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    hPSC 诱导分化中脑类器官试剂盒

    hPSC 诱导分化中脑类器官试剂盒

    Catalog Number: abs90475 Brand: Absin
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    $3,333 USD
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    Product Details

    Product Specification

    Usage I. Experimental Instruments and Materials
    Instruments: Biosafety cabinet, cell incubator, horizontal centrifuge, inverted microscope, cryogenic refrigerator
    Material: Centrifuge tube (specifications are 15mL and 50mL ), pipette (specifications are 10μL 、 100μL 、 1000μL and multi-channel 100μL ), sterile tip (specifications are 10μL 、 200μL and 1000μL ), pipette (specifications are 10mL 、 50mL ）、 37μm a reversible cell filter, 200µL/1mL a wide-mouth pipette tip, AggreWell™800 24 orifice plates, 6 Aperture plate ( TC dispose / non-low adsorption), 6 hole ultra-low adsorption plate

    II. Experimental Methods
    1 、 hESC/iPSC cell preparation
    In 6 Aperture plate ( TC dispose / (Non-low adsorption) hESC/iPSC The cell culture kit will hESC/iPSC Cell culture to confluence 70-80% 。
    2 Midbrain organoid formation (No. 0-6 (sky)
    Preparation of Cell Seeding Medium
    Cell seeding medium ( 2.5mL ） : Recovering to room temperature before use, 2.5mL Basic medium 1+5μL supplements A Ready to use. at room temperature ( 15-25°C Thaw the supplement and mix thoroughly. Note: If the supplement is not mixed, put the supplement in -20°C Save. Do not exceed the shelf life of the supplement. Use immediately after thawing. Do not freeze or thaw repeatedly.
    ①Day0
    （ 1 Prepare one AggreWell™800 24 Aperture plate. If other culture vessels are used, adjust the specifications accordingly for subsequent experiments. Heat the culture dish, medium and reagents to room temperature before use ( 15-25°C ）。
    a. Add to each well 500µL anti-adhesion lubricating solution .
    b. On a centrifuge equipped with trays, 1300xg centrifugal plate 5 minutes. Note: Plates must be level.
    c. The plate was observed under a microscope to ensure that air bubbles in the microwells had been removed. If the bubbles are still trapped in the micropores, 1300 x g Centrifuge again 5 minutes.
    d. Absorb the anti-adhesion lubricant from the hole . 。
    （ 2 ) in AggreWell™800 24 Add to the well plate 1mL Cell seeding medium. Put the plate aside.
    （ 3 ) Take out the well-trained hESC/iPSC Cells, will 6 The culture medium inside the wells in the well plate is aspirated and added. 1mL/ hole DPBS (Does not contain calcium or magnesium), shake gently and sip. Note: 4-6 The cell density of the well is the volume we need for subsequent experiments.
    （ 4 ) Join 1mL of Accutase ， 37℃ incubation 3 minutes. Note: Incubation times may vary when different cell lines or other non-enzymatic cell dissociation reagents are used.
    （ 5 ) Use 1mL Pipette slowly up and down 3-5 Next, gently resuspend the cells. Transfer the cell suspension to sterility 15ml In a conical tube.
    （ 6 ) Use 1-2mL Basic medium 2 The wells were rinsed and this rinse solution was added to the tube containing the cells.
    （ 7 Cells were viable using trypan blue and an automated cell counter. Calculated 4.5 × 106 The volume required for the total number of cells (this will be diluted in the next step to obtain 3 × 106 microscopic cell /mL (Final concentration).
    （ 8 ） 300 x g centrifuged cells 5 minutes.
    （ 9 Carefully aspirate the supernatant and resuspend the cells. 1mL The prepared cell seeding medium was used to obtain a final concentration of 3 ×106 microscopic cell /mL 。
    （ 10 ) This 1mL Single cell suspension (i.e. 3 × 106 (individual cells) added to the containing 1mL Cell seeding medium (step 2 (Prepared in) AggreWell™800 In the hole of the plate. This will produce 10000 microscopic cell / Hole.
    Note: By gently pipetting up and down several times, ensure that the newly grafted cells are evenly distributed over the entire well surface.
    （ 11 ） AggreWell™800 The board is 100 x g Lower centrifugation 3 Minutes, the cells in the microwell were captured. Note: Plates must be level.
    （ 12 ) Examine under a microscope AggreWell™800 The plate ensures that the cells are evenly distributed in the microwells. 37°C ， 5%CO2 Incubation.
    ②Day1-Day5
    Preparation of Midbrain Organoid Formation Medium
    Midbrain organoid formation medium ( 15mL ） : Recover to room temperature before use, i.e. 15mL Basic medium 1 。 Note: If not used immediately, at most... 2-8°C store 3 Weeks, do not exceed the shelf life of the basic medium, use it immediately after thawing, do not freeze and thaw repeatedly.
    （ 13 Carefully remove from the incubator. AggreWell™800 Be careful not to interfere with the contents of the board. Note: Plates must be handled with care to avoid organoids coming out of the wells, leading to premature fusion and reduced yield.
    （ 14 ) Use 1mL The pipette was gently removed from the hole. 2x750µL Culture medium and discard.
