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• • 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
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  • 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
  • C12N5/0695—Stem cells; Progenitor cells; Precursor cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • C—CHEMISTRY; METALLURGY
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  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/02—Drug screening
• • C—CHEMISTRY; METALLURGY
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/70—Polysaccharides
  • C12N2533/80—Hyaluronan

Definitions

• the present invention generally relates to a method for culturing a population of cancer stem cells.
• the present invention also relates to use of a gel as a cell culture substrate.
• Cancer stem cells are a subpopulation of cancer cells (found within tumors or hematological cancers) that are similar to normal stem cells in the sense that they are able to give rise to all cell types found in a particular cancer. This includes characteristics such as self-renewal, propagation of cancer or differentiation into the various types of cancer cells. Such cells are believed to be able to persist in tumors as a distinct population, causing cancer relapse and metastasis due to the formation of new tumors.
• CD44 is one of the most commonly studied CSC markers.
• the subpopulation of cancer cells with CD44 high /CD24 low , CD44 high /CD133 high , CD44 hig Valdehyde dehydrogenase 1 family, member Al (ALDH1A1) hi9h and CD44 high /epithelial cell adhesion molecule (EpCAM) high showed chemoresistance and tumorigenesis .
• such a population is generally small and unstable in culture, making it difficult to perform standard high through-put cell viability assays.
• the CSC enriched population which was prepared by FACS sorting, rapidly decreased during in vitro culture .
• a method for culturing a population of cancer stem cells comprising introducing the cancer stem cells on or in a cell culture substrate, wherein the cell culture substrate is in the form of a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine .
• cancer stem cells that contain a marker that interacts with the glycosaminoglycan may be selectively cultured on the gel.
• the expression level of the marker may control the propagation of the population of cancer cells on the gel.
• the cancer stem cells may be present in a cancer cell line and hence the method for culturing cancer stem cells can also be used to culture a cancer cell line enriched in cancer stem cells that have a high expression of the marker.
• the method for culturing a population of cancer stem cells may include a method for culturing a cancer cell line comprising introducing the cancer cell line on or in a cell culture substrate, wherein the cell culture substrate is in the form of a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine.
• the stiffness or crosslink density of the gel may be altered in order to promote the growth and maintenance of the cancer stem cells or cancer cell line.
• the stiffness or crosslink density of the gel may also affect the chemoresistance of the cancer stem cells or cancer cell line to a selected chemotherapeutic drug.
• a method for selectively separating a population of cancer stem cells from a plurality of cancer cell lines comprising the steps of: (a) subjecting the plurality of cancer cell lines to a cell culture substrate, , wherein the cell culture substrate is in the form of a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine; and (b) allowing the cancer stem cells to interact with the cell culture substrate to thereby separate the cancer stem cells from the plurality of cancer cell lines.
• a method of screening drugs for a population of cancer stem cells comprising the step of culturing the cancer stem cells on or in a cell culture substrate, wherein the cell culture substrate is in the form of a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine, and wherein the gel has a stiffness that is equal to or less than 100 kPa.
• a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine as a cell culture substrate.
• a cell culture substrate in the form of a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine having cancer stem cells cultured on or in the cell culture substrate for screening anti-cancer drugs .
• cancer stem cell marker or “marker” are to be interpreted broadly to refer to genes and their protein products that can be used to isolate and identify the cancer stem cells.
• selection or “selectively” as well as grammatical variants thereof are to be interpreted broadly to refer to the isolation of a desired or target type of cancer cells or cancer stem cells from a plurality or mixture of various types of cancer cells, cancer stem cells or cancer cell lines.
• the term "about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically stated value, more typically, +/ - 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3 , from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, .1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
• the method comprises introducing the cancer stem cells on or in a cell culture substrate, wherein the cell culture substrate is in the form of a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine.
• the cancer stem cells may contain a marker (also known as a cancer stem cell marker) .
