[go: up one dir, main page]

WO2016123683A1 - Trousse, son procédé d'obtention et procédé in vitro pour évaluer la viabilité et/ou la prolifération cellulaire par rapport à un agent actif - Google Patents

Trousse, son procédé d'obtention et procédé in vitro pour évaluer la viabilité et/ou la prolifération cellulaire par rapport à un agent actif Download PDF

Info

Publication number
WO2016123683A1
WO2016123683A1 PCT/BR2015/050008 BR2015050008W WO2016123683A1 WO 2016123683 A1 WO2016123683 A1 WO 2016123683A1 BR 2015050008 W BR2015050008 W BR 2015050008W WO 2016123683 A1 WO2016123683 A1 WO 2016123683A1
Authority
WO
WIPO (PCT)
Prior art keywords
container
cells
kit
asset
proliferation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/BR2015/050008
Other languages
English (en)
Portuguese (pt)
Inventor
Daniela Bauman CORNELIO
Caroline Brunetto DE FARIAS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ziel Biosciencias Pesquisa Desenvolvimento E Diagnostico Ltda
Original Assignee
Ziel Biosciencias Pesquisa Desenvolvimento E Diagnostico Ltda
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ziel Biosciencias Pesquisa Desenvolvimento E Diagnostico Ltda filed Critical Ziel Biosciencias Pesquisa Desenvolvimento E Diagnostico Ltda
Priority to PCT/BR2015/050008 priority Critical patent/WO2016123683A1/fr
Publication of WO2016123683A1 publication Critical patent/WO2016123683A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/16—Apparatus for enzymology or microbiology containing, or adapted to contain, solid media
    • C12M1/18—Multiple fields or compartments
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C12Q1/20—Testing for antimicrobial activity of a material using multifield media

