Membrane-based Stem Cell Culture

Co-culture in Millicell Cell Culture Inserts

Indirect Co-culture of Embryonic Stem Cells with Embryonic Fibroblasts

Note: This protocol is designed to grow undifferentiated embryonic stem cells in an indirect co-culture with the fibroblast feeder layer. Although it is targeted for use with Millicell-24 and Millicell-96 plates, this protocol can be used with Millicell single-well inserts as well.

Materials and Reagents

• Millicell-24 cell culture insert plates — Millipore cat. nos. PSHT 010 R5, PSRP 010 R5
• Millicell-96 cell culture insert plates — Millipore cat. no. MACA C02 B5
• Primary mouse embryo fibroblasts (DMEF) — Millipore cat. no. PMEF-CFL
• 129/S6 Murine embryonic stem cells (ESC) — Millipore cat. no. SCR012
• ESC Media:
• Knock out DMEM — Millipore cat. no. SLM-220-B
• 20% ES qualified Serum — Millipore cat. no. ES-009-B
• 1% Glutamax-1
• 1% PenStrep — Millipore cat. no. TMS-ABZ-C
• 1% Non Essential Amino Acids — Millipore cat. no. TMS-001-C
• 0.1% ESGRO® (LIF) — Millipore cat. no. ESG1106
• 0.1% 2-mercaptoethanol — Millipore cat. no. ES-007-E
• MEF Media:
• DMEM — Millipore cat. no. SLM-022-B
• 10% Fetal Bovine Serum — Millipore cat. no. ES-009-B
• 1% Glutamax-1
• 1% PenStrep — Millipore cat. no. TMS-ABZ-C
• 1% Non Essential Amino Acids — Millipore cat. no. TMS-001-C
• Gelatin 2% Solution — Millipore cat. no. SF008
• Mitomycin C powder — Sigma cat. no. M4287
• DPBS — Millipore cat. no. BSS-1005-B
• TrypLE™ Select (1X), liquid — Invitrogen cat. no. 12563
• Alkaline phosphatase detection kit — Millipore cat. no. SCR004
• Tissue culture flasks and tubes
• Fibronectin Solution, 1mg/mL — Millipore cat. no. FC010

  Note: All steps should be performed in a laminar flow hood using sterile techniques.

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Protocol

i. Fibronectin Coating and MEF Seeding of Single Well Feeder Trays

1. Coat Single well feeder trays with Fibronectin for 45 minutes at room temperature
2. Stock is 0.1% (1000μg/mL)
3. Working concentration is 25μg/mL in sterile DPBS
4. 5-10mL of Fibronectin solution per single well tray
5. Remove excess Fibronectin, trays are now ready for use
6. Thaw MEF from -80°C freezer
7. Gently shake MEF vial in a 37°C water bath
8. Transfer MEF to 15mL tube containing 10mL pre-warmed MEF media
9. Pellet cells at 4°C, 1000rpm for 4 minutes.
10. Remove supernatant, resuspend cells in fresh pre-warmed MEF media
11. Seed Fibronectin coated single well feeder trays with MEF feeder cell suspension
12. 1.7 x 106 MEF cells per single well tray (approximately 2 x 104 cells per cm2) will result in 95% confluence within 24 hours
13. Cover with lid and incubate single well trays at 37°C overnight

ii. ESC/MEF Indirect Co-culture on Membrane-based Cell Culture Plate

1. Harvest ES cells from MEF-ESC direct co-cultures [previously cultured as described (Robertson 1987)]
2. Wash cells 2X with pre-warmed DPBS 10mL per T75 flask (let sit for 1-2 minutes per wash)
3. Add 0.25% trypsin, 3mL per T75 flask, should take about 3 minutes, observe under microscope when cells are balled up
4. Add ESC media to inactivate trypsin
5. Mix well and wash flask wall to remove all cells
6. Enrichment of ESCs from MEF feeder layer cells by selective adhesion
7. Add cell suspension to new TC flask
8. Incubate at 37°C for 30-45 minutes
9. Remove cell suspension and repeat steps a. and b. Three incubations will typically remove most MEFs leaving a majority of ES cells in the cell suspension
10. Seed cell culture filter plate (or insert) wells with ESC suspension
11. Remove MEF media from single well feeder trays (or companion wells) and replace with ESC media
12. Add ESC seeded cell culture filter plates to single well trays (or companion wells)
13. Incubate assembly at 37°C until appropriate confluence level for passage

iii. Passage of ESCs from Membrane-based Cell Culture Plate

1. Wash cells 2X with pre-warmed DPBS (let sit for 1-2 minutes per wash)
2. Add 0.25% trypsin, 100μL per 12mm insert, 500μL per 30mm insert should take about 3 minutes, observe under microscope when cells are balled up
3. Add ESC media to each well to inactivate trypsin
4. Gentle scraping may be necessary to detach cells from the membrane surface. Using a P200 pipette scrape colonies off of insert with pipette tip taking care not to puncture the membrane
5. Mix well to remove all cells
6. Seed membrane cell wells with ESC suspension
7. Incubate at 37°C overnight
8. Repeat as desired when colony morphology dictates need to passage

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