Isolation of primary hGSCs from human stomach sample
1. Prepare 10 mL of hGSC isolation medium per stomach tissue sample (~2 cm3).
2. Warm the isolation medium at 37℃ in water bath.
3. (CRITICAL) Cut tissue into tiny pieces that can go through 10-mL pipet.
4. Coat inner surface of a 10-mL pipet and a P1000 tip with BSA (1% in PBS).
5. Wash tissue with 10 mL of cold DPBS with pipetting using the coated pipet.
6. Let the tissue sink to the bottom.
7. Discard supernatant.
8. Repeat step 5-7 until supernatant looks clean.
9. Spin down at 100 g for 3 min.
10. Discard supernatant.
11. Resuspend pellet in the warmed isolation medium using the coated pipet.
12. (CRITICAL) Incubate tissue at 37℃ in the water bath for 15-30 min with pipetting rigorously using the coated 10 mL pipet (when the size of tissue allows, use the coated P1000 tip instead) until clusters of crypt cells released. NOTE: duration of this step should be adjusted case by case.
13. (Optional) Let stand for 1 min and collect the tissue that sink to the bottom and repeat step 11-12 in a separate tube.
14. Neutralize cells with F12K supplemented with 10% FBS.
15. Spin down at 500 g for 5 min.
16. Discard supernatant.
17. Resuspend pellet in hGSC medium and seed cells on 1 well of 6-well plate that coated with confluent inactivated MEF feeder cells.
18. Maintain cells in hGSC medium at 37 °C in a 5-7.5% CO2 humidified incubator.
19. Change medium every other day.
Expansion and cryopreservation of hGSCs
Note: hGSC colonies should be passaged when the colonies begin to make contact to each other (~70% confluency).
1. Wash cells twice with DPBS.
2. Incubate cells in TrypLE for 10-12 min.
3. Detach cells by pipetting.
4. Transfer cells into a centrifuge tube that contains DMEM complete medium.
5. Centrifuge cells at 300 x g for 5 min.
6. Resuspend pellet in hGSC medium and then seed cells on an inactivated-MEF-coated dish. Note: Split hGSCs every 4-6 days at a ratio between 1:3 and 1:5.
7. Maintain hGSCs in hGSC medium at 37 °C in a 5-7.5% CO2 humidified incubator.
8. Change medium every other day.
9. For hGSC cryopreservation, pellet from step 6 should be resuspended in freezing solution (10% DMSO in FBS) and frozen using standard mammalian cell cryopreservation protocol.
Generation of Ngn3ER-hGSC line
Note: Ngn3ER-hGSCs were labeled with mCherry constitutively by incorporation of a polycistronic cassette EF1a-Ngn3ER-PuroR-mCherry (PuroR, puromycin resistant gene).
1. Seed 105 hGSCs in 1 well of 6-well plate coated with confluent inactivated MEF 24 hours prior to lentiviral transduction.
2. Wash cells with DPBS once.
3. Overlay cells with 2 mL of hGSC medium containing 10 μg/mL polybrene and ~106 TU of Lenti-EF1a-Ngn3ER-PuroR-mCherry. Note: Lentivirus can be prepared in bulk and titrated in 293FT by mCherry+ cells quantification.
4. Spin the cell culture with lentivirus in plate at 1000 g for 30 min at 37°C.
5. Culture the infected hGSCs at 37°C in a 5-7.5% CO2 incubator for 48 hours.
6. Change culture medium to hGSC medium containing 1 μg/mL puromycin bidaily for 2 weeks.
7. The line can be expanded or cryopreserved.
Generation of GINS organoids
1. Ngn3ER activation (Differentiation to endocrine progenitors, day 0-2)
(1) Seed Ngn3ER-hGSCs 4-5 days prior to differentiation.
(2) Wash cells once with DPBS.
(3) Overlay cells with hGSC medium containing 1 µM 4-OH-TAM.
2. Pdx1-Mafa transduction (Differentiation to GINS precursors, day 2-6)
Note: coat the inner surface of pipet tips and centrifuge tubes with 1% BSA before experiment.
(1) Coat dishes in DMEM containing Fibronectin (1:50) and Matrigel (1:50) for 2 hours or overnight.
(2) Gently wash cells with DPBS twice.
(3) Incubate cells in DPBS for 10 min.*
(4) Detach cells by pipetting vigorously using BSA-coated pipet tips. *
(5) Transfer the cells in a BSA-coated centrifuge tube. *
(6) Centrifuge at 100 g for 3 min.*
(7) Remove supernatant carefully. * Note: cell debris should be removed if they retain in supernatant.
(8) Dissociate cells in TrypLE at 37°C for 10-15 min with pipetting every 3-5 min.
(9) (CRITICAL) Neutralize cells in complete DMEM when most of the cells are single cells, doublets or small clusters that contain 3-5 cells.
(10) Spin cells at 100 g for 5 min.
(11) Resuspend cells in medium composed of 50% of hGSC medium, 50% of GINS medium, and 10 μg/mL polybrene.
(12) Infect cells with Lenti-CMV-Pdx1-2A-Mafa at a multiplicity of infection (MOI) of 10. Note: Lentivirus should be prepared in bulk and titrated in 293FT by immunostaining for Mafa and quantification.
(13) Spin the cell culture with lentivirus in plate at 1000 x g for 30 min at 37°C.
(14) Transfer infected cells to Fibronectin/Matrigel coated dishes (~107 cells per 10-cm dish).
(15) Two days post infection, change medium to 75% GINS medium and 25% hGSC medium.
*Note: for extremely large-scale production, consider skipping step 3-7, which was designed to remove sticky cell debris and undifferentiated hGSCs.
3. GINS organoid formation (day 6-21)
(1) Dissociate cells in TrypLE for 5 min.
(2) Neutralize cells in DMEM complete medium.
(3) Spin cells at 300 g for 5 min.
(4) Resuspend pellet in GINS medium and count cells.
(5) Aggregate cells in AggreWell400 (typically 2.0-2.4 million cells/well) using the manufacturer’s recommended protocol. Note: Aggregates normally form within 24 hours.
(6) Change medium every 2-3 days.
(7) Maintain organoids in GINS medium at 37 °C in a 5-7.5% CO2 humidified incubator.