Definitions

  • the present invention is in the field of biology, biochemistry and chemistry. More specifically, the present invention provides products and processes useful as basic research tools for experimental cancer cell proliferation control tests and for testing active combinations and / or novel pharmaceutical compositions for treating cancer. In a preferred embodiment, the products and processes of the invention are useful in detecting / quantifying chemoresistance and / or chemosensitivity in vitro, which contributes to better treatment selection for patients with various types of cancer.
  • Chemotherapy is a fundamental part of tumor management and is often the only cure option for patients.
  • disease progression and death are still major problems that largely result from intrinsic and acquired drug resistance.
  • drug response assessment systems have been tested to determine the potential activity of therapeutic agents prior to their administration to patients.
  • Chemoresistance tests are designed to identify drugs to which the tumor is resistant, while chemosensitivity tests aim to identify drugs to which the tumor is most sensitive. Both assays are performed to assess whether tumor growth is inhibited by a drug or, more commonly, a combination drug panel. However, chemoresistance assays focus on negative predictive value (the ability of the test to identify ineffective agents) rather than positive predictive value (the ability of the test to identify agents capable of inducing a clinical response).
  • Shoesnthal and Kern (1991) reported that the negative predictive value for in vitro drug response assays ranged from 90% to 99%, while the positive predictive value was lower, between 50% and 70%.
  • Haroun, et al. (2002) described a drug resistance profile (extreme, intermediate or low) based on a statistical comparison of a database of brain tumor samples tested against the same panel of chemotherapeutic agents.
  • the authors stated that through continuous analysis and compilation of data from multiple institutions, chemoresistance profiles could help future researchers with the development of clinical trials and treatment regimens for brain tumor patients.
  • chemosensitivity and chemoresistance testing can be used to select chemotherapy in situations where there are several options of equivalent chemotherapy available.
  • NCN National Comprehensive Cancer Network
  • chemoresistance and chemosensitivity tests in the clinical setting faces some obstacles, such as restrictions on testing only by the manufacturer who has adequate infrastructure to handle controlled manipulation of substances such as chemotherapeutic agents.
  • other obstacle lies in the transport of fresh tissue samples. These samples are often in small quantities and need to be transported quickly and cautiously so that they do not degrade. This factor makes it impossible to perform the tests in patients located in areas distant from the appropriate laboratories to perform the tests.
  • the present invention aims to solve these problems.
  • US 2010/0143948 is about assays and methods for monitoring the individual progression of a human neoplasm. It presents a system that evaluates cellular and secretor markers and detects genotypic and phenotypic alterations. Thus, it predicts the therapeutic response of a patient's tumor cells to one or more therapeutic agents. That disclosed herein differs from the present invention, among other technical reasons, because said assay cells are incubated at one or more separate sites prior to contact with the therapeutic agent and controlled manipulation of the therapeutic agent by the tester is required. These difficulties / problems are solved by the present invention.
  • Fruehauf (2002) reviews major in vitro drug response trials and clinical validation of various technologies currently available to assist clinicians in choosing the best treatment for each patient.
  • in vitro drug response assays have emerged to improve treatment response and patient survival by determining the potential activity of a patient's chemotherapeutic agents prior to initiation of treatment.
  • the premise is to build a drug response profile for each individual patient, where differences are based on genetic diversity and the development of tumor subclones with different phenotypes.
  • the in vitro tests described in the article differ from the present invention, among other technical reasons, in that they do not disclose or even suggest prior application of the chemotherapeutic agents to a recipient or cell culture site, which is of great utility and practicality. and safety for the user.
  • the technique remains the need for an in vitro test that detects cell viability and / or proliferation in relation to an active that can be performed simply and effectively, without the need for controlled manipulation and risks of contamination and biosecurity. , enabling the broad realization of the process. Moreover, the various tests performed by the inventors demonstrate the difficulty of achieving this goal.