• the cancer stem cells may be present in a cancer cell line and hence the method for culturing cancer stem cells can also be used to culture a cancer cell line enriched in cancer stem cells that have a high expression of a cancer stem cell marker.
• the method for culturing a population of cancer stem cells may include a method for culturing a cancer cell line comprising introducing the cancer cell line on or in a cell culture substrate, wherein the cell culture substrate is in the form of a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine.
• the expression level of the cancer stem cell marker may control the propagation of the population of cancer cells on the gel.
• the expression level of the cancer stem cell marker is defined as the number of cancer stem cell marker molecules in a single cell.
• the expression level may be expressed as an average value of relative expression level of the cancer stem cell marker, when compared to a comparative cell.
• the average relative expression level may be measured by flow cytometric analysis (FACS) .
• FACS flow cytometric analysis
• the average value of the relative expression level of the cancer stem cell maker may be a value that is above 20, when compared to the comparative cell.
• the average value of the relative expression level of the cancer stem cell maker may be above 30, above 40, above 50 , above 60 , above 70 , above 80 , above 90 , above 100, above 110 or above 150, when compared to a comparative cell.
• the expression level may also be obtained by determining the mRNA expression level of the cancer stem cell marker.
• the comparative cell may be BT-474 (ATCC HTB-20 , obtained from ATCC of Manassas, Virginia of the United States of America) .
• the cancer stem cells may be incubated on the cell culture substrate.
• the incubation conditions may be at a temperature of about 36°C to about 37°C, at a duration of about 5 minutes to about 24 hours, and in a humidified atmosphere.
• the incubation conditions may be at 1 hour at 37°C in a humidified atmosphere of 5% carbon dioxide .
• any cancer cells that are not attached to the cell culture substrate may be removed.
• the unattached cells may be removed by washing the cells with a suitable buffer for a number of times.
• the cells may be washed with an exemplary buffer such as phosphate buffer saline (PBS) for 1 to 5 times, or 3 times.
• PBS phosphate buffer saline
• the cancer stem cell marker may interact with the glycosaminoglycan present in the gel.
• the cancer stem cell marker may be a receptor for the glycosaminoglycan.
• the marker may be selected from the group consisting of CD44, Receptor for HA-mediated motility (RHAMM) and intracellular adhesion molecule-1 (ICAM-1) .
• the growth and maintenance of the cancer stem cells may be determined by the mRNA expressions of Nanog, Sox ⁇ -2 or EpCAM.
• the cancer stem cells may have a mRNA expression level that is at least 2 folds that of the same cells but grown on a polystyrene cell culture plate control.
• the cancer stem cells may have a mRNA expression level that is at least 1.25 folds that of the same cells but a polystyrene cell culture plate control.
• EpCAM the cancer stem cells may have a mRNA expression level that is at least 2.25 folds that of the same cells but grown on a polystyrene cell culture plate control.
• the stiffness or crosslink density of the gel may act as an alternative or additional control to the selection of cancer stem cells (or cancer cells) that can be cultured on the gel.
• the stiffness or crosslink density of the gel may also control the growth and/or maintenance of the cancer stem cells (or cancer cells) that do selectively grow on the gel.
• the stiffness or crosslink density of the gel may also affect the chemoresistance of the cancer stem cells (or cancer cells) to a selected chemotherapeutic drug.
• the stiffness of the gel may be a value selected from a range of about 0.1 kPa to about 100 kPa, about 0.1 kPa to about 1 kPa, about 0.1 kPa to about 2 kPa, about 0.1 kPa to about 3 kPa, about 0.1 kPa to about 4 kPa, about 0.1 kPa to about 5 kPa, about 0.1 kPa to about 6 kPa, about 0.1 kPa to about 7 kPa, about 0.1 kPa to about 8 kPa, about 0.1 kPa to about 9 kPa, about 0.1 kPa to about 20 kPa, about 0.1 kPa to about 30 kPa, about 0.1 kPa to about 40 kPa, about 0.1 kPa to about 50 kPa, about 0.1 kPa to about 60 kPa, about 0.1 kPa to about 70 k
• the crosslink density of the gel may be a value selected from the range of about lxlO "6 to about lxlO "3 mol/cm 3 , lxlO "5 to about lxlO "3 mol/cm 3 , lxlO "4 to about lxlO "3 mol/cm 3 , lxlO "6 to about lxlO "5 mol/cm 3 , or lxlO "6 to about lxlO "4 mol/cm 3 .
