hPSC-Derived Liver Organoid Differentiation Kit
hPSC-Derived Liver Organoid Differentiation Kit
Price:
Regular price
$6,905 USD
Regular price
Sale price
$6,905 USD
Unit price
per
For shipping services or bulk orders, you may request a quotation.
Secure checkout with
View full details
Product Details
Product Details
Product Specification
| Usage |
1. Experimental instruments and materials: Instruments: biological safety cabinet, cell incubator, horizontal centrifuge, inverted microscope, cryogenic refrigerator Materials: Centrifuge tubes (sizes: 15 mL and 50 mL), pipettes (sizes: 10 μL, 100 μL, 1000 μL, multichannel 100 μL), sterile tips (sizes: 10 μL, 200 μL, and 1000 μL), wide-bore pipette tips (sizes: 10 μL, 100 μL), pipettes (sizes: 10 mL, 50 mL), 6-well plates (TC-treated, non-low-adherent), 6-well ultra-low-adherent plates, ultra-low-adherent 96-well U-bottom plates 2. Experimental contents and methods: This kit can generate more than 196 whole-brain organoids. It is recommended to start differentiation with half a 6-well plate to form at least 98 uniform embryoid bodies (EBs) for the whole-brain induction workflow. Preparation of hESC/iPSC cells Culture hPSC on TC-treated non-low-adherent 6-well plates with hESC/iPSC culture kit until cell confluence reaches 70%-80%. 1. Embryoid Body (EB) Formation (Day 0–Day 2) Preparation of EB Formation Medium: Bring all reagents to room temperature (15–25°C) before use. Mix 12 mL Basal Medium 1 + 24 μL Supplement A (500X) to prepare EB formation medium for 3 wells, prepare freshly before use. Thaw Supplement A at room temperature and mix thoroughly. If not used immediately, it can be stored at 2–8°C for up to 2 weeks, do not exceed the shelf life. Do not repeatedly freeze-thaw Supplement A. (1) Bring culture plates, medium and all reagents to room temperature (15–25°C) before operation. (2) Observe good cell morphology under microscope when hPSC reaches 70%-80% confluence. (3) For 3 wells of 6-well plate with 80% confluent hPSC, prepare EB formation medium (12 mL for 3 wells), 3 mL Accutase, 50 mL Ca2+/Mg2+-free sterile DPBS, and 10 mL room-temperature DMEM/F12. (4) Wash 3 wells once with room-temperature Ca2+/Mg2+-free DPBS, aspirate DPBS completely, add 1 mL Accutase per well, incubate at 37 °C for 3 min. (5) When colonies turn white and bright, tap the plate gently on each side twice to detach cells. Transfer to biosafety cabinet, add 1 mL DMEM/F12 to terminate digestion, pipette up and down gently to dissociate cells into single-cell suspension. Perform cell counting with trypan blue, proceed to next step only when cell viability exceeds 90%. (6) Collect 6 mL cell suspension and centrifuge at 160 g for 5 min, discard supernatant and resuspend cell pellet with 12 mL EB formation medium. Re-count cells; optimal cell density is 50,000–80,000 cells per mL. (7) Prepare ultra-low-adherent 96-well U-bottom plate, transfer 12 mL cell suspension into reagent reservoir, dispense 100 μL cell suspension per well. Seal plate with parafilm after seeding, centrifuge on horizontal plate centrifuge at 1200 rpm for 3 min. (8) Remove parafilm and incubate overnight in cell incubator; add another 100 μL Basal Medium 1 per well on Day 1. (9) After 48 h, suspended cells aggregate into EBs with smooth and round edges, ready for subsequent induction. Note: Discard abnormal PSC colonies with spontaneous differentiation or floating cells; recover and amplify a new batch of cells instead of direct induction. 2. Neuroectoderm Differentiation (Day 2–Day 6) Bring all consumables and medium to room temperature (15–25°C) before use. (1) Transfer mature EBs to ultra-low-adherent 6-well plates with 10 EBs per well, wash once with DPBS to remove residual medium, aspirate DPBS completely, add 2 mL Basal Medium 2 per well. Change medium every 2 days for continuous 4 days induction. (2) Qualified EBs show transparent edges with expanded neuroectoderm, diameter >400 μm, dense interior without cavities, morphology similar to primary mouse neurospheres. 