    Note: Do not interfere with organoids. When pipetting, align the tip of the pipette with the upper surface of the medium in the well.
    （ 15 ) use 1mL The pipette was slowly added to the well. 2 × 750µL Midbrain organoid formation medium.
    Note: It is important not to interfere with organoids. Do not add media directly to the surface of the microwells. On the surface of the remaining medium in the well, gently touch the side of the well to support the pipette tip. Make the release of the culture medium more controllable. slowly releasing the medium into the microwells; Rapid release of the medium removes the organoid from the well.
    （ 16 ） 37℃ ， 5%CO2 incubation 24 Hours.
    （ 17 ) in the 2-5 day repeat step 13-16 。
    （ 18 On the sixth day, the next experimental operation was conducted.
    3 Midbrain organoid amplification (No. 6-25 (sky)
    Return the culture dish, medium and reagents to room temperature before use ( 15-25°C ）。
    If ultra-low adsorption plates are not available, tissue culture-treated dishes may be used, which need to be treated with an anti-adhesion lubricant to prevent cell attachment. Add to each well 1 ml of anti-adhesive rinsing solution. After repeated rinsing, remove the solution from the well and discard it.
    ①Day6
     Preparation of Midbrain Organoid Expansion Medium
    Midbrain organoid expansion medium ( 250mL ): Recover to room temperature before use. 245mL Basic medium 3+5mL supplements B+0.5mL supplements C+0.5mL supplements D Note: If not used immediately, at most... 2-8°C store 3 Weeks, do not exceed the shelf life of the basic medium and supplements, use immediately after thawing, do not freeze and thaw repeatedly. Basic medium 3 It is normal to have some stickiness, shake well and pipette slowly before use to ensure effective transfer of media. If the supplement is not mixed, put the supplement in -20°C Save. Do not exceed the shelf life of the supplement. Use immediately after thawing. Do not freeze or thaw repeatedly.
    （ 1 ) in 6 Add to each well of the ultra-low adsorption plate 2mL Midbrain organoid expansion medium.
    Note: From AggreWell™80024 The organoids of one well of the well plate can be uniformly distributed to 6 In the orifice plate. The recommended range is per hole. 25-40 An organoid. Controlling the number of organoids in the well is essential to avoid yield loss due to premature fusion events.
    （ 2 ) will 37µm The reversible filter is placed in 50ml The top of the conical tube. The conical tube is marked with waste liquid.
    Note: The arrow on the reversible filter should point up. For each AggreWell™800 The wells were harvested using new filters and new tubes.
    （ 3 The upper part of the culture medium is aspirated from the wells containing the organoids, and used... 1mL The wide-mouth pipette tip firmly aspirates the substrate medium out of the well.
    （ 4 Using a wide-mouth pipette tip of the same diameter, aspirate the suspension and pass through... 37 μ m Reversible cell filter filtration. The organoids will remain on top of the filter and the single cells will flow into the waste tube.
    （ 5 Use a wide-mouth pipette tip of the same diameter to take... 1mL of DMEM/F-12 And firmly discharge it to the same AggreWell™800 In the hole. When the organoid is in suspension, the suspension is quickly removed from step... 4 Transfer to a filter.
    （ 6 Repeat the steps. 5 , until all organoids have been removed from the well. One or two repetitions should be sufficient to clear all organoids. The wells were examined under a microscope to ensure that all organoids had been removed.
    （ 7 Invert the reversible cell filter into a new one. 50ml On a conical tube and added to a reversible cell filter 2mL Midbrain organoid expansion medium and collection of all organoids adsorbed on the filter.
    Note: Midbrain organoid expansion media is somewhat viscous and may require high liquid absorption to effectively collect organic matter from the filter.
    （ 8 Gently shake the conical tube to produce an organoid suspension, and use... 1mL The wide-mouth pipette tip transfers the organoid to 6 In the orifice plate.
    6 Each hole in the orifice plate shall not exceed 40 An organoid.
    （ 9 Gently shake the plate, making a cross-shake to ensure that the organoids are evenly distributed in the wells. Plates were observed microscopically to ensure minimal contact between organoids.
    （ 10 Carefully place the board on 37°C ， 5%CO2 On the horizontal surface of the incubator. incubation 2 heaven.
    ②Day8-Day25
    Return the culture dish, medium and reagents to room temperature before use ( 15-25°C ）。
    （ 11 Gently tilt 6 Well plate, waiting for the organoids to sink to the bottom of the well (approx. 15-30 seconds).
    （ 12 Carefully level the tablet, use 1mL A pipette removes the medium from the top of each well.
