WO2016146893A1 - Matrice extracellulaire à base de tumeur humaine pour des études de cellules in vitro - Google Patents

Matrice extracellulaire à base de tumeur humaine pour des études de cellules in vitro Download PDF

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WO2016146893A1
WO2016146893A1 PCT/FI2016/050163 FI2016050163W WO2016146893A1 WO 2016146893 A1 WO2016146893 A1 WO 2016146893A1 FI 2016050163 W FI2016050163 W FI 2016050163W WO 2016146893 A1 WO2016146893 A1 WO 2016146893A1
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myogel
cells
human
matrigel
cell
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Tuula Salo
Meeri SUTINEN
Elias SUNDQUIST
Fumi SUOMI
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University Of Oulu
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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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • 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
    • C12N2533/70—Polysaccharides
    • C12N2533/76—Agarose, agar-agar
    • 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
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention is directed to the field of three-dimensional cell cultures.
  • the present invention provides a cell culture composition for studying human cells in vitro comprising homogenized human uterus benign tumor, leiomyoma, tissue.
  • the present invention also provides a method for producing extracel lular matrix homogenate from discarded leiomyoma tissue removed from patients during routine surgical operations.
  • Matrigel ® the mouse Engelbreth-Holm-Swarm (EHS) tumor -derived commercial prod uct (see US 4,829,000 and Kibbey 1994), is widely used particularly for in vitro adhesion, invasion and capillary formation assays of human cancer cells.
  • EHS Engelbreth-Holm-Swarm
  • TM EM human tumor microenvironment matrix
  • Rat tail tendon derived type I collagen is probably the most abundant ECM mimicking matrix used in organotypic cultures.
  • Other commercially available ECM molecules derived from different animal species, like fibronectin (Mao & Schwarzbauer 2005), fibrin (Ho et al. 2006) and hyaluronic acid (Gu rski et al. 2009), are also available for in vitro studies.
  • ECM or peptide matrices are available from d ifferent manufacturers.
  • one separate molecule, or even a mixture of them, or totally synthetic matrices cannot properly simulate the complex effects of natural ECM, since they obviously lack e.g. the hundreds of cell-adhesion signals, cytokines or protease cleavage sites identified in natural tumor ECM.
  • TM E tumor microenvironment
  • myogel for an extracellular matrix material which is derived from human, mouse, rat or pig normal skeletal muscles using similar procedures as Kibbey (1994) for preparing EHS tumor extraction.
  • the material was shown to be adipogenic (Abberton et al. 2008, Ting et al. 2014) and to support the ex vivo amplification of corneal epithelial cells (Francis et al. 2009).
  • H u Biogel an ECM gel derived from normal human amnion tissue containing laminin, collagen types I and IV, entactin, tenascin and heparan sulfate
  • proteoglycan but lacking endogenous growth factors ( EG F, TG F-a, TG F- ⁇ , FG F and PDG F) as well as M M P-2 and -9 (Yuan et al. 2008).
  • the present invention provides a novel Myogel product, prepared from human uterus benign leiomyoma tumor tissue. A solution/gel of the total protein extracts was formulated and the protein contents and characteristics of Myogel were compared with Matrigel ® using a set of in vitro experiments.
  • the tumor tissue solution/gel derived from human leiomyoma offers an excellent human TM EM tool for analyzing human carcinoma cells in vitro.
  • Th is novel Myogel product can also be combined with a low melting agarose, Myogel- LMA, and provides thus an easy to use, practical method for analyzing e.g. cancer cell invasive, adhesive or migratory properties or various potential chemotherapeutic compounds, as well as to test capillary formation.
  • One of the objects of the present invention is to provide a cell culture composition comprising homogenized human leiomyoma tissue.
  • Another object of the present invention is to provide a method of preparing extracellular matrix homogenate comprising the steps of: (a) homogenizing human leiomyoma tumor at least twice in a NaCI-buffer and discarding the soluble fraction after each homogenization;
  • step (c) separating the extract from the residual leiomyoma tumor fraction and saving the first extract while repeating step (b) with the residual leiomyoma tumor fraction and saving the second extract resulting therefrom;
  • step (d) combining the first and second extracts from step (c) and dialyzing against a buffer comprising a sterilizing component;
  • the present invention is also directed to a use of a cell culture composition comprising homogenized human leiomyoma tissue for invasive, migratory, adhesive or capillary formation cultures of human cells.
