Lamina Propria Mononuclear Cell Isolation: A Silica-based Density Gradient Centrifugation Technique to Obtain Mononuclear Cells from Murine Colon

The intestinal lamina propria, located below the intestinal epithelium, consists of loose connective tissue along with a dense network of collagen fibers containing a diverse array of cells such as fibroblasts, mononuclear cells, including lymphocytes and monocytes. These cells play a crucial role in maintaining homeostasis.

To isolate mononuclear cells, begin by taking mouse colon fragments. Treat the fragments with a digestion buffer containing collagenase enzymes. Collagenase enzymes digest the collagen fibers within the tissue, releasing the cells into suspension. 

Collect the supernatant and filter it through cell strainers to remove undigested tissue and debris and obtain a single-cell suspension. Add a chilled buffer to the filtrate to inactivate the collagenase enzymes. 

Centrifuge to separate the cells. Discard the supernatant containing the enzymes and resuspend the cell pellet in a low-density silica-based separation media. Next, layer the suspension over a high-density silica-based separation media to create a separation gradient.

Centrifuge the mixture. The mononuclear cells sediment and form a white layer at the interface between the density gradient layers. Carefully transfer the white interface layer into a fresh tube containing a suitable buffer.

Centrifuge to obtain a pellet containing purified mononuclear cells. Resuspend the pellet in buffer for further downstream analysis.

To begin, add 20 milliliters of collagenase digestion buffer to the tube containing the washed colon fragments. Seal the tube and place it in an orbital shaker at 37 degrees Celsius with a rotation rate of 2 x g for 60 minutes. Ensure the tissue fragments are in constant motion during agitation.

Pour 5 milliliters of 66% silica-based density separation media into each of three separate 15-milliliter polypropylene tubes. Next, use a 25-milliliter serological pipette to collect only the supernatant from Collagenase Digestion 1. Filter the supernatant through a 40-microliter-pore filtration fabric cell strainer placed into a clean 50-milliliter polypropylene conical tube. Fill the tube completely with chilled colon buffer to quench the collagenase digestion buffer. Then, spin the cells, aspirate the supernatant, and keep the pellet on ice.

Continue to perform Collagenase Digestion 2 as outlined in the text protocol. After Collagenase Digestion 2 is completed, flush the tissue fragments vigorously back and forth between the tube and a 10-milliliter syringe through an 18G blunt-end needle. Repeat this flush for at least seven to eight passages, continuing until no gross tissue fragments or debris are visible.

Next, pass the tissue disaggregation suspension through a 40-micrometer-pore filtration fabric cell strainer placed into a clean 50-milliliter polypropylene tube. Fill the tube to the rim with chilled colon buffer to quench the digestion and centrifuge at 800 x g and at 4 degrees Celsius for 5 minutes.

Discard the supernatant via vacuum aspiration and pull the resuspended pellet from Collagenase Digestion 1 to its corresponding tube. After this, resuspend each pellet in 24 milliliters of 44% silica-based density separation media per colon. Use a 10-milliliter serological pipette to slowly layer 8 milliliters of this media onto each of three tubes containing 66% silica-based density separation media.

Carefully balance the tubes within centrifuge buckets using a weigh scale or a balance. Centrifuge at 859 x g and at 20 degrees Celsius for 20 minutes without the break. Allow the rotors to come to complete rest before removing tubes, taking care not to disrupt the cells at the gradient interface.

Visualize the gradient interface near the 5-milliliter mark where typically a 1-2 millimeter thick white band is present. Vacuum aspirate and discard the top 7 milliliters of the gradient to allow easier pipette access to the interface. Using continuous manual suction and steady rotating wrist motion, collect the interface layer of cells. Collect until the interface between the two gradients is clear and refractile, and transfer the interface to a new 50-milliliter polypropylene conical tube.

• Intestinal Lamina Propria - A loose connective tissue below the intestinal epithelium; contains various cells to maintain homeostasis.
• Mononuclear Cells - Cells crucial for maintaining homeostasis; can be extracted from the lamina propria.
• Collagenase Enzymes - Aid in the digestion of collagen fibers, thus releasing cells into suspension.
• Density Gradient Separation - Process used for separating cells based on their densities.
• Silica-Based Separation Media - Substances used in creating a density gradient for cell separation.

• Introduce Intestinal Lamina Propria as a crucial component of immune system's functions. (e.g., homeostasis)
• Mononuclear cells play a vital role in maintaining homeostasis. (e.g., tissue regulation)
• Collagenase enzymes enable mononuclear cells extraction (e.g., collagen digestion)
• Connect isolation of mononuclear cells to further tissue research and cell behavior studies.

• What is the role of intestinal lamina propria in homeostasis?
• How are mononuclear cells isolated using collagenase enzymes?
• What is the purpose of density gradient separation in cell isolation process?

• Understanding of lamina propria and its cell population has practical implications in healthcare. (e.g., tissue repair)
• Mononuclear cell extraction techniques have potential impact in research sector. (e.g., drug testing)
• Insights gained are important for broader understanding of tissue behaviour (e.g., disease study)
• Extraction of mononuclear cells contribute to advancements in cellular biology.