WO2022187725A1 - Système de culture cellulaire et procédés d'utilisation - Google Patents

Système de culture cellulaire et procédés d'utilisation Download PDF

Info

Publication number
WO2022187725A1
WO2022187725A1 PCT/US2022/019052 US2022019052W WO2022187725A1 WO 2022187725 A1 WO2022187725 A1 WO 2022187725A1 US 2022019052 W US2022019052 W US 2022019052W WO 2022187725 A1 WO2022187725 A1 WO 2022187725A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cultureware
cell culture
days
aspects
Prior art date
Application number
PCT/US2022/019052
Other languages
English (en)
Inventor
Jean-Charles F. NEEL
Original Assignee
Sensorium Bio Inc.
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 Sensorium Bio Inc. filed Critical Sensorium Bio Inc.
Publication of WO2022187725A1 publication Critical patent/WO2022187725A1/fr

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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/22—Transparent or translucent parts
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/06—Means for regulation, monitoring, measurement or control, e.g. flow regulation of illumination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • 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
    • C12N2523/00—Culture process characterised by temperature
    • 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
    • C12N2533/30—Synthetic polymers
    • C12N2533/32—Polylysine, polyornithine

Definitions

  • This invention relates to the field of biologic detection systems.
  • the cells of such systems possess receptors and reporters that provide a detectable signal when contacted by a compound that binds to the cell receptors.
  • the invention therefore relates to detection of molecules of interest, including molecules that are volatile.
  • LC lung cancer
  • cell culture systems for culturing cells at ambient temperature, the systems comprising: (a) a cultureware for culturing the cells, wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and can be tightly sealed; (b) cells adhered to the bottom of the cultureware, wherein the bottom of the cultureware is optically transparent; (c) a layer of non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and (d) a cell culture medium covering the cells, wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture.
  • [6] Disclosed herein are methods of culturing cells at ambient temperature, the methods comprising: (a) depositing cells in a cultureware, wherein the cultureware has a top, a bottom, an inner side and an outer side and is gas impermeable and wherein the bottom of the inner side of the cultureware has a coating of a synthetic polymer, wherein the cells passively adhere to the bottom of the inner side of the cultureware on the coating of synthetic polymer in the absence of carbon dioxide; (b) adding non-biodegradable cytocompatible hydrogel around the adhered cells, wherein the cells are immobile within the hydrogel; and (c) adding a cell culture medium to the cells in step b), wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture; and (d) Culturing the cells at ambient temperature without incubating the cells at 37°C and 5% CO2.
  • the methods for culturing cells according to the invention provide for maintaining viable and functional cells outside of a controlled temperature environment without having supplemental carbon dioxide provided to the cell’s surrounding environment typically found in a cell culture incubator. Moreover, the cells cultured according to the invention are able to be maintained as viable and functional outside of an incubator and without temperature control or supplemental carbon dioxide for periods of at least 3 days.
  • the cells cultured according to the invention may be used in the devices and detection methods disclosed herein. As such, the invention disclosed herein provides a closed system in which cells remain viable and functional without supplemental carbon dioxide and wherein the amount of medium present with the cells after sealing is an amount sufficient to maintain viability and functioning of the cells without further supplementation.
  • the system may remain closed without such supplemental nutrients until cells are used for detecting a target molecule, including volatile organic molecules.
  • the methods comprising: a) contacting a cell culture with a fluid sample, wherein the fluid sample comprises one or more volatile organic molecules, wherein the cell culture comprises cells in a cultureware having a top, an optically clear bottom, an inner side and an outer side and is gas impermeable, wherein the cells are adhered to the bottom of the inner side of the cultureware, are functional at room temperature, and present in a cell culture medium that is buffered by a high buffering capacity carbon dioxide-independent buffer; wherein the cells comprise one or more G- protein coupled receptors capable of binding to the one or more volatile organic molecules, wherein the one or more G-protein coupled receptors comprises a reporter; b) exposing the cell culture to a light; and c) detecting the presence of a fluorescence emitted by the
  • cell culture systems for culturing cells at a temperature from about 15°C to about 30°C, the systems comprising: a cultureware for culturing the cells, wherein the cultureware comprises a top, an optically transparent bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and wherein the cultureware has at least one access region to add or remove contents within the cultureware, and wherein the access region is sealable to be gas impermeable; cells adhered to the bottom of the cultureware; a layer of non- biodegradable cytocompatible hydrogel, wherein said hydrogel sufficiently surrounds the adhered cells to maintain the cells in a position within the hydrogel; and a cell culture medium covering the adhered cells, wherein the cell culture medium is buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide.
  • cell culture devices for culturing anchorage dependent, target molecule detecting cells in a sealable culture device in the absence of controlled temperature and supplemental carbon dioxide or nutrients, and wherein said cells remain adhered to a cultureware surface of said culture device for at least 5 days
  • said cell culture device comprising: (a) a top, an optically transparent bottom, an inner side and an outer side and wherein the cultureware of said cell culture device is gas impermeable and wherein the cell culture device has at least one access region to add or remove contents within the cell culture device, and wherein the access region is sealable to be gas impermeable; (b) target molecule detecting cells adhered to the bottom of the cultureware for at least 5 days; and wherein a layer of non-biodegradable cytocompatible hydrogel sufficiently surrounds the cells to maintain the cells in a position within the hydrogel; and (c) a culture medium buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide and wherein the culture medium is
  • the systems and methods for culturing cells disclosed herein can be performed at ambient temperature and in ambient air without incubating the cells at 37°C and 5% CO2.
  • the cell culture system and methods described herein provides a prolonged longevity and functionality of the cells.
  • the cells cultured using the systems and methods disclosed herein can be used in a cell-based assay to detect molecules of interest, including but not limited to metabolites from a living organism.
  • FIG. 1 is a schematic diagram of a cross section of the present invention showing a cultureware having multiple wells with the top of the cultureware open and the cultureware is totally covered and tightly sealed with a gas impermeable film.
  • FIGS. 2A-B show the effects of temporal changes in osmolality on cellular morphology.
  • FIG. 2B shows the maximum ligand- induced fluorescence in cells expressing a surface receptor.
  • FIG. 3 shows the average area (pm 2 ) measurement of cells post exposure to carbon dioxide independent media + 10% FBS + gentamicin (CO2I10) for 1 to 8 days in vitro. The gradual decrease in average cellular area indicative of a loss of cellular spreading onto the substrate. This is indicative of anchoring points decreasing over a period of 7 days.
  • CO2I10 carbon dioxide independent media + 10% FBS + gentamicin
  • FIG. 4 shows the effects of temporal changes in osmolarity on cellular morphology.
  • HEK cells were seeded at a density of 20,000 cells/well and exposed to conditioned media evaporated for 1 day and 8 days to assess the effect of osmolarity.
  • Media was prepared and conditioned separately in polystyrene 96-well plates for 8 days, filtered, and added to respective experimental wells and allowed to incubate for 30 minutes at 37°C, 5% CO2, and 95% humidity.
  • FIG. 5 shows the functional response profile of cells cultured from day 3 to day 12 in carbon dioxide independent media.
  • HEK cells expressing a GPCR were challenged with propylbenzene and cytosolic calcium increase was measured via a genetically-encoded calcium indicator.
  • the mean fluorescence intensity was acquired by imaging 20X fields of cells via an epifluorescence microscope. Images were acquired every minute for a total of 15 minutes and data are represented as a fold induction normalized to the baseline value prior to ligand challenge.
  • FIG. 6 shows the effect of ambient air on pH of DMEM and carbon dioxide independent media.
  • FIGS. 7A-B show the effect of temporal changes in osmolarity on cellular morphology.
  • FIG. 7A shows the average area (pm 2 ) measurement of HEK cells post exposure to carbon dioxide independent media + 10% FBS + gentamicin (CO2I10) for 1 day and 8 days in vitro.
  • FIG. 7B shows the average circularity measurements of cells post exposure to CO2I10 for 1 day and 8 days in vitro.
  • Media was prepared and conditioned separately in polystyrene 96-well plates for 8 days, filtered, and added to respective experimental wells and allowed to incubate for 30 minutes at 37°C, 5% CO2, and 95% humidity.
  • n > 20 cells/well were assayed for n 3 wells (n > 60 per condition). Data is represented as mean +/- STD and between group differences measured via a two-tailed t-test. *P ⁇ 0.05, **P ⁇ 0.01, and ***P ⁇ 0.001.
  • FIG. 8 shows the influence of ambient air on pH of DMEM and carbon dioxide independent media.
  • Data is represented as mean +/- STD and between group differences measured via a two-tailed t-test.
  • FIGS. 10A-B show the distribution of baseline fluorescence across a healthy adherent cell population.
  • FIG. 10A shows that nearly 70% of cells have an initially low baseline of GCaMP7s fluorescence.
