maintaining stem cells or cancer stem-like cells which is essentially serum free.
Accordingly, in an embodiment, the present invention provides a composition for a culture medium for selectively enriching and maintaining stem cells or cancer stemlike cells comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount, wherein the composition is essentially serum free.
In another aspect the present invention provides a composition for feeding media for selectively enriching and maintaining stem cells or cancer stem-like cells.
In one of the embodiments in accordance with the above aspect, the present invention provides a composition for a feeding media for selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, and optionally growth factors or other supplementary elements in an effective amount wherein the composition is essentially serum free.
In another aspect the present invention provides a culture media for selectively enriching and maintaining stem cells or cancer stem-like cells.
In one embodiment in accordance with the above aspect, the present invention provides a culture medium for selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount wherein the culture medium is essentially serum free.
In another aspect the present invention provides a feeding media for selectively enriching and maintaining stem cells or cancer stem-like cells.
In one embodiment in accordance with the above aspect, the present invention provides a feeding medium for selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, and optionally growth factors or other supplementary elements in an effective amount wherein the feeding medium is essentially serum free.
In one additional aspect of the present invention there is provided a cell culture which comprises the stem cells or cancer stem-like cells and any of the culture media of the present invention.
In one embodiment in accordance with the above aspect, the present invention provides a cell culture comprising cancer stem-like cells in essentially serum free culture medium comprised of basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an amount sufficient for maintaining stem cells or cancer stem-like cells.
In one embodiment in accordance with the above aspect, the present invention provides a cell culture comprising stem cells in essentially serum free culture medium comprised of basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an amount sufficient for maintaining stem cells or cancer stem-like cells.
In another aspect the present invention provides a method for isolation and proliferation of stem cells or cancer stem-like cells.
In one embodiment in accordance with the above aspect, the present invention provides a method comprising steps of culturing the single cell suspension of cancer cells or normal cells in the culture medium of the present invention comprising the basic nutrients, heparin, gelatin and methyl cellulose at 37°C in a 5% C02 atmosphere and adding feeding medium to the growing culture intermittently to obtain spheres of stem cells or cancer stem-like cells.
In another aspect the present invention provides a kit for use in the method of the present invention.
In one embodiment in accordance with the above aspect, the present invention provides a kit comprising the culture medium or components thereof, feeding medium or components thereof, the cell culture system comprising the stem cells or cancer stem-like cells, cell culture containers. The kit may also further comprise written instructions for how to perform the cell culture method.
Brief Description of the Drawings
The invention is herein described with reference to the accompanying drawings. With specific reference now to the drawings, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. The various objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:
Figure 1: Shows photomicrograph of spheroids formed from the cultured MCF7 breast cancer cells as per Example 1.
Figure 2: Shows photomicrograph of spheroids formed from the cultured LNCAP prostate cancer cells as per Example 2. Figure 3: Shows photomicrograph of spheroids formed from the cultured MCF-IOA breast cancer cells as per Example 3.
Figure 4: Shows photomicrograph of spheroids formed from the cultured MDA-MB- 231 breast cancer cells as per Example 4.
Figure 5: Shows photomicrograph of spheroids formed from the cultured L 929 mouse connective tissue cells as per Example 5.
Figures 6A - 6C: shows RT-PCR - gene expression analysis of spheroids of cells obtained in Example 3-5.
FIG 6A:
Upper Gel: Lane 1: Marker 1- lOObp, Lane 2: Marker 2- 1Kb, Lane 3-4: primer quantification of primers 1 & 2 FOXC2 I, Lane 5-6: Lane(3-6) primer quantification of primers 3 & 4 GADPH 1.
Lower Gel : Lane 1: Marker 1 lOObp, Lane 2: Marker 2 - 1Kb, Lane 3: cDNA : negative control : 1 ul :no band, Lane 4: Enzyme negative control: no band, Lane 5: Enzyme + water negative control :no band, Lane 6: cDNA :MCF 10 A - faint band seen near the periphery of well, Lane 7: cDIMA : L929 - faint band seen near the periphery of well, Lane 8: cDNA: MDA MB 231 : faint band seen near the periphery of well.
FIG 6B:
Upper Gel: Lane 1: Ladder - indicating 494bps, Lane 2: Control 1: good prominent band, Lane 3: Control 2: good prominent band, Lane 4: Negative control : enzyme negative, Lane 5: MCF 10 A: good prominent band, Lane 6: L929 : good prominent
band, Lane 7: MDA MB 231: good prominent band
Lower Gel: Lane 1: Ladder - indicating 494bps, Lane 2: Blank Lane, Lane 3: Control : good prominent band, Lane 4: Negative control*: enzyme negative, Lane 5: MCF 10 A: good prominent band, Lane 6: L929 : good prominent band, Lane 7: MDA MB 231: good prominent band, Lane 8: Negative: negative .
FIG 6C:
Upper gel: Lane 1: Ladder - indicating 494bps, Lane 2: Control 2: good prominent band, Lane 3: Negative control : enzyme negative: no band seen, Lane 4: MCF 10 A: with P1/P2 2ul cDNA .-Faint band, Lane 5: L929 : with P1/P2 : 2ul cDNA No band, Lane 6: MDA MB 231: with P1/P2: 2ul cDNA faint band, Lane 7: Control 2: 5ul cDNA good prominent band, Lane 8: Negative control : enzyme negative: no band seen, Lane 9: MCF 10 A: with P1/P2 5ul cDNA:Faint band, Lane 10: L929 : with P1/P2 5ul cDNA: No band, Lane 11: MDA MB 231: with P1/P2 5ul cDNA: faint band.
Lower Gel: Lane 1: Ladder - indicating 494bps, Lane 2: Control 2: good prominent band, Lane 3: Negative control : enzyme negative: no band seen, Lane 4: MCF 10 A: with P3/P4 2ul cDNA : prominent band, Lane 5: L929 : with P3/P4 : 2ul cDNA prominent band, Lane 6: MDA MB 231: with P3/P4: 2ul cDNA : prominent band, Lane 7: Control 2: 5ul cDNA good prominent band, Lane 8: Negative control : enzyme negative: no band seen, Lane 9: MCF 10 A: with P3/P4 5ul cDNA: prominent band, Lane 10: L929 : with P3/P4 5ul cDNA:Faint band, Lane 11: MDA MB 231: with P3/P4 5ul cDNA: prominent band.