• the storage modulus of the gel may be a value selected from a range of about 30 to about 100,000 Pa, about 30 to about 1,000 Pa, about 30 to about 10,000 Pa, about 30 to about 50,000 Pa, about 50,000 to about 100,000 Pa, about 1,000 to about 10,000 Pa, or about 10,000 to about 100,000 Pa.
• the gel may be a hydrogel.
• the gel or hydrogel may be a conjugate of a glycosaminoglycan and a substituted phenalkylamine .
• the glycosaminoglycan may be a non- sulfated glycosaminoglycan such as hyaluronic acid (HA) .
• the substituted phenalkylamine may be a substituted phenmethylamine, phenethylamine, phenpropylamine , phenbutylamine or phenpentylamine .
• the phenethylamine may be tyramine such as a meta-tyramine or a para-tyramine .
• the gel may be an enzymatically cross- linked gel.
• the gel may be composed of a HA-tyramine conjugate, which may be formed using oxidative coupling of tyramine moieties catalyzed by catalysts such as hydrogen peroxide and horseradish peroxidase .
• the degree of substitution (defined as the number of substituted phenalkylamine molecules per 100 repeating units of glycosaminoglycan) may be a value selected from a range of about 1 to about 20, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 5 to about 20, about 10 to about 20 or about 15 to about 20.
• the degree of substitution may be about 6.
• the stiffness of the cell culture substrate or gel is less than or equal to lOOkPa, or 1.0 kPa, the cancer stem cells may become resistant to an anti-cancer drug.
• the cells may have at least 70% viability when in the presence of- the anti-cancer drug.
• the anti-cancer drug may be cisplatin or doxorubicin.
• step (b) may comprise the step of binding the cancer stem cells to the glycosaminoglycan of the cell culture substrate via receptor-ligand binding.
• the cancer stem cells For receptor- ligand binding to occur, the cancer stem cells contain a marker that is a receptor for the glycosaminoglycan.
• the expression level of the cancer stem cell marker may also affect the ability of the cancer stem cells (or cancer cell, line containing such cancer stem cells) to bind with the glycosaminoglycan.
• a method of screening drugs for a population of cancer stem cells comprising the step of culturing said cancer stem cells on or in a cell culture substrate, wherein said cell culture substrate is in the form of a gel comprising a conjugate, of a glycosaminoglycan and a substituted phenalkylamine, and wherein said gel has a stiffness that is equal to or less than 100 kPa.
• the stiffness may be equal to or less than 1.0 kPa, 0.5 kPa, 0.4 kPa, 0.2 kPa or 0.1 kPa.
• a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine as a cell culture substrate.
• the cell culture substrate may be selective for a population of cancer stem cells.
• the cancer stem cells may express a marker that may interact with the glycosaminoglycan via receptor- ligand binding.
• a cell culture substrate in the form of a gel comprising a conjugate of a glycosaminoglycan and a substituted phenalkylamine having cancer stem cells cultured on or in the cell culture substrate for screening anti-cancer drugs.
• the anti- cancer drug to be screened may not be particularly limited and may include cisplatin or doxorubicin as well as any other anti-cancer drugs.
• Fig. 1 is a schematic diagram showing a gel for supporting the selection and culture of a population of cancer cells that contain a cancer stem cell marker.