  • the present invention provides a solution for the safe and practical application of chemosensitivity and chemoresistance testing, bypassing current restrictions on controlled substance handling and fresh tissue sample transport.
  • the invention enables such tests to be performed on patients located in areas distant from the appropriate laboratories.
  • the inventive concept common to the objects of the invention is the enabling in vitro evaluation, locally and without the need for manipulation of potentially hazardous substances, viability and / or cell proliferation in relation to an asset.
  • Said assessment may be qualitative / detection or quantitative / dosage and said active may be selected from, for example, chemotherapy in situations where there are several options of equivalent chemotherapy available.
  • It is therefore an object of the present invention to provide a process for preparing a cell viability and / or proliferation assessment kit for an asset comprising preparing a container for stably and actively containing at least two assets, each active or combination of assets occupying at least one well or distinct region of the container.
  • kit preparation process one or more colorimetric compound (s) may also be incorporated into the container to distinguish viable from non-viable cells.
  • the kit container is polymeric and treated with chemical and / or physical means to alter the attachment of the assets to its surface.
  • surface functionalization is done by electromagnetic radiation and more preferably with UV irradiation.
  • said actives are added to the container at concentrations of 1 nM to 20 ⁇ and then dried.
  • semisolid media is additionally included in the container, preferably gelatin and / or agar culture medium.
  • two or more actives are added to the kit container in the same well or region, which configuration is particularly suited for asset synergism or antagonism evaluation tests.
  • Another object of the present invention is a kit for assessing cell viability and / or proliferation with respect to an asset comprising:
  • the kit additionally comprises digestive enzymes, culture medium suitable for cell culture, solubilizer and / or sample collection vessels.
  • the container is of polymeric material, preferably polystyrene.
  • the colorimetric compound is a tetrazolic dye, preferably MTT, MTS, WST and / or XTT.
  • a culture medium for growing said cells and / or one or more colorimetric compound (s) is also added to said container prior to or subsequent to said contacting to distinguish viable from nonviable cells.
  • said cells comprise tumor cells.
  • the active is selected from the group consisting of chemotherapeutic, biological, hormonal agents or combinations thereof. In another preferred embodiment, more than one active is present in the same region of the recipient, enabling evaluation of synergism and / or antagonism.
  • Figure 1 Illustrates cells within normal growth rate and morphology on the polymer plate matrix of a kit, viewed under an inverted microscope. A): cells in smaller increase, B): cells in larger increase.
  • Figure 2 Illustrates cells on the polymer matrix along with discs containing antitumor agents after 48 hours of cultivation, followed by the addition of dye. Wells that only have the dye-staining on the periphery had a significant halo formation, while discs that were stained have cells around them, indicating that there is resistance of the cells to the drug tested.
  • Figure 3 shows a schematic illustrating a 6-well plate kit, where A1 is the culture plate holder, A2 is the culture plate cover, B is the polymer matrix and C is the discs containing the antitumor agents.
  • Figure 4 shows a schematic illustrating a petri dish kit, where A1 is the culture plate holder, A2 is the culture plate cover, B is the polymer matrix and C is the discs containing the antitumor agents.
  • Figure 5 shows the adhesion and cell proliferation in plaque that has been sandblasted.
  • B Visualization of a well by inverted microscope treated with sandblasting. In the bright area no blasting was performed and cell adhesion and proliferation can be noted according to the pattern. However, it was not possible to observe cell growth over the grooves (dark area).
  • FIG. 6 UV-treated polystyrene FTIR-ATR.
  • Sample 1 25 ⁇ _ of gelatin plus chemotherapeutic (25 ⁇ _) before and after washing with saline, placed in oven at approximately 60 ° C. Spectra have been shifted from their original position for better viewing.
  • FIG. 7 UV-treated polystyrene FTIR-ATR, sample 2: chemotherapeutic (25 ⁇ _) before and after washing with saline and oven-drying at approximately 50 ° C. Spectra have been shifted from their original position for better viewing. Subtitles: T: transmittance; N: Wave Number; Circle: Polystyrene; Triangle: Polystyrene + Chemotherapy + Serum + High Temperature; Star: Polystyrene + Chemotherapy + serum + elevated temperature + washed.