  • FIG. 10B shows the distribution of cellular assay responsiveness as a function of baseline fluorescence.
  • 70% of cells have a very low baseline fluorescence and these cells perform best in a fluorescence induction assay as determined by the ligand-induced fluorescence fold induction.
  • Cells that have a higher baseline fluorescence do not register a proportionally increased fluorescence upon stimulation with a ligand as assessed by an imaging system. As a result, maintaining lower baseline fluorescence allows for a larger response range.
  • FIG. 11 shows an example of a microarray with adjustable dimensions.
  • Left image shows microfabricated microarrays on a coverslip.
  • Right image shows an electron micrograph of the same 400 pm by 400 pm array taken with an Apreo 2.
  • Right image shows fluorescent cells adhering to non-fluorescent protein spot arrays.
  • FIG. 12 shows HEK cells sedimenting and adhering in the nano-wells.
  • the left panel shows fluorescent cell size polymer microbeads dispensed into 150 pm nano-well.
  • the right panel shows untransfected cells seeded at confluence into microarray nano-wells.
  • the term “ambient temperature” refers to the actual temperature of the air in any particular place. It can vary from place to place. In some aspects, the ambient temperature can be from about 15°C to about 25°C or even to about 30°C. In some aspects, the ambient temperature can be about 10°C to about 35°C. In some aspects, the ambient temperature can be from about 20°C to about 22°C. In some aspects, the ambient temperature can be from about 15°C to about 30°C, about 20°C to about 28°C, about 20°C to about 25°C or about 20°C to about 24°C.
  • adherent cells refers to a cell or a cell line or a cell system, that remain(s) associated with, immobilized on, or in certain contact with the inner surface of a substrate (e.g., the bottom of the inner side of a cultureware).
  • the adherent cells are anchorage dependent, and, thus, need solid support for its growth.
  • the systems and methods disclosed herein can provide a higher degree of selectivity and sensitivity than existing technologies and are significantly less expensive to manufacture. Accurate real-time actionable output and a low price point make a point-of-care solution for early cancer (e.g., lung cancer) screening.
  • early cancer e.g., lung cancer
  • primary cells are cells that are directly extracted from a living tissue and have not been adapted to be cultured in the laboratory.
  • Primary cells have to undergo adaptation to culture to establish a cell line from the living tissue to allow for long-term study of the tissue.
  • the adaptation to culture of the primary cells in the laboratory can be such that the cells continue to divide to allow studies to be conducted.
  • a difference between a primary cell culture and a cell line is the number of passing times each of them possesses.
  • the primary cell culture is directly isolated whereas a cell line is prepared by passing a primary cell culture for several times thereby selecting for cells best adapted for in vitro culture.
  • Cell lines are typically maintained in a growth medium with sufficient density at 37°C and 5% CO2 in a humidified incubator. Some cell lines can be maintained at temperatures other than 37°C and with gas supplementation other than 5% CO2 (i.e., some cells grow best under slightly hypoxic conditions where the incubator provides 5% CO2, 85% N2 and 10% O2).
  • a commonly used cell growth medium is the Minimum Eagle’s Medium (MEM) supplemented with 5% to 10% Fetal Bovine Serum (FBS) which provides many growth factors.
  • MEM is commercially available (e.g., Sigma-Aldrich, St. Louis, MO).
  • the base cell growth medium provides salts, sugars, vitamins and amino acids, which can vary in compositions.
  • the carbon dioxide independent media can further comprise one or more antibiotics.
  • the one or more antibiotics can be gentamicin, penicillin, streptomycin, amphotericin or a combination thereof.
  • the base cell growth media disclosed herein are carbonate-buffered media that use exogenous CO2 provided by the incubator to maintain appropriate medium pH.
  • the cells When the cell line is removed from the incubator and kept in ambient conditions, the cells rapidly lose pH-sensitive active anchorage, such as integrin-mediated anchorage, from the cultureware and adopt a rounded morphology.
  • the medium is removed from the 5% CO2 incubator, the dissolved CO2 degases out of the medium according to the reaction below:
  • HCO3- + H + H2CO3 H2O + C0 2 thereby causing the pH to increase to a more basic condition and the cells gradually lose their ability to actively attach to the cultureware resulting in cellular stress and limiting the longevity of anchorage-dependent cells.
  • cells cultured under normal and standard conditions for example, being incubated at 37°C and 5% CO2 are under severe stress and do not grow well when incubated at temperatures below 37°C (such as ambient temperature) and/or without exogenous CO2.
  • Examples of evidence of stress may include, but not be limited to, reduction of anchorage to the substrate, cell death, altered metabolism, reduced division and changes in expression of particular phenotypic characteristics or functionality.
  • the cell culture needs to be transported from one location to another, it is desirable that it takes place at ambient temperature while the cells are still maintaining the health and functionality particularly the ability of the cells to register an increase in cellular signaling as measured by a reporter activity upon stimulation by the appropriate stimulus; and, for example, when the cells are used in a cell-based assay to detect one or more molecules of interest, including but not limited to a metabolite from a living organism.
  • Such assays are often carried out at ambient temperatures, such as 20-22°C and without 5% CO2 supplementation.
  • the cells cultured using the systems and methods disclosed herein maintain their function and remain alive for an extended period of time. If the cells are alive but unable to perform their signaling function involving for example a ligand-induced increase in intracellular Ca 2+ which can be measured by a fluorescent reporter, for example, or in another example if the cell signaling pathway fails to deliver an expected readout, the cells are not functional and are described as non-functional. [41] Disclosed herein are systems and methods for culturing cells at ambient temperature without incubating the cells at 37°C and 5% CO2 such that the cells have a prolonged longevity and functionality by limiting one or more causes of cellular stress(es).
  • the systems and methods disclosed herein are superior to and distinguishable from the standard cell culture system or method by: (1) altering the surface coating where the cells initially bind; (2) altering the environment in which the cells are in immediate in contact; (3) altering the temperature in which the cells operate; and (4) the insulation of the system.
  • cell culture systems for culturing cells at ambient temperature.
  • the cultured cells remain viable and functional outside of a controlled temperature environment without carbon dioxide supplementation for at least 3 days. Further, throughout the process of culturing the cells, the cells are not supplemented with carbon dioxide nor are the cells incubated.
  • the cell culture systems and devices disclosed herein can be a closed system in which cells remain viable and functional without supplemental carbon dioxide and wherein the amount of medium present with the cells after sealing is an amount sufficient to maintain viability and functioning of the cells without further supplementation.
  • the system may remain closed without such supplemental nutrients until cells are used for detecting a target molecule, including volatile organic molecules.
  • the cell culture system comprises: a cultureware for culturing the cells, wherein the cultureware has a top, a bottom, an inner side and an outer side.
  • the cultureware is gas impermeable and can be tightly sealed.
  • the cell culture system comprises cells adhered to the bottom of the cultureware.
  • the bottom of the cultureware is transparent.
  • a layer of non-biodegradable cytocompatible hydrogel can be placed around, or over, the cells, wherein the cells remain in a stationary position within the hydrogel. In some aspects, the cells are sufficiently stationary to be locked in position within the hydrogel.
  • the cell culture system comprises a cell culture medium covering the cells, wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide- independent (generally speaking a non-volatile) buffer to maintain a physiologically relevant pH of from about 7 to 8 in the medium throughout the culture.
  • a high buffering capacity carbon dioxide- independent generally speaking a non-volatile
  • the system comprises: (1) a cultureware for culturing the cells wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and can be tightly sealed; (2) a coating of synthetic polymer at the bottom of the inner side of the cultureware wherein the synthetic polymer having a positive surface electrostatic charge to allow passive adhesion of the cells; (3) cells adhered to the bottom of the inner side of the cultureware on top of the coating of synthetic polymer wherein the cells having a surface G-protein coupled receptor, an intracellular G-protein and an intracellular reporter; (4) a layer of optically clear non-biodegradable cytocompatible hydrogel around of the cells locking the cells in position within the hydrogel; and (5) a cell culture medium covering the hydrogel and submerging the cells wherein the cell culture medium has a high buffer
  • the systems can comprise: a cultureware for culturing the cells.
  • the cultureware comprises a top, an optically transparent bottom, an inner side and an outer side.
  • the cultureware is gas impermeable.
  • the cultureware has at least one access region to add or remove contents within the cultureware, and the access region is sealable to be gas impermeable.
  • the systems comprise cells adhered to the bottom of the cultureware.
  • the systems comprise a layer of non-biodegradable cytocompatible hydrogel.
  • the hydrogel sufficiently surrounds the adhered cells to maintain the cells in a position within the hydrogel.
  • the systems comprise a cell culture medium covering the adhered cells.
  • the cell culture medium is buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide.
  • cell culture devices for culturing anchorage dependent, target molecule detecting cells in a sealable culture device in the absence of controlled temperature and supplemental carbon dioxide or nutrients, and wherein said cells remain adhered to a cultureware surface of said culture device for at least 3 days.