    （ 13 Fresh midbrain organoid expansion medium was added to each well. 2mL 。 Before incubation, the petri dish was gently shaken, cross-shaking, to distribute the organoids in the wells. Plates were observed microscopically to ensure minimal contact between organoids. Carefully place the board on 37°C ， 5%CO2 On the horizontal surface in the incubator, ensure that the plate is undisturbed.
    （ 14 ) in the 8 Tianzhi Di 25 Change the fluid every two days (steps 11-13 ）。
    （ 15 )th 25 Today, the next experimental operation will be conducted.
    4 Midbrain organoid differentiation (No. 25-43 (sky)
    ①Day25-43
    Preparation of Midbrain Organoid Differentiation Medium
    Midbrain organoid differentiation medium ( 250mL ): Recover to room temperature before use. 245mL Basic medium 3+5mL supplements B+0.25mL supplements E Note: If not used immediately, at most... 2-8°C store 3 Weeks, do not exceed the shelf life of the basic medium and supplements, use immediately after thawing, do not freeze and thaw repeatedly. Basic medium 3 It is normal to have some stickiness, shake well and pipette slowly before use to ensure effective transfer of media. If the supplement is not mixed, put the supplement in -20°C Save. Do not exceed the shelf life of the supplement. Use immediately after thawing. Do not freeze or thaw repeatedly. Return the culture dish, medium and reagents to room temperature before use ( 15-25°C ）。
    （ 1 Gently tilt 6 Well plate, waiting for the organoids to sink to the bottom of the well (approx. 15-30 seconds).
    （ 2 Carefully level the tablet, use 1mL A pipette removes the medium from the top of each well.
    （ 3 Fresh midbrain organoid differentiation medium was added to each well. 2mL ， 37°C ， 5%CO2 Incubate in an incubator.
    （ 4 ) in the 27 Tianzhi Di 43 Change the fluid every two days (steps 1-3 ）。
    （ 5 )th 43 Today, the next experimental operation will be conducted. Midbrain organoid maintenance (No. 43+ (sky)
    ①Day43+
    Preparation of Midbrain Organoid Maintenance Medium
    Midbrain organoid maintenance medium ( 100mL ): Recover to room temperature before use. 98mL Basic medium 3+2mL supplements B Note: If not used immediately, at most... 2-8°C store 3 Weeks, do not exceed the shelf life of the basic medium and supplements, use immediately after thawing, do not freeze and thaw repeatedly. Basic medium 3 It is normal to have some stickiness, shake well and pipette slowly before use to ensure effective transfer of media. If the supplement is not mixed, put the supplement in -20°C Save. Do not exceed the shelf life of the supplement. Use immediately after thawing. Do not freeze or thaw repeatedly.
    Return the culture dish, medium and reagents to room temperature before use ( 15-25°C ）。
    （ 1 Gently tilt 6 Well plate, waiting for the organoids to sink to the bottom of the well (approx. 15-30 seconds).
    （ 2 Carefully level the tablet, use 1mL A pipette removes the medium from the top of each well.
    （ 3 Fresh midbrain organoid maintenance medium was added to each well. 2mL 。
    （ 4 ) each 2-3 Change the liquid every day.
    Note: Once the organoid becomes larger (i.e., the first 50 (More than 10 days), increase the midbrain organoid maintenance medium to each well 3mL To ensure that all organoids are covered by the culture medium.
    （ 5 )th 50 Immunofluorescence staining of midbrain organoids can detect phenotype-specific markers of midbrain-related cells in the peripheral regions of midbrain organoids, including FOXA2+/LMX1A+ Midbrain progenitor cell markers, TH+/GIRK2+ Markers of dopaminergic neurons. Midbrain organoid maintenance media can be used 50 More than a day.
    Description This product is hPSC Midbrain organoid kit for inducing differentiation, human pluripotent stem cells hPSC The kit can induce differentiation to obtain midbrain organoids, whose cellular composition and structural organization represent the developing human midbrain and can stably express midbrain neuron markers, thereby helping customers achieve neuron-related drug screening, disease model construction, and other basic scientific research.
    This kit requires the operator to have hPSC Cultivation experience and a certain understanding of organoids.
    Product Composition:
    Research Phase Product Name Product Specifications store
    Midbrain organoid formation stage Basic medium 1 20ml -20℃ ， 18 Month
    supplements A 1mg -20℃ ， 12 Month
    Basic medium 2 100ml 2-8℃ ， 12 Month
    Midbrain organoid expansion stage Basic medium 3 245ml 2-8℃ ， 18 Month
    supplements B 5ml -20℃ ， 24 Month
    supplements C 500ul -20℃ ， 12 Month
    supplements D 500ul -20℃ ， 12 Month
    Midbrain organoid differentiation stage Basic medium 3 245ml 2-8℃ ， 18 Month
    supplements B 5ml -20℃ ， 24 Month
    supplements E 250ul -20℃ ， 18 Month
    Midbrain organoid maintenance stage Basic medium 3 98ml 2-8℃ ， 18 Month
    supplements B 2ml -20℃ ， 24 Month
    Quantity of specifications: More than [number missing] can be 500 An organoid.
    Storage Temp. Please refer to the product composition for details.