  • Figure 8 UV-treated polystyrene FTIR-ATR, gelatin sample 3: 25 ⁇ _ plus chemotherapeutic (25 ⁇ _) before and after washing with saline. Spectra have been shifted from their original position for better viewing. Subtitles: T: transmittance; N: Wave Number; Circle: Polystyrene; Triangle: Polystyrene + Chemotherapy + Gel; Star: Polystyrene + Chemotherapy + Gel + Washed.
  • FIG. 9 UV-treated polystyrene FTIR-ATR, sample 4: 25 ⁇ chemotherapy in saline before and after washed with saline. Spectra have been shifted from their original position for better viewing. Subtitles: T: transmittance; N: Wave Number; Circle: Polystyrene; Triangle: Polystyrene + Chemotherapy + Saline; Star: Polystyrene + Chemotherapy + Saline + Washed.
  • Figure 10 Polystyrene Petri dishes after ultraviolet irradiation, followed by the addition of 5-fluoracil and cell inoculation. The absence of inhibitory halo formation around the irradiated central area is observed in all plates.
  • Figure 1 1 Quantitative evaluation comparing HT-29 cells treated with 5-fluoracil (5-FU; 12 ⁇ ) with control cells.
  • Figure 12 Quantitative evaluation comparing Oxaliplatin-treated HT-29 cells (24 ⁇ ) with control cells.
  • Figure 13 Quantitative evaluation comparing Docetaxel-treated OVCAR-3 cells (12 ⁇ ) with control cells.
  • Figure 14 Quantitative evaluation comparing Doxorubicin-treated OVCAR-3 cells (6 ⁇ ) with control cells. A) Week 1. B) Week 2. C) Week 3. D) Week 4.
  • Figure 15 Quantitative evaluation comparing Cisplatin-treated OVCAR-3 cells (12 ⁇ ) with control cells. A) Week 1. B) Week 2. C) Week 3. D) Week 4.
  • Figure 16 Quantitative evaluation comparing Carboplatin-treated OVCAR-3 cells (12 ⁇ ) with control cells. A) Week 1. B) Week 2. C) Week 3. D) Week 4.
  • Figure 17 Quantitative evaluation comparing OVCAR-3 cells treated with combinations of A) Paclitaxel (12 ⁇ ), B) Carboplatin (12 ⁇ ), and C) The combination of Paclitaxel (12 ⁇ ) and Carboplatin (12 ⁇ ).
  • Figure 18 Quantitative evaluation comparing HT-29 cells treated with reduced dose combinations of A) 5-fluoracil (3 ⁇ ), leucovorin (9 ⁇ ) and oxaliplatin (6 ⁇ ) with control cells. B) 5-fluoracil (3 ⁇ ), leucovorin (9 ⁇ ) and irinotecan (3 ⁇ ).
  • Figure 19 Illustrative photo of HT-29 colorectal cancer cell line plaques after dye postponement (72 hours after cell inoculation). A): week 1; B): week 2; C): week 3 and D): week 4 after plate preparation.
  • Figure 20 Illustrative photo of plaques of ovarian cancer cell line, OVCAR-3 (A to D), after dye postponement (72 hours after cell inoculation).
  • Figure 21 Illustrative photo of cells observed under a microscope after 72 hours of inoculation. A) untreated cells; B) cells after doxorubicin treatment; C) cells after treatment with methotrexate; D) cells after asparaginase treatment.
  • Figure 22 Illustrative photo of 96-well plates after dye addition (72 hours after cell inoculation). In both controls were placed on lines D and E.
  • Figure 23 Quantitative results after treatment for 72 hours. ( * ) indicates drugs that showed significant difference from untreated cells (control). A) and C) Trypan blue exclusion cell count analysis; B) and D) Absorbance analysis after dye addition.
  • the process for preparing a cell viability and / or proliferation assessment kit for an asset solves the practical problem of making the assessment available in vitro, locally and without the need for manipulation of potentially hazardous substances, viability and / or cell proliferation relative to an asset. From this process it is possible to implement a practical kit in hospitals, clinics and other health units that need this type of assessment quickly and safely.
  • the kit of the invention comprises a container previously containing the assets and / or combinations among the assets, each asset or combination occupying a well / region.
  • the kit of the invention further comprises: cell culture media; one or more colorimetric compound (s); one or more digestive enzyme (s); one or more solubilizer (s) for subsequent solubilization step of the crystals formed by the colorimetric compound; and / or patient sample storage container (s).
  • the in vitro process of the present invention provides for the evaluation of cell viability and / or proliferation with respect to an active process. comprising contacting one or more cells whose assessment of viability and / or cell proliferation with respect to an asset is desired, with at least two distinct assets present in a container that previously contains said assets.
  • a patient's cells are removed by a surgical procedure determined and performed by the physician and assistant staff. After biopsy or surgery, the tissue is placed in culture medium suitable for transport (when necessary) and kept at room temperature. The tissue is then subjected to the action of digestive enzymes, obtaining the tumor tissue separated from excess adjacent non-tumor tissue. Tumor cells are washed with culture medium and centrifuged, preferably at 3000g for 5 minutes. The culture medium is then removed and only the cell-containing pellet is homogenized again in appropriate culture medium.
  • the suitable container may contain wells in its structure, called "wells", where the active and / or inoculum are inserted.
  • Container wells may be of varying number.
