Co-Culture in Millicell Cell Culture Inserts

Millicell Cell Culture Inserts

Co-culturing procedures involve growing two or more cell types in culture simultaneously. Cell types can be co-cultured in direct contact with each other on the membrane or indirectly where one cell line is cultured on the apical side of the membrane and the other is cultured on either the underside of the membrane (basolateral) or on the surface of the plastic feeder or receiver trays.

Two indirect co-culturing methods are described below:

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.

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

Method 1

Basic Indirect Co-culture on Both Sides of Millicell Cell Culture Insert Membranes

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.

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.
Incubate in a 37°C CO2 incubator for 1 to 4 hours to allow the cells to attach to the membrane.
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.
Incubate in a 37°C CO2 in the incubator for 1 to 4 hours to allow the second cell type to attach.
Add appropriate volume of media to the apical or basolateral chambers and return to incubator.

Method 2

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

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