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    hPSC Induced Differentiation Lung Organoid Kit

    hPSC Induced Differentiation Lung Organoid Kit

    Catalog Number: abs90128 Brand: Absin
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    $7,508 USD
    $7,508 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
    Materials: Cell culture plates (specifications are: 6 hole, 12 hole, 24 Hole), centrifuge tube (specifications are 15 mL and 50 mL ), pipette (specifications are 10μL 、 100 μL 、 1000 μL ), sterile tip (specifications are 10 μL 、 200 μL and 1000 μL ), pipette (specifications are 10mL 、 50mL ）

    II. Experimental content and methods:
    1 、 hPSC Differentiation into endoderm cells ( DE ） (Press 6 orifice plate 1 (Kong Suan)
    （ 1 Matrix glue in 4℃ Thawing below, with DMEM/F12 Mix evenly, lay on a board, and set aside.
    （ 2 Remove the basal culture medium one day in advance. 1 , supplements A , supplements B In 4℃ Thaw the basic culture medium. 1 With supplements B After thoroughly mixing, prepare a base culture 1-B Culture medium, remove 12ml Jipei 1-B Media and Supplements A Mix thoroughly.
    （ 3 ) when hiPSC The convergence degree reaches 75%-85% Aspirate off the culture medium, use 1mL 1x room temperature PBS (Excluding Ca2+/Mg2+) cleaning hiPSC , suck away PBS 。
    （ 4 ) Join 1mL room temperature Accutase , transfer to 37℃ 5% CO2 In the cell culture box 10-20min To dissociate them into single cells.
    （ 5 ) will hESC/iPSC Complete medium was added directly in equal volume Accutase In, and use P-1000 The tip gently blows the cells up and down to create a single-cell suspension, which is then collected into... 15mL In a centrifuge tube, at room temperature 300g centrifuge 5min 。
    （ 6 Carefully draw the coating liquid from the matrix-coated plate without damaging the matrix-coated surface.
    （ 7 After centrifugation is complete, the supernatant is fully removed. 1mL preheated hESC/iPSC Complete culture medium (including 10μMY-27632 Resuspend the cells and gently pipette up and down to ensure uniformity of the single-cell solution.
    （ 8 Cells were counted using an automated cell counter, trypan blue was used to exclude dead cells, and the density was... 2.0*105 microscopic cell /mL The cells were seeded to the step 1 The prepared matrix glue coated 6 On the hole plate, in 6 Final volume per well in well plate 2mL Transfer the well plate to 37℃ 、 5%CO2 Incubation in cell culture box 24h 。
    （ 9 ） 24h After (the 1 (day) Aspirate off the culture medium and add to each well. 2mL Jipei 1-B Media and Supplements A Configured medium.
    （ 10 ) in the 2 Heaven and First 3 One day, aspirate the culture medium and add it to each well. 2mL Jipei 1- supplements B , begins to differentiate into endoderm cells.
    2 、 DE Induced differentiation into foregut endoderm cells (AFE)
    （ 1 Remove the basal culture medium one day in advance. 2 , supplements C In 4℃ Thaw the basic culture medium. 2 With supplements C Mix thoroughly before preparation AFE Culture medium for backup.
    （ 2 )th 4 Days later, the medium was aspirated from the culture and added to each well. 2mLAFE culture medium.
    （ 3 Place the culture plate in 37℃ 5% CO2 Cultivated in an incubator, each 24h Change the medium once and culture continuously 3 heaven.
    3 、 AFE Induced differentiation into lung progenitor cells (LPC) (with 12 orifice plate 4 Take a single hole as an example ）
    （ 1 Remove the basal culture medium one day in advance. 3 , supplements D , supplements E In 4℃ Thaw the basic culture medium. 3 , supplements D With supplements E Mix thoroughly before preparation LPC Culture medium for backup.
    （ 2 )th 7 Days later, the culture medium was aspirated from the culture. 6 Each hole in the hole plate is used 1mL 1x room temperature PBS (Excluding Ca2+/Mg2+) Wash the culture and aspirate. PBS 。
    （ 3 Add to each well 1mL room temperature Accutase Place the culture in 37℃ 5% CO2 Incubation in cell culture box 10min 。
    （ 4 ） 10min Then add to each well 1mL DMEM/F12 Gently blow the cells to remove them from the bottom of the well.
    （ 5 Collect the cell suspension into 15mL In a centrifuge tube, at room temperature 300g centrifuge 5min 。
    （ 6 Carefully aspirate the supernatant without disturbing the cells. 1mL Restore to room temperature LPC Induction medium (containing 10μMY-27632 Resuspend the cells and gently pipette up and down to ensure uniformity of the single-cell solution.
    （ 7 Cells were counted using an automated cell counter, trypan blue was used to exclude dead cells, and the density was... 2.0x105 microscopic cell /mL The cells were inoculated into pre-prepared 12 On the plate coated with porous matrix adhesive, 12 The final volume of medium per well in the well plate is 1mL 。