  • said cell culture device can comprise: a top, an optically transparent bottom, an inner side and an outer side.
  • the cultureware of said cell culture device is gas impermeable.
  • the cell culture device has at least one access region to add or remove contents, including a sample comprising a target molecule, within the cell culture device.
  • the access region is sealable to be gas impermeable.
  • said cell culture device can comprise target molecule detecting cells adhered to the bottom of the cultureware for at least 3 days.
  • a layer of non- biodegradable cytocompatible hydrogel sufficiently surrounds the cells to maintain the cells in a position within the hydrogel.
  • the cell culture device can comprise a culture medium buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide and wherein the culture medium is present in the device in an amount sufficient for the cells to remain viable in said device for at least 3 days in the absence of controlled temperature or supplemental carbon dioxide.
  • said cells comprise a surface G protein-coupled receptor, an intracellular G-protein and an intracellular reporter that provides a detectable signal when said cells contact the target molecule.
  • the cell culture systems described herein can be used for culturing cells at ambient temperature.
  • the system comprises: (1) a cultureware for culturing the cells wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and can be tightly sealed; (2) cells adhered to the bottom of the cultureware; (3) a layer of non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and (4) a cell culture medium covering the cells wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to 8 in the medium throughout the culture.
  • the ambient temperature can be 15, 20, 25, 30°C, 35°C or any temperature in between. In some aspects, the ambient temperature can be 20, 21, or 22°C. In some aspects, the ambient temperature can be between 10°C to about 35°C. In some aspects, the ambient temperature can be between 15°C to about 30°C. In some aspects, the ambient temperature can be between 20°C to about 25°C. In some aspects, the ambient temperature can be from about 15°C to about 30°C, about 20°C to about 28°C, about 20°C to about 25°C or about 20°C to about 24°C.
  • the cell can be cultured at a first temperature or temperature range, but adherent, viable, and functional at a second temperature (e.g., room temperature, 37°C).
  • the cells can be cultured at a first temperature (e.g., between 15°C and 30°C) to reduce metabolic rate and limit cellular division while preserving cellular viability and adherence and ability of the cells to respond in the presence of molecules that bind to the GPCR and generate a detectable reporter response.
  • the cells can be any cells.
  • the cells can be primary cells.
  • the cells can be derived from a cell line.
  • the cells can be prone to apoptosis.
  • the cells can include, but not limited to, animal cells (such as cells derived from salmon), human cells, plant cells, insect cells, etc.
  • the cells can be animal cells.
  • the cells can be mammalian cells.
  • the cells can be human embryonic kidney cells.
  • the cells can be fish cells.
  • the cells can be any fish cell.
  • the fish cells can be from freshwater, marine or brackish water fish. Examples of fish cells include but are not limited to salmon, trout, minnow, catfish, bluegill, flounder, sea bream, cobia, grouper, snakehead, sea bass, pompano, and the like.
  • the cells can be primary cells or cells derived from cell lines such as, but not limited to, tumor cells, hybridoma cells, etc.
  • the cell is an immortalized human embryonic kidney (HEK) cell.
  • the animal cells can be mammalian cells, including by not limited to bovine, canine, feline, hamster, mouse, porcine, rabbit, rat, or sheep.
  • the mammalian cells can be cells of primates, including but not limited to, monkeys, chimpanzees, gorillas, and humans.
  • the animals cells can be rodent cells (e.g., mouse cells).
  • Animal cells include, for example, fibroblasts, epithelial cells (e.g., renal, mammary, prostate, lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, hematopoietic cells.
  • the animal cells can be adult cells (e.g., terminally differentiated, dividing or non-dividing) or stem cells.
  • Mammalian cell lines can also be used.
  • the cell lines can be derived from Chinese hamster cells, Human kidney cells, or Monkey kidney cells. The cell lines disclosed at the web-sites for ThermoFisher, ATCC, and Charles River Laboratories are incorporated by reference in their entirety for all purposes.
  • the fish cells can be coho salmon-derived cell lines.
  • salmon-derived cell lines include but are not limited to Salmon Head Kidney- 1, Anterior Salmon Kidney (ATCC Cat# CRL-2747TM), and Atlantic salmon gill cells ASG-10 and ASG-13.
  • ATTC CRL-1681 CHSE-241, chinook salmon embryo
  • ATTC CCL-55 RGT-2, rainbow trout gonad
  • epithelioma papulosum cyprinid ATTC CCL-42 (fathead minnow); ATTC CCL-59 (brown bullhead); ATTC CCL-91 (bluegill fry); WSSK-1 (white sturgeon skin); WSS-2 (white sturgeon spleen); CCO (channel catfish ovary); ATTC CRL-2747 (ASK, atlantic salmon kidney); and SHK-1 (salmon head kidney).
  • the cells can be cancerous cells, non-cancerous cells, tumor cells, non tumor cells, healthy cells or any combination thereof.
  • the cells can be transformed cells.
  • the cells can be immortal.
  • the cells lose contact inhibition.
  • the cells have a cancer cell phenotype.
  • the cells are adherent at around 20°C. In some aspects, the cells can be genetically modified to be adherent at around 20°C. In some aspects, the cells are adherent around 12°C to 22°C. In some aspects, the cells are adherent around 22°C to 38°C. In some aspects, the cells are adherent at around 15°C to about 25°C or even to about 30°C. In some aspects, the cells are adherent at around 10°C to about 35°C. In some aspects, the cells are adherent at around 20°C to about 22°C. In some aspects, cells are adherent at around 15°C to about 30°C, about 20°C to about 28°C, about 20°C to about 25°C or about 20°C to about 24°C.
  • plant cells can be cells of monocotyledonous or dicotyledonous plants, including, but not limited to, alfalfa, almonds, asparagus, avocado, banana, barley, bean, blackberry, brassicas, broccoli, cabbage, canola, carrot, cauliflower, celery, cherry, chicory, citrus, coffee, cotton, cucumber, eucalyptus, hemp, lettuce, lentil, maize, mango, melon, oat, papaya, pea, peanut, pineapple, plum, potato (including sweet potatoes), pumpkin, radish, rapeseed, raspberry, rice, rye, sorghum, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, tobacco, tomato, turnip, wheat, zucchini, and other fruiting vegetables (e.g.
  • plants refers to any of the physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks,
  • the cells described herein can be used in cell-based assays to detect a target molecule.
  • the cells are capable of detecting one or more target molecules.
  • the cells are capable of detecting one or more target molecules in a cell-based assay.
  • the target molecule can be a metabolite from a living organism.
  • the metabolite can be a volatile organic compound (VOC).
  • VOC can refer to a molecule having a weight of less than 1000 g/mol.
  • the VOC can be a compound that can bind to an odorant receptor or a modified odorant receptor.
  • the term “compound” or “target molecule” refers to a molecule that can produce a detectable signal in a cell.
  • the VOC or target molecule is capable of acting as a ligand at the GPCR.
  • the signal can be a fluorescent signal.
  • the target molecule can be a lung cancer metabolite.
  • the lung cancer metabolite can be present in breath of a subject.
  • the lung cancer metabolite can be a lung cancer metabolite VOC.
  • the lung cancer metabolite VOC can be 2-decanone, 1 -butanol, limonene, 2-undecanone, n-octane, isopropylamine, methyl cyclopentane, 1,2,4-trimethylbenzene, 2-nonanone, 2-methylpentane, decane, propylbenzene, ethylbenzene, pentane, heptane, 1-hexanol, 1,2,3-trimethylbenzene, acetophenone, 2,2,4,6,6-pentamethylheptane, 3-methylhexane, 2-methylhexane, 2- pentadecanone, nonadecane, pentanal, octanal, nonanal, 4-heptanone, thiophene, 2,3-butadione, or a combination thereof.
  • the lung cancer metabolite VOC can be acetone, 2- butanone, n-propanol, hexane, 2-methylpentane, trimethyl heptane, isoprene, benzene, toluene, ethylbenzene, cumene, trimethyl benzene, alkylbenzene, styrene, naphthalene, 1- methylnaphthalene, propanal, acetone, 2-butanone, phenol, benzaldehyde, acetophenone, nonanal, ethyl propanoate, methyl isobutanoate, dichloromethane, dichlorobenzene, trichloroethane, trichlorofluoromethane, tetrachloroethylene, styrene, 2,2,4,6,6-pentamethylheptane, 2- methylheptane, decane, n-propyl
  • the lung cancer metabolite VOC can be dimethyl sulphide, 2-hydroxyacetaldehyde, 4- hydroxyhexenal, ethylbenzol, n-dodecane, isopropyl benzene, p-xylene + m-xylene, 5-(2-methyl- )propyl-nonane, 2,6,-ditertbutyl-4-methyl-phenol, 4-hydroxy-2-hexenal, hydroxyacetaldehyde, 4- hydroxy-2-nonenal, n-hexane, n-nonanal, diethyl ether, isothiocyanatocyclohexane, hydrogen isocyanide, or a combination thereof.
  • the cells can be adherent or adhered cells. In some aspects, cells adhere to the bottom of the inner side of the cultureware.
  • the cells can be a modified cell.
  • the cells can be a genetically modified cell.
  • the modified cell can comprise one or more cell-surface receptors.
  • the modified cell can comprise a modified cell-surface receptor.
  • the modified cell-surface receptors can be modified to increase or decrease their ability to bind to a variety of compounds.
  • modified cell-surface receptors can be modified to increase or decrease their ability to bind to a specific compound.
  • the modified cell can comprise a deletion of one or more endogenous cell-surface receptors.
  • the cells can comprise one or more surface G-protein-coupled receptors (GPCRs).
  • GPCRs surface G-protein-coupled receptors
  • the cell can comprise a GPCR, an intracellular protein and an intracellular reporter.
  • the cells express or can be modified to express a GPCR, an intracellular G-protein and an intracellular reporter.
  • GPCRs are a large family of related receptors that are cell surface receptors that interface the cell with the outside world by detecting molecules outside the cell and activating a cellular response to couple with the intracellular G-protein followed by generation of an intracellular signal.
