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Home / Equine Research Bank / BMC Vet Res / Culture of equine intestinal epithelial stem cells after del...
BMC veterinary research2022; 18(1); 445; doi: 10.1186/s12917-022-03552-6

Culture of equine intestinal epithelial stem cells after delayed tissue storage for future applications.

Abstract: Equine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sample storage has not been described. The objective of this study was to describe the isolation and culture of equine ISCs following delayed tissue storage. Small intestinal full thickness biopsies were collected post euthanasia. Fresh tissue was immediately processed or stored at 4 °C for 24, 48 and 72 h (H) before processing. Intestinal stem cells (crypts) were dissociated and cultured. Size, growth efficiency and proliferation potential were compared between resultant enteroids ("mini-guts") derived from each storage timepoint. In a separate study, growth efficiency of cryopreserved crypts was compared to cryopreserved enteroid fragments to investigate prolonged storage techniques. Results: Intestinal crypts were successfully isolated and cultured from all timepoints. At 72H post initial collection, the intestine was friable with epithelial sloughing; resultant dissociation yielded more partial crypts. Enteroids grown from crypts isolated at 72H were smaller with less proliferative potential (bud units, (median 6.5, 3.75-14.25)) than control (median 25, 15-28, p < 0.0001). No statistical differences were noted from tissues stored for 24H compared to control. Following cryopreservation, growth efficiency improved when cells were stored as enteroid fragments (median 81.6%, 66.2-109) compared to crypts (median 21.2%, 20-21.5, p = 0.01). The main limitations included a small sample size and lack of additional functional assays on enteroids. Conclusions: Equine ISCs can be isolated and cultured after prolonged tissue storage. Resultant enteroids had minimal differences even after 24-48H of whole tissue storage. This suggests that ISCs could be isolated for several days from samples properly stored after procedures, including surgery or necropsy, and used to create ISC repositories for study or therapy of equine intestinal diseases.
© 2022. The Author(s).
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Publication Date: 2022-12-23 PubMed ID: 36564773PubMed Central: PMC9783463DOI: 10.1186/s12917-022-03552-6Google Scholar: Lookup
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  • Journal Article

Summary

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This research investigates the possibility of isolating and storing equine Intestinal Stem Cells (ISCs) for future experimental or therapeutic use, and specifically how this can be achieved after a delayed tissue storage period. The findings suggest that reliable extraction and potent cell growth of equine ISC is possible, even after whole tissue storage for up to 48 hours.

Research Objectives

  • The research primarily aimed to explore the feasibility of isolating and cultivating equine ISCs following delayed tissue storage. This facilitates functionalities like sample repositories and future therapeutic applications among others.
  • Researchers gathered small intestinal tissue samples post-euthanasia. These samples were either processed immediately or stored at 4 °C for 24, 48, and 72 hours prior to processing. The study compared the size, growth effectiveness, and proliferation potential of the stem cell derived enteroids from each storage duration.
  • The study also compared the growth efficiency of cryopreserved crypts with cryopreserved enteroid fragments to understand extended storage techniques.

Research Findings

  • Intestinal crypts (a type of ISCs) were successfully isolated and brought under cultivation from samples stored at all mentioned timepoints.
  • Notably, at 72 hours post initial collection, the intestinal tissue started to degrade with epithelial shedding, resulting in the production of more incomplete crypts.
  • The enteroids grown from crypts isolated at the 72-hour mark were smaller and demonstrated less proliferative potential compared to the control group. However, no significant difference was observed in stems cells stored for 24 hours compared to the control.
  • Upon cryopreservation, a superior growth efficiency yield was observed when cells were preserved as enteroid fragments as compared to crypt cells.

Limits and Conclusions

  • The sample size in this study was minimal, which could potentially impact the how extensive these

Cite This Article

APA
Stewart AS, Schaaf CR, Veerasammy B, Freund JM, Gonzalez LM. (2022). Culture of equine intestinal epithelial stem cells after delayed tissue storage for future applications. BMC Vet Res, 18(1), 445. https://doi.org/10.1186/s12917-022-03552-6

Publication

BMC veterinary research
ISSN: 1746-6148
NlmUniqueID: 101249759
Country: England
Language: English
Volume: 18
Issue: 1
Pages: 445
PII: 445

Researcher Affiliations

Stewart, Amy Stieler
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC, 27607, USA.
Schaaf, Cecilia R
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC, 27607, USA.
Veerasammy, Brittany
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC, 27607, USA.
Freund, John M
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC, 27607, USA.
Gonzalez, Liara M
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC, 27607, USA. lmgonza4@ncsu.edu.

MeSH Terms

  • Animals
  • Cryopreservation / veterinary
  • Epithelial Cells / cytology
  • Horses
  • Intestines / cytology
  • Stem Cells / cytology
  • Time Factors
  • Cells, Cultured
  • Intestinal Diseases / therapy
  • Intestinal Diseases / veterinary

Grant Funding

  • 4T32OD011130-09, Ruth L. Kirschstein NRSA / NIH HHS
  • SERCA K01OD0199 / Office of Research Infrastructure Programs, National Institutes of Health

Conflict of Interest Statement

The authors declare that they have no competing interests.

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Citations

This article has been cited 3 times.
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