FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
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).
Get Full Text
Publication Date: 2022-12-23 PubMed ID: 36564773PubMed Central: PMC9783463DOI: 10.1186/s12917-022-03552-6Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

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.

References

This article includes 24 references
  1. USDA. Part I: Baseline reference of equine health and management.. 2005.
  2. Gonzalez LM, Moeser AJ, Blikslager AT. Animal models of ischemia-reperfusion-induced intestinal injury: progress and promise for translational research.. Am J Physiol Gastrointest Liver Physiol 2015 Jan 15;308(2):G63-75.
    doi: 10.1152/ajpgi.00112.2013pmc: PMC4297854pubmed: 25414098google scholar: lookup
  3. Gonzalez LM. The mother of a gut cell: Intestinal epithelial stem cells.. Equine Veterinary Education 2015;27(11):559–560.
    doi: 10.1111/eve.12456google scholar: lookup
  4. Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, Watanabe M. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5⁺ stem cell.. Nat Med 2012 Mar 11;18(4):618-23.
    doi: 10.1038/nm.2695pubmed: 22406745google scholar: lookup
  5. Fordham RP, Yui S, Hannan NR, Soendergaard C, Madgwick A, Schweiger PJ, Nielsen OH, Vallier L, Pedersen RA, Nakamura T, Watanabe M, Jensen KB. Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury.. Cell Stem Cell 2013 Dec 5;13(6):734-44.
    doi: 10.1016/j.stem.2013.09.015pmc: PMC3858813pubmed: 24139758google scholar: lookup
  6. Stewart AS, Freund JM, Gonzalez LM. Advanced three-dimensional culture of equine intestinal epithelial stem cells.. Equine Vet J 2018 Mar;50(2):241-248.
    doi: 10.1111/evj.12734pmc: PMC5796842pubmed: 28792626google scholar: lookup
  7. Powell RH, Behnke MS. WRN conditioned media is sufficient for in vitro propagation of intestinal organoids from large farm and small companion animals.. Biol Open 2017 May 15;6(5):698-705.
    pmc: PMC5450310pubmed: 28347989doi: 10.1242/bio.021717google scholar: lookup
  8. Mohamed MS, Chen Y, Yao CL. Intestinal stem cells and stem cell-based therapy for intestinal diseases.. Cytotechnology 2015 Mar;67(2):177-89.
    doi: 10.1007/s10616-014-9753-9pmc: PMC4329309pubmed: 24981313google scholar: lookup
  9. Hong SN, Dunn JC, Stelzner M, Martín MG. Concise Review: The Potential Use of Intestinal Stem Cells to Treat Patients with Intestinal Failure.. Stem Cells Transl Med 2017 Feb;6(2):666-676.
    doi: 10.5966/sctm.2016-0153pmc: PMC5442796pubmed: 28191783google scholar: lookup
  10. Fuller MK, Faulk DM, Sundaram N, Mahe MM, Stout KM, von Furstenberg RJ, Smith BJ, McNaughton KK, Shroyer NF, Helmrath MA, Henning SJ. Intestinal stem cells remain viable after prolonged tissue storage.. Cell Tissue Res 2013 Nov;354(2):441-50.
    doi: 10.1007/s00441-013-1674-ypmc: PMC3812345pubmed: 23820734google scholar: lookup
  11. Scott A, Olack B, Rouch JD, Khalil HA, Kokubun BA, Lei NY, Wang J, Solorzano S, Lewis M, Dunn JCY, Stelzner MG, Niland JC, Martín MG. Comparison of Surgical and Cadaveric Intestine as a Source of Crypt Culture in Humans.. Cell Transplant 2020 Jan-Dec;29:963689720903709.
    doi: 10.1177/0963689720903709pmc: PMC7784595pubmed: 32907378google scholar: lookup
  12. Fuller MK, Faulk DM, Sundaram N, Shroyer NF, Henning SJ, Helmrath MA. Intestinal crypts reproducibly expand in culture.. J Surg Res 2012 Nov;178(1):48-54.
    doi: 10.1016/j.jss.2012.03.037pmc: PMC3654833pubmed: 22564827google scholar: lookup
  13. Seiler KM, Schenhals EL, von Furstenberg RJ, Allena BK, Smith BJ, Scaria D, Bresler MN, Dekaney CM, Henning SJ. Tissue underlying the intestinal epithelium elicits proliferation of intestinal stem cells following cytotoxic damage.. Cell Tissue Res 2015 Aug;361(2):427-38.
    doi: 10.1007/s00441-015-2111-1pmc: PMC4530061pubmed: 25693894google scholar: lookup
  14. Taelman J, Diaz M, Guiu J. Human Intestinal Organoids: Promise and Challenge.. Front Cell Dev Biol 2022;10:854740.