3. Neural Budding (Day 6–Day 25) Bring all consumables and medium to room temperature (15–25°C) before use. (1) Perform matrix embedding when EBs reach 400–600 μm in diameter; thaw aliquoted low-growth-factor Matrigel (2 mL) at 4 °C. (2) Prepare a 10 cm × 10 cm parafilm sheet, press concave dots on 200 μL tip box, place on 10 cm culture dish, soak with alcohol for 30 s, then UV sterilize in biosafety cabinet for over 30 min before use. (3) Use wide-bore 10–100 μL pipette tip to transfer EBs onto concave dots. Carefully aspirate residual medium inside dots, embed each EB with 30 μL low-growth-factor Matrigel. If EB adheres to gel edge, gently adjust surrounding gel to center the EB, avoid bubbles. (4) Cover plate tightly, incubate at 37 °C for 15 min to solidify Matrigel. Use sterile tweezers to lift parafilm, flush gel droplets with 1 mL Basal Medium 3 into ultra-low-adherent 6-well plate. Place maximum 10 organoids per well, medium volume per well ≤3 mL. (5) Alternative embedding method: Pre-cool 12 mL Basal Medium 3 for over 30 min, supplement with 10% low-growth-factor Matrigel, vortex thoroughly. Aspirate Basal Medium 2, add 3 mL mixture per well. After 48 h induction, aspirate medium, wash twice with DPBS, add 3 mL Matrigel-free Basal Medium 3. (6) Place plate on horizontal shaker at 80 rpm, incubate at 37 °C for 18 days, refresh medium every 2 days. Early VZ/SVZ-like structures appear on Day 4; budding points gradually converge and darken after Day 12. 4. Neural and Brain Maturation (Day 25+) Bring all consumables and medium to room temperature (15–25°C) before use. (1) When EBs show dense core, loose outer epithelial structure and diameter >1 mm, completely discard Basal Medium 3, add 3 mL Basal Medium 4 per well and culture continuously until Day 38, change medium every 2 days at 80 rpm. When EB diameter exceeds 2 mm, gently remove surrounding Matrigel with 1 mL syringe, add 3 mL Basal Medium 5 per well with daily medium change, adjust shaker speed to 120 rpm to enhance nutrient circulation for long-term culture over 100 days. |
|||||||||||||||||||||||||||||||||||||||
| Description | This product is a kit for inducing differentiation of whole-brain organoids by hPSC. Human pluripotent stem cells hPSC can be induced and differentiated through this kit to obtain whole-brain organoids. The kit realizes classic cerebral organoid differentiation workflow including four stages: neuroectoderm differentiation, neuroepithelial budding and neural tube formation, neural progenitor expansion and brain maturation. EBs need to be embedded in low-growth-factor Matrigel during induction. Whole-brain organoids cultured over 38 days express basic neural markers such as TUJ1, SOX2, Nestin, NeuN, as well as forebrain and cortical markers FOXG1, CTIP2. After long-term culture ≥80 days, small amounts of functional neuron and glial markers including GFAP and TH can be detected. This kit requires the operator to have experience in hPSC culture and some knowledge of organoids. Product composition:
Optional supporting reagents and consumables: hESC/iPSC Culture Kit (Absin, abs90487); Stem Cell Matrigel (Absin, abs9496); DMEM/F-12 (Absin, abs9560); Dulbecco's Phosphate Buffered Saline (DPBS, without Ca2+/Mg2+ and phenol red, Absin, abs970); 6-well plates (TC-treated/non-low-adherent, Absin, abs7033); 6-well ultra-low-adherent plates (Absin, abs7056); Ultra-low-adherent 96-well U-bottom plates (Absin, abs7060); Accutase (Absin, abs47014935); Wide-bore pipette tips (10 μL/100 μL, Absin, abs7075/abs7079); Trypan Blue (Absin, abs50036) |
|||||||||||||||||||||||||||||||||||||||
| Storage Temp. | Stored at -20 ℃. Basal Medium 1 is valid for 6 months; Basal Medium 2, 3, 4, 5 are valid for 24 months; Supplement A and low-growth-factor Matrigel are valid for 6 months. Shipped with dry ice. |