  • a cell culture composition comprising homogenized human leiomyoma tissue for invasive, migratory, adhesive or capillary formation cultures of human cells.
  • HSC-3 cells divided and formed colonies within Myogel combined with LMA (low melting agarose). HSC-3 cells grew similarly in Myogel-LMA than in LMA only (A and B).
  • FIG. 3 Migration and horizontal invasion of HSC-3, MDA-MB-231, PaOlc, Pa02c, Pa03c and Pa04c cells on and through Myogel and Matrigel 9 .
  • HSC-3 cells migrated more efficiently on Myogel coated wells than on Matrigel ® coated wells. In plain wells their migration was the most efficient (A and B). More HSC-3 and pancreatic cancer cells invaded horizontally through Myogel than through Matrigel ® in scratch wound invasion assay (C-E).
  • FIG. 4 Invasion of HSC-3 cells through Myogel and Matrigel 9 . HSC-3 cells invaded more efficiently through Myogel than through Matrigel ® (A). In all different Myogel batches HSC-3 cells invaded more in Myogel than in Matrigel ® (B). In Myogel and Matrigel ® mixtures HSC-3 cells invaded more when the mixture contained more Myogel (C). Invasion pattern of HSC-3 cells through Myogel and Matrigel ® (D).
  • FIG. 5 Invasion of oral squamous cell carcinoma, melanoma and pancreatic cancer cells through Myogel and Matrigel 9 .
  • HSC-3 cells invaded efficiently through Myogel and Matrigel ® mixed with agarose, 09-HSC-3 cells have a higher passage number than 13-HSC-3 cells (A).
  • Invasion pattern of HSC-3 cells through different mixtures of Myogel, Matrigel ® and LMA (B).
  • Oral squamous cell carcinoma cells SCC-9, LN-1 and LN-2 as well as melanoma cells SK-Mel and A2058 invaded more efficiently through Myogel-LMA than through growth factor- reduced Matrigel ® (Matrigel ® -G FR, (C).
  • the term "leiomyoma” or “myoma” refers herein to a benign smooth muscle neoplasm that is very rarely premalignant. Leiomyomas or myomas can occur in any organ but the most commonly in the uterus, small bowel and the esophagus. Leiomyomas of the uterus are common pathologic abnormalities of the female genital tract. Occurrence increases with age, and leiomyomas are found in even 50% of women older than 30 years. Consequently, large amou nts of myoma tissue are constantly removed from patients in routine operations and discarded, providing an unlimited amount of myoma tissue available for cancer research.
  • homogenizing refers herein to application of a homogenizer used for disrupting the structure of various types of organic material, such as tissue. Many different homogenizer models have been developed using various physical technologies for d isruption. The term
  • cryopulverization may also refer to cryopulverization, repeated freezing and thawing, nitrogen decompression or to usage of glass, ceramic or steel beads suspended in aqueous media for the disruption of the sample.
  • total protein extract refers herein to a product of an extraction protocol which solubilizes proteins, including membrane proteins, from a tissue sample and separates them from other cell structures without denaturation of the extracted proteins.
  • agarose refers herein to a linear polymer extracted from seaweed and made up of the repeating unit of agarobiose. Agarose is commercially available and can be used as a gel for culturing motile cells and micro-organisms. The gel's porosity is directly related to the concentration of agarose in the medium.
  • low melting point agarose refers herein to agarose which is modified by hydroxyethylation and thus has lower melting and gelling temperature than standard agaroses.
  • the melting temperature of low melting point agarose is usually under 65 degrees of Celsius, preferably 60-65 degrees of Celsius, and the gelling temperature is under 30 degrees of Celsius, preferably 26-30 degrees of Celsius.
  • cell culture composition refers to extracellu lar matrix providing a two-d imensional or three-dimensional microenvironment in vitro for mammalian cells, preferably human cells, incubated or cultured on or in the matrix.
  • cell culture composition refers herein to extracellular matrix comprising homogenized human leiomyoma tissue (i.e. Myogel).
  • the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived Matrigel ® is the most commonly used tumor microenvironment matrix (TM EM) in research laboratories worldwide.
  • TM EM tumor microenvironment matrix
  • the Matrigel ® is non-human in origin, it contains molecules which are not present in human TM EM and therefore, lack of a human TM EM for in vitro cancer studies has been apparent.
