Isolation and Maintenance of Intestinal Stem Cell

Intestinal stem cells (ISCs) play a pivotal role in the maintenance and repair of the intestinal epithelium. Isolating and maintaining these cells in a controlled laboratory environment is crucial for studying their properties and potential therapeutic applications.

Creative Biolabs outlines a step-by-step procedure for the isolation and maintenance of intestinal stem cells. By following this protocol, researchers can obtain a pure population of ISCs, enabling in-depth investigations into their regenerative capabilities and therapeutic potential.

Materials Required

• Intestinal tissue samples
• Sterile phosphate-buffered saline (PBS)
• Hanks' balanced salt solution (HBSS)
• Dulbecco's modified eagle's medium (DMEM)
• Fetal bovine serum (FBS)
• Dispase II, Collagenase IV
• Cell culture media supplements

Procedure

Tissue Collection and Preparation

Obtain fresh intestinal tissue samples, ensuring timely processing of samples to maintain cell viability. Rinse tissue samples with sterile PBS to remove debris and blood. Transfer the tissue sample to a Petri dish containing HBSS, remove the mucosal layer and cut into small pieces.

Enzymatic Digestion

Transfer the mucosal fragments to a centrifuge tube containing Dispase II solution. Incubate for enzymatic digestion. Gently pipette the sample up and down to dissociate the cells. Transfer the digested cell suspension to a new centrifuge tube.

Cell Dissociation

Add Collagenase IV solution to the digested cell suspension. Incubate and shake the tube intermittently. Pipette samples up and down every 10-15 minutes to promote dissociation of cells. Filter the dissociated cell suspension to remove any remaining tissue debris. Collect the filtrate into a new centrifuge tube.

Centrifugation and Cell Resuspension

Centrifuge the cell suspension, discard the supernatant, and collect the cell pellets. Resuspend the cell pellets in DMEM and transfer them to a cell culture flask for culture.

Maintenance and Expansion of ISCs

Change the medium with fresh DMEM every 2-3 days. Monitor cell growth and proliferation under an inverted microscope. Once the cells reach approximately 70-80% confluency, passage the cells using dissociation reagent. Repeat the process of cell expansion and passage as necessary to maintain an adequate supply of ISCs.

Notes

• Strict aseptic techniques should be followed throughout the entire procedure to prevent contamination.
• Adjust the digestion and dissociation times according to the tissue source and age of the animals.
• The cell culture flasks should be coated with extracellular matrix proteins to enhance ISC attachment and growth.
• Avoid excessive pipetting or shaking, as it may compromise HSC viability and functionality.
• Careful handling and manipulation of the cells are crucial to avoid excessive stress and potential damage to the ISCs.

This protocol serves as a guide for obtaining a pure population of ISCs from intestinal tissue samples, allowing for further exploration of their molecular characteristics and therapeutic potential. Creative Biolabs is committed to advancing our understanding of ISC biology and contributing to the development of innovative approaches for intestinal repair and regeneration.

If you have any questions or comments, please do not hesitate to contact us.

Reference

1. Jung P, et al. Isolation and in vitro expansion of human colonic stem cells. Nature medicine, 2011, 17(10): 1225-1227.

For Research Use Only. Not For Clinical Use.