  • the container pre-contains the active prior to addition of the inoculum with the cell of interest for cell viability and / or proliferation detection.
  • the container may consist of transparent polymeric material such as transparent rigid polystyrene.
  • a non-limiting example of a suitable container in the context of the present invention is a 6, 12, 24, 96, 384-well polystyrene microtiter plate, among others.
  • the actives consist of substances which are intended to evaluate the effect (sensitivity or resistance) on the cells of interest.
  • the evaluation results from the detection and / or quantification of cell viability and / or proliferation after exposure of the active cells for a certain time.
  • the actives correspond to chemotherapeutic, biological, hormonal agents and combinations thereof.
  • the assets and / or combinations thereof are pre-applied to the wells / regions of the container. The advantage of doing this beforehand, ie prior to the addition of the cell-containing inoculum of interest to the wells is that in this way, the in vitro process of the invention can be largely performed without the restriction of controlled handling of toxic substances that could occur given the choice of certain assets.
  • the inoculum consists of cells in which the sensitivity or resistance to the active is to be evaluated.
  • the cells are tumor cells.
  • Tumor cells are cancer cells with altered proliferation rate and / or no physiological function in normal tissue, which originate and were a tumor.
  • the inoculum containing the cells of interest is placed in at least one non-active well, establishing a control well for assessing viability and / or cell proliferation under standard conditions without the active.
  • the colorimetric compound is a compound that provides for the visual differentiation of viable cells from nonviable cells. Cell viability and / or proliferation can be detected with the aid of a spectrophotometer or similar equipment.
  • the wavelength of radiation that hits the container containing active, inoculum and colorimetric compound varies according to the colorimetric compound used.
  • the colorimetric compound is a tetrazolic dye which, when reduced by dehydrogenase and reductase enzymes present in viable cells, gives formazan, a colorant compound.
  • the process for preparing a cell viability and / or proliferation assessment kit for an asset comprises preparing a container for stably and actively containing at least two assets, each active or combination of assets occupying at least one well or distinct region of the container.
  • the container comprises two or more actives in the same well or region, which configuration is particularly suited for asset synergism or antagonism evaluation tests.
  • a culture medium concentration curve was performed, where a buffer solution was homogenized with bacto-agar and gelatin, followed by 15 minutes of autoclave sterilization. Afterwards, the same volume of RPMI 1640 culture medium (LGC, Brazil) containing 10% fetal bovine serum was added to the sterile solution, keeping as final concentrations: 0.1%; 0.25%; 0.5%, 1%; 1, 25% 1, 5% and 2%, always at a ratio of 1: 1 buffer solution and RPMI 1640 medium and 1: 1 gelatin and bacto-agar. At the time of placing the medium on the culture plate, it was observed that concentrations of 0.1% and 0.25% did not become completely solid even after a few days of plating.
  • kit procurement process was designed as follows:
  • dyes were also prepared to facilitate cell visualization.
  • the kit was used as follows: Cells were previously centrifuged and placed with 100 to 400 ⁇ _ of suitable culture medium (using a loop or 10 to 100 ⁇ _ pipettes) on the plate. were placed directly on the polymeric medium of the plate. The cell plate was left in a CO 2 incubator and 5% humid atmosphere for one hour. Then, the discs containing the anti-tumor agents were added from 48 hours to 96 hours. After this period, the formation of an inhibition halo was evaluated. Each antitumor agent tested had a distinct halo size. For ease of viewing, the kit dyes were placed on the plate for up to one hour, followed by measuring the halo with a ruler. Figures 1-4 illustrate the results of such experiments.
  • Example 1 Due to the difficulties in measuring halos, and the experimental variability of matrix formation (based on the conditions set forth in the tests described above), further tests have been conducted for the development and preparation of a practical kit, that is, that of the present invention.
  • different techniques for the deposition of the active ingredients / drugs in the plates were evaluated.
  • a 6-well polystyrene plate procedure was performed.
  • a metal mask has been developed to form circles of 5 mm in diameter equally distant from each other. This mask was placed over each of the wells and sandblasted. The plate was then sterilized with hydrogen peroxide and different tests were performed on these plates:
  • the drugs were placed directly over the circles at concentrations ranging from 1 nM to 20 ⁇ .