  • the present invention provides a cell culture composition comprising homogenized human leiomyoma tissue for studying human cells in vitro or an extract thereof such as an extracellular matrix ( ECM) extract obtained from human leiomyoma tissue.
  • ECM extracellular matrix
  • leiomyoma tissue for the cell culture composition or extracellular matrix is from uterus as availability of the material is relatively high.
  • a preferred cell culture composition is made of a total protein extract from human leiomyoma tissue.
  • Said total protein extract can be prepared by multiple homogenization and purification methods known for a person skilled in the art, although the method described by Kibbey 1994 with modifications as explained in the Experimental Section below is the most preferred.
  • the cell culture composition preferably comprises agarose, since it provides improved manageability and reproducibility for human cell culture studies.
  • low melting point agarose is particularly preferred.
  • the melting temperature of said low melting point agarose is under 65 degrees of Celsius.
  • the gell ing temperature of said low melting point agarose is usually under 30 degrees of Celsius, preferably 26-30 degrees of Celsius, more preferably 30 degrees of Celsius.
  • the present invention is also directed to a method of preparing extracellular matrix homogenate as described above.
  • the method comprises the steps of: (a) homogenizing human leiomyoma tumor at least twice in a NaCI-buffer and discarding the soluble fraction after each homogenization;
  • step (b) extracting the residual leiomyoma tumor in a urea buffer and stirring the extract for about 12-18 hours; (c) separating the extract from the residual leiomyoma tumor fraction and saving the first extract while repeating step (b) with the residual leiomyoma tumor fraction and saving the second extract resulting therefrom;
  • step (d) combining the first and second extracts from step (c) and dialyzing against a buffer comprising a sterilizing component such as chloroform; and (e) further dialyzing against a buffer not comprising a sterilizing component and recovering a dialysate.
  • a sterilizing component such as chloroform
  • Th is method may also comprise further steps of
  • step (f) dialyzing the dialysate obtained from step (e) against a serum-free medium
  • the NaCI-buffer of step (a) is a buffer containing at least or about 3.4 M NaCI and the urea buffer of step (b) is a buffer containing at least or about 2 M urea.
  • the buffer of step (a) is a 3.4 M NaCI buffer having a pH of about 7.4 and the urea buffer of step (b) is a 2 M urea buffer having a pH of about 7.4.
  • the method may comprise a further step of mixing the dialysate from step (g) with agarose, such as low melting point agarose, which preferably has melting temperature under 65 degrees of Celsius.
  • agarose such as low melting point agarose
  • the present invention is thus directed to the use of the described cell culture composition for studying or incubating mammalian cells, preferably human cells, more preferably human cancer cells, mesenchymal stem cells, epithelial cells, fibroblasts, endothelial cells, neurons, and most preferably invasive cancer cells.
  • the present invention is also directed to the use of the cell culture composition for invasive, migratory or adhesive cultures of human cells, or for capillary formation culture of human umbilical vein endothelial cells.
  • Another preferred use of the invention is in drug discovery, particularly cancer drugs.
  • the described Myogel is able to simulate the tumor environmental conditions of human tissues similarly as the myoma disc disclosed by the prior art (N diligentnniemi et al. 2009).
  • the Myogel is easier to prepare and handle than the myoma disc of the prior art. Also it provides more repeatable results since extracel lular matrix can be prepared from mixture of several myomas whereas myoma disc is from tissue isolated from one patient.
  • the Myogel provides a natural-like 3D environment for human cancer cells so that they show their migration characteristics
  • the matrix is suitable for functional irradiation studies and for other cancer study applications in vitro, e.g. angiogenesis studies, and improves the prediction of preclinical human cancer studies.
  • Suitable cell types for such studies are, e.g. cancer cell l ines, such as carcinomas, adenocarcinomas, melanomas and sarcomas.
  • the study time in Myogel is considerably shorter than in myoma disc (a few days vs. a few weeks) and the invasion results from Myogel can be obtained by measuring absorbance, which is much faster and easier than histologic sampling from myoma disc.
  • Hu man uterus leiomyoma tissue leftover pieces were received from the Oulu U niversity Hospital, Department of Gynegology (see Nu rmenniemi et al. 2009) after patient's written informed consent.
