hPSC Induced Differentiation Lung Organoid Kit
hPSC Induced Differentiation Lung Organoid Kit
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1. Experimental instruments and materials: Instruments: biological safety cabinet, cell incubator, horizontal centrifuge, inverted microscope, cryogenic refrigerator Materials: Cell culture plates (sizes: 6-well, 12-well, 24-well), centrifuge tubes (sizes: 15 mL and 50 mL), pipettes (sizes: 10 μL, 100 μL, 1000 μL), sterile tips (sizes: 10 μL, 200 μL, and 1000 μL), pipettes (sizes: 10 mL, 50 mL) 2. Experimental contents and methods: 1. hPSC differentiated into endodermal cells (DE) (calculated as 6-well plate and 1 well) (1) Thaw the matrigel at 4 °C, evenly mix with DMEM/F12, lay the plate, and set aside. (2) When the confluence degree of hiPSC reaches 75%-85%, aspirate the medium and use 2mL 1x room temperature PBS (without Ca2+/Mg2+) The hiPSC was washed and PBS was aspirated. (3) Add 1mL of room temperature Accutase containing Y-27632 (10μM) and transfer to 37 °C 5% CO2In the cell incubator for 20 min to dissociate into single cells. (4) After 20 min, the hESC/iPSC complete medium was directly added to Accutase in an equal volume, and the cells were gently pipped up and down with a P-1000 pipette tip to make a single cell suspension, and the cell single suspension was collected into a 15mL centrifuge tube and centrifuged at 300 g at room temperature for 5 min. (5) The coating liquid was carefully aspirated from the matrigel-coated plate without damaging the matrigel-coated surface. (6) At the end of centrifugation, the supernatant is fully removed, and the cells are resuspended with 1 mL of preheated hESC/iPSC complete medium (containing 10μM Y-27632), and gently pipetted up and down to ensure uniform single cell solution. (7) Cells were counted using an automatic cell counter, dead cells were excluded using trypan blue, and the density was 2.0 * 105Cells/mL of cells were seeded onto the 6-well Matrigel-coated plate prepared in Step 1 with a final volume of 2 mL per well in the 6-well plate, and the plate was transferred to 37 °C, 5% CO2Incubate in the cell incubator for 24 h. (8) After 24 h (day 1) the medium was aspirated and 1.97 mL of basal medium 1 +10 μL of supplement A +20 μL of supplement B was added. (9) On days 2 and 3, the medium was aspirated and 1.98 mL of basal medium 1 +20 μL of supplement B was added to start differentiation into endoderm cells. 2. DE induces differentiation into foregut endoderm cells (AFE) (1) On day 4, the medium was aspirated from the culture, and 2 mL of 1x room temperature PBS (Ca-free) per well in a 6-well plate2+/Mg2+) The culture was washed, then PBS was removed from the wells and 2 mL of basal medium 2 was added per well. (2) Place the culture plate in 37 °C 5% CO2Culture in an incubator, change the medium every 24 hours, and culture continuously for 3 days. 3. AFE induces differentiation into lung progenitor cells (LPC) (taking 4 wells of 12-well plate as an example) (1) 29.92 mL basal medium 3 + 80 μL supplement C to prepare LPC induction medium (2) On day 7, the medium was aspirated from the culture, and 2 mL of 1x room temperature PBS (Ca-free) per well in a 6-well plate2+/Mg2+) Cultures were washed and PBS was aspirated. (3) Add 1mL of room temperature Accutase containing Y-27632 (10μM) per well, and place the culture at 37 °C 5% CO2Incubate in the cell incubator for 10 min. (4) After 10 minutes, add 1mL DMEM/F12 (containing 10μM Y-27632) to each well, and gently blow the cells to separate the cells from the bottom of the well. (5) Collect the cell suspension in a 15 mL centrifuge tube and centrifuge at 300 g for 5 min at room temperature. (6) Carefully aspirate the supernatant without interfering with the cells, resuspend the cells with 1mL of LPC induction medium (containing 10μM Y-27632) returned to room temperature, and gently pipette up and down to ensure uniform single cell solution. (7) Cells were counted using an automated cell counter, dead cells were excluded using trypan blue, and the density was 2.0 x105Cells/mL were seeded onto pre-prepared 12-well Matrigel-coated plates with a final volume of 1 mL of media per well in the 12-well plate. (8) The next day, change to LPC induction medium without Y-27632. The media was changed every other day for 11 days. 4. LPC-induced differentiation into 3D lung organoids (taking 6 wells of 24-well plate as an example) (1) On day 18, 14.965 mL of basal medium 4 +35 μL of supplement D was formulated into 3D organoid induction medium (2) Thaw the Matrigel on ice and place the pipette