    doi: 10.3389/fcell.2022.854740pmc: PMC8962662pubmed: 35359445google scholar: lookup
  15. Blutt SE, Klein OD, Donowitz M, Shroyer N, Guha C, Estes MK. Use of organoids to study regenerative responses to intestinal damage.. Am J Physiol Gastrointest Liver Physiol 2019 Dec 1;317(6):G845-G852.
    doi: 10.1152/ajpgi.00346.2018pmc: PMC7132322pubmed: 31589468google scholar: lookup
  16. Khalil HA, Lei NY, Brinkley G, Scott A, Wang J, Kar UK, Jabaji ZB, Lewis M, Martín MG, Dunn JC, Stelzner MG. A novel culture system for adult porcine intestinal crypts.. Cell Tissue Res 2016 Jul;365(1):123-34.
    doi: 10.1007/s00441-016-2367-0pmc: PMC4919165pubmed: 26928041google scholar: lookup
  17. Gonzalez LM, Williamson I, Piedrahita JA, Blikslager AT, Magness ST. Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.. PLoS One 2013;8(6):e66465.
    doi: 10.1371/journal.pone.0066465pmc: PMC3696067pubmed: 23840480google scholar: lookup
  18. Chandra L, Borcherding DC, Kingsbury D, Atherly T, Ambrosini YM, Bourgois-Mochel A, Yuan W, Kimber M, Qi Y, Wang Q, Wannemuehler M, Ellinwood NM, Snella E, Martin M, Skala M, Meyerholz D, Estes M, Fernandez-Zapico ME, Jergens AE, Mochel JP, Allenspach K. Derivation of adult canine intestinal organoids for translational research in gastroenterology.. BMC Biol 2019 Apr 11;17(1):33.
    doi: 10.1186/s12915-019-0652-6pmc: PMC6460554pubmed: 30975131google scholar: lookup
  19. Meneses AMC, Schneeberger K, Kruitwagen HS, Penning LC, van Steenbeek FG, Burgener IA, Spee B. Intestinal Organoids-Current and Future Applications.. Vet Sci 2016 Oct 21;3(4).
    doi: 10.3390/vetsci3040031pmc: PMC5606586pubmed: 29056739google scholar: lookup
  20. Hamilton CA, Young R, Jayaraman S, Sehgal A, Paxton E, Thomson S, Katzer F, Hope J, Innes E, Morrison LJ, Mabbott NA. Development of in vitro enteroids derived from bovine small intestinal crypts.. Vet Res 2018 Jul 3;49(1):54.
    doi: 10.1186/s13567-018-0547-5pmc: PMC6029049pubmed: 29970174google scholar: lookup
  21. Derricott H, Luu L, Fong WY, Hartley CS, Johnston LJ, Armstrong SD, Randle N, Duckworth CA, Campbell BJ, Wastling JM, Coombes JL. Developing a 3D intestinal epithelium model for livestock species.. Cell Tissue Res 2019 Feb;375(2):409-424.
    doi: 10.1007/s00441-018-2924-9pmc: PMC6373265pubmed: 30259138google scholar: lookup
  22. Beaumont M, Blanc F, Cherbuy C, Egidy G, Giuffra E, Lacroix-Lamandé S, Wiedemann A. Intestinal organoids in farm animals.. Vet Res 2021 Feb 25;52(1):33.
    doi: 10.1186/s13567-021-00909-xpmc: PMC7905770pubmed: 33632315google scholar: lookup
  23. Zachos NC, Kovbasnjuk O, Foulke-Abel J, In J, Blutt SE, de Jonge HR, Estes MK, Donowitz M. Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology.. J Biol Chem 2016 Feb 19;291(8):3759-66.
    doi: 10.1074/jbc.R114.635995pmc: PMC4759158pubmed: 26677228google scholar: lookup
  24. Cook VL, Blikslager AT. The use of nonsteroidal anti-inflammatory drugs in critically ill horses.. J Vet Emerg Crit Care (San Antonio) 2015 Jan-Feb;25(1):76-88.
    doi: 10.1111/vec.12271pubmed: 25521286google scholar: lookup

Citations

This article has been cited 3 times.
  1. Yang R, Qi Y, Zhang X, Gao H, Yu Y. Living biobank: Standardization of organoid construction and challenges. Chin Med J (Engl) 2024 Dec 20;137(24):3050-3060.
    doi: 10.1097/CM9.0000000000003414pubmed: 39663560google scholar: lookup
  2. Windhaber C, Heckl A, Csukovich G, Pratscher B, Burgener IA, Biermann N, Dengler F. A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium. Vet Res 2024 Mar 16;55(1):30.
    doi: 10.1186/s13567-024-01283-0pubmed: 38493107google scholar: lookup
  3. Hellman S, Martin F, Tydén E, Sellin ME, Norman A, Hjertner B, Svedberg P, Fossum C. Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection. Vet Res 2024 Feb 27;55(1):25.
    doi: 10.1186/s13567-024-01266-1pubmed: 38414039google scholar: lookup
Subscribe to our newsletter
Join 99,357 horse owners like you who receive our email updates on horse health and nutrition.