Proximal Culture System: An In Vitro Culture Technique to Study Paracrine Signaling Between Cells

In paracrine signaling, cells produce and secrete signaling molecules that induce responses in neighboring cells. To mimic paracrine signaling, begin by preparing single-cell suspensions of ovarian cancer cells and mesothelial cells in their appropriate medium.

Next, take an inverted porous membranous insert in a sterile culture dish. Pipet the cancer cell suspension onto the bottom surface of the insert membrane to form a liquid dome. Incubate to allow the cells to settle down and attach to the membrane.

Invert the insert to drain the media. Transfer the insert into a fresh culture dish containing growth media to nourish the cancer cells adhered to the bottom surface of the membrane. Now, add the mesothelial cell suspension into the insert well. Incubate to allow the cells to adhere to the opposite side of the membrane, denying any direct contact between the two cell types.

During culture, both cell types secrete signaling factors. These molecules diffuse through the porous membrane towards the neighboring cells. They bind to specific cell receptors and induce complex signaling cascades in both cell types.

Begin by detaching the ovarian cancer cells from an 80% confluent mixture with 1 milliliter of 0.25% trypsin for 40 to 60 seconds, followed by neutralization of the reaction with 6 milliliters of complete growth medium. Collect the cells by centrifugation and resuspend the pellet in 5 milliliters of fresh complete growth medium.

After counting, dilute the cells to a 100,000 ovarian cancer cells per milliliter of complete growth medium concentration, and place three inverted 0.4-micrometer pore tissue-culture-treated polycarbonate membrane cell culture inserts per experimental condition in an appropriately sized sterile tissue culture dish. Label the inserts according to the experimental condition, and carefully pipet the cancer cells onto the bottom of each insert in concentric circles, starting from the middle of the membrane and moving outward, to form an 800 microliter dome.

Be extremely careful when seeding the cells onto the bottom surface of the insert so that the dome of the medium-containing cells does not run off the edges of the insert during the incubation.

When all of the inserts have been seeded, cover the dish and carefully place the inserts into the cell culture incubator. After about 4 hours, detach the HPMC with 2 milliliters of 0.25% trypsin for one to two minutes, followed by neutralization of the reaction with 12 milliliters of complete growth medium.

Collect the HPMC by centrifugation and resuspend the pellet in 10 milliliters of complete growth medium for counting. Dilute the cells to a 300,000 HPMC per milliliter of complete growth medium and add 2.5 milliliters of fresh complete growth medium to each well of a 6-well culture plate.

Using sterile forceps, place each insert right side up into each well of the 6-well plate so that the lower ovarian cancer cell attached surfaces are immersed in the media. Then, seed 1.5 milliliters of HPMC into the well of each insert and place the plate in the cell culture incubator for 72 hours.

• Paracrine Signaling - Cellular communication through secreted molecules affecting neighboring cells.
• Mesothelial Surface - The membrane where the mesothelial cells adhere and function.
• Culture System - Set-up where cells are grown under controlled conditions, often in vitro.
• Corning 10-013-CV - Specific type of culture dish used for cell adhesion and growth experiments.
• In Vitro Cell Culture Techniques - Methods used to cultivate cells in controlled environments outside of the living organism.

• Introduce Paracrine Signaling – Paracrine signaling is the process by which cells communicate and influence the behavior of neighboring cells via secreted molecules (e.g., paracrine).
• Key Concepts - We can simulate this biological process in an in vitro cell culture system using human mesothelial cells and cancer cells (e.g., culture system).
• Underlying Mechanisms - Both cell types secret signaling factors which bind to receptors and trigger complex responses (e.g., mesothelial surface).
• Connect to Experiment - To study this, cells are cultivated on either side of a porous membrane in a culture dish (e.g., corning 10-013-CV).

• What is paracrine signaling and how does it work in an in vitro culture system?
• How are mesothelial and cancer cells cultured for this study?
• What are the key concepts and mechanisms underlying paracrine signaling?

• Practical Applications - Understanding paracrine signaling could improve diagnosis and treatment of diseases like cancer (e.g., in vitro cell culture techniques).
• Industry Impact - The knowledge acquired is beneficial for the medical and pharmaceutical sectors (e.g., culture system).
• Societal Importance - The broader implications extend to improving patient outcomes and quality of care (e.g., mesothelial surface).
• Link to Scientific Advancements - The use of in vitro cultivation techniques continues to advance our understanding of cell communication (e.g., corning 10-013-CV).