tip in a-20 ℃ refrigerator in advance. (3) Aspirate the LPC induction medium and use 1 mL 1xPBS (without Ca2+/Mg2+) washed once. (4) Add room temperature Accutase (0.5 mL/12 wells) containing 10 μM Y-27632 and treat it at 37 °C, 5% CO2Incubate in an incubator for 10 min; After 10 minutes, 1mL of DMEM/F12 (containing 10μM Y-27632) returned to room temperature was added to each well, and the cells were gently pipped to separate the cells from the bottom of the well. (5) Collect the cell suspension in a 15 mL centrifuge tube and centrifuge at 300 g for 5 min at room temperature. (6) Carefully aspirate the supernatant without interfering with the cells, resuspend the cells with 1 mL of 3D organoid induction medium, and gently pipette up and down to ensure uniform 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; Within 18 days of differentiation, the cells should not become hyperconfluent. (8) Centrifuge at 300 g for 5 min at room temperature, aspirate the medium and resuspend the cells in cold Matrigel, 4.0 * 10 per well for ESC4200 μL of Matrigel was added to each cell, and for iPSC, 8.0 * 104 cells per well, 200 μL of Matrigel was added and the Matrigel was placed on ice with the cells. (9) 200 μL of Matrigel/cell mixture was seeded into a 24-well plate at 30 μL per well. (10) Place the plate in a 37 °C, 5% CO2 incubator for 20-30 minutes to solidify the matrigel. (11) 700 μL of 3D organoid induction medium was added per well and the medium was changed every other day for 6 days. (12) On day 23, 14.96 mL of basal medium 5 +40 μL of supplement E was formulated into 3D organoid differentiation medium, and the medium in the wells was replaced with 700 μL of 3D organoid differentiation medium returned to room temperature, and the medium was replaced every other day for 6 days. (13) On day 29, 199.48 mL of basal medium 6 +520 μL of supplement F was formulated into 3D organoid maturation medium, and the medium in the wells was replaced with 700 μL of 3D organoid maturation medium returned to room temperature, and the medium was replaced every other day for long-term culture. 5. 3D lung organoid passage (1) Take out the frozen matrigel and thaw it at 4 °C, and place the gun tip at-20 °C for precooling for 1h in advance; (2) Aspirate the culture medium, add 1mL of pre-cooled Tryple (containing 1% BSA), and let stand for 1min; (3) Use a 1mL pipette gun and blow the mixture in the hole 40-50 times. Note: do not produce bubbles; (4) 1 mL of pre-cooled PBS was added to each well and the mixture was transferred to a 15 mL centrifuge tube; (5) Clean the culture plate with 1mL of pre-cooled PBS, add the cleaning solution to the centrifuge tube of the previous step, and supplement the pre-cooled PBS to make the final volume in the centrifuge tube 12mL; Centrifuge at 300g for 5min, remove the supernatant; (6) Use the pre-cooling gun tip to add an appropriate amount of matrigel to the centrifuge tube in an amount of 30μL per well, blow 5-8 times, evenly mix the single cell-matrigel, and pay attention to avoid bubbles during the blowing process; (7) Take out the 24-well plate placed in the incubator in advance, add 30μL gel droplets to the center of the well, and gradually move the gun tip upward when slowly injecting the mixed solution to make the cells evenly distributed in the gel; (8) Place the culture plate in an incubator for 20-30min to solidify the glue droplets; (9) Carefully add 700μL of 3D organoid maturation medium returned to room temperature along the well wall, and change the medium every other day (the medium can be changed on Monday, Wednesday, and Friday, and 800μL can be added on Friday, and the medium can be added on Saturday and Sunday. Do not change the medium), note: do not touch the glue droplets, put the well plate in the incubator, and continue culturing. |
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| Description | This product is a hPSC-induced differentiation lung organoid kit. Human pluripotent stem cell hPSC can be induced and differentiated through this kit to obtain lung organoids. This kind of organ is composed of cells similar to alveoli. The mature organoids include airway cells and alveolar cells. This product requires the operator to have experience in hPSC cell culture and some knowledge of organoids. Product composition: < td style = "width: 13.8429%; text-align: center; height:22px; "> 512 uL
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| Storage Temp. | Stored at-20 ℃, the validity period of the basal medium is 12 months, and the validity period of the supplement is 6 months. |
