Co-Culture in Millicell Cell Culture Inserts

Millicell Cell Culture Inserts

ESC colonies grown on Millicell-96 cell culture insert 1.0 PET membrane, stained for alkaline phosphatase activity, after culturing via indirect co-culture with ESC in apical well, at a 200 cell per well seeding density, and MEF in the single-well feeder tray.

• Method 1 is a basic protocol for seeding cells on both sides of the membrane.
• Method 2 is a detailed protocol for seeding embryonic stem cells on the apical side of the membrane and embryonic fibroblasts on the plastic feeder plate.

Method 1

Note: Depending on the cell lines and the nature of the co-culture, the researcher can decide which side of the membrane is seeded first. To maintain sterile incubations of cells seeded on the underside of the membranes, use sterile Petri dishes for the Millicell single-well inserts and sterile feeder trays for Millicell 24- and 96-well cell culture insert plates.

1. Using an optimized seeding density, seed the first cell type in either the apical wells or on the basolateral underside of the membranes. Refer to the recommended working volumes chart (see page 42) for appropriate apical volumes. For basolateral seeding volumes, use approximately 200 ìL for Millicell 12 mm single-well inserts and Millicell-24 insert plates, and approximately 30 µL for Millicell-96 insert plates.
2. Incubate in a 37°C CO2 incubator for 1 to 4 hours to allow the cells to attach to the membrane.
3. Gently turn the Millicell device over and seed the second cell type in either the apical well or on the basolateral underside of the membrane.
4. Incubate in a 37°C CO2 in the incubator for 1 to 4 hours to allow the second cell type to attach.
5. Add appropriate volume of media to the apical or basolateral chambers and return to incubator.

Method 2

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.

• 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

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