  • the ethical permission for using the myoma tissue was approved by the Ethics Committee of the Oul u U niversity
  • tissue powder was suspended in 20 ml of ice cold 3.4 M, pH 7.4 NaCI buffer. After centrifugation, the pellet was homogenized in another 20 ml of the same NaCI buffer as in a previous step using T18 U ltra-Turrax (I KA ® -Werke GmbH & Co. KG). T18 U ltra-Turrax was used also in all the following homogenizations.
  • the protein concentration in each preparation was measured using DC Protein Assay (Bio-Rad) according to manufacturer's instructions. The absorbances were read at 590 nm using Victor 3 V 1420 Multilabel Counter and Wallac 1420 Manager.
  • Protein concentrations in various Myogel batches were diluted using cell culture med ia described in cell culture-section (see below) accordingly to match Matrigel ® protein concentration in every experiment.
  • Myogel solution was stored in small ( ⁇ 1 ml) aliquots at -20 °C.
  • Matrigel ® (BD Matrigel Matrix, BD Biosciences Cat. N umber 354234) was diluted in proportions of 1+1 (one part of Matrigel ® + one part of serum free medium), and here the same amount of total protein for Myogel was obtained by diluting it accordingly (10+6).
  • the pH of the gels was measured in the beginning, after 17 h, and at the end of the 48 h experiment from both samples. The gels were incubated at 37 °C in a 5% CO2 humidified cell culture chamber with or without HSC-3 cells on top of the gels. Gradient SDS-PAGE of Myogel and Matrigel ® and proteomic analyses of Myogel
  • the amount of protein in four different Myogel batches (3, 4, 6, 9) was determined using Bradford assay (Bio-Rad) according to the manufacturer ' s instructions. Absorbances were measured using Biochrom Asys Expert plus Microplate Reader (Biochrom) at 595 nm. 20 ⁇ of each sample was loaded on gradient (4%, 8%, 15%) SDS-PAG E gel and ran using 15 mA for 90 min. PageRuler Prestained Protein Ladder (Thermo Scientific) was ran together with the samples. Proteins were stained using Coomassie Bl ue and the gel was washed with elution buffer to remove excess staining. The gel was viewed over a stripping table and individual bands were cut and collected from the gel. After digestion (0.3 ⁇ g of trypsin was used/band) each band was resuspended with formic acid on three selected Myogel samples (3, 6, 9) and stored in -20 °C until gel digestion.
  • SDS-PAG E was performed overn ight in polyacrylamide gels (12.5% T, 2.6% C) with the Ettan DALT I I system (G E Healthcare) at 1-2 W per gel and 12 °C.
  • the gels were silver stained as described earlier (Ohlmeier et al., 2008) and analyzed with the 2-D PAG E image analysis software Melanie 3.0 (GeneBio).
  • Biosciences Cat. N umber 354234 were detected by zymography method using fluorescently labeled gelatin (Pirila et al. 2007). Prestained low-range SDS-PAG E Standards (Bio-Rad) as well as purified control M M P-2 and M M P -9 samples were ru n in adjacent wells to the samples. After electrophoresis, gelatinases were activated by incubating the gels with zymography buffer (50 m M Tris-HCI, 5 m M CaCI 2 , 1 ⁇ ZnCI 2 , 0.02% NaN3, pH 7.5) overnight at 37 °C. Gelatin degradation was visualized under long wave UV l ight and photographed using AlphaDigiDoc ® RT Gel Documentation System.
  • zymography buffer 50 m M Tris-HCI, 5 m M CaCI 2 , 1 ⁇ ZnCI 2 , 0.02% NaN3, pH 7.5
  • Hu man oral tongue squamous cell carcinoma cell lines HSC-3, SAS (both from Japan Health Sciences Foundation, Japan) and SCC-25 (American Type Cu lture Collection. ATCC) with different aggressive potential were cultured in a 1: 1 DM EM/F-12 medium (Life Technologies) supplemented with 100 U/ml penicillin (Sigma-Aldrich or Life Technologies), 100 ⁇ g/ml streptomycin (Sigma-Aldrich or Life Technologies), 250 ng/ml fungizone (Sigma-Aldrich), 50 ⁇ g/ml ascorbic acid (Sigma-Aldrich or Applichem)and 0.4 ⁇ g/ml hydrocortisone (Sigma- Ald rich) and 10% heat inactivated fetal bovine serum (FBS; Life technologies).