  • the G-protein signaling pathway can comprise G-protein-mediated activation of adenylate cyclase with the resultant production of cAMP as a second messenger.
  • the cAMP can interact with a cAMP activated cation channel.
  • the G-protein can be comprised of three subunits the Ga subunit (e.g., Uniprot P38405), GP subunit (e.g., Uniprot P62879) and Gy subunit (e.g., Uniprot P63218).
  • the adenylate cyclase and the G protein can be from the same species.
  • the adenylate cyclase and the G protein can be from different species.
  • the G protein subunits can be from the same or from different species.
  • the intracellular reporter can be cAMP.
  • the intracellular G proteins can interact directly with the intracellular reporter to produce a detectable signal, e.g., adenylate cyclase can produce cAMP. In some aspects, the cAMP molecule itself can be detected.
  • the G proteins can interact with adenylate cyclase to induce a reporter. In some aspects, the G proteins can interact with adenylate cyclase to produce a first signal, and a second system amplifies the first signal when the reporter responds to the first signal.
  • the GPCRs can comprise one or more reporters.
  • a heterologous gene encoding a reporter protein can be introduced into the cells disclosed herein so that the cells express the reporter, and the G-protein activates the reporter when an appropriate interaction (e.g., binding event) occurs at the GPCR.
  • the cells described herein can be engineered or genetically modified to express a single reporter.
  • different cells can each express a different reporter, and can be used to enhance signal detection.
  • the cells described herein can be engineered to express two or more reporter products, for example by using a single vector construct encoding two or more reporters.
  • the reporter or reporters can be one or more of a fluorescent reporter, a bioluminescent reporter, or a combination thereof.
  • fluorescent reporters include, but are not limited to, green fluorescent protein from Aequorea victoria or Renilla reniformis , and active variants thereof (e.g., blue fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, etc.); fluorescent proteins from Hydroid jellyfishes, Copepod, Ctenophora, Anthro50 zoas, and Entacmaea quadricolor, and active variants thereof; and phycobiliproteins and active variants thereof.
  • fluorescent reporters include, for example, small molecules such as CPSD (Disodium 3-(4-methoxyspiro ⁇ l,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.137] decan ⁇ -4-yl) phenyl phosphate, ThermoFisher Catalog #T2141).
  • CPSD Disodium 3-(4-methoxyspiro ⁇ l,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.137] decan ⁇ -4-yl) phenyl phosphate
  • ThermoFisher Catalog #T2141 ThermoFisher Catalog #T2141.
  • Bioluminescent reporters include, but are not limited to, aequorin (and other Ca 2+ regulated photoproteins), luciferase based on luciferin substrate, luciferase based on Coelenterazine substrate (e.g., Renilla, Gaussia, and Metridina), and luciferase from Cypridina, and active variants thereof.
  • the bioluminescent reporter can be, for example, North American firefly luciferase, Japanese firefly luciferase, Italian firefly luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, railroad worm luciferase, Renilla luciferase, Gaussia luciferase, Cypridina luciferase, Metrida luciferase, OLuc, and red firefly luciferase.
  • North American firefly luciferase Japanese firefly luciferase, Italian firefly luciferase, East European firefly luciferase, Pennsylvania firefly luciferase
  • Click beetle luciferase railroad worm luciferase, Renilla luciferase, Gaussia luciferase, Cypridina lucifer
  • the reporter can be a fluorogenic reaction that can participate in the quenching or suppressing of fluorescence or luminescence. Changes in light intensity or wavelength can be used to optically detect the presence or activation of the reporter. Such reporter activity can result from ionic changes including but not limited to pH changes, quenching, presence or absence of enzyme substrates, or ability to bind a Second reagent, such as an antibody conjugate, which itself participates in generating an optically detectable signal.
  • light-generating reporters typically are two-component systems, where light emitted by one component undergoes either a spectral or an intensity change due to a physical interaction of the first component with the second component.
  • the term “signal” can refer to a signal in response to a binding event.
  • a signal can be generated by a compound binding to a cell-surface receptor of a cell, and activating adenylate cyclase which in turn activates cAMP that leads to the generation of a signal that can be visually detected.
  • the signal can be a signal measured over a period of time.
  • the signal can be a measurement of amplitude, period or a frequency or a combination thereof.
  • the signal is an intracellular change, for example, emission of light or change of pH that activates a detectable signal such as fluorescence.
  • the signal can be measured in real-time.
  • the reporter can emit light or produce a molecule that can be detected with an optical sensor.
  • Real time measurements can be obtained through the cultureware comprising the cells disclosed herein by recording the change in light emission over time as the cells comprising the GPCR interacts with a sample and wherein a sample comprising a volatile organic compound or a target molecule will cause the activation of light as a detectable signal.
  • the real time measurements can be used to quantify the binding interaction by an absolute measurement or a relative measurement.
  • the real time signal can be compared to a standard to determine the binding activity of the GPCR.
  • Known amounts of the volatile organic compound or a target molecule for the GPCR can be used to generate a standard binding curve for receptor occupancy versus reporter gene output. Binding of a sample comprising a target molecule can then be compared to the standard curve to quantify interaction of the target molecule at the GPCR. For example, the amount of signal (e.g., light) produced correlates with the amount of calcium present in the cell(s) upon GPCR activation such that the light intensity is proportional to the concentration of the target molecule (e.g., VOC).
  • the cells disclosed herein can include internal references that allow differences in interactions at the GPCR to be compared.
  • a reference GPCR can be included in any of the cells disclosed herein, and a known amount of the reference ligand can be added to the reference receptor to act as a standard.
  • the reference receptor can be coupled to a different reporter, e.g., a reporter that provides a different optical signal from the GPCR reporter.
  • the reference and test receptors can be coupled to different fluorescent proteins such as green fluorescent protein (GFP), and red fluorescent protein (RFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • the ratio of green fluorescence to red fluorescence can be compared for different target molecules at the same GPCRs, or to compare binding of the same test ligand to different GPRCs.
  • GFP’s sensitive to different pH changes may also be used. See, U.S. patent 6,670,449.
  • the GPCR, G-protein or intracellular reporter can be already present, e.g., endogenous, in the cells, or one or more of them can be genetically engineered to be expressed by the cells. Any one or all of them can be naturally occurring or they can be genetically engineered (referred to as synthetic).
  • the G-protein or the intracellular reporter is artificial and engineered to be expressed by the cell.
  • the GPCR, the G protein or the reporter can be natural (e.g., endogenous) or synthetic (e.g., genetically engineered).
  • the artificial G- protein or intracellular reporter is also known as a synthetic G-protein or synthetic intracellular reporter, respectively.
  • the cells can be genetically modified to express one or more GPCRs.
  • the cells also can be genetically modified to express the human G protein subunits Ga, Gp, and Gy.
  • the genes encoding the human Ga, Gp, and Gy subunits can be placed under the control of appropriate control sequences (promoters, enhancers, translation start sequences, polyA sites, etc.) for the desired cell, and these constructs for the human Ga, Gp, and Gy subunits can be placed into the desired cell.
  • the human G-protein also can be associated with adenylate cyclase.
  • the gene for an appropriate adenylate cyclase can be placed under the control of appropriate control sequences for the desired cell, and this construct can be placed into the desired host cell.
  • the Ga subunit can be a Gq protein alpha subunit.
  • the cell can express or can be genetically modified to express Gq protein alpha subunit.
  • Gq proteins couple to GPCRs to activate beta-type (phospholipase C-beta) enzymes.
  • PLC-beta in turns hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to diacyl glycercol (DAG) and inositol triphosphate (IP3), which activates Ca 2+ .
  • the reporter activity can result from changes (increases) in intracellular Ca 2+ levels.
  • the reporter activity can be detected by a fluorescent or bioluminescent reaction and/or changes in light intensity or wavelength.
  • the reporter can be a genetically-encoded Ca 2+ biosensor.
  • the reporter can be GCaMP7s.
  • the reporter can be a resonance energy transfer pairs or self- quenching fluorophores.
  • the report can be fluorescence resonance energy transfer (FRET) or a bioluminescence resonance energy transfer (BRET) pair.
  • FRET pairs include but are not limited to CFP and YFP; ECFP and EYFP ; EBFP and GGFP; mCerulean and mVenus; SBFP2 and EBFP2; EBFP2 and mEGFP, MiCy and mKO; TFP1 and mVenus; EGFP and mCherry; Venus and mCherry; Venus and TdTomato; and Venus and mPlum.
  • BRET pairs are not limited to RLuc and Topaz; RLuc and GFP; Aequorin and GFP; Firefly luciferase and red fluorescent protein; and Firefly luciferase and D-luciferin.
  • the GPCRs can detect one or more VOCs.
  • the cells can express a single type of compound-sensing receptors or a combination of compound-sensing receptors.
  • a single VOC can bind to different GPCRs with different binding affinities. In some aspects, the binding of a single VOC to a GPCR can activate a signaling pathway within the cell.
  • a gas impermeable foil can be applied to seal the cultureware. Such application with the gas impermeable foil can prevent osmolarity drift. Under increased osmotic conditions, cells lose their active anchorage and adopt a rounded morphology and detach from the cultureware.