Detailed Description of the Invention
In the following detailed description, various specific details are set forth in order to provide a better understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present
invention.
The present invention is directed towards obviating the problems associated with initiation, proliferation and maintenance of cultures of stem cells or cancer stem-like cells. More particularly the present invention is directed to selectively enriching and maintaining the population of stem cells or cancer stem-like cells and to make them accessible for further research, regenerative therapies, clinical investigations or drug screening for anticancer drug therapeutics.
Accordingly, the present invention is directed towards providing compositions for media for example culture medium and feeding medium, for culturing, selectively enriching and maintaining stem cells or cancer stem-like cells.
In an embodiment, the present invention provides a composition of a medium for selectively enriching and maintaining stem cells or cancer stem-like cells originating from cancerous and non-cancerous tissue comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount wherein the composition is essentially serum free.
As used herein the phrase "serum free" refers to being devoid of a human or an animal serum.
According to some embodiments of the invention, the serum free media compositions do not comprise of serum or portions thereof. The basic nutrients refers to a mixture comprising of salts, amino acids, vitamins that provide cells with water and certain bulk inorganic ions essential for normal cell metabolism, maintain intra- and extra-cellular osmotic balance, provide a carbohydrate as an energy source, and provide a buffering system to maintain the medium within the physiological pH range. The basic nutrients may be formulated into a base media by mixing the above mentioned nutrient components. Alternately,
basic nutrients may be incorporated based on the known basal media as such, or after modification. Non-limiting examples of basic nutrients to be incorporated based on the known basal media include Dulbecco's Modified Eagle's Medium (DMEM), DMEM/F-12, or KO-DMEM, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), BGJb Medium, RPM1 1640, Ham's F-10, Ham's F-12, ex- Minimal Essential Medium (ctMEM), Brinster's BMOC-3 Medium, C02-lndependent Medium, CMRL Medium, Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium, Waymouth's MB 752/1 Media, Williams Media E, Medium NCTC-109, neuroplasma medium, Leibovitz's L-15 Media, McCoy's 5A Media (modified), MCDB 131 Mediumor the likes and/or mixtures thereof.
According to some embodiments of the invention, gelatin is present typically from about 0.001% to about 0.01% w/v, for example from about 0.001% to about 0.5% w/v, for example from about 0.001% to about 0.05% w/v, or for example from about 0.001% to about 0.01% w/v. Preferably gelatin is incorporated in an amount from about 0.001% to about 0.01% w/v.
According to some embodiments of the invention, methyl cellulose is present typically from about 0.01% to about 10% w/v, for example from about 0.01% to about 5% w/v, for example from about 0.05 % to about 2.5% w/v, or for example from about 0.05 % to about 1.5% w/v. According to some embodiments of the invention, the composition further comprises heparin. According to some embodiments the concentration of said heparin is from about 0 ng/ml to about 100 ng/ml, for example from about 2 ng/ml to about 50 ng/ml, for example from about 2.5 ng/ml to about 25 ng/ml, for example from about 5 ng/ml to about 15 ng/ml, for example from about 4 ng/ml to about 10 ng/ml, for example from about 4 ng/ml to about 8 ng/ml.
The composition of a medium for selectively enriching and maintaining stem cells or cancer stem-like cells of the present invention further comprises growth factors selected from the group consisting of but not limited to, members of the epidermal growth factor family (EGFs), members of the fibroblast growth factor family (FGFs), members of the hydrocortisone growth factor family, B27 supplement, insulin and/or other suitable growth factors.
Preferably, one or more growth factors and insulin to be incorporated in the composition are recombinantly produced molecules. Alternately, they may be isolated from natural sources. With regard to the growth factors and insulin proteins, the invention also contemplates the use of homologs, or proteins having sequence identity of at least about 70% and the receptor activating activity of the respective naturally occurring protein that is growth factors or insulin, artificial analogs, polypeptide fragments that activate the respective growth factors or insulin receptor and/or downstream signaling, and other molecules that activate one or more growth factors or insulin receptors and/or their downstream signaling.
According to some embodiments of the invention, the composition comprises Basic fibroblast growth factor (also known as bFGF, FGF2 or FGF-β) a member of the fibroblast growth factor family. In some embodiments the concentration of said bFGF is from about 5 ng/ml to about 100 ng/ml, for example from about 10 ng/ml to about 50 ng/ml, for example from about 10 ng/ml to about 25 ng/ml, for example from about 15 ng/ml to about 25 ng/ml.
According to some embodiments of the invention, the composition comprises Epidermal growth factor (also known as EGF). In some embodiments the concentration of said EGF is from about 1 ng/ml to about 100 ng/ml, for example from about 2.5 ng/ml to about 50 ng/ml, for example from about 2.5 ng/ml to about 25 ng/ml, for example from about 5 ng/ml to about 15 ng/ml.
According to some embodiments of the invention, the composition comprises insulin. In some embodiments the concentration of said insulin is from about 1 §/ηιΙ to about 100 μg/ml, for example from about 2.5 μg/ml to about 50 μg/ml, for example from about 2.5 μg/ml to about 25 μg/ml, for example from about 5 μg/ml to about 15 μg/ml.
According to some embodiments of the invention, the composition comprises hydrocortisone. In some embodiments the concentration of said hydrocortisone is at least about 0.1 μg/ml, for example at least about 2.5 μg/ml, for example at least about 5 μg/ml, for example at least about 7.5 μg/ml, for example at least about 1 g ml.
According to some embodiments of the invention, the composition comprises B27 supplement. One of the commercially available B27 supplement is without vitamin A which is available from Gibco-lnvitrogen, Corporation, Grand Island, NY USA, Catalogue No. 12587-010. The B27 supplement is a serum-free formulation which includes d-biotin, fatty acid free fraction V bovine serum albumin (BSA), catalase, L- carnitine HC1, corticosterone, ethanolamine HC1, D-galactose (Anhyd.), glutathione (reduced), recombinant human insulin, linoleic acid, linolenic acid, progesterone, putrescine-2-HCI, sodium selenite, superoxide dismutase, T- 3/albumin complex, DL alpha-tocopherol and DL alpha tocopherol acetate. However, the use of B27 supplement is limited since it includes albumin from an animal source. When incorporated, it may be included at the concentration of IX to 5X.