  • DM EM/F-12 medium Life Technologies
  • penicillin Sigma-Aldrich or Life Technologies
  • streptomycin Sigma-Aldrich or Life Technologies
  • 250 ng/ml fungizone Sigma
  • HSC-3 cells labeled with RFP were generated by stable transduction with commercial lentiviral particles containing a non-coding control sequence (Amsbio) and selected with puromycin. They were cultured as normal HSC-3 cells.
  • HSC-3 cells labeled with G FP were generated by stable transduction with non-silencing G I PZ lentiviral sh RNAmir control particles (pG I PZ vector contains G FP in order to track sh RNAmir expression; Thermo Fischer Open Biosystems) with puromycin (Sigma-Aldrich) selection according to manufacturer's instructions.
  • N uclear histone-2B (H2 B)-coupled mCherry expression vector pLenti6.2V5/DEST (a gift from Dr. Cindy E.
  • HSC-3 cells Dieteren, Department of Cell Biology, Radboud U MC, Netherland was introduced to the HSC-3 cells with cytosolic G FP labeling using lentivirus mediated infection and selected in culture media containing 5 ⁇ g/ml blasticidin-S (Merck Millipore).
  • HSC-3 cells expressing cytoplasmic G FP and H2B-cou pled mCherry were cultured in DM EM/F12 medium
  • SCC-9 cell line (American Type Culture Collection, ATCC) was maintained in DM EM/F-12 med ium (Invitrogen) supplemented with 10% FBS (Cultilab), 400 ng/mL hydrocortisone, and antibiotic/antimycotic solution (Invitrogen).
  • SCC-9 cells were labeled with ZsGreen protein and implanted subcutaneously into the footpads of the left front limb of BALB/c nude mice and LN-1 and LN-2 cell lines with increased metastatic potential were derived by in vivo selection from axillary lymph nodes with metastatic cells as described earlier (Agostini et al. 2014). Both LN-1 and LN-2 cells were maintained in culture as SCC-9.
  • Hu man melanoma cell line Bowes was cultured in DM EM medium (high glucose, Life technologies) supplemented with 100 U/ml penicillin (Life tech nologies), 100 ⁇ g/ml streptomycin (Life technologies), 250 ng/ml amphotericin B (Sigma-Aldrich), 50 ⁇ g/ml ascorbic acid (Applichem), 1 mmol/L sodium pyruvate (Sigma-Aldrich) and 10% heat- inactivated fetal bovine serum (Life technologies).
  • G F Normal oral gingival fibroblasts
  • Hu man umbilical vein endothelial cells were cultured in a 1: 1 mixture of DM EM/F12 medium (Invitrogen) supplemented with 10% FBS and 400 ng/ml hydrocortisone (Sigma-Aldrich).
  • TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
  • the TU N EL assay was performed using a commercially available kit (In Situ Cell death Detection Kit, Roche). At the end of the assay, samples were counterstained with DAPI for 10 min at RT. After coverslip mounting with a water based medium, 100 cells were counted under confocal microscope using blue channel (405 nm) and apoptosis was detected using green channel (488 nm).
  • Cell adhesion assay was conducted to determine how many cells bind to Myogel compared to Matrigel ® (BD Matrigel Matrix, BD Biosciences Cat. Nu mber 354234).
  • HSC-3 cells were cu ltured to subconfluence.
  • Wells in 96-well plate were coated either with 100 ⁇ of PBS, BSA (bovine serum albumin, 10 ⁇ g/ml, Sigma), Matrigel ® or Myogel .
  • Matrigel ® was diluted to 1: 10 in PBS and Myogel was diluted to the same protein concentration.
  • the wells were coated 24 h before adding the cells. At the same time, the cell cu lture medium was changed to serum-free.
  • HSC-3 cells (6 000) in 100 ⁇ of serum-free medium were added to each well and the wel ls were incubated at 37 °C in 5% CO2 humidified atmosphere for 2 h.
  • the non-adherent cells were rinsed off, and the remaining cells were fixed with 10% trichloroacetic acid (TCA), stained with crystal violet and quantified using ELISA reader at 540 nm.
  • TCA trichloroacetic acid
  • Ad hesion of G Fs on top of Myogel was studied using 6-wel l plates coated at 37 °C in 5% CO2 humidified atmosphere with 0.62 mg/ml Myogel diluted with DM EM without supplements. After 2 h the excess liquids were removed and 150 000 G Fs were added in their normal culture medium. The cultures were photographed with Olympus CKX41 inverted microscope after 2.5 h and 9.5 h with 20 x magnification to record the morphology of the cells.