  • the cultureware can be in the form of cassette.
  • the cassette can be inserted, for example, into a device and positioned to be in contact with a sample comprising one or more VOCs.
  • FIG. 14 shows an example of a microfabricated microarray on a coverslip.
  • FIG. 15 shows that nano-wells can be individually addressable, by dispensing 10 pm fluorescent polymer microbeads using a Nanoject micro injector under stereotactic control. Horizontal lines in the empty wells are microfabrication photopolymerization artifacts. Cells were seeded cells onto a microarray, and were sedimented and adhered in the nano-wells (see, FIG. 15). The microarrays can be imaged by a fluorescence microscope.
  • the lifespan of the cells within the cultureware can be at least 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 15 days, 20 days or longer. In some aspects, the lifespan of the cells within the cultureware can be at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer. In some aspects, the cells can be functional within the cultureware for at least at least 3 days, 4 days, 5 days, 7 days, 10 days, 15 days, 20 days or longer.
  • the cells can be functional within the cultureware for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer.
  • the cells remain viable and adherent to the substrate for at least at least 3 days, 4 days, 5 days without supplemental CO2. In some aspects, the cells remain viable for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer without supplemental CO2.
  • the cells within the cultureware can be adherent for at least 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 15 days, 20 days or longer. In some aspects, the cells within the cultureware can be adherent for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer. In some aspects, the cells within the cultureware can be adherent for at least at least 3 days, 4 days, 5 days, 7 days, 10 days, 15 days, 20 days or longer.
  • the cells within the cultureware can be adherent for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer.
  • the cells remain viable and adherent to the substrate for at least 3 days without supplemental CO2.
  • the cells remain viable and adherent to the substrate for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer without supplemental CO2.
  • the cells remain viable and adhered to the bottom of the cultureware for at least 3 days without supplemental CO2. In some aspects, the cells are adherent for at least 12 days at ambient temperature without CO2 supplementation. In some aspects, the cells remain viable and adherent to the substrate indefinitely without supplemental CO2
  • the cultureware can be a single chamber, such as a culture flask.
  • the cultureware can be a plate with a plurality of chambers or wells.
  • multi well plates can be used include but are not limited to the 96-Well Black/Clear Bottom Plate or White/Clear Bottom Plate available from ThermoFisher (Chicago, IL).
  • the cultureware has a plurality of chambers that are in the form of wells with the top of the cultureware open and wherein the cultureware is entirely sealed on the outer side with a gas impermeable film to provide gas impermeable chambers.
  • the gas impermeable film can function as a reversible sealable opening to allow one or more substances to be added or removed from the cultureware.
  • the one or more substances can be a target molecule.
  • Gas impermeability of the cultureware can be achieved by constructing the cultureware with a gas impermeable material.
  • the gas impermeable material is glass or metal.
  • the gas impermeable material is transparent.
  • the gas impermeable material is a UV-transparent substrate.
  • the UV-transparent substrate can be made of optically clear plastic polymers.
  • the optically clear plastic polymer can be cyclic olefin copolymers (COC).
  • the gas impermeable material is such that the adhered cells remain in the focal plane for the imaging system to capture the signal (e.g., fluorescent response).
  • the cultureware can be tightly sealed by capping the flask with a cap.
  • the flask contains a one-way opening that can be used to allow passage into the flask of ambient gas from the environment so that VOC present in the gas may dissolve in the cell medium so that the VOC’s may contact the cells comprising one or more GPRCs.
  • the cultureware can be plastic.
  • the cultureware can be polystyrene.
  • the cultureware is a plate with a plurality of wells, the top of the wells is open for passing the materials into the wells.
  • the plate can be made gas impermeable by covering the entire outer side of the cultureware with a gas impermeable film.
  • the gas impermeable film can provide a gas impermeable seal to the cultureware.
  • the gas impermeable film is light insulating.
  • the gas impermeable film is light insulating when the culture medium contains light sensitive components such as L- glutamine or other aromatic components.
  • the gas impermeable film is an aluminum film.
  • the Gas Impermeable Aluminum Adhesive Cat# A2350, Sigma, St. Louis, MO
  • This adhesive provides gas impermeability and a tight seal while also being light insulating.
  • the cells described herein adhere to the bottom of the inner side of the cultureware.
  • the adhesion can be “active” or “passive”.
  • the cells sediment to the bottom of the inner side of the cultureware and secrete an extracellular matrix to allow the cells to adhere.
  • the binding of the cells to the secreted extracellular matrix proteins may be temperature-, osmolarity- or pH-dependent and therefore prone to failure when environmental conditions change over time. “Actively” adhered cells tend to lose adhesion within a few days when kept at ambient temperature, limiting their longevity.
  • the adhesion is at least in part “passive”. “Passive” adhesion does not involve any cellular activities of the cells for the adhesion. “Passive” adhesion can be accomplished by providing a positive surface electrostatic charge to the bottom of the inner side of the cultureware. This positive surface electrostatic charge can be provided by a coating of a synthetic polymer having the charge. In some aspects, the bottom of the inner side of the cultureware can have a positive surface electrostatic charge to allow passive adhesion of the cells to the bottom of the cultureware. In some aspects, the positive surface electrostatic charge is provided by a coating of a synthetic polymer with a positive surface electrostatic charge.
  • the cell surface proteins are mainly negatively charged under physiological conditions allowing the cells to adhere electrostatically to the positively-charged coating.
  • Commercially available synthetic polymers can be used to provide this function. Examples of these polymers include, but not limited to, poly-D-lysine, poly-L-lysine, poly-D-omithin or polyetherimide.
  • the synthetic polymer is poly-D-lysine (e.g., Cat# 7886, Sigma, St. Louis, MO). Spontaneous loss of adhesion by “passively” adhered cells tends to be substantially delayed even at ambient temperature.
  • the coating of positive surface electrostatic charge can be supplied by the vendor with the cultureware, or it can be applied to the cultureware before the culture by the user.
  • the cultureware or substrate can be pre-coated with the synthetic polymer.
  • the synthetic polymer can be a polycationic polymer.
  • the polycationic polymer can be polylysine, polyomithine or polyethyleneimine.
  • the application of the synthetic polymer can limit the lowered temperature impact (e.g., about 22°C instead of 37°C).
  • the applied synthetic polymer e.g., hydrogel
  • the applied synthetic polymer can lock the cells in place because the synthetic polymer (e.g., hydrogel) is applied as a liquid and then turns into a conformal solid upon gelation.
  • Example 2 in the Examples Section describes an example of coating the bottom of the inner side of the cultureware with poly-D-lysine.
  • Passive adhesion of the cells may require incubating the cells in MEM and 37°C for a period of time. This allows the freshly seeded cells to gradually sediment to the bottom of the cell suspension until the cells adhere to the coating.
  • hydrogels are non-biodegradable and cytocompatible.
  • the non-biodegradable cytocompatible hydrogel can be optically clear.
  • Hydrogel is formed when the monomer of the hydrogel polymerizes in the presence of an appropriate catalyst. Water (or more generally medium) is trapped within the polymer during polymerization to form the hydrogel.
  • cytocompatible is that neither the resulting hydrogel, nor the monomer nor the catalyst causes either short-term or long-term toxicity to the cells. While some hydrogels are non-cytotoxic, the monomer and/or catalyst used can be cytotoxic.
  • hydrogel-entrapped cells can be incubated in a wash solution to allow for passive diffusion of catalyst and/or monomers to exit the hydrogel. Suitable hydrogels for the present invention are commercially available.
  • hydrogels examples include but are not limited to the QGel Hydrogel from QGel SA (Cat# NS22-A; Lausanne, Switzerland) and the TruGeBDTM (Sigma, St. Louis, MO).
  • the components for making the hydrogel come as a kit and the hydrogel is prepared according to the manufacturer instruction.
  • the cells are used for cell-based assays and the intracellular signal is emission of light. In some aspects, the cells are imaged to detect the emitted light.
  • the hydrogel is optically clear.
  • An example of an optically clear cytocompatible hydrogel is the QGel Hydrogel.
  • the hydrogel can be formed inside the cultureware in the presence of the adhered cells so that the hydrogel is conformal around the cells locking the cells in position within the hydrogel. Locking the cells in position within the hydrogel prevents the cells from movement and further limiting division which otherwise leads to eventual apoptosis, thereby prolonging the longevity and functionality of the cells.
  • the hydrogel is non-biodegradable so that it can maintain its structural integrity during the entire culture.
  • the culture system can be used for cell-based assays.
  • the hydrogel is receptor ligand permeable so that the ligand can penetrate the hydrogel to reach the cells.
  • Hydrogels can be ligand permeable due to mesh size of the hydrogel being larger than molecular size.
  • the hydrogel can be sufficiently permeable to allow nutrients and waste to diffuse in and out of the cells.
  • Formation of the hydrogel inside the cultureware around the cells can be accomplished by incubating the culture at 37°C for a period of time.