    （ 8 The next day, it was changed to excluding... Y-27632 of LPC Induction medium. Change the culture medium every other day and continue 11 heaven.
    4 、 LPC induced differentiation into 3D Lung organoids (with 24 orifice plate 6 (Taking a single hole as an example)
    （ 1 )th 18 Day, basic culture medium 4 With supplements F Prepared into 3D organoid induction medium
    （ 2 Thaw the matrix gel on ice and place the pipette tip in advance. -20℃ In the refrigerator.
    （ 3 ) Suction LPC Induction medium, using 1mL 1x room temperature PBS (Excluding Ca2+/Mg2+) washing 1 Next.
    （ 4 Add to room temperature Accutase （ 0.5mL/12 (hole) and in 37℃ 、 5%CO2 Incubation in incubator 10min ； 10min Then add to each well 1mL Restore to room temperature DMEM/F12 The cells are blown away from the bottom of the well.
    （ 5 Collect the cell suspension into 15mL In a centrifuge tube, at room temperature 300g centrifuge 5min 。
    （ 6 Carefully aspirate the supernatant without disturbing the cells. 1mL3D The organoid induction medium resuspended the cells, gently pipetting up and down to ensure homogeneity of the single cell solution.
    （ 7 Cells were counted using an automated cell counter and dead cells were excluded using trypan blue. Note: Each cell line must optimize the number of cells; In differentiation 18 During the day, the cells should not become hyperfused.
    （ 8 At room temperature 300g centrifuge 5min The culture medium is aspirated and the cells are resuspended in cold matrix gel, for ESC , each hole 4.0*104 Microcell addition 200μL Matrix glue, for iPSC , each hole 8.0*104 Microcells, add 200μL Matrigel, which is placed on ice with the cells.
    （ 9 ) will 200μL matrix rubber / The cell mixture was inoculated transwell middle.
    （ 10 Place the plate in 37℃ 、 5% CO2 Incubator 20-30 The matrix gum was allowed to set for minutes.
    （ 11 Add to each well 1mL 3D Organoid induction medium (including 10μMY-27632 The next day, it was changed to "excluding". Y-27632 of 3D Organoid induction medium, changed every other day, continuously 6 heaven.
    （ 12 ) in the 23 Day, basic culture medium 5+ supplements G+ supplements H Prepared into 3D Organoid differentiation medium, replace the medium in the wells with 1mL Restore to room temperature 3D Organoid differentiation medium, change medium every other day, continue 6 heaven.
    （ 13 ) in the 29 Day, basic culture medium 6+ supplements I+ supplements J Prepared into 3D Organoid maturation medium, replace the medium in the wells with 1mL Restore to room temperature 3D The organoid maturation medium was changed every other day for long-term culture.
    （ 14 Testing and identification: For analysis DE The efficiency, in the first 3 celestial markers FOXA2 、 SOX17 Proceed qPCR identification; For analysis AFE The efficiency, the first 6 Heaven SOX2 and FOXA2 Proceed qPCR identification; For analysis LPC Efficiency, in
    No 15-17 Heaven NKX2-1 Proceed qPCR identification; Organoids can be fixed for staining identification after maturation: respiratory markers include Krt5 。 Alveolar markers include SPC 、 SPB , and AGER 。
    DE Markers: SOX17 ， FOXA2
    AFE Markers: FOXA2 ， SOX2
    LPC Markers: NKX2.1
    Description This product is hPSC Lung organoid kit for inducing differentiation, human pluripotent stem cells hPSC Pulmonary organoids can be obtained by inducing differentiation through the kit, the organoids are composed of alveolar-like cells, and the mature organoids are formed by airway cells and alveolar cells.
    This product requires operators to have hPSC Experience in cell culture and a certain understanding of organoids.
    Product Composition:
    Cultivation stage serial number Name Specification Preserve
    definitive endoderm
    (DE) Stage     		L01 Basic medium 1 48.5mL -20℃ ， 12 Month
    L01-A supplements A 50uL -20℃ ， 6 Month
    L01-B supplements B 1.5mL -20℃ ， 6 Month
    foregut endoderm
    (AFE) Stage     		L02 Basic medium 2 50mL -20℃ ， 12 Month
    L02-C supplements C 200uL -20℃ ， 6 Month
    lung progenitor cells
    (LPC) Stage     		L03 Basic medium 3 100mL -20℃ ， 12 Month
    L03-D supplements D 50uL -20℃ ， 6 Month
    L03-E supplements E 50μL -20℃ ， 6 Month
    lung (3D) Organoid formation stage L04 Basic medium 4 50mL -20℃ ， 12 Month
    L04-F supplements F 50uL -20℃ ， 6 Month
    lung (3D) Organoid differentiation stage L05 Basic medium 5 50mL -20℃ ， 12 Month
    L05-G supplements G 50uL -20℃ ， 6 Month
    L05-H supplements H 50uL -20℃ ， 6 Month
    lung (3D) Organoid maturation stage L06 Basic medium 6 200mL -20℃ ， 12 Month
    L06-I supplements I 100uL -20℃ ， 6 Month
    L06-J supplements J 5mL -20℃ ， 6 Month
        Matrigel 5mL -20℃ ， 6 Month
    Storage Temp. See product composition for details.