  • sterile base low melting agarose (LMA, Sea Plaque Low Melting Agarose, Lonza) melted in PBS was mixed with 10 x DM EM/F12, 100% FBS to give a final 0.8% agarose with 1 x medium, 10% FBS. Of the mixture, 0.5 ml was added to 24-well plate and let to solidify for at least 30 min in the laminar. HSC-3 (H2B-G FP) cells were trypsinized and counted.
  • LMA Sea Plaque Low Melting Agarose, Lonza
  • Myogel was centrifuged at 4 000 rpm for 10 min prior to the procedure. Agarose mixtures were gently mixed by swirling and 0.5 ml was added on the top of LMA. The plates were incubated at 37 °C in humidified incubator for 28 days. Cells were fed twice a week with 0.25 ml normal HSC-3 med ium. After 28 days the pictures of colonies were taken with transmitted light, G FP & RFP channel in different objectives (lOx, 20x & 40x) using EVOS inverted microscope. Cells in colonies were calculated from the pictures and ImageJ software (Rasband, W.S., ImageJ, U .S. National Institutes of Health, Bethesda, Maryland, USA,) was used to measure colony area.
  • ImageJ software Rasband, W.S., ImageJ, U .S. National Institutes of Health, Bethesda, Maryland, USA, was used to measure colony area.
  • Transwell ® nylon filter membrane insert (Corning Inc.), incubated at 37 °C in 5% CO2 humidified atmosphere for 30 min after which 50 000 HSC-3 cells suspended in 100 ⁇ of serum-free medium were seeded onto the upper compartment of the Transwell ® chamber.
  • Transwell ® inserts were incubated for 12 h - 48 h at 37 °C in 5% CO2 humidified atmosphere, after which the cells were fixed in 10% TCA for 15 min, rinsed and air dried overnight. Once dry the membranes were stained with crystal violet for 20 min and the excess stain was removed by water rinsing. The uninvaded cells from the upper side of the membrane were removed by carefully sweeping with a cotton swab.
  • 2.4 mg/ml Myogel (with 0.2% LMA) or Matrigel ® was used to study the invasion of oral squamous cell carcinoma cell lines SAS and SCC-25, melanoma cell l ine Bowes and pancreatic cancer cell lines Pa02c, Pa03c and Pa04c.
  • the Transwell inserts were incubated for 72 h after which the invaded cells were stained with 1% Toluidine blue solution. The absorbance of the eluted dye was measured at 570 nm wavelength.
  • collagen/Matrigel ® (BD Matrigel Matrix, BD Biosciences Cat. N umber 354234) (1.5 mg/ml, 1.5 mg/ml) and collagen/Myogel (1.5 mg/ml, 4.3 mg/ml) mixtures were prepared similarly.
  • the hanging drop technique was used to observe the cell movement in 3 D culturing condition.
  • HSC-3 cells were washed with PBS, trypsinized and 70 000 cel ls in 10 ⁇ of F12/DM EM med ium (2% FBS) were mixed with 50 ⁇ of the matrix mixtu re. 20 ⁇ of the cell suspension in each matrix was dropped on the 4 compartment plate. Plate was flipped around after 5 min incubation in culturing conditions and hanged for 3 h in humidified chamber in culturing conditions. Mimosine (200 ⁇ ) was added to the medium to synchronize the cell cycle.
  • HSC-3 cells transduced with RFP were seeded into uncoated or Myogel coated 6-well plates (three wells each). The next day, the cells were harvested for RNA extraction by Qiagen RNA kit. The three samples of each group (on top of plastic or Myogel coating) were pooled with an equal amount of each RNA.
  • Affymetrix GeneChip Hu man Genome U 133 Plus 2.0 Arrays were used for microarray analysis and experimental proced ures were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 1 ⁇ g of total RNA was used as a template to synthesize biotinylated cRNA by means of the GeneChip 3'IVT Express kit (Affymetrix) according to the
  • the cRNA was fragmented to 35 to 200 nt prior hybridizing to Affymetrix H u man Genome U 133 Plus 2.0 arrays containing approximately 55 000 human transcripts. The array was washed and stained with streptavidin-phycoerythrin (Molecular Probes). Finally, biotinylated anti-streptavidin (Vector Laboratories Inc.) was used to amplify the staining signal and a second staining was performed with streptavidin-phycoerythrin. The arrays were scanned on GeneChip Scanner 3000. The expression data was analyzed to find genes with fold change (FC) 1.5 or more using dChip software (Li & Wong 2001). The genes with FC 1.2 or more were divided into Gene Ontology (GO) categories using DChip enrichment analysis tool.