• Fig. 3 is a number of graphs showing the flow cytometric analysis (FACS) of the amount of cancer cells that contain the cancer stem cell marker in (a) MDA- B- 231; (b) MCF-7; and (c) BT-474 cancer cell lines. Isotype controls were conducted for all cell lines.
• FACS flow cytometric analysis
• Fig. 4(a) is a graph showing the FACS of MCF-7, HCC193 and MDA-MB-231 cells.
• Fig. 6(a) shows a number of phase contrast microscopic images of MDA-MB-231 cells adhered to polystyrene control and gels of varying stiffness at 24 hours after cell seeding.
• Fig. 8(a) are a series of phase contrast microscopic images of MDA-MB-231 cells and their colonies on HA-Tyr hydrogels (I-V, VII) and polystyrene (VI) at 14 days after cell seeding.
• the stiffness of the various hydrogels are 0.1 kPa (I) and (VII) , 0.2 kPa (II), 0.5 kPa (III), 1 kPa (IV), 4 kPa (V) .
• the scale used in .(I) is 300 pirn while that in (VII) is 100 ⁇ .
• Fig. 10 (a) is a graph showing the relative mRNA expression levels of CD44 (CD44s, CD44v3-10 and CD44v8- 10) .
• Fig. 11 is a graph showing the prevention of MDA-MB- 231 cell adhesion on HA-Tyr hydrogel due to the presence of blocking antibodies against CD44.
• Fig. 12(a) is a graph showing the relative mRNA expression levels of CD44 (CD44s, CD44v3-l0, CD44v8-10) , EpCAM and ALDH1A1 in MDA-MB-231 cells, which were selected after 1 hour cell seeding by 3 times wash with PBS.
• FIG. 1 there is provided a schematic diagram showing a gel 2 for supporting the selection and culture of a population of cancer cells that contain a cancer stem cell marker 4.
• the gel 2 composed of a . HA-tyramine conjugate, was formed using oxidative coupling of tyramine moieties catalyzed by hydrogen peroxide (H 2 0 2 ) and horseradish peroxidase (HRP) .
• H 2 0 2 hydrogen peroxide
• HRP horseradish peroxidase
• HA hyaluronate
• Tyramine hydrochloride (Tyr-HCl) , N- hydroxysuccinimide (NHS), l-ethyl-3- (3- dimethylaminopropyl) -carbodiimide hydrochloride (EDC-HC1) , hyaluronidase from bovine testes (400-1,000 units/mg) and cisplatin were all purchased from Sigma-Aldrich (Minnesota of the United States of America) .
• Horseradish peroxidase (HRP, 100 units/mg) was purchased from Wako Pure Chemical Industries (Osaka, Japan) .
• Hydrogen peroxide (H 2 0 2 ) was obtained from Lancaster.
• Doxorubicin was obtained from Boryung pharmaceutical (Seoul, South Korea) . AlamarBlue, CyQUANT ® cell proliferation assay kit, TRIzol ® and Taqman ® gene expression master mix were provided by Life Technologies (Singapore) , respectively.
• Mouse monoclonal antibody to human CD44 and FITC-conjugated rat antimouse IgG2a secondary antibody were obtained from GeneTex (Hsinchu, Taiwan) .
• Rat monoclonal anti-CD44 antibody (Hermes- 1) were purchased from Abeam (Cambridge, United Kingdom). Trypsin-EDTA (0.025%) and penicillin/streptomycin were purchased from PAN Biotech GmbH (Aidenbach, Germany) .
• Heat-inactivated fetal bovine serum was purchased from GE Healthcare (Buckinghamshire, United Kingdom) .
• Phosphate buffered saline (PBS, 150 mM, . pH 7.3), Dulbecco' s modified eagle medium (DMEM) and RPMI-1640 medium were all supplied by the media preparation facility in Biopolis (Singapore).
• MDA-MB-231, MCF-7, HCC1937 and BT-47 breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, Virginia of the United States of America) .
• MDA-MB-231, HCC1937 and BT-474 cells were grown in RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin.