The composition may further comprise of supplementary nutrients for example L- glutamine or stable glutamine. The composition of a medium according to the invention can also be supplemented with any compound(s) that will not interfere with, and preferably supports the enrichment and/or maintenance of stem cells or cancer stem-like cells over time. Preferred examples of such compounds include
non-essential amino acids, anti-oxidants, reducing agents, vitamins, organic compounds, inorganic salts, transferring, and albumins.
Preferably, all ingredients included in the composition of the present invention are substantially pure, with a tissue culture grade. In one embodiment, the present invention provides a culture medium. The culture medium for culturing, selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount wherein the culture medium is essentially serum free. As used herein the phrase "culture medium" refers to a solid or a liquid substance used to support the growth of stem cells and selectively enriching and maintaining stem cells or cancer stem-like cells. Preferably, the phrase "culture medium" as used herein refers to a liquid substance.
In one of the preferred embodiments the present invention provides a medium for initiating, selectively enriching and maintaining stem cells or cancer stem-like cells originating from cancerous and non-cancerous cells comprising gelatin, heparin, the basic nutrients comprising of an approximately 50:50 mixture of DMEM and Ham's F12 nutrients, L- glutamine or stable glutamine, basic Fibroblast Growth Factor- FGF, Epidermal Growth Factor -EGF, recombinant growth factor Insulin, B27 supplement, and hydrocortisone. The medium when used as a culture medium is supplemented with methyl cellulose prior to use. The medium may be used as such, as a feeding medium.
In another preferred embodiments the present invention provides a medium for initiating, selectively enriching and maintaining stem cells or cancer stem-like cells originating from cancerous and non-cancerous cells comprising gelatin present at concentration from about 0.001% to about 0.01% w/v, methyl cellulose present at
concentration from about 0.05 % to about 1% w/v, heparin present at the concentration from about 4 ng/ml to about 8 ng/ml, the basic nutrients comprising of an approximately 50:50 mixture of DMEM and Ham's F12 nutrients, basic Fibroblast Growth Factor- bFGF present at concentration from about 15 ng/ml to about 25 ng/ml, Epidermal Growth Factor -EGF present at concentration from about 5 ng/ml to about 15 ng/ml, Insulin present at concentration from about 5 g/ml to about 15 μg/ml, B27 supplement IX to 5X, and hydrocortisone present at concentration of at least about 1 g/ml. The medium when used as a culture medium is supplemented with prior to use. The medium may be used as such, as a feeding medium.
The normal cells as well as cancer cells of various origins may be used in the present invention as a source of stem cells or cancer stem-like cells. In one of the embodiments, stem cells or cancer stem-like cells of the present invention are derived from a cell line. In another embodiment, stem cells or cancer stem-like cells of the present invention are derived from a primary cell culture.
In another embodiment, stem cells of the present invention are derived using a well known protocol from blood, or from tissues or from various organs comprising the stem cells such tissues or organs may be selected from but not limiting to liver, bile duct, cardiac tissue, skeletal muscles, neural tissue from brain and spinal cord, epidermal tissue, skin, hair follicle or any other organ or tissue comprising the stem cells.
In another embodiment, the primary cell culture comprising cancer stem-like cells is derived from a tumor or cell metastasis. In another embodiment, tumors and cell metastasis are derived from but not limited to: prostate cancer, breast cancer, carcinoid tumor, carcinoma, cervical cancer,
colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewings family of tumors (pnet), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, laryngeal cancer, adrenocortical carcinoma/ anal cancer, bladder cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal, pineal tumors, hypothalamic glioma, leukemia, acute lymphoblastic, leukemia, oral cavity cancer, liver cancer, lung cancer, small cell, lymphoma, AJDS- related, lymphoma, central nervous system (primary), lymphoma, cutaneous T-cell, lymphoma, hodgkin's disease, non-hodgkin's disease, malignant mesothelioma, melanoma, merkel cell carcinoma, metasatic squamous carcinoma, multiple myeloma, plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, exocrine, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary cancer, plasma cell neoplasm, rhabdomyosarcoma, rectal cancer, renal cell cancer, salivary gland cancer, sezary syndrome, skin cancer, cutaneous T-cell lymphoma, skin cancer, kaposi's sarcoma, skin cancer, melanoma, small intestine cancer, soft tissue sarcoma, soft tissue sarcoma, testicular cancer, thymoma, malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, unusual cancer of childhood, vaginal cancer, vulvar cancer, or wilms' tumor. In one additional aspect of the present invention there is provided a cell culture which comprises the stem cell or cancer stem-like cells and any of the culture media described hereinabove.
The stem cell or cancer stem-like cells or cell cultures comprising the same are capable of keeping their self-renewal potential during 1-100 passages of in-vitro cultivation. In another embodiment, stem cell or cancer stem-like cells are capable of keeping their self-renewal potential during 1-90 passages of in-vitro cultivation. In another embodiment, stem cell or cancer stem-like cells are capable of keeping their self-renewal potential during 20-60 passages of in-vitro cultivation.
In another embodiment of the present invention, a method is provided for isolation and proliferation of stem cells or cancer stem-like cells, in which, a method comprises steps of cuituring the single cell suspension of cancer cells or normal cells in culture medium as per the present invention comprising the basic nutrients, heparin, gelatin and methyl cellulose at 37°C in a 5% C02 atmosphere and adding feeding medium to the growing culture intermittently to obtain spheres of stem cells or cancer stem-like cells. The propagation time for generation of spheroids of stem cells or cancer stem-like cells by the culture method and using the culture medium with methyl cellulose as well as feeding medium of the present invention is shorter compared to the other known protocols. Spheres or spheroids of stem cells or cancer stem-like cells may be obtained by the method and media of the present invention in 5-10 days depending upon the type of cells / cell line and cell density plated. Unless stated otherwise the terms "spheroid" and "spheres" are used interchangeably herein and refer to a certain "ball-shaped" globular structure, consisting of more than a single cell that has initially developed from a single or from multiple cells.