  • Example 3 in the Examples Section provides an example of forming the hydrogel inside the cultureware in the presence of the adhered cells.
  • the cells are used for cell-based assays, and wherein the cells are imaged to detect the emitted light, the cells can be locked in position in the focal plane by limiting their movement allows better quality of the images.
  • Locking or maintaining the adhered cells in a stationary position by the hydrogel can be applicable to cells that are prone to apoptosis.
  • the cells grow and divide until they are in contact with each other (known as contact inhibition) then the resulting senescence precedes initiation of apoptosis events leading to cell death.
  • contact inhibition is prohibited and thus delaying the apoptosis process.
  • Cells from cell lines do not exhibit contact inhibition but they grow and divide indefinitely and eventually undergo apoptosis and cell death.
  • their growth is mechanically controlled to delay apoptosis and cell death.
  • Cells in cell cultures that are normally cultured optimally at 37°C can detach from the cultureware and undergo apoptosis due to loss of anchorage when they are exposed to lower temperatures, such as ambient temperature. By locking the cells in position within the hydrogel, the cells no longer detach from the cultureware at the lower temperatures to delay the apoptosis process. Yet in anchorage-dependent cells, they undergo anoikis when they detach from the surroundings. Anoikis is a form of programmed cell death. Thus, these anchorage-dependent cells can benefit from being locked in position by the hydrogel. [88] Disclosed herein is a cell culture medium that can be in the cell culture. In some aspects, the medium is carbon dioxide-independent.
  • the pH of the medium is independent of the presence of carbon dioxide. This is in contrast to standard cell cultures that require incubating the culture in presence of 5% CO2 in the incubator. Reduction in the level of CO2 in the culture causes the pH of the culture to rise and induces stress to the cells.
  • the culture medium as described herein is buffered by a high buffering capacity medium which is independent of the presence of carbon dioxide. Thus, the culture is not incubated in the presence of 5% CO2 as required in many standard cell culturing methods.
  • the high buffering capacity of the buffer maintains a pH of about 7 to about 8 for the culture medium during the entire culture. In some aspects, the high buffering capacity of the buffer can prevent pH drift. Suitable buffers include, but not limited to, MOPS.
  • HEPES, MES, BICINE or phosphate Commercially available culture media with high buffering capacity and carbon dioxide-independent include but are not limited to carbon dioxide- independent medium from ThermoFisher (Cat# 18045088; Chicago, IL), HibemateTM-E Medium and Leibovitz’s L-15 Medium from Sigma (both from ThermoFisher, Chicago, IL).
  • PCT Publication No. 1991/000451 by VisticaD. T. etal. discloses a carbon dioxide- independent growth medium for maintenance and propagation of cells.
  • the high buffering capacity of the cell culture medium can be accomplished by using an increased amount of the medium, such as topping the cultureware with the medium. In some aspects, the high buffering capacity of the cell culture medium can be achieved by having an excessive amount of the medium.
  • the cell culture medium can comprise growth factors.
  • the cell culture medium can comprise Fetal Bovine Serum (FBS).
  • FBS Fetal Bovine Serum
  • the FBS can be in the range of about 1% to about 20%.
  • FIG. 1 is a schematic diagram of a cross-section of cultureware that can be useful in the methods described herein.
  • the cultureware can be a plate having multiple wells with the top of the wells open for passage of materials into the wells.
  • the cell culture system 10 has multiple wells 20.
  • a coating of synthetic polymer 30 provides a positive surface electrostatic charge to allow passive adhesion of the cells 40 to the bottom of the inner side of the cultureware.
  • a layer of non-biodegradable cytocompatible gel 50 surrounds the cells 40 locking the cells 40 into position.
  • the well 20 of the cultureware is filled with a culture medium 60 buffered with a high buffering capacity and carbon dioxide independent buffer.
  • the entire cultureware is covered tightly with a gas impermeable light insulating film 70 to prevent the evaporation of the culture medium 60 during the culture.
  • the system comprises: (1) a cultureware for culturing the cells, wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and can be tightly sealed; (2) a coating of synthetic polymer at the bottom of the inner side of the cultureware wherein the synthetic polymer having a positive surface electrostatic charge to allow passive electrostatic adhesion of the cells; (3) cells adhered to the bottom of the inner side of the cultureware on top of the coating of synthetic polymer wherein the cells having a surface G-protein coupled receptor, an intracellular G-protein and an intracellular reporter; (4) a layer of optically clear hydrogel around of the cells locking the cells in position within the hydrogel; and (5) a cell culture medium covering the cells wherein the cell culture medium is buffered by a high buffer capacity to maintain pH of from about 7 to about 8 in the medium throughout
  • the methods described herein provide a means for culturing cells such that the cultured cells remain viable and functional outside of a controlled temperature environment without supplemental carbon dioxide for at least 3 days. Further, the cultured cells use the methods described herein. Additionally, the methods used to culture cells include a sufficient amount of medium that after sealing the cells in the system or device disclosed herein, the cultured cells remain viable and functioning without further supplementation.
  • the method comprises the steps of: (1) providing a cultureware for culturing cells wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the bottom of the inner side of the cultureware having a coating with a synthetic polymer to provide a positive surface electrostatic charge to allow passive adhesion of the cells to the bottom of the cultureware; (2) depositing the cells to the bottom of the inner side of the cultureware on the coating of synthetic polymer; (3) allowing the cells to adhere to the bottom of the inner side of the cultureware on the coating of synthetic polymer; (4) providing a layer of non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and (5) covering the cells with a cell culture medium wherein the cell culture medium is buffered by a high capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture; and (6) culturing the cells at ambient temperature without incubating the cells at 37°C and 5% CO
  • the ambient temperature can be from about 15°C to about 25°C or even to about 30°C. In some aspects, the ambient temperature can be from about 20°C to about 22°C. In some aspects, the ambient temperature can be from about 15°C to about 30°C, about 20°C to about 28°C, about 20°C to about 25°C or about 20°C to about 24°C. In some aspects, the cell can be cultured at a first temperature or temperature range, but adherent, viable, and functional at a second temperature (e.g., room temperature, 37°C).
  • a first temperature or temperature range but adherent, viable, and functional at a second temperature (e.g., room temperature, 37°C).
  • the cells can be cultured at a first temperature (e.g., between 15°C and 30°C) to reduce metabolic rate and limit cellular division while preserving cellular viability and adherence and ability of the cells to respond in the presence of molecules that bind to the GPCR and generator a detectable reporter response.
  • a first temperature e.g., between 15°C and 30°C
  • the cells remain viable and adherent to the substrate for at least 3 days without supplemental CO2.
  • the methods comprise the steps of: (1) providing a cultureware for culturing cells wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the bottom of the inner side of the cultureware having a coating with a synthetic polymer to provide a positive surface electrostatic charge to allow passive adhesion of the cells to the bottom of the cultureware; (2) depositing the cells to the bottom of the inner side of the cultureware on the coating of synthetic polymer; (3) allowing the cells to adhere to the bottom of the inner side of the cultureware on the coating of synthetic polymer; (4) providing a layer of non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and (5) covering the cells with a cell culture medium wherein the cell culture medium is buffered by a high capacity carbon dioxide- independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture; and (6) culturing the cells at ambient
  • the methods can comprise adding non-biodegradable cytocompatible hydrogel around the adhered cells, wherein the cells are immobile within the hydrogel; and adding a cell culture medium to the cells, wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture; and culturing the cells at ambient temperature without incubating the cells at 37°C and 5% CO2.
  • said method can comprise: maintaining cells in a cell culture device, wherein the cell culture device comprises a top, an optically transparent bottom, an inner side and an outer side.
  • the cultureware of said cell culture device is gas impermeable.
  • the cell culture device has at least one access region to add or remove contents within the cell culture device.
  • the access region is sealable to be gas impermeable.
  • the methods can comprise maintaining the cells adhered to the bottom of the cultureware for at least 3 days.
  • a layer of non-biodegradable cytocompatible hydrogel sufficiently surrounds the cells to maintain the cells in a position within the hydrogel.
  • the methods can comprise covering the cells in the culture device with a culture medium buffered to maintain a pH of from about 7 to about 8 in in the absence of supplemental carbon dioxide and wherein the cells remain viable in said device for at least 3 days.
  • the methods can comprise contacting a cell culture with a fluid sample.
  • the fluid sample comprises one or more volatile organic molecules.
  • the cell culture can comprise cells in a cultureware having a top, an optically clear bottom, an inner side and an outer side and is gas impermeable.
  • the cells are adhered to the bottom of the inner side of the cultureware.
  • the cells are functional at room temperature.
  • the cells are present in a cell culture medium that is buffered by a high buffering capacity carbon dioxide-independent buffer.