  • the drugs were placed on the circles in the presence of 1% gelatin (before the drugs, together or after the drugs).
  • the drugs were placed on the circles in the presence of 1% gelatin and dye (before the drugs, together or after the drugs).
  • the kit container was polymeric and was treated with physical means to alter the attachment of the assets to its surface. More specifically, such surface functionalization is done by UV irradiation at a center point of approximately 8 mm in diameter on polystyrene petri dishes. Through the surface functionalization technique assisted by electromagnetic irradiation, the objective was to increase the hydrophilicity of the polystyrene surfaces in order to evaluate whether the drugs could be retained in the irradiated area. After irradiation, the drugs were placed on the irradiated area and waited until the plate was completely dried, either in an oven at 60 Q C or at room temperature.
  • the treatment outcome was evaluated by water contact angle (WCA) measurements by the sessile drop method and using a digital electron microscope connected to a computer. After photochemical treatments the WCA ranged from approximately 85 Q (untreated polystyrene plate) to approximately 50 Q. After the treatments, four conditions were evaluated:
  • Sample 1 25 ⁇ _ gelatin + 25 ⁇ _ 5-fluoracil (20 ⁇ ) was placed over the modified area over the modified area. Subsequently the sample was introduced into an oven at approximately 60 ° C to Q Evaporation of the solvent for 2 hours.
  • Sample 2 25 ⁇ _ saline (without gelatin) + 25 ⁇ _ 5-fluoracil (20 ⁇ ) was placed over the modified area. Subsequently the sample was also introduced in an oven at 60 Q C to evaporate the solvent for 2 hours. Sample 3: Same as sample 1 but solvent evaporation was performed leaving the sample overnight on the bench (room temperature).
  • Sample 4 Same as sample 2, but solvent evaporation was performed by leaving the sample overnight on the bench (room temperature).
  • HT-29 colorectal cancer cells (5x10 5 cells per plate) were inoculated onto the polystyrene plate. It was observed that the cells were able to adhere to and grow on this plate. However, there was no halo formation around the irradiated plaque area, as the drug was diffused evenly throughout the plaque area, confirming the experiments previously described with FTIR-ATR ( Figure 10). Given that the previously tested methodologies were not able to sufficiently retain the drugs in specific portions of the plate, we proceeded to a new methodology using larger well-sized polystyrene plates (96-well plates). It was observed that the drug applied directly to the plates, and not to the inoculated cells, maintained its efficacy, even when kept at temperatures ranging from 21 Q C to 60 Q C.
  • said kit comprises:
  • a container containing at least two distinct well assets contained in said container, wherein each asset or combination of assets occupies at least one well in the container;
  • the kit contains at least one well without prior addition of active, a control well.
  • a suitable container in the context of the present invention would be a 6, 12, 24, 96 or 384-well polystyrene microtiter plate.
  • the kit further comprises a container for storing patient samples.
  • a culture medium suitable for cell culture and digestive enzymes is an integral part of the Kit.
  • the tumor tissue sample is subjected to digestive enzymes, which promote the separation of the tumor tissue from the adjacent non-tumor tissue, followed by centrifugation, washing and homogenization with appropriate culture medium.
  • digestive enzymes which promote the separation of the tumor tissue from the adjacent non-tumor tissue, followed by centrifugation, washing and homogenization with appropriate culture medium.
  • Such procedures ensure that the inoculum preferably consists of tumor cells and a suitable culture medium.
  • the kit further comprises a solubilizer for subsequent solubilization step of the crystals formed by the colorimetric compound, providing better reading on a spectrophotometer or similar equipment.
  • dose curves were performed in ovarian cancer cell lines, OVCAR-3, and colorectal cancer, HT-29, with drugs already used in clinical practice.
  • the tests were performed according to the conventional technique of cell culture, that is, the cells were previously inoculated in the plates, for later application of the drugs. Once a dose was effective in inhibiting the growth and proliferation of these cells, this dose was established and used in subsequent experiments.
  • paclitaxel (12 ⁇ ) paclitaxel
  • cisplatin 12 ⁇
  • carboplatin 12 ⁇
  • docetaxel (12 ⁇ )
  • doxorubicin 6 ⁇
  • Polystyrene plates were prepared with the above drugs all at the same time and stored at room temperature. Weekly, cells in a fixed amount (3 x 10 3 for OVCAR-3 and 7 x 10 3 for HT-29) were inoculated into each of these plates. Over a month, it can be observed that the drugs remained stable, showing similar efficacy weekly, ie, there was no drug degradation after one month of plaque preparation ( Figures 1 1, 12, 13, 14, 15 and 16).
