Ex vivo Organoid Cultures from Intestinal Stem Cells

Introduction: This poster presentation explores recent developments in ex vivo intestinal organoid cultures, shedding light on their potential applications in therapeutic interventions. Optimal standardized methodologies for the growth and maintenance of intestinal organoids were used, emphasizing key factors that contribute to the success of these cultures. This project is a preliminary study to establish a successful baseline intestinal organoid culture for future experimentation. Organoid growth, morphology, and viability were all evaluated.

Methods: Techniques from STEMCELL Technologies Intestinal Epithelial Organoid Culture protocol were employed, aiming to recapitulate the complex cellular diversity of the intestinal epithelium. Intestinal crypts were isolated from two male mice. These crypts were then resuspended and plated in wells with an undiluted Matrigel matrix to allow for 3D culturing, a technique demonstrated to improve emulation of the in vivo environment. To each well, growth medium was added that contained specific growth factors shown to be necessary for the differentiation and proliferation of intestinal stem cells (ISCs). The media were changed every 48 hours to give the organoids longevity and promote continued growth and differentiation. The organoids were passaged at 7-day intervals to ensure proper proliferation and fresh matrix. Light microscopy was used to image organoid growth and count organoids with proper development from the crypt stem cells. Organoid total RNA was isolated for mRNA expression analyses of ISC and cell differentiation markers using reverse transcription quantitative PCR (RT qPCR) Cultures were also prepared for detection of cell proliferation and ISC protein markers by immunocytochemistry.

Results: Intestinal crypts were successfully isolated from the donor mice and then used to grow intestinal organoids. Imaging revealed typical organoids grown in the Matrigel matrix. RNA was successfully isolated and RT qPCR showed expression ISC and cell differentiation markers. Immunocytochemistry staining is in progress to examine protein expression.

Conclusions: Insights into the recreation of in vivo conditions within these “miniaturized organ” cultures offer a promising platform for studying intestinal disorders and evaluating potential therapeutic interventions. This research contributes to the evolving landscape of regenerative medicine, personalized therapy, and disease modeling.