The single cell suspension of the normal cells or cancer cells is cultured as per the present invention method at an appropriate cell density. The cell density may range from about 1X104 cells /ml to 1X106 cells/ml. For obtaining the single cell suspension of normal cells or cancer cells for culturing as per the method of the present invention, normal cells or cancer cells are expanded by incubating them in a complete minimal essential medium supplemented with antibiotic, vitamins and
serum for period of 24-48 hours at about 37 degree centigrade in a 5% C02 atmosphere. On attaining the 90% confluency, cells are washed twice with DPBS and trypsinised using 0.25% trypsin-EDTA at 37°C in a 5% C02 atmosphere for 2-5 minutes and cells are dissociated into a single cell suspension, which are initially cultured in a 96 well non-treated plate with the medium as per the present invention.
It will be appreciated that although single-cell suspensions of stein cells are usually seeded, small clusters may also be used. While using the small clusters, enzymatic digestion (such as with type IV collagenase) utilized for cluster disruption is terminated before stem cells become completely dispersed and the cells are triturated with a pipette such that clumps (i.e., 10-200 cells) are formed. However, measures are taken to avoid large clusters which may cause cell differentiation.
For further enriching the stem cells or cancer stem-like cells, the spheres or spheroids comprising stem cells or cancer stem-like cells are isolated from the primary growing sphere cultures and passaged in the culture medium of the present invention, further supplemented periodically with the feeding medium to obtain large spheres comprising of stem cells or cancer stem-like cells. In one of the embodiments, the methods of the present invention provide that stem cells or cancer stem-like cells can be passaged without loosing their respective phenotype for at least 5 passages. In other embodiments, the methods of the present invention provide that stem cells or cancer stem-like cells can be passaged without loosing their respective phenotype for at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 passages.
According to some embodiments of the invention, when cultured according to the teachings of the present invention, the growth of the stem cells or cancer stem-like cells is monitored. Monitoring the formation of spheres or spheroids comprising stem cells or cancer stem-like cells is within the capabilities of those skilled in the art
and can be effected by morphological evaluations and determination of expression of differentiation-specific markers [e.g., using immunological techniques or RNA- based analysis (e.g., RT-PCR, cDIMA microarray)].
In some embodiments, the growth of the stem cells or cancer stem-like cells is monitored to determine their differentiation state. The differentiation state can be determined using various approaches including, for example, morphological evaluation (e.g., as shown in Figures 1-5) and/or detection of the expression pattern of typical markers of the undifferentiated state using immunological techniques such as flow cytometry for membrane-bound markers, immunohistochemistry or immunofluorescence for extracellular and intracellular markers and enzymatic immunoassay, for secreted molecular markers. For example, the level of transcripts of specific markers e.g., FOXC2 or undifferentiation markers e.g., Oct 4, IManog, Sox2, Rexl, Cx43, FGF4 or differentiation markers e.g., albumin, glucagons, -cardiac actin, β-globulin, Flkl, AC133 and neurofilament can be detected using RNA-based techniques such as RT-PCR analysis and/or cDNA microarray analysis.
In one additional embodiment, the present invention provides a kit comprising the culture medium or components thereof, feeding medium or components thereof, the cell culture system comprising the normal cells or stem cells or cancer cells or cancer stem-like cells, cell culture containers. Optionally, the kit also comprises written instructions for how to perform the cell culture method.
Compositions of the culture and feeding media, the use of the culture and feeding media of the present invention and the method of culturing as per the present invention allows selectively enriching and maintaining stem cells or cancer stem-like cells in liquid suspension as spheroids and thereby provides ease in separating said stem cells or cancer stem-like cells from differentiated cells.
In particular, the present invention employs passaging suspended cells in liquid serum-free medium to enrich the cells with non-differentiated cells. Additionally, the system is an ideal in vivo mimicking study model for stem cells or cancer stemlike cells. The compositions of media and methods of culturing of the present invention provides advantages of a well-defined culture system for obtaining spheroids of stem cells or cancer stem-like cells of different origin; shorter culture cycle; reduced exposure to pathogens and ability for the isolation and proliferation of stem cells or cancer stem-like cells. Due to the serum-free nature of the media as per the present invention toxic effects of serum are avoided. Further, sensitive proteins are not degraded by serum proteases. Also, downstream processing of products from such cultures of stem cells or stem-like cells is easier.
The selectively enriched stem cells or cancer stem-like cells or cell cultures comprising the same as obtained by the present invention can provide a means of exploring basic mechanisms in cancer cell biology and disease, the same can be used advantageously in cancer stem cell research, clinical research and drug screening of anticancer therapeutics. The selective enrichment and maintains of stem cells or cancer stem-like cells from a particular tumor or metastatic lesion is useful, for example, in diagnosing a pathology and/or developing a rational therapeutic treatment that targets a developing pathology. In some instances, enrichment and maintains of stem cells or cancer stem-like cells is desirable for further in-vitro studies exploring physiological and molecular mechanisms, wherein in other instances, these cells can be used to inoculate a test animal for further studies of cancer progression or therapy. Effects of the test compound on cancer stem cells can also be measured for example by determining the number of cancer stem cells that persist in culture or in the tumors in vivo after treatment with the test compound. In addition to determining the number of cancer stem cells, the effects of the test compound on cancer stem cells, cell cycle status and marker expression can also be
determined by flow-cytometry.
The invention will be further described by way of examples providing specific details thereof. It should be noted that the examples are only for the purpose of illustration and do not in any way limit the scope of the invention. The various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above description illustrate the invention in a non limiting fashion. Many changes and modifications may be made within the scope of the embodiments and examples disclosed herein without departing from the true spirit of the invention. The invention herein includes all such modifications. Example 1:
Cells from breast cancer cell line MCF 7 were expanded in Minimal essential Medium MEM containing IX antibiotic, L - glutamine and 10 % FBS in a cell culture plate and incubated at 37°C in a 5% C02 atmosphere. After attaining 90% confluency the cells were washed twice with PBS and trypsinised with 0.25% trypsin -EDTA. Later the cells were dissociated into single cells using a trypsin solution and lOOul of cell suspension containing a pre-defined number of (1000) cells were seeded into 96 well low attachment plates in a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 8ng/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 20ng/ml, Epidermal Growth Factor -EGF lOng/ml, recombinant Insulin lOug/ml, growth factor B27- IX and hydrocortisone lug/ml, and incubated at 37°C in a 5% C02 atmosphere. Feeding
medium comprising of all the constituents of the culture medium except methyl cellulose was added at three days intervals.
