Application Protocol: Guidelines for MCF7 cells growth and maintenance

Transcription

1 REPRODUCTION AND USE This document is protected by copyright and cannot be used or shared without permission from Vortex Biosciences, Inc. Such permission is given on condition that Vortex Biosciences is acknowledged whenever this protocol is reproduced or is published in whole or in part. PURPOSE This Application Protocol describes guidelines to properly grow and maintain MCF7 human breast cancer cell line, to be used for VTX-1 Liquid Biopsy System evaluation. DEFINITIONS Adherent cells: Cells proliferating on a surface, such as a flask. MATERIALS Consumables Long pipette tips (p10, p200, p1000), with filter (ART Tips from ThermoFisher #214005, and or equivalent). 15 ml Falcon polypropylene tubes or equivalent. 50 ml Falcon polypropylene tubes or equivalent. Eppendorf tubes or equivalent. 5 ml sterile serological pipettes. 10 ml sterile serological pipettes. 50 ml sterile serological pipettes. Sterile disposable aspirating pipettes. Cell culture flasks - vented cap - 25 cm 2 (VWR # or equivalent). Cell culture flasks - vented cap - 75 cm 2 (Corning # or equivalent). Paper towels. Disposable Nitrile gloves. Disposable gown. Equipment Inverted bright-field microscope (CKx31, Olympus Life Science or equivalent). BSL II Biosafety cabinet. CO2 incubator (HERACell 150i, ThermoScientific or equivalent). Vacuum system (Evac Waste System, EV310, Argos Technologies Inc. or equivalent). Water bath, set to 37 C (Digital Lab-line AquaBath, Thermo Scientific or equivalent). Page 1 of 5

2 Benchtop centrifuge with swinging-bucket rotor and 15 ml/50 ml conical adapters (Allegra X-14R or GS-6R, Beckman Coulter or equivalent). Neubauer hemocytometer, automated cell counter or equivalent + glass slides (if necessary). Digital counter or hand tally counter. All-purpose upright 4 C freezer (Isotemp Refrigerator, Fischer Scientific or equivalent). All-purpose upright -20 C freezer (Isotemp Freezer, Fischer Scientific or equivalent). Tube rack. Pipettes (p10, p200, p1000), Gilson or equivalent. Pipet-aid (Drummond or equivalent). Timer. Tube rack. Sharpie. Chemicals and Reagents (these should NOT be replaced without further validation) MCF7 breast cancer cell line (ATCC #HTB-22). Sterile HBSS Ca2+ free, Mg2+ free, phenol red (Gibco ). Sterile TrypLE Express non-enzymatic cell dissociation reagent (Gibco, # ). RPMI-1640 Medium, GlutaMAX Liquid Supplement (Gibco # or equivalent). Eagle's Minimum Essential Medium (EMEM) (ATCC # or equivalent). Fetal Bovine Serum (Corning #35010CV, heat inactivated). Penicillin-Streptomycin Solution, 100X (Corning # CI). Insulin Recombinant Human Zn (Life Technologies # ). Trypan Blue Solution 0.4% (Gibco, # or equivalent). Virkon-S or equivalent. Concentrated regular household bleach (Clorox or equivalent). 10 % Bleach solution prepared daily (from Clorox or equivalent). 70 % Ethanol, laboratory grade (Carolina Biological Supply or equivalent). CaviCide (Metrex). Deionized water. REFERENCES PB004_ Processing of cancer cells spiked in human blood through VTX-1 Liquid Biopsy System. GUIDELINES 1) Recovery of cryopreserved cells If the vial is to be received from ATCC or another supplier, thaw the vial and initiate the culture as soon as possible upon receipt to ensure the highest level of viability. Page 2 of 5

3 If upon arrival continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -80 C. Storage at -80 C could result in loss of viability. It is strongly advised to use the medium recommended by the cell supplier. For MCF7 cells, if purchased from ATCC, the medium should be Eagle's Minimum Essential Medium (EMEM) (ATCC ) supplemented with: - 10% of Fetal Serum Bovine (FBS) heat inactivated at 56 C for 30 min, - 10 µg/ml of human recombinant insulin (final volume), - 1% of 100X Penicillin and Streptomycin solution (final concentration 1X), if using antibiotics. 2) Changing medium 2.1. Regular medium change The cells should be inspected regularly and the culture medium replaced with fresh media every 2 to 3 days until the cells are ready to be passaged, harvested for an experiment or banked. When growing cells over the weekend, the medium should be changed on Friday afternoon, and changed again on Monday morning. If the medium color in the culture flask radically differs from the color of the medium stock, ph has been altered and cells are in stressed conditions. The flask should be checked under the microscope for overgrowth or contamination Transitioning from EMEM to RPMI-1640 medium MCF7 cells growing in complete EMEM (cemem) usually form multi-layer domes, which increases the presence of cell clumps once cells are harvested. These domes also make the estimation of cell confluency difficult. When processing MCF7 cells through the VTX-1 Liquid Biopsy System, cell confluency and clumping are 2 crucial variables directly impacting the performance and its reproducibility. Therefore, it is recommended to transition MCF7 cells to a type of medium in which they grow in a monolayer. MCF7 cells can be transitioned from cemem (composition in section 1) to complete RPMI-1640 medium, (crpmi) which consists of RPMI-1640 medium supplemented with: - 10% of Fetal Serum Bovine (FBS heat inactivated at 56 C for 30 min, - 10 µg/ml of human recombinant insulin (final volume), - 1% of 100X Penicillin and Streptomycin solution (final concentration 1X), if using antibiotics. After thawing, MCF7 cells should be grown in cemem for 1 passage before gradually transitioning to crpmi Passage 2: 75% cemem + 25% crpmi. - Passage 3: 50% cemem + 50% crpmi. - Passage 4: 25% cemem + 75% crpmi. - Passage 5: 100% crpmi. Page 3 of 5

