Conditioned medium (CM) is a nutrient-rich solution that contains various enzymes, growth factors, cytokines, hormones, and other soluble mediators that are secreted from cell lines.¹ CM plays a crucial role in supporting cell growth and viability by supplying the necessary factors for epithelial cell growth without the direct presence of the feeder cell type, particularly useful in cell culture applications or downstream analytics of the target cell population.

This protocol describes the process of generating CM for in vitro expansion of primary non-keratinocyte epithelial cells derived from either normal or diseased tissues. CM is generated from 3T3-J2 Irradiated Feeder Cells (3T3-J2 Cells) cultured in Conditional Reprogramming Medium (CRM). Over the course of 96 hours of incubation, the 3T3-J2 Cells will release factors into the medium that, when collected, may be used to expand epithelial cells without the need of a 3T3-J2 feeder layer. This protocol provides instructions for generating CM over 96 hours in T-225 cm² flasks; however, the protocol can be scaled up or down to generate the desired volume of CM.

Materials

• 3T3-J2 Irradiated Feeder Cells (Catalog #100-0353), 3 cryovials
• Conditional Reprogramming (CR) Medium (Catalog #100-0352)
• Cholera toxin (Sigma Catalog #C8052) reconstituted to 1 mg/mL in CR Medium
• T-225 cm² Tissue Culture-Treated, Vented Flasks (Corning Catalog #431082)
• 15 mL Conical Tubes (Catalog #100-0092)
• 50 mL Conical Tubes (Catalog #100-0090)
• 250 mL collection bottle (e.g. Catalog #38084)
• 0.4% Trypan Blue (Catalog #07050)

Part I: Preparation of 3T3-J2 Irradiated Feeder Cells for Culture in CR Medium

1. Remove 3 cryovials of 3T3-J2 Cells from liquid nitrogen storage.
2. Wipe the outside of the vials with 70% ethanol or isopropanol.
3. In a biosafety hood, twist the cap a quarter-turn to relieve internal pressure and then re-tighten.
4. Quickly thaw cells in a 37°C water bath by gently shaking the vials. Remove the vials when a small frozen cell pellet remains. Do not vortex cells.
   NOTE: Working quickly in the following steps is important to ensure high cell viability and recovery.
5. Wipe the outside of the vials with 70% ethanol or isopropanol.
6. Transfer the cell suspension to a 15 mL conical tube.
7. Rinse each vial with 1 mL of medium at a time, and add it dropwise to the cells while gently swirling the 15 mL tube for a total of 3 mL.
8. Wash by adding 10 mL of CR Medium dropwise, while gently swirling the tube.
9. Centrifuge the cell suspension at 300 x g for 10 minutes at room temperature (15 - 25°C).
10. After centrifugation, remove and discard the supernatant without disturbing the cell pellet. Add 3 mL of CR Medium to resuspend the cell pellet.
11. Remove a 20 μL aliquot of cells for counting. If using Trypan Blue to assess viability, add 20 μL of Trypan Blue and count the number of viable cells under a hemocytometer. See this protocol on How to Count Cells with a Hemocytometer.
12. Cells are now ready for use in generating CM.

Part II: Generating Conditioned Medium

1. Add 6.75 x 10⁶ viable 3T3-J2 Cells to 45 mL of cholera toxin-free CR Medium, and plate the mixture into a T-225 flask.
   NOTE: This number of 3T3-J2 Cells will result in a seeding density of 3 x 10⁴ viable cells/cm².
2. Incubate the vessel at 37 °C in a 5% CO2 humidified incubator.
3. After 48 hours of incubation (Day 2 following cell seeding), collect 45 mL of CM from the vessel and transfer it to a 50 mL centrifuge tube.
   1. Add 45 mL of fresh cholera toxin-free CR medium to the T-225 flask. Return the culture vessel to the humidified incubator.
   2. Centrifuge 45 mL of the collected Day 2 CM at 300 x g for 10 minutes at room temperature (15 - 25°C). After centrifugation, collect the supernatant while being careful not to disturb the cell pellet, and transfer it to a 250 mL bottle. Store at 4°C protected from light. Discard the cell pellet.
4. After an additional 24 hours of incubation (Day 3 following cell seeding), collect 45 mL of CM from the vessel and transfer it to a 50 mL centrifuge tube.
   1. Add 45 mL of fresh cholera toxin-free CR medium to the T-225 flask. Return the culture vessel to the humidified incubator.
   2. Centrifuge 45 mL of the collected Day 3 CM at 300 x g for 10 minutes at room temperature (15 - 25°C). After centrifugation, collect the supernatant while being careful not to disturb the cell pellet, and transfer it to the same 250 mL bottle containing CM from Day 2. Store at 4°C protected from light. Discard the cell pellet.
5. After an additional 24 hours of incubation (Day 4 following cell seeding), collect 45 mL of CM from the vessel and transfer it to a 50 mL centrifuge tube.
   1. Centrifuge 45 mL of the collected Day 4 CM at 300 x g for 10 minutes at room temperature (15 - 25°C). After centrifugation, collect the supernatant while being careful not to disturb the cell pellet, and transfer it to the same 250 mL bottle containing the CM from Days 2 and 3. Store at 4°C protected from light. Discard the cell pellet.
   2. The T-225 flask containing the feeder layer can now be discarded.
6. If using Conditioned Medium right away:
   1. Supplement the final volume of CM (approximately 135 mL final volume) with cholera toxin to a final concentration of 8.6 - 20 ng/mL.
      NOTE: To obtain a final concentration of 8.6 ng/mL in 135 mL of CM, add 1.2 µL of 1 mg/mL cholera toxin in CR media.
   2. Seed epithelial cells as desired with CM, replacing with fresh CM with cholera toxin every 2 - 3 days.
   3. Use CM with cholera toxin within 2 weeks, keeping media stored at 4°C, and protected from light when not in use.
7. If not using CM right away, freeze aliquots without Cholera toxin:
   1. Aliquot CM as desired into appropriate vessels for storage at - 20°C.
   2. Store for up to 6 months at - 20°C.
   3. Thaw overnight at 4°C prior to use.
   4. Supplement the thawed CM with cholera toxin to obtain a final concentration of 8.6 - 20 ng/mL prior to culture use.
      NOTE: The use of CM in place of co-culture directly with 3T3-J2 Cells has been demonstrated to be sufficient to promote robust epithelial cell growth. However, the level of epithelial cell expansion observed in CM may not be equivalent to the level of epithelial cell expansion in CR Medium in the presence of 3T3-J2 Cells.
      NOTE: The generation of CM using this protocol does not include supplementation of the CR Medium with cholera toxin during the conditioning step. Instead, cholera toxin is added to the CM immediately prior to epithelial cell culture. Alternatively, including cholera toxin in the CR Medium during the conditioning step can be tested, and it is recommended that researchers evaluate each protocol to identify which one is optimal for their research purpose.