The cell culture split up into adherent cells and non-adherent cells during growth and enrichment, wherein the non-adherent cells developed close packed and hollow acinaric spheres. After spheres formation was visibly detected, they were isolated, passaged and newly seeded into the culture medium initially and further supplemented with feeding medium periodically. Wherein, the composition of the culture medium and the feeding medium was the same as disclosed above for culturing and feeding. It was observed that after such passaging, again new spheres were formed. The obtained spheres were dissociated enzymatically with 0.25% trypsin-EDTA and seeded onto a 96 well plate to a density of an average of 1000 cells per well and incubated at 37°C in a 5% C02 atmosphere. This gave large spheroids comprising of breast cancer stem cells or cancer stem-like cells as seen in FIG. 1.
Example 2:
Cells form LNCaP prostate cancer cell line were firstly expanded in Rose Parker Memorial Institute 1640 (RPMI 1640) containing 2mM sodium Pyruvate, 0.075% sodium bicarbonate, IX antibiotic, 2 mM L-glutamine and 10 % FBS in a cell culture plate 100mm and incubated for 24-48 hrs at 37°C in a 5% C02 atmosphere. After attaining 90% confluency, the cells were washed twice with PBS and trypsinised with 0.25% trypsin - EDTA. Later the cells were dissected into single cells using a trypsin solution and lOOul of cell suspension containing 2.5xl04 /ml cells were seeded into 96 well low attachment plates with the medium as per the present invention comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 4 ng/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 15 ng/ml, Epidermal Growth Factor -EGF 12 ng/ml, recombinant Insulin 15 ug/ml,
growth factor B27- 0.5X and hydrocortisone 2 ug/ml, and incubated at 37°C in a 5% C02 atmosphere. Feeding medium comprising of all the constituents of the culture medium except methyl cellulose was added at three days intervals.
At around 5-10 days formation of spheres was detectable. Spheres were extracted, passaged three times and newly seeded into the culture medium initially and further supplemented with feeding medium periodically, wherein the composition of culture medium and feeding medium was the same as disclosed above for culturing and feeding. It was observed that after such passaging again new spheres were formed. The obtained spheres were dissociated enzymatically with 0.25% trypsin- EDTA and seeded onto a 96 well plate to a density of an average of 1000 cells per well and incubated at 37°C in a 5% C02 atmosphere. This gave large spheroids comprising of prostate cancer/ stem cells or cancer stem-like cells as seen in FIG. 2.
Example 3:
Cells from breast cancer cell line MCF-IOA were expanded in DMEM/F12 500 ml, supplemented with FBS 10%, ΙΟΟμΙ EGF (100 mg/ml), 250μΙ Hydrocortizone (lmg/ml), 50μΙ CholeraToxin (lmg/ml), 500μΙ Insulin (lOmg/ml and 5.0ml Pen/Strep in a cell culture plate and incubated at 37°C in a 5% C02 atmosphere. After attaining 90% confluency the cells were washed twice with PBS and trypsinised with 0.25% trypsin -EDTA. Later the cells were dissociated into single cells using a trypsin solution and lOOul of cell suspension containing a pre-defined number of (1000) cells were seeded into 96 well low attachment plates in a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 3 ng/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 16 ng/ml, Epidermal Growth Factor -EGF 8 ng/ml, recombinant Insulin 12 g/ml, growth factor B27- IX and hydrocortisone lug/ml, and incubated at 37°C in a 5% C02
atmosphere.
The cell culture split up into adherent cells and non-adherent cells during growth and enrichment, wherein the non-adherent cells developed close packed and hollow acinaric spheres. At around 5-10 days formation of spheres was detectable. Spheres were extracted, isolated, passaged and newly seeded into the culture medium initially and further supplemented with feeding medium periodically. Wherein, the composition of the culture medium and the feeding medium was the same as disclosed above for culturing and feeding. It was observed that after such passaging, again new spheres were formed. The obtained spheres were dissociated enzymatically with 0.25% trypsin-EDTA and seeded onto a 96 well plate to a density of an average of 1000 cells per well and incubated at 37°C in a 5% C02 atmosphere. This gave large spheroids comprising of breast cancer stem cells or cancer stem-like cells as seen in FIG. 3. Example 4:
Cells from breast cancer cell line MDA-MB-231 were expanded in DMEM/F12 500 ml, supplemented with containing IX antibiotic, L - glutamine and 10 % FBS in a cell culture plate and incubated at 37°C in a 5% C02 atmosphere. After attaining 90% confluency the cells were washed twice with PBS and trypsinised with 0.25% trypsin -EDTA. Later the cells were dissociated into single cells using a trypsin solution and lOOul of cell suspension containing a pre-defined number of (1000) cells were seeded into 96 well low attachment plates in a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 5 ng/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 20 ng/ml, Epidermal Growth Factor -EGF 10 ng/ml, recombinant Insulin 14 ug/ml, growth factor B27- IX and hydrocortisone lug/ml, and incubated at 37oC in a 5% C02 atmosphere.
The cell culture split up into adherent cells and non-adherent cells during growth and enrichment, wherein the non-adherent cells developed close packed and hollow acinaric spheres. At around 5-10 days formation of spheres was detectable. Spheres were extracted, isolated, passaged and newly seeded into the culture medium initially and further supplemented with feeding medium periodically. Wherein, the composition of the culture medium and the feeding medium was the same as disclosed above for culturing and feeding. It was observed that after such passaging, again new spheres were formed. The obtained spheres were dissociated enzymatically with 0.25% trypsin-EDTA and seeded onto a 96 well plate to a density of an average of 1000 cells per well and incubated at 37°C in a 5% C02 atmosphere. This gave large spheroids comprising of breast cancer stem cells or cancer stem-like cells as seen in FIG. 4.