  • the test was based on a kit containing a polystyrene plate with wells containing drugs previously deposited at pre-set doses, as well as control wells (untreated). After obtaining and preparing the cells, they were inoculated into the wells of the plate, remaining for approximately 72 hours in culture in a humidified incubator containing 5% carbon dioxide. After this period, a dye was added to the wells for up to 4 hours, which reacted with viable cells. Then the total volume was removed from each well. With the plate completely dry, a visual analysis of the colorimetric density of each plate was performed. After visual analysis, a quantitative analysis of the cells in each plate was performed.
  • solubilizing reagent dimethyl sulfoxide
  • said process comprises the steps of: adding inoculum with cell (s) of interest to wells / regions of the container which previously comprises said; and
  • the inoculum is inoculated into the container that already contains the active ingredients / drugs and follows an incubation time t, which varies according to the amount of inoculated cells, cell type, whether or not tumor, size and amount of cells placed in the plate, and consists of the required action time of the active in the cell of interest.
  • the inoculum containing the cells of interest is placed in at least one well that does not contain the active, establishing a standard well for assessing viability and / or cell proliferation under standard conditions without the active.
  • the cells are tumor cells obtained from an individual's tumor tissue by biopsy or surgery and stored in sterile conical tubes.
  • the colorimetric compound is added to the container after time t after inoculation of the cells of interest. After the addition of colorimetric compound, follows a time interval t ', which varies according to the nature of the colorimetric compound. Cell viability and / or proliferation can be detected with the aid of a spectrophotometer or other similar equipment.
  • the wavelength of radiation that hits the container containing active, inoculum and colorimetric compound varies according to the colorimetric compound used.
  • the colorimetric compound may be a tetrazolic dye which, when reduced by dehydrogenase and reductase enzymes present in viable cells, yields formazan, a staining compound.
  • tumor cell inoculation was done on a polystyrene plate that previously had the active ingredients / drugs. Immediately after inoculation of the cells in the plate, the cells were cultured in a humidified 5% carbon dioxide incubator for 72 hours. After this period, the colorimetric compound was added to detect cell viability and / or proliferation within a time period of 4 hours. After this time, the medium was removed and the plate was dried. Solubilizer was added, and the reading was performed in a spectrophotometer at 492 nm, followed by analysis of the results to evaluate cell viability and proliferation. Results are shown in figures 1 1 to 16.
  • Figures 19 and 20 show an example of pre-prepared polystyrene plaques with the drugs of choice containing HT-29 cell lines ( Figure 19) and OVCR-3 ( Figure 20). Visually, a marked difference can be observed between the treatments that induced resistance (darker wells) and those that induced less resistance in cells (light wells).
  • the cells were obtained after bone marrow or peripheral blood collection from patients diagnosed with leukemia. Afterwards, the samples were placed in a 3: 4 FICOLL-PAQUE-containing tube (3 mL of FICOLL to 4mL of sample), followed by centrifugation at 1300 rpm for 30 minutes. Then the plasma layer was removed so that the lymphocyte layer was accessible. The lymphocyte layer was then placed in another tube and washed with saline, followed by centrifugation at 900 rpm for 10 minutes. The supernatant was discarded and the pellet homogenized with saline again, followed by centrifugation at 670 rpm for 10 minutes.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Sustainable Development (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des produits et des procédés utiles, tels que des outils de recherche de base, destinés à réaliser des tests de contrôle expérimental de la prolifération de cellules cancéreuses ainsi que des tests de combinaisons d'agents actifs et/ou de nouvelles compositions pharmaceutiques pour le traitement du cancer. Dans un mode de réalisation préféré, les produits et les procédés de l'invention sont utiles dans la détection/quantification de la chimiorésistance et/ou de la chimiosensibilité in vitro, contribuant à améliorer la sélection du traitement de patients présentant divers types de cancer.
PCT/BR2015/050008 2015-02-02 2015-02-02 Trousse, son procédé d'obtention et procédé in vitro pour évaluer la viabilité et/ou la prolifération cellulaire par rapport à un agent actif Ceased WO2016123683A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/BR2015/050008 WO2016123683A1 (fr) 2015-02-02 2015-02-02 Trousse, son procédé d'obtention et procédé in vitro pour évaluer la viabilité et/ou la prolifération cellulaire par rapport à un agent actif

