Animals and sample collection
All animal use was approved by the Institutional Animal Care and Use Committee at North Carolina State University. Tissues were obtained from a total of 15 horses (12 geldings, 3 mares) that were not euthanized for this study but were free from overt signs of systemic disease, and euthanized for reasons unrelated to gastrointestinal disease such as orthopedic or behavioral conditions. Of note, at the time of euthanasia, several horses (n = 5) had been recently administered a non-steroidal anti-inflammatory (NSAID) medication. All jejunal tissue appeared grossly normal and was used in this study as reported adverse effects of NSAIDs are most often localized to the stomach and right dorsal colon . Horses ranged from 5–15 years of age and included 4 Thoroughbreds, 2 Warmbloods, 2 Quarterhorses, 2 Arabians and 1 of each of the following: Saddlebred, Haflinger, Morgan, Draft and Paint. Our previous work demonstrated no correlation between age and resultant enteroid efficiency . Immediately following euthanasia with pentobarbital sodium (Fatal-Plus, Vortech Pharmaceuticals, MI), several 20 cm long sections of mid-jejunum were removed through a celiotomy incision and placed into cold PBS. For storage studies, tissue sections were stored in PBS and placed into a 4 °C refrigerator for 24, 48 or 72 H prior to processing as described.
Intestinal epithelial crypt isolation, enteroid culture, and analysis
Intestinal epithelial stem cells (ISCs, crypt units) were isolated as described previously . Briefly, the excised jejunum was washed in PBS and opened longitudinally. The intestine was sectioned into smaller squares measuring 1–2 cm in diameter. Several small pieces of full thickness tissue were incubated for 30 min (min) in a 50 ml-conical tube with PBS containing 30 mM Ethylenediaminetetraacetic acid (EDTA), 10 mM Y-27632, 1 mM DTT, 1X antibiotic–antimycotic and Primocin (InvivoGen US, San Diego, CA). The conical tube was maintained on ice on an orbital shaking platform moving at 60 rpm in addition to vigorous shaking performed at 5 min intervals. Tissue pieces were transferred into a 37˚C pre-warmed PBS solution containing 30 mM EDTA, 10 mM Y-27632, 1X antibiotic–antimycotic and Primocin. The tissue was incubated in this solution at 37˚C for 10 min and shaken vigorously to help mobilize the crypt/villi units. Following this incubation, the tissues were placed in an ice-cold PBS wash containing 1X antibiotic–antimycotic for 5 min. Tissue was then transferred into additional washes and shaken until crypt/villi units were seen with minimal background debris as previously described . Following the final wash, the remnant intestine was removed and the remaining solution was filtered using a 100-micron sterile cell strainer to remove the villi. A desired crypt yield of approximately 50 crypts/50 µL was determined by examining several 50 µL aliquots at 10X. The crypts were then pelleted in preparation to plate.
The pelleted crypts were re-suspended directly into a growth factor reduced extracellular matrix (Matrigel®, Corning®, Corning, NY) supplemented with 100 ng/mL recombinant human Noggin, 500 ng/mL recombinant human R-Spondin, 50 ng/mL recombinant human EGF, 100 ng/mL recombinant human Wnt3a, 10 mM Y-27632, 1 mM Nicotinamide, 10 nM Gastrin, 10 mM SB202190, 500 nM LY2157299 and 2.5 μM glycogen synthase kinase 3 inhibitor (GSK3i, CHIR99021). Between 25–50 crypt units were plated in 50 µL of Matrigel on a 24 well plate. After allowing the matrix to polymerize for 30 min at 37˚C, each well was overlaid with 500 mL of Advanced DMEM/F12 containing the supplements 1X N-2 supplement, 1X B-27 supplement minus vitamin A, 1X Glutamax, 100 mg/mL penicillin/streptomycin and 1 mM Hepes buffer. Growth factors were added to the media 48H after plating and subsequent 48H intervals. The entire volume of media was changed 96H following plating and every subsequent 96H interval.
Plating efficiency was determined by first counting the number of enterospheres or enteroids formed every 24H for 5 days (120H) and then dividing the total count by the starting number of crypt units initially plated within each Matrigel matrix.
Intestinal stem cell rich bud units were counted per enteroid after 120H in culture to determine proliferative potential as each bud is capable of independently forming a new enteroid . For each timepoint, 5–10 enteroids were counted per horse per storage group.
Cryo-preservation of equine crypts and enteroid fragments
To assess the effect of freezing on growth efficiency of crypts, freshly isolated equine jejunal crypts were suspended in 1 ml freeze-preservation medium consisting of 10% dimethylsulfoxide (DMSO) in 1X DMEM/F12 culture medium with no growth factors and were frozen slowly over 12-18H using a cryo freezing container (Mr. Frosty ™, Thermo Fisher Scientific, Waltham, MA), containing isopropanol placed in a -80 ˚C freezer. This method is designed to achieve an optimum cooling rate for cell preservation of close to -1 °C/minute. Following removal from the -80 ˚C freezer, tubes were stored in liquid nitrogen. As previously described, cryopreservation tubes were obtained from liquid nitrogen storage and warmed in a 37˚C water bath to thaw . The tubes were centrifuged to pellet the crypts at 200G for 5 min. The freezing media was removed and the crypt pellet was resuspended in cooled Matrigel and plated as above on a 24 well plate . Efficiency was counted after 48 H in culture as described above.
To assess the effect of freezing on growth efficiency of enteroid fragments, mature, complex enteroids grown in culture for 120-168H were split using aggressive pipetting techniques and suspended in freeze preservation medium as described above. Fragments were then thawed and plated. Efficiency of resultant enteroids was calculated at 48H as described above.
Measurements and statistics
One author (ASS) was responsible for all enteroid measurements and cell counting. Enteroid area was measured using the “Measurement and ROI” feature from the Olympus micro imaging software cellSens (Olympus Corporation, Tokyo, Japan). Enteroids were identified at 24H post plating, tagged, and followed over a period of 120H. Five to ten enteroids were measured per horse at each storage time point. All measurements were performed in triplicate and averaged for each experiment. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). Outliers were identified using ROUT analysis. Samples were assessed for normality using the Shapiro–Wilk test and found to not be normally distributed. Subsequent nonparametric analysis was performed using the Kruskal–Wallis test with p < 0.05 significant and subsequent post-hoc analysis performed using Dunns multiple comparisons. Growth efficiency of cryopreserved crypt units compared to enteroid fragments was analysed using a Welch’s t-test. Based on our previous growth data, to achieve a power of 0.8, a sample size of 4 horses was required to detect a significant difference between sample means.