Example 5: Cells from mouse connective tissue cell line L929 were expanded in DMEM supplemented with 1% Penicillin Streptomycin / L-glutamine and 10% heat inactivated Fetal Bovine Serum at 56°C for 30 min. and 7.5% C02 atmosphere. After attaining 90% confluency the cells were washed twice with PBS and trypsinised with 0.25% trypsin -EDTA. Later the cells were dissociated into single cells using a trypsin solution and lOOul of cell suspension containing a pre-defined number of (1000) cells were seeded into 96 well low attachment plates in a culture medium comprising of basic nutrients of DMEM:F12 medium emulsified with 1% methyl cellulose and further supplemented with gelatin 0.001%, heparin 6 g/ml, and other growth factors including stable glutamine 1 %, Fibroblast Growth Factor- FGF 18 ng/ml, Epidermal Growth Factor - EGF 8ng/ml, recombinant Insulin 13 ug/ml, growth factor B27- IX and hydrocortisone lug/ml, and incubated at 37°C in a 5% C02 atmosphere. C02 atmosphere. Feeding medium comprising of all the
constituents of the culture medium except methyl cellulose was added at three days intervals.
The cell culture split up into adherent cells and non-adherent cells during growth and enrichment, wherein the non-adherent cells developed close packed and hollow acinaric spheres. At around 5-10 days formation of spheres was detectable. Spheres were extracted, isolated, passaged and newly seeded into the culture medium initially and further supplemented with feeding medium periodically. Wherein, the composition of the culture medium and the feeding medium was the same as disclosed above for culturing and feeding. It was observed that after such passaging, again new spheres were formed. The obtained spheres were dissociated enzymatically with 0.25% trypsin-EDTA and seeded onto a 96 well plate to a density of an average of 1000 cells per well and incubated at 37°C in a 5% C02 atmosphere. This gave large spheroids comprising of breast cancer stem cells or cancer stem-like cells as seen in FIG. 5.
Example 6:
RT-PCR Analysis:
10-14 day-old spheres were harvested for RNA isolation and cDNA synthesis.
RT-PCR of cells cultured on 3-D culture systems - Total RNA was isolated from 10-14 day-old spheres on three-dimensional culture systems in the culture media having composition as mentioned above in examples 3, 4 and 5 respectively for respective cell types MCF10A, MDA MB 231 and L929. RNA was isolated by following protocol provided in instruction manual pages 25 - 30 of Qiagen RneasyMini Kit. cDNA was synthesized as per the protocol provided in instruction manual of PureExtreme® by Fermentas Life Science.
Primer sets used were:
Primer 1 :hFOXC2 (Forward) : lOOmM stock- GCCTAAGGACCTGGTGAAGC
Primer 2 :hFOXC2 (Reverse) : lOOmM stock-TTG ACG AAG CACTCGTTG AG
Primer 3 :hGADPH (Forward): lOOmM stock- ACCCAGAAGACTGTGGATGG
Primer 4 :hGADPH{ (Reverse) : lOOmM stock-TCTAGACGGCAGGTCAGGTC
The primers were quantified upon re-constitution and appropriate enzyme and positive and negative controls were used in the PCR assay.. RNA was extracted from breast and prostrate cancer stem cells harvested in the existing medium, using the RNeasy Mini Kit with an on-column DNase I digest (Qiagen). First-strand cDNA was synthesized from 1 μg total RNA, which was synthesized in a 20 μΙ reaction by reverse transcription using Superscript II (Fermentas). The following conditions for cDNA synthesis were applied: 37°C for 60 minutes and cooling for 1 minute at 4°C, then 42°C for 50 minute and 72°C for 15 minutes. In a 50 μΙ PCR reaction mixture, 2 μΙ cDNA (total 100 ng RNA) were amplified in eppendorf master cycler with incubation at 94°C for 3 minutes, followed by 30 to 40 cycles of a three temperature program of 30sec at 94°C, 30 seconds at 58°C, and 45 seconds at 72°C. The PCR„ reaction was terminated after a 10 minute extension at 70°C and stored at 4°C until analysis.. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control (Table 2). All inhouse primers were synthesized by SIGMA. PCR products were size- fractionated using 1 % agarose gel electrophoresis using ethidium bromide and 10X orange G dye. DNA markers were used to confirm the size of the resultant fragments. For quantitative PCR (Q-PCR), densitometry of tested genes was normalized to GAPDH. Three repeats were conducted for each tested line.
Results : As seen in Gel picture (FIG. 6A) primer quantification shows that intact primers were used for PCR.
As seen in gel picture (FIG. 6B) GADPH primer P3 & P4 (Fermentas) as well as PI & P2 designed in-house helped in normalization of mRNA used in assay/ served as good house keeping genes for all the cell lines.
As seen in gel picture (FIG. 6C) FOXC2 results show that FoxC2 expression which is prominent in cancer stem-like cells is highly expressed in -human breast cell line MDA MB 231 and non transformed breast cell line MCF 10 A cultured using the media and the method of the present invention.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Thus, it is understood that any dependent claim among the appended claims merely represents particular embodiments within the scope of the subject matter bounded by the claim(s) from which the claim depends, and the inventors reserve the right to pursue subject matter that is within the scope of a more broad claim but is not specifically recited in an appended claim.
Claims
Claims :
A culture medium for selectively enriching and maintaining stem cells or cancer stem-like cells comprising basic nutrients, gelatin, methyl cellulose and optionally growth factors or other supplementary elements in an effective amount wherein the culture medium is essentially serum free.
The culture medium as claimed in claim 1, wherein said gelatin is present from about 0.001% to about 0.01% w/v
The culture medium as claimed in claim 1, wherein said methyl cellulose is present from about 0.01% to about 10% w/v,
The culture medium as claimed in claim 1, wherein said medium further comprises heparin at concentration from about 0 ng/ml to about 100 ng/ml. The culture medium as claimed in claim 1, wherein said medium further comprises growth factors selected from the group consisting of but not limited to, members of the epidermal growth factor family, members of the fibroblast growth factor family (FGFs), members of the hydrocortisone growth factor family, B27 supplement and insulin.