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/BR2015/050008 WO2016123683A1 (fr) 2015-02-02 2015-02-02 Trousse, son procédé d'obtention et procédé in vitro pour évaluer la viabilité et/ou la prolifération cellulaire par rapport à un agent actif

Publications (1)

Publication Number Publication Date
WO2016123683A1 true WO2016123683A1 (fr) 2016-08-11

Family

ID=56563249

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/BR2015/050008 Ceased WO2016123683A1 (fr) 2015-02-02 2015-02-02 Trousse, son procédé d'obtention et procédé in vitro pour évaluer la viabilité et/ou la prolifération cellulaire par rapport à un agent actif

Country Status (1)

Country Link
WO (1) WO2016123683A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5242806A (en) * 1990-05-07 1993-09-07 Baxter Diagnostics Inc. Method for conducting the cytotoxicity assays on tumor cells
WO1993025705A1 (fr) * 1992-06-09 1993-12-23 Barranco Sam C Iii Procede et kit servant a tester la sensibilite de tumeurs a des medicaments
WO2009124997A1 (fr) * 2008-04-11 2009-10-15 Universität Leipzig Procédé et kit pour l’évaluation ex vivo de la réponse d’une tumeur à des conditions à évaluer
US20100028863A1 (en) * 2005-01-21 2010-02-04 Falcon Genomics, Inc. High throughput assay for cancer cell growth inhibition

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5242806A (en) * 1990-05-07 1993-09-07 Baxter Diagnostics Inc. Method for conducting the cytotoxicity assays on tumor cells
WO1993025705A1 (fr) * 1992-06-09 1993-12-23 Barranco Sam C Iii Procede et kit servant a tester la sensibilite de tumeurs a des medicaments
US20100028863A1 (en) * 2005-01-21 2010-02-04 Falcon Genomics, Inc. High throughput assay for cancer cell growth inhibition
WO2009124997A1 (fr) * 2008-04-11 2009-10-15 Universität Leipzig Procédé et kit pour l’évaluation ex vivo de la réponse d’une tumeur à des conditions à évaluer

Similar Documents

Publication Publication Date Title
Goh et al. Diagnosis and treatment of culture-negative periprosthetic joint infection
EP2989460B1 (fr) Procédé pour un test de criblage de médicaments à base de cellules et son utilisation
Folkesson et al. High-throughput screening reveals higher synergistic effect of MEK inhibitor combinations in colon cancer spheroids
Bayat et al. The anti-angiogenic effect of atorvastatin in glioblastoma spheroids tumor cultured in fibrin gel: in 3D in vitro model
BRPI0922952B1 (pt) métodos de detectar e identificar um microorganismos em meios sólidos ou semi-sólidos
JP2016001184A (ja) 細胞分析の進歩した方法および装置
Crespo et al. The chick embryo chorioallantoic membrane as an in vivo model to study metastasis
Spanswick et al. Measurement of drug-induced DNA interstrand crosslinking using the single-cell gel electrophoresis (comet) assay
WO2025236610A1 (fr) Procédé de détection de la sensibilité d'une tumeur aux médicaments
US20130071874A1 (en) System and method for anti-cancer drug candidate evaluation
Griner et al. Generation of high-throughput three-dimensional tumor spheroids for drug screening
Shie et al. Highly mimetic ex vivo lung‐cancer spheroid‐based physiological model for clinical precision therapeutics
WO2017150366A1 (fr) Sphéroïdes régulièrement disposés et de taille uniforme, et utilisation de ces derniers
US20240069011A1 (en) Predicting efficacy of or resistance to treatment of colon cancer
WO2016123683A1 (fr) Trousse, son procédé d'obtention et procédé in vitro pour évaluer la viabilité et/ou la prolifération cellulaire par rapport à un agent actif
Offenbacher et al. Polyamine depletion by d, l-alpha-difluoromethylornithine inhibits ewing sarcoma metastasis by inducing ferroptosis
Stegeman et al. Hypoxia, metabolism, and growth factor signaling in head and neck squamous cell carcinoma: Correlation between primary and xenograft tumors
BR102013019219A2 (pt) kit, seu processo de obtenção, e processo in vitro para avaliação de viabilidade e/ou proliferação celular em relação a um ativo
US20140295483A1 (en) Apparatus and method for in-vitro drug screening for cancer metastasis
US20190011434A1 (en) Methods to determine cancer treatment using robotic high-throughput drug sensitivity testing
EP3236239A1 (fr) Appareil et procédé de mesure de présence de matière organique ou de vie/mort de matière vivante
Yılmaz et al. Determination Of Metastatic Capacity in Primary Lung Cancer Cells: Reflection of Patient Profile In The Clinic Using in vitro Methods
BR102021003612A2 (pt) Kit, seu processo de obtenção, e processo in vitro para avaliação de crescimento e/ou viabilidade e/ou proliferação e/ou morte celular em relação a um ativo
Frongia et al. 3D imaging of the response to CDC25 inhibition in multicellular spheroids
US20110244503A1 (en) System and Method for Anti-Cancer Drug Candidate Evaluation

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: 15880678

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15880678

Country of ref document: EP

Kind code of ref document: A1