• MCF-7 cells were grown in DMEM containing 10% FBS and 1% penicillin/streptomycin . All cell lines were cultured on polystyrene tissue culture flask, and were passaged when reach to 80% confluent.
• the degree of substitution (the number of tyramine molecules per 100 repeating units . of HA) was 6 as determined by X H NMR.
• the stiffness and gelation rate of the gel could be independently tuned by H 2 0 2 and HRP concentrations, respectively.
• G' of the HA-Tyr gel was well controlled by H 2 0 2 concentration, and ranged from 70 to 4,000 Pa when an aqueous solution of HA-Tyr conjugate (1.75 % (w/v) ) was utilized.
• G" increased with increasing H 2 0 2 concentration from 0.22 m -to 1.15 mM, suggesting that a higher crosslinking density was achieved when the H 2 0 2 concentration increased.
• the average molecular weight between crosslinks ⁇ M c ) and crosslinking density (v e ) of the HA-Tyr hydrogels were determined. From the results of the storage modulus (C) measurement, the average molecular weight between crosslinks (M c ) and crosslinking density (v e ) . were calculated by rubber-elasticity theory. As shown in Fig. 2 (b) , the M c of the HA-Tyr hydrogels decreased with increasing H 2 0 2 concentration, while the v e of the HA-Tyr hydrogels increased with increasing H 2 0 2 concentration.
• Example 2 To evaluate the cancer cell adhesion (Example 2) , proliferation (Example 3) , maintenance (Example 4) , inhibition of HA-CD44 interaction (Example 5) and chemoresistance (Example 6) , a number of gels of varying stiffness (0.1, 0.2, 0.5, 1.0, 2.5 and 4.0 kPa, where appropriate) were used. As will be seen from the Examples below, breast cancer cell adhesion on HA-Tyr hydrogel was strongly modulated by the stiffness of the hydrogels, and was dependent on CD44-HA interaction. HA-Tyr hydrogels, as compared to polystyrene, provided different culture environments in maintaining the round shape cell morphology, low cell proliferation and colony formation.
• the HA-Tyr hydrogels outperformed the polystyrene with better enhancement of CD44 variant isoforms, Sox-2 and ALDH1A1 mRNA expression levels.
• the mechanical properties of the hydrogel (such as the components and stiffness of the hydrogel) as well as expression levels of CD44 variant isoforms are factors that affect the cell adhesion, proliferation and malignancy of the cancer cells. Controlling the stiffness of the HA-Tyr hydrogel is a simple and effective means to change the malignancy of breast cancer cells.
• FACS Fluorescein isothiocyanate
• the cells were then analyzed and sorted using a BD LSRII flow cytometry analyser (BD Biosciences, of New Jersey of the United States of America) . As shown in Fig. 3, it can be seen that 99.5%, 44.2% and 0.5% of CD44 positive cells were detected in MDA-MB- 231 (Fig. 3(a)), MCF-7 (Fig. 3(b)) and BT-474 (Fig. 3(c)), respectively, using FACS . The CD44 protein expression levels (average) of MDA-MB-231, HCT116 and MCF-7 cells were 103, 12 and 5 times higher than that on BT-474 cells.
• CD44 expression level is defined as the number of CD44 molecules in a single cell. Different expression levels of CD44 among these breast cancer cell lines were evaluated based on protein expression level on cell surfaces (Fig. 4(a)) as well as mRNA expression level (Fig. 4(b)). The mRNA expression levels of CD44 in MDA-MB-231 and HCC1937 cells were 8.5 and 4.5 times higher than that of MCF-7 cells. These results were closely related to the results of the CD44 protein expression levels as shown in Fig. 4(a).
• HA-Tyr gels 250 ⁇
• stiffness 0.1, 0.2, 0.5, 1.0, 2.5 and 4.0 kPa, where appropriate
• the gels were allowed to settle overnight. Gels were then washed three times with PBS and once with culture medium. 250 ⁇ of breast cancer cells in culture medium at cell density of 1.0 10 5 cells/ml was seeded onto the gels.