The culture medium as claimed in claim 5, wherein said fibroblast growth factor family member is bFGF present at concentration from about 5 ng/ml to about 100 ng/ml.
The culture medium as claimed in claim 5, wherein said epidermal growth factor family member is epidermal growth factor (EGF) present at concentration from about 1 ng/ml to about 100 ng/ml.
The culture medium as claimed in claim 5, wherein said insulin is present at concentration from about 1 g/ml to about 100 μg/ml.
The culture medium as claimed in claim 5, wherein said hydrocortisone growth factor family member is hydrocortisone present at concentration of least about 0.1 g/ml.
10. The culture medium as claimed in claim 1, wherein said basic nutrients are in the form of basal medium selected from but not limiting to Dulbecco's Modified Eagle's Medium (DMEM), DMEM/F-12, or KO-DMEM, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), BGJb Medium, RPMl 1640, Ham's F-10, Ham's F-12, a-Minimal Essential Medium (ctMEM), Brinster's
BMOC-3 Medium, C02-lndependent Medium, CMRL Medium, Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium, Waymouth's MB 752/1 Media, Williams Media E, Medium NCTC-109, neuroplasma medium, Leibovitz's L-15 Media, McCoy's 5A Media (modified), MCDB 131 Mediumor and/or combination thereof.
11. A culture medium for selectively enriching and maintaining stem cells or cancer stem-like cells comprising gelatin present at concentration from about 0.001% to about 0.01% w/v, methyl cellulose present at concentration from about 0.05% to about 1.5% w/v, heparin present at the concentration from about 4 ng/ml to about 8 ng/ml, the basic nutrients comprising of an approximately 50:50 mixture of DMEM and Ham's F12 nutrients, basic Fibroblast Growth Factor- bFGF present at concentration from about 15 ng/ml to about 25 ng/ml, Epidermal Growth Factor -EGF present at concentration from about 5 ng/ml to about 15 ng/ml, Insulin present at concentration from about 5 g/ml to about 15 μg/ml, B27 supplement IX to 5X, and hydrocortisone present at concentration of at least about 1 μg/ml.
12. The culture medium as claimed in claim 1 or 11, wherein said culture medium does not contain human or an animal serum or portion thereof.
13. A feeding medium for selectively enriching and maintaining stem cells or cancer stem-like cells comprising components as claimed in claim 1 or 11 except methylcellulose.
14. A cell culture comprising stem cells or cancer stem-like cells and the culture medium of claim 1 or 11.
A method for selectively enriching and maintaining stem cells or cancer stemlike cells comprising steps of culturing the single cell suspension of cancer cells or normal cells in the culture medium of claims 1 or 11 at 37°C in a 5% C02 atmosphere and adding feeding medium of claim 12 to the growing culture intermittently to obtain spheres of stem cells or cancer stem-like cells.
The method as claimed in claim 16, wherein, said single cell suspension of the normal cells or cancer cells is cultured at the cell density ranging from about 1X104 cells /ml to 1X106 /ml.
The method as claimed in claim 16, wherein, said spheres or spheroids of stem cells or cancer stem-like cells are obtained in around 5-10 days.
A kit comprising the culture medium as claimed in claim lor 11 or components thereof, feeding medium as claimed in claim 13 or components thereof, the cell culture system comprising the normal cells or stem cells or cancer cells or cancer stem-like cells, cell culture containers and optionally the written instructions for how to perform the cell culture method of claim 15.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN1085/MUM/2011 | 2011-03-31 | ||
IN1085MU2011 | 2011-03-31 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2012131733A2 true WO2012131733A2 (en) | 2012-10-04 |
WO2012131733A3 WO2012131733A3 (en) | 2015-06-18 |
Family
ID=46932019
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IN2012/000234 WO2012131733A2 (en) | 2011-03-31 | 2012-03-30 | Media compositions, method of initiating and enriching cultures of stem cells and/or cancer stem-like cells |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2012131733A2 (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103898043A (en) * | 2014-03-11 | 2014-07-02 | 山东大学附属千佛山医院 | Cell culture layer and application in culture of human primary cancer cells thereof |
WO2014097318A3 (en) * | 2012-12-18 | 2015-03-19 | Godavari Biorefineries Limited | Agents for eliminating tumour-initiating cells |
CN104630140A (en) * | 2013-11-06 | 2015-05-20 | 吉林济惠生物科技有限公司 | Isolation and culture method of placenta mesenchyma precursor stem cells |
WO2015111734A1 (en) * | 2014-01-23 | 2015-07-30 | 日産化学工業株式会社 | Undifferentiated maintenance culture material |
WO2017207737A1 (en) * | 2016-06-02 | 2017-12-07 | Stemtek Therapeutics Sl | Methods for producing cancer stem cell spheroids |
GB2543374B (en) * | 2015-05-08 | 2018-11-07 | Imagen Therapeutics Ltd | Personalised media |
WO2019060629A1 (en) * | 2017-09-21 | 2019-03-28 | Codiak Biosciences, Inc. | Production of extracellular vesicles in single-cell suspension using chemically-defined cell culture media |
CN110878286A (en) * | 2019-12-24 | 2020-03-13 | 江苏信安佳医疗科技有限公司 | Culture medium for culturing liver cancer organoid cell balls |
US10959952B2 (en) | 2015-06-10 | 2021-03-30 | Board Of Regents, The University Of Texas System | Use of exosomes for the treatment of disease |
CN113862216A (en) * | 2021-09-17 | 2021-12-31 | 深圳市旷逸生物科技有限公司 | Purification reagent and method for purifying cells |
CN115261325A (en) * | 2021-04-29 | 2022-11-01 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium and culture method of oral cancer primary cells |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE444078T1 (en) * | 2000-06-07 | 2009-10-15 | Merck Serono Sa | HUMAN GROWTH HORMONE FOR STIMULATING MOBILIZATION OF PLURIPOTENT HEMAPOIETIC STEM CELLS |
JP4374419B2 (en) * | 2002-10-31 | 2009-12-02 | 独立行政法人理化学研究所 | Composition for pluripotent stem cell culture and use thereof |