  • the cells comprise one or more G-protein coupled receptors capable of binding to the one or more volatile organic molecules. In some aspects, wherein the one or more G-protein coupled receptors comprises a reporter. In some aspects, the methods can comprise exposing the cell culture to a light. In some aspects, the methods can comprise detecting the presence of a fluorescence emitted by the reporter after binding of the one or more G-protein coupled receptors to the one or more volatile organic molecules, an increase in brightness of the fluorescence, or a combination thereof. In some aspects, the cells in the cultureware are non-mammalian cells. In some aspects, the non-mammalian cells can be fish cells. In some aspects, the fluid sample can be a gaseous sample from an exhaled breath of a mammalian subject. In some aspects, the cells can be maintained in the culture for at least 3 days in an uncontrolled temperature environment and without supplemental carbon dioxide.
  • Example 1 Preparation of coating on bottom of the inner side of the cultureware using poly-D-lysine
  • the synthetic polymer poly-D-lysine (PDL)
  • PDL poly-D-lysine
  • Other synthetic polymers that can be used include but are not limited to poly-L-lysine (PLL) and poly-L-omithin (PLO), or polyetherimide (PEI). These synthetic polymers can be used interchangeably. Described herein is a method of coating the bottom of the inner side of the cultureware using a synthetic polymer (e.g., poly-D-lysine). Any suitable synthetic polymers can be used.
  • the cells could be deposited at this stage. If the poly-D-lysine coated cultureware was to be used later, the inner side of the cultureware could be rinsed with deionized water before being stored at 4°C to avoid crystallization of salts from PBS.
  • Example 2 Depositing the cells onto the bottom of the inner side of the cultureware coated with poly-D-lysine
  • the cells are deposited into a cultureware not coated with the synthetic polymer, the cells sediment to the bottom of the cultureware due to gravity and roll on the bottom until they secrete enough natural extracellular matrix proteins to coat the cultureware to allow their adhesion to the cultureware.
  • the coating of the cultureware with synthetic polymers speeds up the initial adhesion of the cells to the cultureware.
  • HEK cells were dispensed in suspension into the poly-D-lysine coated cultureware. After the cells were allowed to passively sediment to the bottom of the suspension to the poly-D-lysine coated inner side of the cultureware, the cells no longer moved independently as floating cells. They moved in unison once they were adhered to the bottom of the cultureware. When the side of the cultureware was lightly tapped, no cell continued to move with inertia as cells were now locally adhered to the surface of the inner side of the cultureware following sedimentation.
  • Cells per unit area under the experimental conditions described herein is about 5,000/mm 2 . This number is dependent on the cell type as larger cells would be over-crowded at the density of 5,000/mm 2 . This number is for standard size cells like Human Embryonic Kidney (HEK) or Chinese Hamster Ovary (CHO).
  • HEK Human Embryonic Kidney
  • CHO Chinese Hamster Ovary
  • Hydrogel is formed inside the cultureware in the presence of the adhered cells according to manufacturer instructions.
  • QGel Hydrogel (from QGel SA, Lausanne, Switzerland) was used.
  • the lyophilized monomer is mixed with a HEPES solution (0.3 M) until the monomer dissolves entirely.
  • Culture medium is then added (1 part of culture medium to 4 parts of hydrogel monomer in HEPES solution).
  • the polymerization reaction starts immediately and the QGel monomer solution is dispensed onto the adhered cells before it gels.
  • the optically clear hydrogel can then be allowed to form around the cells at 37°C for one hour in a conformal way thereby locking the cells in position.
  • the hydrogel is not biodegradable so the cells are limited in their movement.
  • Example 4 Effect of temporal changes in osmolality on cellular morphology
  • FIGs. 2 A and 2B show the effect of temporal changes in osmolality on cellular morphology.
  • FIG. 2A shows the average area (pm 2 ) measurement of HEK cells post exposure to carbon dioxide independent media + 10% FBS + gentamicin (carbon dioxide-independent medium supplemented with 10% FBS, abbreviated as CO2I10) for 1 to 8 days in vitro.
  • FIG. 2B shows the average circularity measurements of cells post exposure to CO2I10 for 1 to 8 days in vitro.
  • Circularity 4 (area/peri meter 2 ). As the value approaches 1.0, it is indicative of a perfect circle. As the value approaches 0, it indicates an increasingly elongated polygon.
  • n > 20 cells/well were assayed for n 3 wells (n > 60 cells per condition) Data is represented as mean +/- STD.
  • Example 5 Protective effect of the microenvironment on cell-based assay stability
  • FIG. 3 shows the response profile of biosensor (e.g., cells cultured using the disclosed methods) detecting 1 mM propylbenzene with a 2.5-fold increase of fluorescence following a 15- minute exposure compared to baseline fluorescence. Over 12 days at room temperature outside the laboratory setting, the propylbenzene response of cells maintained in the engineered microenvironment remains steady.
  • biosensor e.g., cells cultured using the disclosed methods
  • FIG. 4 shows phase micrographs of HEK cells exposed to carbon dioxide independent media + 10% FBS (CO2I10) allowed to evaporate for different amounts of time hereby referred to as the different conditions.
  • Cells were seeded at a density of 20,000 cells/well where they were given 24 hours at 37°C, 5% CO2 , 95% humidity to adopt the reference morphology. Cells were then exposed to conditioned media evaporated for 1 day and 8 days to assess the effect of osmolality.
  • CO2I10 was prepared and conditioned separately in polystyrene 96-well plates for 8 days, sterilized by 0.22 pm filtration after collection, and 50 pL were added to respective experimental wells and allowed for 30 minutes at 37°C, 5% CO2 , 95% humidity.
  • Example 7 Functional response profile of cells cultured from day 3 to day 12 in carbon dioxide-independent media
  • FIG. 5 shows HEK cells expressing a GPCR were challenged with propylbenzene and cytosolic calcium increase was measured via a genetically-encoded calcium indicator. The mean fluorescence intensity was acquired every minute for a total of 15 minutes and data are represented as a fold induction normalized to the baseline value prior to ligand challenge. By tracking the amount of 517 +/- 23 nm emitted green light under 480 +/- 17 nm excitation light, the amount of calcium in the cell can be estimated and the amount of propylbenzene that activates the cell surface receptor can be measured.
  • Example 8 The influence of ambient air on pH of DMEM and carbon dioxide independent-media
  • FIG. 6 shows in the control measurements, media was procured directly from commercial bottles.
  • Example 9 Microenvironment conditions for maintaining functionality of adherent cells for 12 days at ambient temperature without the need for CO2 supplementation
  • adherent HEK cells were cultures in standard laboratory conditions using high glucose DMEM (Gibco Cat# 11965118) supplemented with 10% FBS. The cells were cultured using a humidified CO2 incubator. In the absence of a 5% excess of incubator CO2, dissolved buffering CO2 immediately degassed and the pH increased within minutes to toxic levels as shown in FIG. 8. The pH rapidly increased from pH 7.4 to pH 8.0. Next, the culture medium was replaced from DMEM to CCh-independent medium to remove the acute pH drift. After culturing the cells out of the incubator for 8 days, cell morphology was tracked daily and a gradual change was observed. As shown in FIGS.
  • the cell morphology changed over time with a decrease in observed cell area from about 450 pm 2 to about 200 pm 2 as well as an increase in circularity from 0.45 to 0.90. These changes were not associated with a corresponding change in cell viability as assessed with an ATP assay. These findings show that gradual osmolarity changes were leading the cells to lose anchoring points and adopt a round morphology. The increase in cell circularity was associated with an increase in cytoplasmic Ca 2+ concentrations indicative of cellular stress.
  • the substrate was pre-coated with polycationic polymers (e.g., poly-L-lysine) to limit evaporation by insulating the culture. As shown in FIG.
  • FIG. 5 shows the functional response profile of cells cultured from day 3 to day 12 in carbon dioxide independent media, by limiting passive pH increase, osmolarity increase and assisting passive cell adhesion with the use of polycationic polymers.
  • the results show a sustained cell assay performance over a 12 day period.
  • the cells were excited with 418/17 nm and read at 517/23nm.
  • the ZOE imager BioRad was used.
  • a 96-well plate (Greiner Bio-One Cat# 655809) was pre-treated overnight at 37°C and 5% CO2 with 50 pL per well of Poly-D-lysine (Sigma Cat# P6407) at 75 ng/mL in UltraPure water (Invitrogen Cat# 10977023). After an initial coating step, the excess Poly-D-lysine coated microwells were rinsed twice with 100 pL ambient temperature PBS at pH7.4 (Gibco Cat#10010072) and once with lOOpL UltraPure water.
  • Coated microwells were then seeded with HEK cells dissociated with Trypsin-EDTA 0.05% (Gibco Cat# 25300062) at a viability of >97% as assessed diluting cell suspension to an equal volume of 0.4% Trypan Blue (BioRad Cat# 1450021) and using automated cell counting with a TC20 (BioRad). After seeding, cells were allowed to sediment and adhere at 37°C under 5% CO2 for 24 hrs. After confirming adherent cell morphology, the overlaying medium was replaced with non-HEPES buffered CO2- independent medium (Gibco Cat# 18045088) supplemented with 10% FBS and gentamicin (Gibco Cat# 15710064). After replacing the culture medium, plates were sealed with gas impermeable foil and stored at ambient temperature outside of the CO2 incubator.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Cell Biology (AREA)
  • Clinical Laboratory Science (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un système de culture cellulaire et un procédé de culture de cellules. Plus particulièrement, la présente invention concerne un système et un procédé de culture de cellules à température ambiante sans incubation des cellules à 37 °C en présence de 5 % de CO2.
PCT/US2022/019052 2021-03-05 2022-03-05 Système de culture cellulaire et procédés d'utilisation WO2022187725A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163157445P 2021-03-05 2021-03-05
US63/157,445 2021-03-05