  • FC fold change
  • FC Gene Ontology
  • 96-well culture plates (BD Biosciences) were coated with Myogel with 2% low melting agarose (Myogel-LMA), Matrigel ® -G FR (BD Matrigel Matrix, Cat. N umber 35430 - BD, Lot. 3270647) or ECMatrixTM (ECMatrix - In vitro Angiogenesis Assay Kit, Cat. N umber ECM625 - Millopore, Lot. 2383502) previously thawed overnight on ice, in a total vol ume of 50 ⁇ /well and allowed to solidify overnight at 37 °C.
  • Myogel-LMA Myogel with 2% low melting agarose
  • Matrigel ® -G FR Matrigel Matrix
  • ECMatrixTM ECMatrix - In vitro Angiogenesis Assay Kit, Cat. N umber ECM625 - Millopore, Lot. 2383502
  • UVEC cells were trypsinized, neutralized with DM EM/F12 with 10% FBS, washed once with PBS and resuspended in DM EM/F12 at a density of 450 000/ml, and 100 ⁇ of this cell suspension was added into each well. The cells were incubated at 37 °C for 12 h . Tube formation was observed under an inverted microscope (N ikon Eclipse Ti-S, x4), photos were taken and analysed using the motic images plus 2.0 program. Three visual fields were randomly selected from each well to count the tubes, and the average value was taken for statistical analysis. Tubule perimeter was assessed by drawing a line around each tubule and measuring the line.
  • DSRT Drug sensitivity and resistance testing
  • SPSS for Windows software program version 21.0 SPSS Inc. was used for statistical analyses. To establish the statistical significance of differences between the two independent cell culture groups, a Student's t-test or Mann-Whitney U test was used depending on the normalization of the distributions. Results
  • Protein content in different Myogel batches varies slightly and is different from Matrigel ®
  • the pH of Myogel is neutral and more stable than the pH of Matrigel ®
  • Matrigel ® Matrigel ® protein content was summarized from Vukicevic et al . (1992), Hughes et al. (2010) and Talbot & Caperna (2014). Based on the comparison, e.g. laminin, type IV collagen, heparan sulfate proteoglycans, nidogen and epidermal growth factor were found in both. As compared to Matrigel ® Myogel was lacking enactin, fibroblast growth factor, insulin- like growth factor 1, platelet-derived growth factor and nerve growth factor. Myogel had e.g. tenascin-C, collagen types XI I and XIV, etc. which were lacking in Matrigel ® .
  • Myogel contains both latent and active forms of M M P-2, whereas in Matrigel ® latent and active forms of both M M P-2 and M M P-9 were present.
  • 1030 proteins identified in Matrigel ® 40 were characterized as RI KEN cDNA and 45 represented predicted genes. Hence with these data we cannot directly indicate the presence of these proteins in Myogel .
  • Myogel has much more proteins visible in 2-DE than Matrigel ®
  • HSC-3 cells stayed alive up to 28 days, divided and formed colonies within Myogel combined with LMA.
  • the results with HSC-3 cells were rather similar using either Myogel-LMA than conventional LMA method (Fig. 2A). However, more colonies with the lowest cell number were present in LMA, while the highest cell number/colony was present in Myogel-LMA (Fig. 2B). According to nuclear mCherry expression, 92% of the cancer cells were alive in Myogel- LMA colonies, whereas 85% were alive in LMA colonies. The total average area of cell colonies in Myogel-LMA was three percentages less than in LMA (not shown).
  • HSC-3 cells invade more efficiently through Myogel than through Matrigel ®
  • HSC-3 oral tongue carcinoma cell line
  • Myogel solidified with agarose (Myogel-LMA) is suitable TME matrix for invasion assay
  • Myogel-LMA was compared with Matrigel ® -G FR.
  • all the oral squamous cell carcinoma cell lines used for this invasion assays (HSC-3, LN-1, LN-2 and SCC-9) seemed to prefer Myogel-LMA to Matrigel ® -G FR d uring the invasion ( Figure 5C ).