• the plates were returned to an incubator (at 37°C in a humidified atmosphere of 5% C0 2 ) for an appropriate period of time ranging from 1 to 9 hours. At selected time intervals, the media with unattached cells were aspirated and the wells were washed three times with PBS. A cell culture plate without the gel served as a comparison.
• the cells attached to the gels were incubated in culture medium containing 10% Alamar Blue dye at 37?C for 4 hours.
• the fluorescence measurement of the Alamar Blue dye was performed using a microplate reader with excitation and emission at 545 and 590 nm, respectively.
• 100 ⁇ of AlamarBlue solution was transferred to 96 well plate to measure the fluorescence intensity.
• the fluorescence intensity of the Alamar blue dye was regarded as the number of attached cells in this study.
• Fig. 5 shows the cell adhesion on the surface of HA- Tyr gels.
• a significant number of MDA-MB-231 cells were attached to the surfaces of HA-Tyr gels (Fig. 5(c)).
• the attached MDA-MB-231 cell number was dependent on HA-Tyr hydrogel stiffness, and the attached cell number increased with decreasing hydrogel stiffness.
• Fig. 5(a) and Fig. 5(b) also show that the cell adhesion of MCF-7 and HCC1937, respectively, increased with decreasing hydrogel stiffness.
• Fig. 5(a) to Fig. 5(c) showed that the number of attached cells on HA-Tyr hydrogels appeared to be closely related to CD44 expression level (Fig. 4) .
• Breast cancer cell adhesion on HA-Tyr hydrogel increased with increasing expression levels of CD44.
• the growth rate of MDA-MB-231 cells cultured on HA-Tyr gels was much slower than that cultured on culture plate. Only soft HA-Tyr gels with stiffness of 0.1 kPa, 0.2 kPa and 0.5kPa showed cell growth during 13 days, while no cell growth was observed from the HA-Tyr gels of other stiffness.
• the mRNA expression levels of Nanog, Sox-2 and EpCAM in MDA-MB-231 cells after 13 days of cultivation on HA-Tyr gel were investigated. Additionally, the transcription levels of CD44s (CD44 standard), CD44v3-10 (CD44 variant isoform), CD44v8-10 (CD44 variant isoform) , Sox-2, EpCAM and ALDHD1A1 genes in MDA-MB-231 cells on HA-Tyr hydrogels (0.1, 0.2, 0.5, 1.0 and 4.0k Pa stiffness) were also investigated.
• the MDA-MB-231 cells on the various HA-Tyr hydrogels were collected by treating with hyaluronidase (1,000 units/ml) and trypsin-EDTA for subsequent RNA extraction.
• the total RNA of MDA-MB-231 cell was extracted using a TRIzol (Life technologies, Singapore) after 13 days of culture.
• RT reaction mixture (Thermo Scientific, China)-, which contains RT buffer, random hexamer primer, deoxynucleotide triphosphate (dNTP) mixture, RNase inhibitor and reverse transcriptase, followed by DNase treatment .
• Singapore contained 10 TaqMan PCR master mix, 1.0 ]iL of each primer, 2.0 L of cDNA, and 7.0 L ' of distilled water.
• the various primers used were Hs01075861_ml (CD44 total), Hs01081473_ml (CD44s) , Hs01081480_ml (CD44v3 -vlO) , Hs01081475_ml (CD44v8-vlO) ) , Nanog (Hs02387400_sl) , Sox-2
• CD44V3-10 and CD44v8-10 on HA-Tyr hydrogels were significantly higher than that on polystyrene, while the expression levels of CD44s were almost the same as that on polystyrene.
• the enhanced expression levels of CD44v3-10 and CD44v8-10 increased with increasing the HA-Tyr hydrogel stiffness (Fig. 10a) .