Publications (1)

Publication Number Publication Date
WO2022187725A1 true WO2022187725A1 (fr) 2022-09-09

Family

ID=83154642

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/019052 WO2022187725A1 (fr) 2021-03-05 2022-03-05 Système de culture cellulaire et procédés d'utilisation

Country Status (1)

Country Link
WO (1) WO2022187725A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117070438A (zh) * 2023-06-13 2023-11-17 中国水产科学研究院长江水产研究所 一种中华鲟鳃细胞系及其构建方法和应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030096414A1 (en) * 2001-03-27 2003-05-22 Invitrogen Corporation Culture medium for cell growth and transfection
US7037580B2 (en) * 2001-09-25 2006-05-02 Ali Razavi Pattern adhesive sealing films and mats for multi-well plates
US20130035256A1 (en) * 2010-04-20 2013-02-07 Addex Pharma Sa Chimeric Polypeptides Useful in Proximal and Dynamic High-Throughput Screening Methods
US20130194410A1 (en) * 2010-09-14 2013-08-01 Ramot At Tel-Aviv University Ltd. Cell occupancy measurement
US20130344159A1 (en) * 2011-03-09 2013-12-26 Occlugel Implantable swellable bio-resorbable polymer
US20160348049A1 (en) * 2013-09-11 2016-12-01 Dai Nippon Printing Co., Ltd. Cell culture vessel
US20190322969A1 (en) * 2014-10-29 2019-10-24 Corning Incorporated Devices and methods for generation and culture of 3d cell aggregates
US20200324021A1 (en) * 2019-04-11 2020-10-15 University Of Maryland, College Park 3D Printed Scaffold Structures and Methods of Fabrication
US20200385660A1 (en) * 2019-06-10 2020-12-10 Upm-Kymmene Corporation Cell culture plate, method for preparing thereof and method for detecting a substance

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030096414A1 (en) * 2001-03-27 2003-05-22 Invitrogen Corporation Culture medium for cell growth and transfection
US7037580B2 (en) * 2001-09-25 2006-05-02 Ali Razavi Pattern adhesive sealing films and mats for multi-well plates
US20130035256A1 (en) * 2010-04-20 2013-02-07 Addex Pharma Sa Chimeric Polypeptides Useful in Proximal and Dynamic High-Throughput Screening Methods
US20130194410A1 (en) * 2010-09-14 2013-08-01 Ramot At Tel-Aviv University Ltd. Cell occupancy measurement
US20130344159A1 (en) * 2011-03-09 2013-12-26 Occlugel Implantable swellable bio-resorbable polymer
US20160348049A1 (en) * 2013-09-11 2016-12-01 Dai Nippon Printing Co., Ltd. Cell culture vessel
US20190322969A1 (en) * 2014-10-29 2019-10-24 Corning Incorporated Devices and methods for generation and culture of 3d cell aggregates
US20200324021A1 (en) * 2019-04-11 2020-10-15 University Of Maryland, College Park 3D Printed Scaffold Structures and Methods of Fabrication
US20200385660A1 (en) * 2019-06-10 2020-12-10 Upm-Kymmene Corporation Cell culture plate, method for preparing thereof and method for detecting a substance

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CALIARI ET AL.: "A practical guide to hydrogels for cell culture", NAT METHODS, vol. 13, May 2016 (2016-05-01), pages 405 - 414, XP055392866, DOI: 10.1038/nmeth.3839 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117070438A (zh) * 2023-06-13 2023-11-17 中国水产科学研究院长江水产研究所 一种中华鲟鳃细胞系及其构建方法和应用

Similar Documents

Publication Publication Date Title
Pekkurnaz et al. Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase
Mustafa et al. Identification and profiling of novel α1A-adrenoceptor-CXC chemokine receptor 2 heteromer
JP5943607B2 (ja) 細胞アッセイを行うための方法
Handke et al. Towards long term cultivation of Drosophila wing imaginal discs in vitro
WO2022187725A1 (fr) Système de culture cellulaire et procédés d'utilisation
WO2009052183A1 (fr) Composition et procédé pour mesurer l'entrée et la sortie de thallium
EP2257812A1 (fr) Essai à base de fluorescence pour détecter des composés modulant un « mode inverse » d'un échangeur de sodium/calcium (ncx)
KR100951656B1 (ko) 프로베네시드를 포함하는 ｔｒｐｖ２ 활성제
Getter et al. Identification of small-molecule allosteric modulators that act as enhancers/disrupters of rhodopsin oligomerization
WO2015190529A1 (fr) Procédé pour évaluer l'interaction de protéines
AU2008226101B2 (en) Fluorescence-based assay for detecting compounds for modulating the sodium-calcium exchanger (NCX) in forward mode
Garcia et al. Generation of mixed murine organoids to model cellular interactions
JP6579539B2 (ja) 光識別方法、物質の検出方法、レポーターアッセイ方法、キット、ルシフェリン−ルシフェラーゼ反応阻害剤、ルシフェリン−ルシフェラーゼ反応阻害方法及び装置
Sarker et al. mOrange2, a genetically encoded, pH sensitive fluorescent protein, is an alternative to BCECF-AM to measure intracellular pH to determine NHE3 and DRA activity
Brockmann Implementation of a high throughput screening assay to identify and characterize insecticides acting on ion channels
Rahmouni et al. Evaluating effects of tyrosine phosphatase inhibitors on T cell receptor signaling
Koester The spatial dynamics of insulin-regulated GLUT4 dispersal
TW201305557A (zh) 用於鑑定改變細胞ｄｄｒ活性的化合物之測試系統及方法
Greier Metabolic stress and epithelial-mesenchymal transition in head and neck cancer
US20110117574A1 (en) Fluorescence based assay to detect sodium/calcium exchanger "forward mode" modulating compounds
Ashrafi et al. Effects of different cryopreservation parameters on the differences between trypan blue and fluorescent SYTO 13/GelRed assays
Rojo‐Ruiz et al. Using Fluorescent GAP Indicators to Monitor ER Ca2+
Pozzan et al. Imaging calcium dynamics using targeted recombinant aequorins
Pal Investigating the Impact of FGFR5 Expression on β-cell Metabolism, Maturity and Intracellular Signaling
Liu et al. Establishing a assay for detection of nonylphenol estrogenic effects

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: 22764210

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: 22764210

Country of ref document: EP

Kind code of ref document: A1