Method Article

An Optimized Sequential Isolation of Crypts and Mesenchymal Stromal Cells from Porcine Intestinal Tissue

DOI:

10.3791/69429

⸱

December 19th, 2025

 , 
1Department of Animal and Avian Sciences, University of Maryland

In This Article

Summary

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This study provides a stepwise protocol for the sequential isolation of intestinal crypts and intestinal mesenchymal stromal cells (iMSCs) from porcine jejunal tissue. The method generates 2D enteroid monolayers and iMSCs suitable for developing reproducible, physiologically relevant epithelial-stromal co-culture models to investigate cellular crosstalk during intestinal homeostasis and regeneration.

Abstract

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The intestinal epithelium closely interacts with the underlying intestinal mesenchymal stromal cells (iMSCs) to regulate development, homeostasis, and regeneration. However, standardized methods for sequentially isolating both epithelial and stromal fractions from porcine tissue are limited. This protocol describes an optimized stepwise process for isolating crypts and iMSCs from the same jejunal segment of piglets. The method uses an ethylenediaminetetraacetic acid/dithiothreitol (EDTA/DTT)-based dissociation buffer to release crypts, followed by collagenase/dispase digestion of the extracellular matrix to isolate iMSCs. This approach minimizes spatial variations in the isolated cells. The isolated crypts were successfully cultured and formed typical expanding, two-dimensional (2D) enteroid monolayers on collagen hydrogel-coated plates. The cultured iMSCs adhered to culture flasks and expressed stromal markers such as platelet-derived growth factor receptor alpha (PDGFRα) and cluster of differentiation 81 (CD81), as determined by immunostaining. Overall, this optimized isolation protocol yields both epithelial and stromal cells from the same piglet jejunal segment, providing a reproducible foundation for future research involving intestinal organoids and iMSC co-culture systems to elucidate mechanisms controlling epithelial-stromal interactions in pigs, a translationally relevant model for replicating human physiology and advancing animal sciences.

Introduction

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The intestinal epithelium is a dynamic tissue that undergoes continuous self-renewal and differentiation to maintain intestinal homeostasis and facilitate regeneration after mucosal injuries1,2. Intestinal epithelial cells originate from intestinal stem cells (ISCs), and they interact with diverse microenvironment factors3,4,5,6,7,8. The subepithelial compartments, mainly the lamina propria and muscularis mucosa, c....

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Protocol

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A 5 cm segment was dissected from the excised proximal jejunum of 21-day-old piglets housed in the pig facility at the University of Maryland, College Park. The procedure (R-JUN-22-30) used to sacrifice the piglet followed guidelines for animal care and use policies approved by the university's Institutional Animal Care and Use Committee. Before proceeding with the isolation steps, the protocol outline is presented in Figure 1, and the list of reagents and equipment used for the sequential isolation of the porcine intestinal crypts and iMSCs is provided in the Table of Materials.

1. P....

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Results

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During epithelial dissociation, ethylenediaminetetraacetic acid (EDTA) chelates calcium ions to disrupt calcium-dependent cell-cell adhesions, while dithiothreitol (DTT) cleaves disulfide bonds in mucin glycoproteins to facilitate mucolysis. Thus, the combined use of a chelating compound (EDTA) and a reducing agent (DTT) enables effective release of crypts from the epithelial membrane without disrupting the extracellular matrix (ECM) structure2,32 (