MX2007001772A (en) * | 2004-08-13 | 2007-07-11 | Univ Georgia Res Found | Compositions and methods for self-renewal and differentiation in human embryonic stem cells. |
US9029146B2 (en) * | 2005-09-02 | 2015-05-12 | Agency For Science, Technology And Research | Mesenchymal stem cell conditioned medium |
JP5646990B2 (en) * | 2007-04-23 | 2014-12-24 | ストワーズ インスティテュート フォー メディカル リサーチ | Methods and compositions for stem cell self-renewal |
-
2012
- 2012-03-30 WO PCT/IN2012/000234 patent/WO2012131733A2/en active Application Filing
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014097318A3 (en) * | 2012-12-18 | 2015-03-19 | Godavari Biorefineries Limited | Agents for eliminating tumour-initiating cells |
CN104630140A (en) * | 2013-11-06 | 2015-05-20 | 吉林济惠生物科技有限公司 | Isolation and culture method of placenta mesenchyma precursor stem cells |
WO2015111734A1 (en) * | 2014-01-23 | 2015-07-30 | 日産化学工業株式会社 | Undifferentiated maintenance culture material |
CN106103700A (en) * | 2014-01-23 | 2016-11-09 | 日产化学工业株式会社 | Undifferentiated property maintains culture materials |
JPWO2015111734A1 (en) * | 2014-01-23 | 2017-03-23 | 日産化学工業株式会社 | Undifferentiated maintenance culture material |
US10316292B2 (en) | 2014-01-23 | 2019-06-11 | Nissan Chemical Industries, Ltd. | Material for undifferentiated state-maintaining culture |
CN103898043A (en) * | 2014-03-11 | 2014-07-02 | 山东大学附属千佛山医院 | Cell culture layer and application in culture of human primary cancer cells thereof |
GB2543374B (en) * | 2015-05-08 | 2018-11-07 | Imagen Therapeutics Ltd | Personalised media |
US10959952B2 (en) | 2015-06-10 | 2021-03-30 | Board Of Regents, The University Of Texas System | Use of exosomes for the treatment of disease |
WO2017207737A1 (en) * | 2016-06-02 | 2017-12-07 | Stemtek Therapeutics Sl | Methods for producing cancer stem cell spheroids |
WO2019060629A1 (en) * | 2017-09-21 | 2019-03-28 | Codiak Biosciences, Inc. | Production of extracellular vesicles in single-cell suspension using chemically-defined cell culture media |
CN110878286A (en) * | 2019-12-24 | 2020-03-13 | 江苏信安佳医疗科技有限公司 | Culture medium for culturing liver cancer organoid cell balls |
CN110878286B (en) * | 2019-12-24 | 2023-07-18 | 江苏信安佳医疗科技有限公司 | Culture medium for culturing liver cancer organoid cell balls |
CN115261325A (en) * | 2021-04-29 | 2022-11-01 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium and culture method of oral cancer primary cells |
CN115261325B (en) * | 2021-04-29 | 2023-12-12 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium and culture method of primary cells of oral cancer |
CN113862216A (en) * | 2021-09-17 | 2021-12-31 | 深圳市旷逸生物科技有限公司 | Purification reagent and method for purifying cells |
CN113862216B (en) * | 2021-09-17 | 2024-03-19 | 深圳市旷逸生物科技有限公司 | Purifying reagent and method for purifying cells |
Also Published As
Publication number | Publication date |
---|---|
WO2012131733A3 (en) | 2015-06-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2012131733A2 (en) | Media compositions, method of initiating and enriching cultures of stem cells and/or cancer stem-like cells | |
Bottenstein | Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes. | |
US5814511A (en) | Human breast epithelial cell type with stem cell and luminal epithelial cell characteristics | |
JP4532493B2 (en) | Cell culture media | |
US7932084B2 (en) | Methods and compositions for growing adipose stem cells | |
Savickiene et al. | Human amniotic fluid mesenchymal stem cells from second-and third-trimester amniocentesis: differentiation potential, molecular signature, and proteome analysis | |
Xie et al. | Isolation and expansion of human limbal stromal niche cells | |
JP5872046B2 (en) | Preparation of basic culture medium for mesenchymal stem cells | |
KR20130112028A (en) | Serum-free chemically defined cell culture medium | |
Jin et al. | GD2 expression is closely associated with neuronal differentiation of human umbilical cord blood-derived mesenchymal stem cells | |
JP6238265B2 (en) | Cell aggregate production method | |
WO2021185198A1 (en) | Serum-free and heterologous component-free mesenchymal stem cell culture medium and use thereof | |
KR20090038439A (en) | Differentiation of stem cells from umbilical cord matrix into hepatocyte lineage cells | |
JP6990659B2 (en) | Chemically defined medium for culturing cancer stem cell (CSC) -containing cell populations | |
WO2007020611A2 (en) | Adult human neural stem/progenitor cells from the olfactory epithelium and olfactory lamina propria, isolation method, proliferation and differentiation in serum free culture medium and utilization for transplantation | |
JP6677648B2 (en) | Regulation of cell proliferation and pluripotency | |
WO2007056505A2 (en) | Systems and methods for selection and maintenance of homogeneous and pluripotent human embryonic stem cells | |
Ferrari et al. | Investigation of growth conditions for the expansion of porcine mesenchymal stem cells on microcarriers in stirred cultures | |
EP2951291A1 (en) | Serum-free culture medium for cancer stem cells | |
US20080213892A1 (en) | Self-renewal of neural stem cells is promoted by wnt proteins | |
WO2017093418A1 (en) | Methods for differentiating cells into hepatic stellate cells | |
US20200131480A1 (en) | Method For Enhancing Proliferation Capability Of Stem Cells Using Ethionamide | |
US9423404B1 (en) | Method of screening for an agent that enhances beige fat adipogenesis | |
Mariya et al. | Mammary gland cell culture of Macaca fascicularis as a reservoir for stem cells | |
KR102359675B1 (en) | Composition for inducing differentiation into hepatocyte-like cells having increased urea synthesis activity |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12762823 Country of ref document: EP Kind code of ref document: A2 |
|
NENP | Non-entry into the national phase in: |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 12762823 Country of ref document: EP Kind code of ref document: A2 |