4 3) Sub-culturing and passaging cells from a monolayer Cells should be sub-cultured when culture has reached 70-80% confluency. The operator should avoid passaging under (<20%)- or over confluent (>80%) cells. Not respecting this guideline could dramatically affect the cell integrity. Examples of under confluent and over confluent cells are presented in figures 1 and 2. Figure 1: Example or under- (left) and over- (right) confluent MCF7 cells when grown in cemem. Cells grow in multi-layers and form domes. Scale bar: 100 µm. Figure 2: Example or under- (left) and over- (right) confluent MCF7 cells when grown in crpmi. Cells grow in mono-layer. Scale bar: 100 µm. MCF7 cells should be split at a ratio between 1:3 and 1:6. Splitting cells at a higher or a lower ratio could lead to drastic changes in the cell proliferation rate. The plating density recommended by ATCC is cells for a T25 flask, and cells for a T75 flask (40,000 cells/cm 2 ). The cell culture should be discarded after 20 passages or 2 months in culture, whichever is the shortest. A P followed by a number indicates the passage number of a cell line. NOTE: The passage number is continued from the freezing passage number. For example, cells that were frozen at P5 can be passaged until P20 or 2 months, whichever is reached first. The passage number increases by one-unit every time the cells are sub-cultured. Page 4 of 5

5 4) Harvesting cells for downstream assay using the VTX-1 Liquid Biopsy System When harvesting cells for downstream assays where cell surface protein integrity is critical, such as for cell separation and enrichment using Vortex Biosciences technology or flow cytometry, we strongly recommend against the use of trypsin as the experiment outcome may be negatively impacted. For downstream assays, cells are to be dissociated using a gentler reagent such as TrypLE express. IMPORTANT NOTES 1. How to avoid contamination Work under a biosafety hood, and use it as specified by manufacturer. Be careful to stay within its sterile zone. Do not manipulate above the air flow grids. When placed in the water bath, do not submerge completely reagent containers. Bottles and tube caps must stay away from the water. If containers float because they are too light, secure them with a weight to avoid them flipping or falling in the water. Clean all the materials and reagents going in the biosafety cabinet with Virkon-S and 70% ethanol and clean paper towels. Clean the cap of reagents meticulously. The operator should clean his/her gloves with 70% ethanol prior to move them under the biosafety cabinet. Clean the biosafety cabinet with 70% ethanol between two cell lines. When opening, or pipetting a liquid in a bottle or a tube, do not touch the inside of the container and the cap with the pipette or a finger. Discard all the biological liquid waste in a special container containing bleach. Use properly maintained and calibrated equipment, in their intended use. Cultures should be visually assessed on a routine basis for evidence of contamination. Culture flasks should be observed under the microscope before medium change, collection or a new passage. The presence of microorganisms in a culture may be detected by examination of cells under a microscope or by changes in the color or turbidity of the medium. Cross-culture contamination is another type of contamination caused by a culture of mammalian cells being contaminated by other mammalian cells. Contamination of one culture with a different type of cell may be extremely difficult to detect and can go un-noticed by the researcher for many years, leading to faulty interpretations of experimental data. Handle only one cell line at a time under the biosafety cabinet. Mycoplasma contamination: Mycoplasma are small (0.2 to 0.3 μm) intracellular bacteria that attach to the cell membrane, inhibit cell growth, and eventually lead to cell death. Because these parasites do not have cell walls, do not grow in colonies, and do not change the ph of the medium, they are difficult to detect visually in cultures. Mycoplasma can multiply to very high concentrations and adversely affect cultures by altering cell growth characteristics, inhibiting cell metabolism, disrupting nucleic acid synthesis, inducing chromosome aberrations, changing cell membrane antigenicity, and altering transfection rates and viral susceptibility. Mycoplasma are spread by cross contamination from infected cultures through aerosolization during pipetting, or via the transfer of contaminated cells or contaminated reagents used in cell culture. A PCR-based mycoplasma detection test is available for routine in-lab testing. It is recommended that mycoplasma testing of all cell cultures be performed on a regular basis, such as every 3 months. 2. Wear personal protective equipment: Always wear lab gown, safety goggles, closed shoes, lab coat, and gloves. Page 5 of 5