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Discussion

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Isolating intestinal crypts and iMSCs from the same porcine jejunal segment offers a novel approach for preserving tissue segment-specific cellular and functional characteristics. This is crucial because intestinal tissues exhibit regional and spatial identities, as demonstrated in previous porcine studies2,36,37,38. Prior organoid studies in pigs or other species, which relied on crypts isolat.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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The authors appreciate financial support from the MAES Competitive Grant Program at the University of Maryland, College Park, USA.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.5M EDTAPromegaPR-V4231
1 M HEPES bufferThermoFisher15630080For collagen hydrogel plate preparation
1000 uL pipet tipsAlkali Scientific FT1000104027-530
10x PBSInvitrogenAM9624For collagen hydrogel plate preparation
1N sodium hydroxideThermoSS266-1For collagen hydrogel plate preparation
1x HBSSThermoFisher14025092
1X PBSHycloneSH30256FS
4 % paraformaldehyde (PFA)ThermoFisherAAJ61899AK
50 mL conical tubesThermoFisher12-565-271
6 well platesFisherbrandFB012927
7.5% Sodium Bicarbonate SolutionCorningMT25035CIFor collagen hydrogel plate preparation
Advance DMEM/F12 Gibco 12634-010
Alexa fluor plus 647InvitrogenA32728Secondary antibody
Alexa plus 555 - A32732 InvitrogenA32732Secondary antibody
Aluminum foilReynoldshttps://www.amazon.com/dp/B00M8ZEAW4?ref=fed_asin_title&th=1
ANTI-ANTIGibco15240096
B27ThermoFisher12587010
BSAFisherbrandBP1600-100
CD81 Proteinintech66866-1-igPrimary antibody
Cell Culture Grade WaterHycloneSH30529FSFor collagen hydrogel plate preparation
Collagen, Type 1Corning356236For collagen hydrogel plate preparation
Collagenase IVGibco17104019
Corning 100 mm petri dishCorning07-202-011
Cryostor cell cryopreservation mediaStemcell Technologies7930
cryovial for cells Fisherbrand1050026
Dako antibody diluentDako/AgilentPart number: S302283-2primary antibody diluent
Dako fluorescent mounting mediaDako/AgilentPart number: S302380-2
DAPIPromocell/VWR10180-470DNA staining
Dispase IIRoche4942078001
DMEM, high glucoseCorningMT10013CV
DTTSigma646563
EGFPeprotech315-09
FBS (HI)Avantor1300-500H
GastrinAnaspecAS-64149
GentamicinMP BiomedicalsICN1676045
GlutaMaxThermoFisher35050061
HEPESThermoFisher15630-080
Inverted flourescent microscrropeNikonECLIPSE Ti2Flourescent imaging
Inverted phase-contrast microscopeNikonEclipse TS100For cultured cells imaging 
iScript Advanced cDNA Synthesis KitBio-Rad1725038RT-PCR kit
iTaq Universal SYBR Green SupermixBio-Rad1725122qPCR kit
LWRN conditional mediaATCCCRL-3276LWRN media was prepared in house according to the ATCC procedure
N-acetyl cysteineMP Bio194603
Nanodrop 2000 spectrophotometerThermoFisherND 2000
NicotinamideSigmaN0636-100G
Optical 384-Well Plates with BarcodeApplied Biosystems4483285qPCR kit
PDGFRαAbcamab230457Primary antibody
Prostaglandin E2 (PGE2)Cayman chemical 14010
QuantStudio 5Applied BiosystemA28570qPCR instrument
SB202190LC LaboratoriesS-1700
Stereo microscopeNikon SMZ745
T 75 flaskFisherbrandFB012937
Triton X100FisherbrandPI85111
Trizol reagent Invitrogen15596026
Tween 20, 100% Nonionic DetergentBio-rad1706531
Y27632ApexBioA3008-200

References

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  1. Gehart, H., Clevers, H. Tales from the crypt: New insights into intestinal stem cells. Nat Rev Gastroenterol Hepatol. 16 (1), 19-34 (2019).
  2. Yin, L., et al. Changes in progenitors and differentiated epithelial cells of neonatal piglets. Anim Nutr....

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Tags

Crypt IsolationMesenchymal Stromal CellsPorcine Intestinal TissueSequential IsolationEDTA DTT DissociationCollagenase DigestionEnteroid MonolayersCollagen HydrogelStromal MarkersEpithelial Stromal Interactions
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