  • HSC- 3 and LN-2 (in this order) seemed to have a higher potential to invade in both Myogel-LMA and Matrigel ® -G FR, compared to LN-1 and SSC-9 cell lines.
  • the cells invading in the Myogel- LMA seemed to keep more of their morphological characteristics, like we see in monolayer culture.
  • Myogel induces efficiently the tube formation
  • ECMatrixTM (not shown), where most of the H UVEC cells even already after 24 h were apoptotic. We found three times higher number of tubules formed in Myogel-LMA compared to ECMatrixTM (Fig. 6B). Otherwise, measu ring the diameters of the capillaries, the tubule parameters in Matrigel ® -G FR, and especially in ECMatrixTM assays, were significantly higher than in tubes formed in Myogel-LMA (Fig. 6C).
  • HSC-3 cells move faster in Myogel-collagen matrix than in Matrigel ® -collagen matrix in 3D culturing condition
  • HSC-3 cells seemed to move relatively similarly in pure collagen, Myogel-collagen and Matrigel ® -collagen matrices.
  • the nuclei count was higher and varied more in all matrices than the cytosomal cell count, the count was highest in pure collagen in both.
  • the nuclei size was about the same and rather even in all the matrices while the cell size varied more, both between the matrices and during the recording in each matrix. Both the nuclei and the cell sizes were largest in Matrigel ® -collagen matrix.
  • the average eccentricity/roundness measured by nuclear stain was about the same and rather even in all the matrices while was highest in pure collagen and varied more when measured by cytosomal stain. In speed the result was rather similar measured by nuclear and cytosomal stain in each matrix. However, the speed was highest in Myogel-collagen matrix and lowest in Matrigel ® -collagen matrix.
  • Myogel is suitable coating for drug testing
  • Viability of the cells to be used in drug testing was first measured on different concentrations of Myogel and Matrigel ® .
  • the viability of the cel ls was significantly higher in Myogel coated wells than in Matrigel ® coated ones (Fig. 7).
  • About one third of the drugs (48) had different IC50 values against Pa02c cells when tested on top of Myogel vs plastic. 20% of the drugs were more effective in Myogel than in plastic, while 16% were more effective in plastic than in Myogel. More than one third of the drugs (56) had different IC50 values when tested on top of Matrigel ® vs plastic.
  • Myogel and especially easy-to-use Myogel- LM A, are both well suited for in vitro cancer studies. They are in some cases superior to the Matrigel ® or to the rat tail type I collagen - based methods. Using easily obtained human uterus leiomyoma tumor tissue, which normally is wasted after histopathological analyses, to produce Myogel mixtu re, the material costs are relatively low.
  • Myogel offers a natural human TM E based matrix to study the behavior of various cancer cell lines and cancer drugs in vitro.
  • this set of instruments may well be usable also in the future for personalized medicine where, after obtaining a fresh tumor tissue biopsy for analyses, the effects of drugs or chemoradiation therapies could be tested for finding the optimal treatment modality for the patients.

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Abstract

La présente invention concerne un homogénat de matrice extracellulaire utilisé pour la culture de cellules humaines in vitro comprenant des tissus de léiomyome humains homogénéisés. La présente invention concerne également un procédé de production d'un homogénat de matrice extracellulaire à partir de tissus de léiomyome éliminés prélevés sur des patients pendant des interventions systématiques.
PCT/FI2016/050163 2015-03-17 2016-03-17 Matrice extracellulaire à base de tumeur humaine pour des études de cellules in vitro WO2016146893A1 (fr)

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CN111705037A (zh) * 2020-06-24 2020-09-25 西交利物浦大学 一种成纤维细胞与癌细胞的3d共培养模型及其制备方法和应用
CN115261302A (zh) * 2022-07-20 2022-11-01 创芯国际生物科技(广州)有限公司 一种基质胶及其制备方法和应用

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CN111705037A (zh) * 2020-06-24 2020-09-25 西交利物浦大学 一种成纤维细胞与癌细胞的3d共培养模型及其制备方法和应用
CN115261302A (zh) * 2022-07-20 2022-11-01 创芯国际生物科技(广州)有限公司 一种基质胶及其制备方法和应用
CN115261302B (zh) * 2022-07-20 2023-06-06 创芯国际生物科技(广州)有限公司 一种基质胶及其制备方法和应用

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