• mR A expression levels of Sox-2 and ALDH1A1 on HA-Tyr hydrogels were also significantly enhanced on HA-Tyr hydrogels compared to those on polystyrene (Fig. 10b).
• the expression level of ALDH1A1 was independent on hydrogel stiffness of HA-Tyr hydrogels. This result suggested that the CD44-HA interaction enhance ALDH1A1 expression in a stiffness independent manner.
• mRNA expression level was significantly increased on 0.2 and 0.5 kPa HA-Tyr hydrogel.
• the enhancement of ALDH1A1 expression which is one of the CSC markers, indicated that the MDA-MB-231 cells that adhered on the HA-Tyr hydrogel are induced CSC property.
• HA-based cell culture substrates can be useful for selection, culture and maintenance of CSCs .
• MDA-MB-231 cell adhesion on HA-Tyr hydrogel using anti-CD44 antibody was evaluated.
• the MDA- MB-231 cells were washed with PBS and harvested from the cell culture flask using trypsin-EDTA.
• the detached cells were washed with RPMI-1640 medium without -FBS, and were incubated with 30 ⁇ g/ml anti-CD44 antibody on ice for 1 hour.
• a cell suspension without the anti-CD44 antibody served as a comparison.
• the cells on the HA-Tyr hydrogels and polystyrene were harvested by incubating with hyaluronidase (1,000 units/ml) and trypsin-EDTA, respectively.
• the cell pellets were washed with PBS twice and were used for measurement of mRNA expression level.
• CSC markers such as CD44S, CD44v3-10, CD44v8-10, EpCAM and ALHD1A1 on 4 kPa HA-Tyr hydrogels were significantly higher than that on polystyrene (Fig 12(a)).
• CD44v8-10 level was significantly higher than that on polystyrene, even on soft (0.1 and 0.2 kPa) HA-Tyr hydrogels.
• CD44v8-10 have been well investigated the correlation with breast cancer metastasis due to enhancement of the resistance against reactive oxygen species (ROS) .
• ROS reactive oxygen species
• EpCAM and ALDH1A1 also are well known to correlate with CSC properties.
• the cell viability of MDA-MB-231 on HA-Tyr gels was measured in the presence or absence of conventional anti-cancer drugs, such as cisplatin and doxorubicin.
• conventional anti-cancer drugs such as cisplatin and doxorubicin.
• 250 ⁇ of MDA-MB-231 cells in culture medium at cell density of 5.2 x 10 3 cells/ml was seeded onto the gels (0.2, 0.5, 1.0, 2.5 and 4.0 kPa) .
• the culture medium was changed every 3 days.
• 10-100 ⁇ cisplatin or doxorubicin was added to cultured cells on gels and incubated ,for 48 hours. Then, the cells were washed with PBS and the fluorescence intensity was measured using the AlamarBlue cell viability assay as described above.
• Cell viability in the presence of anti-cancer drugs was normalized to the untreated control groups (0 ⁇ ) .
• HA-Tyr gels showed much higher, cell viability in a test concentration range compared to cell culture plate. Notably, the cell viability was almost 100% at even 100 mM of cisplatin when HA-Tyr with 0.5 and 1 kPa.
• the disclosed method can be used in cell culture applications to culture a desired cancer stem cell or cancer cell line that contains the cancer stem cell.
• the disclosed method may be used to selectively separate and thereby isolate a desired cancer stem cell (or cancer cell line) from a plurality or mixture of various cancer cells.
• the disclosed gel may be used to support the selection and growth of a desired cancer stem cell.
• the disclosed gel together with the cancer stem cells may be used as a drug screening platform for cancer therapy to determine anti-cancer drugs that the cancer stem cells are chemo-resistant to or which have a therapeutic effect on the cancer stem cells.
• the disclosed method and gel may be used to selectively isolate cancer stem cells that contain a desired marker by receptor- ligand binding.

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• 2014
  • 2014-06-18 WO PCT/SG2014/000290 patent/WO2014204406A1/en not_active Ceased
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